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googleのランキングの要因は伝説のものです。

そこには、googleのランキングを知らせる200の以上のシグナルと噂(この統計は、2006年に始まったが、物事がそれ以来少しも変わってきたと言うことはおそらく安全だ)されており、正確なリストを作る要因だけでなく、彼らの重要な順序は、多年生の議論の対象です。

で、私たちますが検索エンジン・ウォッチは、決してグーグルの完全なリストを主張することができますランキング要因(と彼らはあなたに嘘をついていることができますと言う誰も-そう、彼らはgoogleからの方にも、おそらく)、我々は掘り下げました主題はかなり少しです。

去年私たちの勇敢な編集者christopher ratcliffは、いくつかのgoogleの重要なランク付け要素の詳細を調べる10回シリーズの記事を書きました。このガイドはあなたの参照の便宜のためにそのシリーズからの主要な洞察を要約します、それぞれのランキング要因の完全な説明へのリンク。

コンテンツの鮮度からコンテンツの品質、内部リンクからバックリンクまで、googleの堅実なランキングを達成するために必要な主なポイント、およびそれらのヒット方法について説明しました。

それでさらに面倒なことなしに始めましょう。

にジャンプします。

パート1:ページ要因
パート2:キーワード
パート3:品質の高い内容
パート4:コンテンツの鮮度
パート5:重複するコンテンツとシンジケーション
パート6:信頼、権威および専門知識
パート7:サイトレベルの信号
パート8:内部リンク
パート9:アウトバウンドリンク
パート10:バックリンク
ボーナス:ランクブレイン

パート1:ページ要因
googleのランク付け要因に関するガイドの最初の部分では、googleがページのランク付けに使用する単純で技術的な要素(タイトルタグ、h1タグ、メタ説明)について説明します。

これらはすべてあなたが完全にコントロールできる要素であり、googleがあなたのサイトをランク付けする方法とあなたのサイトがserpに現れる方法の両方に重要な影響を与えます。したがって、それらを適切に最適化する方法を学ぶことは非常に重要です。

タイトルタグ、h1タグ、および検索用のメタ説明を最適化する方法に関するいくつかの重要なポイント:

ランク付けしたいキーワードをタイトルタグに含めます。キーワードがタグの先頭に近いほど、そのキーワードでページがランク付けされる可能性が高くなります。
そうは言っても、あなたのタイトルタグが人間のために書かれていることを確認してください – それは彼らがまだ論理的な意味を成す必要があり、キーワードだけでいっぱいに詰め込まれることを意味します
あなたのウェブサイトでタイトルタグを重複させないでください。
ターゲットキーワードもh1タグに含める必要がありますが、h1はタイトルタグと異なる場合があります
通常は1ページに1つのh1タグしか使用できませんが、h2タグとh3タグを使用してコンテンツをさらに分割することができます。
メタディスクリプションは厳密にはランキングのシグナルではありませんが、優れたメタディスクリプションはクリック率を大幅に向上させることができるため、賢明に使用してください。
seoのタイトルタグとメタ説明の書き方についてさらに深く知りたい場合は、2つの別々のガイドをチェックしてください。

seoのメタタイトルタグの書き方
seoのためのメタ説明を書く方法

パート2:キーワード
ランキング要因ガイドのパート2では、seoに関する議論の永遠の主題であるキーワードについて説明しています。

seoにおけるキーワードの役割は、検索の初期から大きく変化してきましたが、ロングテールキーワードと自然言語検索の進化により、控え目なキーワードは依然として検索最適化の重要な基本要素の1つであり、googleの重要なランキングです。信号。

しかし、前のセクションで説明したように、キーワードが重要であるからといって、それらを狂ったように詰め込むべきではありません。ここでは、キーワードの上手な使い方についてのgoogleのランキング要因に関するガイドからのハイライトをいくつか紹介します。

キーワードの関連性と掲載順位は、頻度よりもはるかに重要です。最初の文ではない場合、あなたのキーワードまたはキーフレーズはあなたのページの最初の100単語に現れるべきです
グーグルは最初にメタ情報とヘッダーを優先し、次にボディーコピー、そして最後にサイドバーとフッターを優先する。
キーフレーズは、検索者が検索エンジンに入力する内容と完全に一致するようにしてください。これは、自然言語検索クエリに最適化したい場合は、キーワードを会話形式で表現することを意味します。
過度のキーワードの繰り返し、および他のコンテンツに関係のないキーワードの使用は、ペナルティを受ける可能性があります。
ドメインのurlにキーワードを含めると、 seoが少し向上します。

パート3:品質の高い内容
あなたのブログやサイトをグーグルで高く評価する方法として「品質の高いコンテンツ」という言葉が使われているのは間違いありません。「品質の高いコンテンツを制作する」。

まあ、それはすべて非常によくて良いです、しかしそれは実際的にはどういう意味ですか?制作しているコンテンツがgoogleにとって十分に高品質であるかどうかをどうやって知ることができますか。

googleのランキング要因に関するガイドの第3部では、スペルや文法から読みやすさ、フォーマット、長さまで、コンテンツの品質を評価するための14のヒントを紹介します。これが私たちのポインタのいくつかです:

前に説明したように、コンテンツは、アルゴリズムだけでなく人間に訴えるように書かれていることを確認し、キーワードで飽和させないでください。
fleschの読みやすさテストでコンテンツの読みやすさのスコアを確認し、60%以上になることを目指します
文章や段落を短くして、改行(モバイルでは読みやすくするための余白が多くなります)や小見出しでそれらを分割してください。
文章や段落を短くしたいのですが、全体的な内容は空想のままにすることができます。詳細な内容は品質の大きな指標です。

パート4:コンテンツの鮮度
ページ上のコンテンツのシグナルを続けていくと、webページが最近公開された時期もランク付けのシグナルです。ただし、最近のイベントの検索、話題のトピック、定期的な定期的なイベントなど、検索の種類は異なります。

googleのアルゴリズムは、検索を最も関連性があり最新の結果と一致させるときに、これらすべてを考慮に入れようとします。

昨年、mozは、コンテンツの鮮度がgoogleのランキングにどのように影響するかについて包括的な調査結果を発表しました。これは、googleの順位要素に関するガイドの第4部にある洞察の基盤となります。主な要点は次のとおりです。

webページには、その発行日に基づいて、コンテンツが古くなるにつれて時間の経過とともに減衰する即時の「鮮度スコア」を付与できます。コンテンツを定期的に更新すると、そのスコアを維持するのに役立ちます。
コンテンツにリンクしている外部サイトの数の増加は、関連性と鮮度の指標と見なすことができます。
「新鮮な」サイトからのリンクはあなたのコンテンツにその新鮮さを伝えるのを助けることができます
最新の結果が常に最良というわけではありません。ニュース価値の低いトピックでは、詳細で信頼性の高い結果が新しいコンテンツよりも細いコンテンツを上回る可能性があります。

パート5:重複するコンテンツとシンジケーション
あなたのウェブサイトで他人のコンテンツを再公開すること、またはあなた自身のコンテンツを社内で再利用することが許容されるのはいつですか?seoコミュニティは、偶然にも「重複コンテンツペナルティ」を犯してしまうという恐怖を共有しており、それを回避する方法についてアドバイスがたくさんあります。

確かに、許可なく他人のコンテンツを盗んで再公開することはひどいやり方であり、これを頻繁に行うことはスパムで質の低いwebサイトの明らかな兆候です。ただし、ann smartyがfaqで重複コンテンツについて説明しているように、「重複コンテンツのペナルティ」というようなことはありません。グーグルの誰もそのようなペナルティの存在を確認したことはなく、また「重複した内容」のアルゴリズムの更新もなかった。

それでは、重複したコンテンツを公開することの危険性は何ですか?つまり、オンラインで同じ投稿に複数のバージョンがある場合、googleはどの記事をランク付けするかについて電話をかけ、最初に公開された記事とは無関係に検索を行います。最高権威。

同様に、ランキングをめぐって競合する同じ内部コンテンツの複数のバージョンがある場合(これには同じサイトの別々のデスクトップバージョンとモバイルバージョンが含まれます)、足を踏み入れて自分を撮影することができます。

どうすればこれをすべて回避できますか。googleのランク付け要素の記事の第5部では、重複して配信されるコンテンツを管理してgoogleが優先urlのみをインデックスに登録するようにする方法について説明しています。いくつかのポイントが含まれます:

自分のサイトに重複したコンテンツがある場合は、301リダイレクトを設定して、googleが自分の優先ページにインデックスを付けるようにする
別の携帯サイトの代わりにレスポンシブwebサイトを使用する
シンジケートコンテンツにrel = canonicalタグまたはmeta noindexタグを使用して、どの記事がオリジナルであるかをgoogleに伝えます。

パート6:信頼、権威および専門知識
特にリンク構築キャンペーンに関しては、高いレベルの権限を持つwebサイトがより大きな重みを持っていることを私たちは知っています。しかし、グーグルはあなたのウェブサイトの信頼、権威そして専門性のレベルをどのように評価しているのでしょうか?

googleのランキングの要因に関するガイドの第6部では、googleがどのように権限、信頼、専門知識を計算するのかと同様に、あなたのサイトのページ品質評価を構成する要因を調べます。主なポイントは次のとおりです。

コンテンツの品質、コンテンツの量、およびwebサイト情報は、すべてページ品質評価の要素です。
論理サイトアーキテクチャは、より高いレベルの権限を手助けすることができます。
ブログを始めることはあなたのビジネスが関連していて信頼できることを示すのを助けることができます – そしてまた、コンテンツの新鮮さを手助けすること
特に総レビュー数が多い場合は、マイナスの顧客レビューがページの品質評価に影響を与えることはありません。googleは実際の評価よりも、コンテンツのレビューを確認する傾向があります。

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パート7:サイトレベルの信号
ページ上のコンテンツから離れて、googleのランキング要因に関するガイドの第7部ではサイトレベルのシグナルに注目しています。

googleは、サイトレベルであなたのランキングに影響を与える可能性のある要因をどのように考慮していますか?ここにいくつかあります…

https:googleは2014年に、httpsを「非常に軽量な信号」として使用し始めたと発表しました。それ以来それが強化されたかどうかはわからないが、特にあなたのウェブサイトが金融取引を扱うならば、httpsの使用も一般的にちょうど良い習慣です
携帯性:2015年の最初の「mobilegeddon」更新以降、携帯性はgoogleの検索結果における重要な要素となっています。それ以来、信号は強化されてきました
サイトの速度:特に携帯電話でサイトの速度を評価して最適化することに時間をかけてください。そうすれば、検索のランキングが向上することに気付くでしょう。

パート8:内部リンク
私たちのランキングの要因のパート8、9、10はすべて、インターネットの神経系を扱うものです:リンク。さまざまな種類のリンクがどのようにあなたのサイトが検索でうまくランク付けするのを助けますか?

まず、内部リンクです。starcomのjason mcgovernによると、内部リンクは、特定のページのコンテンツが重要であることをgoogle(および訪問者)に伝えるために使用できる数少ない方法の1つです。それでは、どのようにあなたはあなたのウェブサイトの他のページに内部的にリンクすることについて行くべきですか?

パート8では、「ハブページ」やその構築方法など、内部リンクを使用してサイトの指標やユーザーエクスペリエンスを向上させる方法について説明します。

重要なポイントを理解したら、seoのための内部リンクの完全ガイド:例とベストプラクティスを必ずチェックしてください。

パート9:アウトバウンドリンク
アウトバウンドリンク、つまり外部リンクは、あなたのサイトから別のウェブサイトへと外側を向いているリンクです。それらはあなたがリンクしているサイトにあなた自身のサイトのランキング力の一部を(リンクがスーパースパムのウェブサイトに行かない限りあなたに何の害もなしに)伝えます。

しかし、これはどのようにあなたに利益をもたらしますか?なぜあなたは他のサイトへのリンクジュースの景品をリンクしているのでしょうか?

実際、googleのgoogleのランキング要因ガイドの第9部で明らかにされているように、関連性のある権威のあるサイトへの外部リンクはあなたのランキングに役立ちます。アウトバウンドリンクとseoに関するその他の重要なポイントは次のとおりです。

pagerankの保持は神話です – あなたのサイトが内部リンクより外部リンクを持つことによってリンクジュースを「漏らす」ことは不可能です
実際、アウトバウンドリンクは信頼のシグナルとしてカウントされます – あなたがあなたのデータと研究をバックアップするために参照にリンクしているならば、あなたは明らかにあなたの仕事をきちんと終えて信頼できます。
アフィリエイトリンクも問題ありませんが、必ずgoogleのベストプラクティスに従ってnofollowメタタグを使用してください。

パート10:バックリンク
なぜバックリンク(第三者からあなたのサイトに戻るリンク)がseoにとって重要なのですか?先ほど説明したように、自分のサイトから別のwebサイトへの外部リンクは、ランク付けの力の一部を通ります – その逆もまた当てはまります。

オンラインで他の場所からあなたのサイトにリンクを張ることはあなたの検索ランキングを改善するための重要な方法です。実際、昨年google irelandのsearch quality senior strategistであるandrey lipattsevによって明らかにされたように、あなたのウェブサイトへのリンクは上位3つのランク付け要因の1つです。

それで、seoでのリンク構築の周りに急成長している取引があります – リンクを構築する方法についてのアドバイスとリンク自体を売買することの両方において。ただし、有料リンクの構築はブラックハットseoと見なされ、ペナルティを受ける可能性があります。

グーグルは、無料ギフトと交換されるブログ上のリンクなど、さまざまな種類の有料リンクをさまざまな時期に締め付けてきた。これにより、多くのseo がリンク構築の実行に慎重になりました。しかし、グーグルは原則的にリンク構築に対して何もしていない – それどころか、グーグルはリンクに頼ってどんなウェブサイトがすべてについてであるか、そして特定の検索においてそれらをどれくらい優先するかを知っている。

では、どのようにして正しい方法でバックリンクを獲得することができるでしょうか。googleランキング要因ガイドの第10部からのヒントをいくつか紹介します。

言うまでもなく、あなたのウェブサイトを参照している個々のドメインの数は、googleのアルゴリズムにおける重要な要素です – しかし、それらの権威もそうです。(グレッグギフォードがあなたに言うように、ローカルseoを除いて)少ない、権威あるバックリンクを持つことは、低品質のリンクをたくさん持つことよりもseo価値の観点からもっと価値があります。
あなたのニッチの関連サイトからのバックリンクもまた、無関係なサイトやページよりもはるかに価値があります。
同じドメインからのリンクが多すぎるとスパムと見なされる可能性があるため、さまざまなwebサイトからのリンクが適しています。
長文の常緑樹コンテンツ内のリンクも、短いニュースベースの投稿内のリンクよりも価値があります。

ボーナス:ランクブレインとseo
googleのランキング要因に私たちのガイドの公式な一部ではありませんが、私はgoogleが正式の一つとしてrankbrain名付けられたように、このラウンドアップの一環としてrankbrainとseoにダン・テイラーの優れたガイドが含まれるだろうと思って最も重要な3つの信号ことウェブサイトのランキングに貢献する。

彼のガイドの中で、dan taylorはrankbrainがどのように機能するのか、またどのような機械学習があるのか​​、そしてassociation rule learning(arl)の基礎となる概念を分解して解き明かします。
ホームページ制作でweb集客なら佐賀のホームページ制作会社株式会社ハートウェブへ

キッチンワゴン キャスター付き 幅30cm 木製 ナチュラル 木製 完成品 カントリー 幅30cm キッチンワゴン ワゴン タンス キッチンカウンターワゴン キッチンカウンター キッチン収納 国産品 日本製:ウッディライフキッチンワゴン キッチンカウンター キッチンキャビネット キッチン収納 ダイニングボード キッチンボード カップボード 食器棚 食器収納棚 水屋

はそれからrankbrainのための「最適化」(ヒント:あなたが信じているほど複雑ではない)と、rankbrainがpandaやpenguinのような「古典的なアルゴリズム」とどう違うかを説明します。グーグルの対策をする上で、様々な考え方やシステムが世の中にはありますが、その中でもグーグルそのものも認めているシステムがあります。それがワードプレスです。

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ワードプレスは、基本的にはブログを構築するソフトではありますが、ワードプレスの中で使用されているテーマというデザインを構成する概念があるのですがそのテーマのデザインやcssなどを駆使する事で、一般企業や、中小企業、商店のホームページとして作る事も可能です。

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Источник: https://crackmix.com/graphpad-prism-crack/

Students

SolisWorkspace

By using SolisWorkspace, you can access software that you need for your studies, such as SPSS, Mathematica, MatLab, NVivo or Stella. You can use the software using anytime, any place, using devices with Windows, Apple or any other operating system. For more information, visit the page SolisWorkspace.

SURFspot

You can also buy software and hardware with a discount on SURFspot.nl. (Click on "English" at the upper right corner on the website.) Log on using your Solis-id and password.

Other Software

Utrecht University also offers some applications and suites which you may install locally (and free of charge) on your own laptop or computer. See below for specific information:

Adobe Creative Cloud is available for all students of Utrecht University on the public student computers on campus. Applications covered by this version include Photoshop, Indesign and Illustrator. 

In order to use the applications, it is necessary to create a Adobe ID. You'll have to accept the privacy terms of Adobe. These terms are not a part of the contract between Utrecht University and Adobe (via SURF); sharing information with Adobe is your own responsibility.

How to create an Adobe ID is described in this manual. Once you have completed the registration, you can use these Adobe applications. You only need to create an Adobe ID once.

If you want to use Adobe Creative Cloud on your privately owned computer, you can buy a license on SURFspot.nl, with a considerable discount.

With ArcGIS Pro, you can explore, visualize, and analyze data; create 2D maps and 3D scenes; and share your work to your ArcGIS Online. 

ArcGIS Pro is available in MyWorkplace. When starting ArcGIS Pro choose 'enterprise login', type uni-utrecht (so now it reads: uni-utrecht.maps.arcgis.com), then type your Solis-id and password.

Local installation on your Windows computer/laptop

The ArcGIS license of Utrecht University allows students, faculty and staff to install ArcGIS Pro software on their personally-owned windows computer. Summary of Steps:

  1. Download the installers. You can find the installers on Blackboard under Communities (top right) - Gis Community - Content - ESRI Software - ArcGisPro. (If the Gis Community is not available in Blackboard, please contact Maarten Zeylmans Van Emmichoven.)
  2. Unpack the 7z file.
  3. Start the installation by double clicking ArcGISPro<version>.exe - confirm licenses and folder destinations etcetera - click Finish.
  4. Execute the second installation by double clicking ArcGIS_Data_Interop<version>.exe
  5. Start ArcGIS Pro, choose 'enterprise login', type uni-utrecht (so now it reads: uni-utrecht.maps.arcgis.com), then type your Solis-id and password.

ArcGIS Pro regularly and automatically checks if updates are available

ArcGIS Online

ArcGIS Online is a cloud-based mapping and analysis solution. Use it to make maps, analyze data, share and collaborate. Get access to workflow-specific apps, maps and data from around the globe, and tools for being mobile in the field. Log in with your Solis-id and password on ArcGIS Online.

Mathematica is a software application focused on mathematics. It uses symbolic language (mathematical formulas).

Use the software via SolisWorkspace (free of charge)

Myworkplace is the easiest way to use the software for free.

Local installation on your PC

You may install Mathematica on your own PC. There are versions available for Windows, Linux and Mac OS.

  1. First, create a Wolfram ID, using your @students.uu.nl or @ucr.nl email address. Other email accounts do not work.
  2. Then you can request an activation code on the portal of Wolfram. You will receive the code and download links directly on the screen via email.

Every year you will receive an email to confirm you are still a student; this prolongs your license for another year.

Need help with the installation? Visit the support page of the supplier. 

MATLAB (developed by MathWorks) is a multi-paradigm numerical computing environment and fourth-generation programming language. MATLAB allows matrix manipulations, plotting of functions and data, implementation of algorithms, creation of user interfaces.

Use the software via SolisWorkspace (free of charge)

Myworkplace is the easiest way to use the software for free.

Local installation on your PC

The MATLAB license of Utrecht University allows students (and faculty and staff) to install MathWorks software on their personally-owned computers.

Summary of Steps:

  1. Log in to the MathWorks Portal, using your @students.uu.nl email address. (If you don't have a MathWorks account, you may need to create one first.)
  2. Download the installer for your selected version of MATLAB and run it.
    1. Windows users: double click on the Installer named MATLAB_<version number>_win64.exe in your download folder.
    2. Mac users: double click on the Installer named MATLAB_<version number>_maci64.zip - which will extract the files in the folder named MATLAB_<version number>_maci64. Start the Installer by double clicking InstallForMacOSX.
  3. In the installer, select Sign in with a MathWorks Account and follow the instructions.
  4. When prompted, select the Academic – Total Headcount license labeled MATLAB (Individual).
  5. In the Product Selection screen, select the products you want to download and then click Begin Install.

When the end date of the license is near, you'll see a pop-up. Start the application as Administrator (Start - click with the alternative (usually right) mouse button - More - Run as Administrator) - Click Help – Licensing – Activate Software.

MATLAB and Simulink Training

All students (and faculty and staff) also have free access to MATLAB and Simulink Training Online. Access through matlabacademy.mathworks.com. (If you don't have a MathWorks account, you may need to create one first on the MathWorks Portal.)

MATLAB Online and Simulink Online

All students (and faculty and staff) also have free access to MATLAB Online and Simulink Online. With MATLAB Online and Simulink Online you can access MATLAB and Simulink in your webbrowser without installation. You can also share files with others or across different applications. Access MATLAB Online and Simulink Online throught matlab.mathworks.com.

MATLAB Mobile

You can also find free MATLAB Mobile apps in the Apple App Store or Google Play.

Office 365 is a collaboration platform developed by Microsoft. Every UU employee and -student has a license and can use it for free. This includes Word, Excel, Access, Teams and PowerPoint, with a licence that allows you to use it on 5 different devices.

Office 365 can be accessed via office.com. You can log on with your email address that ends with @uu.nl or @students.uu.nl and your Solis password, after which you’ll see the Office 365 homepage. From this page you have access to all the online Office 365 applications.

Please note: If you're asked to enter a product key, click on 'sign in to an existing office 365 subscription'.

Students can download Windows 10 Education for free. Windows 10 Education contains more options that the standard Windows 10 Home operating system.

Do you already own a laptop?

  • Yes, it’s a laptop with Windows – you can order the free upgrade on SURFspot.nl.
  • Yes, it’s an Apple MacBook with Windows - you can order the free upgrade on SURFspot.nl.
  • Yes, it’s an Apple MacBook without Windows - first, order the cheapest Windows license, on Microsoft.com or elsewhere. Next, you can order the free upgrade on SURFspot.nl.

You don’t own a laptop, and plan to buy one?

  • You plan to buy a laptop with Windows – in most cases, Windows is installed on the laptop, with an ‘OEM license’. Order the cheapest Windows license. Next, you can order the free upgrade on SURFspot.nl.
  • You plan to buy an Apple MacBook – in most cases, it is possible to obtain an ‘OEM license’ with your laptop. Order the cheapest Windows license. Next, you can order the free upgrade on SURFspot.nl.

The Microsoft Azure DevTools for Teaching (formerly called Dreamspark/Imagine) offers free Microsoft software with a valid Windows licence. Log in with your Solis-id and password.

Please note: this software can only be used by students and teachers of STEM (Science, Technology, Engineering and Mathematics) to learn/teach design, development and testing of software applications, and researchers STEM (Science, Technology, Engineering and Mathematics) to perform non-commercial research.

Access is limited to employees and students of the Faculty of Science.

QSR NVIVO is a software package that can be used for the qualitative analysis of primarily textual information.

Use the software via SolisWorkspace (free of charge)

Myworkplace is the easiest way to use the software for free.

Local install on your own PC

  • Go to https://portal.mynvivo.com e and create an account with your UU email address. You'll have to accept the privacy terms of NVivo, which are accepted by Utrecht University with reservation.
  • After logging on to the website, you can download and install NVivo.
  • The first time you start the application, you will be asked to activate NVivo. Click “provide enterprise key to activate” and use the license code that you'll find on this website (log in using your Solis-id and password)

Please note:

  • It is NOT necessary to share your personal financial/payment information in order to use NVivo.
  • After logging on to the website, you will see advertisements for other QSR products, such as NVivo Transcription*. The use of those products is not allowed under the terms of the Utrecht University license.

*ITS is working on a transcription service for Utrecht University. More information about this will be published in due time.

Qualtrics is a powerful online survey tool that enables UU students and staff to create complex surveys that meet a variety of research needs. You can use this tool to design, distribute and analyze surveys. Log in with your Solis-id, password and two-factor-authentication at https://survey.uu.nl.

More information

Visit the Qualtrics page.

Reference Management tools are used to systematically collect, organise and use references to sources. How to do this in the most efficient way depends on the type of sources you use most frequently and also on your personal workflow and preferences. Read more about Reference Management in the LibGuide by Utrecht University Library. Whatever tool you choose, it is always relatively easy to switch and import your references in another tool.

Name and Wikipedia-linkProducerLicenceAvailable on
ZoteroCorporation for Digital Scholarship (an independent nonprofit organization)Open Source and free of charge
  • MyWorkPlace
  • Private device (please install this yourself, see Wikipedia-link)
MendeleyElsevierFree of charge
  • MyWorkPlace
  • Private device (please install this yourself, see Wikipedia-link)
RefWorksEx Libris Group / ProQuestCommercial. There is a university wide campus license, so you can use this without extra chargeWeb-based, no installation necessary.

Plugin for Microsoft Word is available on

  • MyWorkPlace
  • Private device (please install this yourself, see Wikipedia-link)
EndNoteClarivate Analytics (previously Thomson Reuters)Commercial. There is a university wide campus license, so you can use this without extra charge
  • MyWorkPlace
  • Private device (please install this yourself, see below)

To install Endnote on a private device (pc/laptop):

MacOS:

  • Go to this website, log in using your Solis-id and password.
  • Download the installation file and double click to install Endnote.

Windows:

  • Go to this website, log in using your Solis-id and password.
  • Download the msi-file and License.dat and save them in the same folder/directory.
  • Double click the msi-file to install Endnote.
  • When the installation is completed: start Endnote and click ‘I accept the licence agreement’. Endnote will ask you to create an account; this is not necessary.
  • You can exit Endnote now, and delete the two files you downloaded.

Think-cell facilitates the creation of charts on Microsoft PowerPoint presentation slides from Microsoft Excel data sheets, e.g., bar charts, waterfall charts, Marimekko charts and Gantt charts. It consists of a Microsoft Office add-in for PowerPoint and Excel, which integrates seamlessly with these applications. Some features overlap with those provided by built-in charts in Office 2016. See the Think-cell website.

Employees and students of Utrecht University can use this software for free. Go to this website, log in using your Solis-id and password and download the License Code.

You will find a direct link to Van Dale Online in the start menu of your Solis workspace. You do not have to log in to the site. You have access to the full range of Van Dale dictionaries, including the complete Dutch dictionary and other language dictionaries.

Use the software via SolisWorkspace (free of charge)

Myworkplace is the easiest way to use the software for free.

On campus

On campus, but not using an UU computer? Start the software via MyWorkplace or visit the website uu.vandale.nl.

Access off campus

Are you outside the campus? Start the software via MyWorkplace or browse to www.vandale.nl and log in under ‘Inloggen Onderwijs / SURFconext’ using your Solis-id. 

Do you spend a lot of time behind your laptop or PC? Check out if WORK & MOVE is something for you. With this software, short breaks in your work provide mental and physical moments of exercise. This way you stay focused and energetic! We recommend that you install WORK & MOVE if you regularly work at a computer for more than two hours. WORK & MOVE is a Windows application. Follow these steps to install this application:

  1. Download WORK & MOVE from WorkAndMove.com (or go to this website and choose "Software installation (Click Once)"), and install it by double clicking the downloaded file and following the steps in the installation wizard. Make sure you have an active internet connection during installation.
  2. Go to this website, log in with your Solis ID and password and download the license file License.json from the WORK and MOVE folder.
  3. Start WORK & MOVE, click Help - Show license page (toon licentiepagina) - Import license file (importeer het licentiebestand). Choose the json file there.
Источник: https://students.uu.nl/en/free-software

GraphPad Prism 9.2.0.332 Crack

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GraphPad Prism Crack is a non-linear freehand application to measure the regression unit and analysis from any graph. It refers to the entered data from your tabular notation also with master key points. The preferred analysis and graphing solution is purpose-built for scientific research. Join the world’s leading scientists and discover how you can use GraphPad Prism Pro Crack to save time, make more appropriate analysis choices, and elegantly graph and present your scientific research. Unlike spreadsheets or other scientific graphing programs.

It has eight different types of data tables specifically formatted for the analyses you want to run. This makes it easier to enter data correctly, choose suitable analyses, and create stunning graphs. Avoid statistical jargon. In clear language, GraphPad Prism Keygen presents an extensive library of analyses from common to highly specific—nonlinear regression, t-tests, nonparametric comparisons, one-, two- and three-way ANOVA, analysis of contingency tables, survival analysis, and much more. Each analysis has a checklist to help you understand the required statistical assumptions and confirm you have selected an appropriate test. Reduce the complexity of statistics.

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GraphPad Prism License Key online help goes beyond your expectations. At almost every step, access thousands of pages from the online Prism Guides. Browse the Graph Portfolio and learn how to make a wide range of graph types. Tutorial data sets also help you understand why you should perform certain analyses and how to interpret your results.

No other program simplifies curve fitting like Prism. Select an equation and GraphPad Prism Serial Key does the rest—fits the curve, displays a table of results and function parameters, draws the curve on the graph, and interpolates unknown values. No coding is required. Graphs and results are automatically updated in real-time. Any changes to the data and analyses—adding missed data, omitting erroneous data, correcting typos, or changing analysis choices—are reflected in results, graphs, and layouts instantaneously.

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This software is not dedicated to organizations or scientific researchers. However, it is also useful for individuals, medium, or small organizations. This application has an extensive library for analysis. Extended libraries include t-tests, non-linear regression, one-way, two-way, three-dimensional ANOVA, contingency table analysis, non-parametric comparisons, and survival analysis. Downloading Prism with Torrent 2021 simplifies curve customization that is not possible with other graphics software. You just have to choose an equation and this software will do the rest.

GraphPad Prism activation code is a powerful statistical and scientific 2D graphics software that combines data organization with comprehensible statistics, integrated curve customization, and scientific graphing. GraphPad Prism serial numbers can be used for all kinds of research and scientific research, including analysis, graphing, and presentation of scientific data. One advantage of this tool is that it can simplify nonlinear regression, curve fitting, and interpolation of unknown values.

Key features of GraphPad Prism 9.2.0.332:

  • Paired or unpaired t tests. Reports P values and confidence intervals.
  • Nonparametric Mann-Whitney test, including confidence interval of difference of medians.
  • Kolmogorov-Smirnov test to compare two groups.
  • Wilcoxon test with a confidence interval of the median.
  • Perform many t-tests at once, using False Discovery Rate (or Bonferroni multiple comparisons) to choose which comparisons are discoveries to study further.
  • Ordinary or repeated measures one-way ANOVA followed by the Tukey, Newman-Keuls, Dunnett, Bonferroni or Holm-Sidak multiple comparison tests, the post-test for trend, or Fisher’s Least Significant tests.
  • Many multiple comparisons tests are accompanied by confidence intervals and multiplicity-adjusted P values.
  • Greenhouse-Geisser correction so repeated measures one-way ANOVA does not have to assume sphericity. When this is chosen, multiple comparison tests also do not assume sphericity.
  • Kruskal-Wallis or Friedman nonparametric one-way ANOVA with Dunn’s post-test.
  • Fisher’s exact test or the chi-square test. Calculate the relative risk and odds ratio with confidence intervals.
  • Two-way ANOVA, even with missing values with some post-tests.
  • Two-way ANOVA, with repeated measures in one or both factors. Tukey, Newman-Keuls, Dunnett, Bonferroni, Holm-Sidak, or Fishers LSD multiple comparisons testing main and simple effects.
  • Three-way ANOVA (limited to two levels in two of the factors, and any number of levels in the third).
  • Kaplan-Meier survival analysis. Compare curves with the log-rank test (including test for trend).
  • Calculate min, max, quartiles, mean, SD, SEM, CI, CV,
  • Mean or geometric means with confidence intervals.
  • Frequency distributions (bin to histogram), including cumulative histograms.
  • Normality testing by three methods.
  • One sample t-test or Wilcoxon test to compare the column mean (or median) with a theoretical value.
  • Skewness and Kurtosis.

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  • Identify outliers using Grubbs or ROUT method.
  • Calculate slope and intercept with confidence intervals.
  • Force the regression line through a specified point.
  • Fit to replicate Y values or mean Y.
  • Test for departure from linearity with a runs test.
  • Calculate and graph residuals.
  • Compare slopes and intercepts of two or more regression lines.
  • Interpolate new points along the standard curve.
  • Pearson or Spearman (nonparametric) correlation.
  • Analyze a stack of P values, using Bonferroni multiple comparisons or the FDR approach to identify “significant” findings or discoveries.
  • Bland-Altman plots.
  • Receiver operator characteristic (ROC) curves.
  • Deming regression (type ll linear regression).
  • Simulate XY, Column or Contingency tables.
  • Repeat analyses of simulated data as a Monte-Carlo analysis.
  • Plot functions from equations you select or enter and parameter values you choose.
  • The area under the curve, with confidence interval.
  • Transform data.
  • Normalize.
  • Identify outliers.
  • Normality tests.
  • Transpose tables.
  • Subtract baseline (and combine columns).
  • Compute each value as a fraction of its row, column, or grand total.

What’s New in GraphPad Prism 9.2.0.332 Cracked:

  • It has fixed the issue in which X values unexpectedly disappeared in the Normalize results if the last X values had missing Y values in the source data table.
  • It has fixed the issue in which graphs that contained curves from a nonlinear regression analysis with a specified X range did not display points from the source data that were outside of the defined X range.
  • The new release comes with bug fixes like now, it brings one independent (X) variable
  • It has the ability to Rapidly generate a logistic curve and obtain the most important results from your analysis
  • Also, common in toxicity studies when testing the effect of new drug/formulation concentrations
  • Fixed the issue in which the KI value was missing from results following nonlinear regression using the “One site competition” model (Classic equation)
  • The new version brings many new features such as all dose-response equations now include versions for X as concentration and X as Log(concentration)
  • It has a new  Natural Log (Ln) axis scale option (Prism already allowed log10 and log2 axes)
  • Also, it has the power to add multiple annotations for a single bar.
  • Also, it has improved performance on copying and pasting data sets with huge numbers of rows for Mac

GraphPad Prism Serial Number 2022

  • NHCIE6-VYXQ52-KYV952-8NF62F-HC
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Operating System:

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Prism Academy is an online learning portal, accessed via the Prism application and included with valid, paid Prism subscriptions. In Prism Academy, users can access a wealth of product tutorials and educational content. Prism Academy includes over 125 video tutorials taught by experts and exclusive online access to the leading biostatistics resource for working scientists, Intuitive Biostatistics.

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The users that have activated Prism under your group subscription have access to Prism Academy, included with the valid subscription, on the computer on which they have activated Prism

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Users can access Prism Academy throughout the entire duration of their valid subscription. If a subscription is canceled or expired, Prism Academy will no longer be accessible.  

 

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The resources are available through an online portal, accessed via the Prism software itself. The users of the group subscription can access Prism Academy directly through the installed  Prism application on the computer that has been activated under your group license serial number. Just launch Prism and click the Prism Academy button on the bottom left of the Prism welcome screen. There is also a Prism Academy button in the toolbar. 

 

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Your users can access Prism Academy directly through their Prism application on the machine that is licensed under your serial number. Your users must be using Prism v9.1 or higher to access Prism Academy.

To access, they simply click the Prism Academy button on the bottom left of the Prism welcome screen. They can also access Prism Academy from the Help option in the Prism toolbar.

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When you sign in to your Prism admin account page at graphpad.com/myaccount, you can click the “Activations” tab on your dashboard to view the list of computers activated under your group license. If a user has accessed Prism Academy from a Prism-activated computer, you will see an additional line beneath the Prism activation information that says “Academy” with the associated email address.

Note: This email address has nothing to do with the allowed user email addresses under your Prism license. This is simply the email address/customer account used to sign into the Prism Academy training portal on that particular computer.

 

Can I change the Prism Academy email associated with a specific Prism activation? 

Yes, administrators of Prism group subscriptions have the ability to clear the email associated with a Prism Academy account from their online admin account page. To do this, you will need to log in to https://www.graphpad.com/myaccount/  and select the “Activations” tab. There, you will see a list of activated machines and their associated email addresses. Once Prism Academy has been accessed on a machine, you will see a line under the activation entry that is labeled “Academy”. There will be a blue “(x)” next to the email address associated with the Prism Academy account. Clearing this association does not affect the Prism software activation in any way. 

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Why are some of my users unable to access Prism Academy?

Be sure that all of your users are running at least Prism v9.1.0, which is when we added Prism Academy access to the software itself. If users are running older versions of Prism, they do not have access to Prism Academy. 

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We are also happy to troubleshoot and assist further if needed. Please reach out to us at help.graphpad.com.

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Activation of GPR40 produces mechanical antiallodynia via the spinal glial interleukin-10/β-endorphin pathway

Journal of Neuroinflammationvolume 16, Article number: 84 (2019) Cite this article

  • 2164 Accesses

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Abstract

Background

The G protein-coupled receptor 40 (GPR40), broadly expressed in various tissues such as the spinal cord, exerts multiple physiological functions including pain regulation. This study aimed to elucidate the mechanisms underlying GPR40 activation-induced antinociception in neuropathic pain, nik collection 2019 free download mac - Free Activators related to the spinal glial expression of IL-10 and subsequent β-endorphin.

Methods

Spinal nerve ligation-induced neuropathic pain model was used in this study. β-Endorphin and IL-10 levels were measured in the spinal cord and cultured primary microglia, astrocytes, and neurons. Double immunofluorescence staining of β-endorphin with glial and neuronal cellular biomarkers was also detected in the spinal cord and cultured primary microglia, astrocytes, and neurons.

Results

GPR40 was expressed on microglia, astrocytes, and neurons in the spinal cords and upregulated by spinal nerve ligation. Intrathecal injection of the GPR40 agonist GW9508 dose-dependently attenuated mechanical allodynia and thermal hyperalgesia in neuropathic rats, with Emax values of 80% and 100% MPE and ED50 values of 6.7 and 5.4 μg, respectively. Its mechanical antiallodynia was blocked by the selective GPR40 antagonist GW1100 but not GPR120 antagonist AH7614. Intrathecal GW9508 significantly enhanced IL-10 and β-endorphin immunostaining in spinal microglia and astrocytes but not in neurons. GW9508 also markedly stimulated gene and protein expression of IL-10 and β-endorphin in cultured primary spinal microglia and astrocytes but not in neurons, originated from 1-day-old neonatal rats. The IL-10 antibody inhibited GW9508-stimulated gene expression of the β-endorphin precursor proopiomelanocortin (POMC) but not IL-10, whereas the β-endorphin antibody did not affect GW9508-stimulated IL-10 or POMC gene expression. GW9508 increased phosphorylation of mitogen-activated protein kinases (MAPKs) including p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), and its stimulatory effects on IL-10 and POMC expression were blocked by each MAPK isoform inhibitor. Spinal GW9508-induced mechanical antiallodynia was completely blocked by graphpad prism tutorial - Free Activators minocycline, IL-10 neutralizing antibody, β-endorphin antiserum, and μ-opioid receptor-preferred antagonist naloxone.

Conclusions

Our results illustrate that GPR40 activation produces antinociception via the spinal glial IL-10/β-endorphin antinociceptive pathway.

Introduction

The orphan G protein-coupled receptor 40 (GPR40), also known as free fatty acid (FFA) receptor 1, was first discovered in 1997 [1] and deorphanized in 2003 [2]. Belonging to the family A of the G protein-coupled receptor, it is activated by saturated/unsaturated medium- and long-chain FFA [2, 3]. GPR40 is widely expressed in various tissues, such as the brain, pancreas islet, heart, and spinal cord, with preferential expression in pancreatic β-cells [2,3,4,5]. GPR40 activation exerts multiple physiological functions in glucose metabolism, including regulation of the secretion of insulin [3], incretin [6], and glucagon [7]. Accumulative studies have proved GPR40 to be a promising target molecule for the treatment of type 2 diabetes graphpad prism tutorial - Free Activators, and its ligands are under clinical investigations as antidiabetic drugs [8,9,10,11]. In addition, GPR40 produces anti-neuroinflammation [12] and neuroprotection [13, 14]. GPR40 activation directly led to ERK activation, which subsequently performed profound functions in neuronal plasticity and long-term memory [15, 16]. The GPR40 agonist GW9508 significantly improved amyloid β-impaired cognitive performance in a mouse model of Alzheimer’s disease [17].

GPR40 has been extensively demonstrated to be implicated with pain regulation. The GPR40 endogenous agonist docosahexaenoic acid (DHA) or exogenous agonist GW9508, given orally, intraperitoneally, or intracerebroventricularly, remarkably reduced pain behaviors in the Steganos Privacy Suite 2021 CrackActivation - Free Activators models of inflammatory pain or visceral pain induced by acetic acid, formalin, complete Freund’s adjuvant (CFA), carrageenan, or cyclophosphamide [14, 18,19,20,21,22]. Given intracerebroventricularly, they also significantly attenuated mechanical hyperalgesia induced by bilateral carotid artery occlusion in the mouse model of central post-stroke pain, which was reversed by the GPR40 antagonist GW1100 [23]. Furthermore, intrathecal injection of GW9508, DHA, and MEDICA16 significantly reduced mechanical allodynia and thermal hyperalgesia in the mouse models of neuropathic pain induced by spinal nerve injury and inflammatory pain induced by CFA and carrageenan [24], although a study reported these agents, driver easy pro crack 2021 intrathecally, were ineffective in reducing formalin-induced pain [18].

The mechanisms underlying GPR40 activation-induced antinociception remain unclear. It was reported that the descending inhibitory serotonergic and noradrenergic systems contributed to supraspinal GPR40 antinociception since intracerebroventricular injection of GW9508 facilitated the release of noradrenaline and 5-HT in the spinal cord and that the serotonergic and noradrenergic depleting and blocking agents 6-hydroxydopamine, dl-p-chlorophenylalanine, yohimbine, and WAY100635 inhibited GW9508-induced antinociception in the mouse formalin test [25]. DHA-induced antinociception also possibly occurred through the inhibition of microglia and astrocyte activation-induced spinal neuroinflammation and the secretion of inflammatory cytokines [23, 26]. On the other hand, the secretion of β-endorphin has been assumed to be associated with GPR40 activation-induced antinociception, as the GPR40 agonists accelerated β-endorphin release from the hypothalamus, and pretreatment with the β-endorphin antiserum, opioid receptor antagonist naloxone, and μ-opioid receptor antagonists β-funaltrexamine and CTOP blocked DHA- and GW9508-induced antinociception in the mouse inflammatory pain models of acetic acid, formalin, and CFA [18, 20, 21, 27, 28].

We recently uncovered that various glucagon-like peptide-1 (GLP-1) receptor agonists (exenatide, WB4–24, and shanzhiside methylester), Cynanchi-derived cynandione A, and Aconitum-derived bulleyaconitine A, bullatine A, and lappaconitine effectively relieved chronic pain through the microglial secretion of β-endorphin and dynorphin A [29,30,31,32,33,34,35]. In addition, interleukin-10 (IL-10) is a well-known anti-inflammatory and immunosuppressive cytokine, expressed in astrocytes [36, 37] and microglia [38, 39], and its antinociception has been markedly confirmed in various pain models [40,41,42,43,44,45]. Intrathecal injection of exogenous IL-10-induced mechanical antiallodynia was mediated by the spinal microglial expression of β-endorphin [39]. Moreover, the GLP-1 receptor agonist exenatide stimulated the spinal expression of β-endorphin and produced mechanical antiallodynia in neuropathic pain through the autocrine microglial secretion of IL-10, via a cAMP/PKA/p38β/CREB signal pathway [46]. Thus, it is possible that GPR40 activation-induced β-endorphin secretion, likewise GLP-1 receptor activation, is mediated by IL-10 expression.

The present study aimed to investigate the mechanisms underlying GPR40 activation-induced antinociception in neuropathic pain, especially related to the spinal glial IL-10/β-endorphin antinociceptive pathway. We first tested the effect of GW9508, given intrathecally, on mechanical allodynia and thermal hyperalgesia in neuropathic rats. Furthermore, we studied the glial expression of IL-10 and β-endorphin in the spinal cord and cultured primary cells and the signal transduction pathways. Lastly, we explored the association between the spinal glial expression of IL-10/β-endorphin and GW9508-induced mechanical antiallodynia. Our study, for the first time, reveals that GPR40 activation produces antinociception in neuropathic pain through the spinal glial expression of IL-10 and subsequent β-endorphin, highlighting the role of the newly discovered glial IL-10/β-endorphin pathway in pain transmission and transduction.

Materials and methods

Drugs and reagents

GW9508, naloxone, GW1100, and AH7614 were purchased from Target Mol (Shanghai, China), Sigma-Aldrich (St. Louis, MO, USA), Cayman Chemical Company (Ann Arbor, Michigan, USA), and ApexBio (Houston, TX, USA), respectively. SP600125, SB203580, and U0126 were from Selleck Chemicals (Houston, TX, USA). Polyclonal antiserum against β-endorphin was purchased from Abcam (Cambridge, UK), and polyclonal antibodies against IL-10 and β-endorphin were obtained from Thermo Fisher Scientific (Vienna, Austria) and Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA), respectively. GW1100 or AH7614 was dissolved in 30% dimethyl sulfoxide (DMSO) and 70% polyethylene glycol (PEG) 400 or 50% PEG 400 in 0.9% normal saline. All other drugs or reagents were dissolved or diluted in normal saline.

Experimental animals

Male or female 1-day-old neonatal and male adult (160–180 g) Wistar rats were from the Shanghai Experimental Animal Institute for Biological Sciences (Shanghai, China). The adult rats were kept in the Laboratory Animal Center of Shanghai Jiao Tong University in a temperature- and humidity-controlled environment with an automatic 12-h light/dark cycle and access to food and water ad libitum. The animals were under 3–5 days’ adaptation before being used for any experiments. All the research protocols were approved by the Animal Care and Welfare Committee of the Shanghai Jiao Tong University (Shanghai, China) and carried out in accordance with the animal care minitool power data recovery for android of the US National Institutes of Health.

Primary cell culture

Primary microglia, astrocytes, and neurons were isolated from the spinal cords of 1-day-old neonatal rats. The isolated spinal cords were minced and incubated with 0.05% trypsin-EDTA (Invitrogen, Carlsbad, CA, USA) for 7 min. The dissociated cells were suspended in DMEM supplemented with 10% (v/v) fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). The cell suspension was filtered by a 40-μm cell strainer and then plated into 75-cm2 tissue cultural flasks (1 × 107 cells/flask) pre-coated with poly-l-lysine (0.1 mg/ml) and maintained at 37 °C in a 5% carbon dioxide incubator. For microglial cell preparation, the aliquots were collected after shaking the flasks at 260 rpm, 37 °C for 1.5–2 h after 10 days of culture. The cell suspension was then transferred to new plates, and unattached cells were removed by washing with phosphate-buffered saline (PBS) before drug treatments. Harvested microglial cells exhibited a purity > 95%, as determined by the microglial cellular marker ionized calcium binding adaptor molecule-1 (Iba-1) immunoreactivity [29, 39].

For the astrocyte preparation, the mixed cells were incubated with 4 ml of 0.05% trypsin-EDTA in the cell incubator for 3 min to separate the oligodendrocytes. After neutralization, the floating cell suspension was discarded. The remaining monolayer of astrocytes was then trypsinized and sub-cultured in new plates for further experiments. Harvested astrocytes exhibited a purity > 90%, as determined by the astrocytic cellular marker glial fibrillary acid protein (GFAP) immunoreactivity [29].

For neuronal cells, the cell suspension was plated into a 10-cm dish after being filtered by 40 μm for about 30 min to remove non-neural cells, and then, the cell suspension was plated into poly-l-lysine pre-coated plates. After incubation with complete DMEM for 1–2 h, the medium was changed to the Neurobasal (Invitrogen) containing 2% B27 supplement and 0.5 mM glutamine. The following experiments were performed 3–4 days later. Harvested neurons exhibited a purity of 85%, as determined by the neuronal cellular marker neuronal specific nuclear protein (NeuN) immunoreactivity [29].

Intrathecal catheterization and injection in rats

An 18-cm PE-10 catheter (OD 0.55 mm, ID 0.30 mm; AniLab Software & Instruments Co., Ningbo, China) was inserted into the lumbar level of the spinal cord under inhaled isoflurane anesthesia (4% for induction and 2% for netbalancer 9.12.8 crack - Free Activators run by a gas anesthesia system (Ugo Basile, Comerio, Italy). The intrathecal catheterization was checked after 1-week recovery from surgery by intrathecal administration of 10 μl 4% lidocaine with 15 μl normal saline flush. Intrathecally catheterized rats that had no motor impairments after surgery and developed instant bilateral hindlimb paralysis after lidocaine injection were included in further research. For intrathecal injection, 10 μl of the drug solution was administered in a 50-μl syringe (Shanghai Anting Micro-Injector Factory, Shanghai, China) followed by a 15-μl saline flush.

Rat model of neuropathic pain and behavior assessment

After environmental adaption, L5/L6 spinal nerve ligation surgery was performed on rats under inhaled isoflurane audials one 2019 - Activators Patch as described previously [47, 48]. Briefly, the left L5 and L6 spinal nerves were isolated and tightly ligated with 6–0 silk thread. After ligation, the wound was sutured after giving antibiotic and the rats were allowed to recover for about 2 weeks. As intrathecal injection was needed in the research, intrathecal catheterization was performed at the same time just before nerve ligation. After recovery, only rats with significant unilateral allodynia to mechanical stimulation (hindlimb withdrawal thresholds in the operated side < 8 g) and with no motor deficits were included in the further experiments.

To evaluate mechanical allodynia, the animals were acclimatized to transparent plexiglass boxes on a metal grid for about 30 min. The hindlimb withdrawal threshold was measured by using a 2450 CE Electronic Von Frey hair (IITC Life Science Inc., Woodland Hill, CA, USA). The electronic handheld transducer with a no. 15 monofilament (with forces ranging from 0.1 to 90 g) was applied perpendicularly to the base of the third and fourth toe of the hindpaws with increasing force until a sudden withdrawal. The lowest force to induce the withdrawal response was considered as the threshold. The average of three repeated measurements at a 2-min interval was recorded as each hindlimb for each time point.

To assess thermal hyperalgesia, the rats were placed in plexiglass boxes on a heated glass surface (about 37 °C) for about 30 min for adaption. After that, a radiant heat source (setting at a low density of 50) was applied to the plantar medial surface of each hindpaw. The hindpaw withdrawal latency was measured by a 390G Plantar Test Analgesia Meter (IITC Life Science Inc.). The paw withdrawal latency was defined as the time from the onset of radiant heat application to the withdrawal of the hindpaw. The cutoff latency was set at 30 s to avoid tissue damage. Each value at a time point was the mean value of three repeated measurements at a 2-min interval between tests.

Neuropathic rats, starting the drug testing 2–3 weeks after the spinal nerve ligation, were randomly assigned to experimental groups, and the behavior observations were performed in a manner blind to the experimental conditions.

RNA extraction and quantitative real-time PCR measurement

For tissue samples, spinal lumbar enlargements (L3–5) from rats were isolated and homogenized using an electronic homogenizer at 4000 rpm for 15 s in TRIzol (Invitrogen) on ice. For cell samples, cells were collected after drug treatment and lysed in TRIzol for 5 min. Total RNAs extracted from the spinal cords and cells were reversely transcribed into cDNA using a ReverTra Ace qPCR RT kit (Toyobo Co., Osaka, Japan) according to the manufacturer’s protocols. Quantitative real-time PCR was then carried out by using the Realmaster Mix (SYBR Green I) (Toyobo Co.) avast internet security license file till 2050 - Crack Key For U a Mastercycler ep realplex (Eppendorf, Hamburg, Germany). The fold change was calculated using the 2−ΔΔCt method after normalization to GAPDH. The primers were 5′-CCA AGG TCA TCC ATG ACA AC-3′ and 5′-TCC ACA GTC TTC TGA GTG GC-3′ for GAPDH [29], 5′-GGC TCA GCA CTG CTA TGT TGC C-3′ and 5′-AGC ATG TGG GTC TGG CTG ACT G-3′ for IL-10 [49], 5′-CTT TCC GCG ACA GAG CCT-3′ and 5′-CCA GCT CCA CAC GTC TAT GG-3′ for the β-endorphin precursor proopiomelanocortin (POMC) (NM_139326.2), and 5′-CCT GTC CTT GTG TTC CCT GT-3′ and 5′-AGA GGC AGT CAG GGT GAG AA-3′ for the dynorphin precursor prodynorphin (PDYN) [32]. All the primers were synthesized by Synbio Technologies (Suzhou, China).

Protein extraction and Western blotting

Cultured primary microglia were seeded in 6-well plates. The cells were treated with GW9508 (100 μM) for about 30 min on the next day, and then harvested with cold PBS and lysed in the radioimmunoprecipitation assay (RIPA) lysis buffer with the addition of the phosphates inhibitor cocktail and the protease inhibitor cocktail (Biotool, Houston, USA) after being centrifuged. Cell lysates were denatured at 100 °C for 10 min and separated in 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk in 1 × TBS containing 0.1% Tween-20 (TBST) and incubated with different primary antibodies against phospho-p38, phospho-ERK1/2, phospho-JNK (1:1000, rabbit polyclonal, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:2500, mouse monoclonal, Proteintech Group, Rosemont, IL, USA) diluted in the antibody diluent (Beyotime Biotechnology, Shanghai, China) at 4 °C overnight. On the following day, the membrane was thoroughly washed in TBST for 4 times (10 min each time) and further incubated with the Dylight 680-conjugated anti-mouse IgG and Dylight 800-conjugated anti-rabbit IgG (H + L) (Cell Signaling Technology) at room temperature for 1 h. The fluorescent density of each protein band was scanned by using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA) after being washed 4 times in TBST over 40 min. The relative expression of each protein was analyzed and quantified by using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) for the band ratios of phospho-p38/GAPDH, phospho-ERK1/2/GAPDH, and phospho-JNK/GAPDH.

Immunofluorescence staining

Double immunofluorescence staining of GPR40, β-endorphin or IL-10, and cellular biomarkers of microglia, astrocytes, and neurons was undertaken and observed using a TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) in cultured primary cells and the spinal cord sections using modified protocols [29].

For immunocytochemistry, cultured primary microglia, astrocytes, and neurons were seeded on poly-l-lysine pre-coated coverslips placed at the bottom of 12-well plates (5 × 104 cells/well). After culture overnight, cells were washed with PBS and fixed in cool 4% paraformaldehyde for 1 h and were further blocked in the PBS containing 10% goat serum and 0.5% Triton X-100 for 1 h after being washed three times for 15 min in total with PBS. The cell flasks were then incubated with various primary antibodies (Table 1) in blocking buffer at 4 °C overnight, followed by incubation with Alexa 555-conjugated goat anti-rabbit secondary antibody (1:200; Life Technologies, Carlsbad, CA, USA) and Alexa 488-conjugated goat anti-mouse secondary antibody (1:200; Life Technologies) for 1 h at 37 °C. Nucleic dye reagent 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, 0.1 μg/ml; Beyotime, Shanghai, China) was used to stain cell nuclei. The expression of GPR40, Iba-1, GFAP, and NeuN was visualized under a laser scanning confocal microscope. Colocalization analysis was carried out by utilizing a computer-assisted image analysis program (ImageJ Software, National Institutes of Health, USA) equipped with a colocalization finder, under which colocalized pixels appeared as white points [29].

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For immunohistochemistry, rats were anesthetized by intraperitoneal pentobarbital sodium (40 mg/kg) and then intracardially perfused with 100 ml normal saline, followed by 60 ml of 4% paraformaldehyde. The spinal lumbar enlargements (L3–5) were isolated and fixed in paraformaldehyde Beyond Compare 4.3.7 License key Crack Free 12 h and dehydrated in gradient sucrose solutions (10%, 20%, and 30% in PBS) at 4 °C until precipitated at the bottom. Tissue sections were embedded in the OCT embedding agent (Leica Microsystems) and cut into 30-μm slices. The tissue sections were then incubated with 10% goat serum (v/v) and 0.5% Triton X-100 (v/v) in PBS for 1 h. The following steps were the same as the immunocytochemistry, with various primary antibodies (Table 1) and secondary antibodies.

To quantify the relative intensity of GPR40, IL-10, and β-endorphin or their co-expression in microglia, astrocytes, and neurons in the spinal cord, photomicrographs of the medial threefourths of the dorsal horn (laminas I–V) were captured under × 10 or × 30 magnification. The positively stained surface area was measured by using the ImageJ Software after the low and high thresholds were set to exclude the background fluorescence. Only immunofluorescent intensity measurements from positively stained areas were included. The same setup configurations were used to measure all the surface areas in each experimental group at the same time. The averaged value of the positive immunofluorescence area fraction from three non-adjacent sections of each spinal cord was recorded as the relative expression or co-expression.

IL-10 and β-endorphin measurement by ELISA kits

IL-10 and β-endorphin were measured both in primary cells and spinal enlargements of neuropathic rats. For primary cells, cell culture supernatants were collected after treatment with GW9508 for 2 h and then centrifuged at 5000 rpm at 4 °C for 5 min. The supernatants after being centrifuged were prepared as the protein sample for ELISA. For the spinal enlargements, they were isolated from neuropathic rats after drug administrations, homogenized at 4000 rpm for 15 s with a homogenizer (Fluko Equipment) in 10 mM Tris-HCl (1 g of tissue/5 ml), and centrifuged at 4000 rpm at 4 °C for 15 min. The total protein concentration was measured by using the standard BCA assay (Beyotime Institute of Biotechnology, Shanghai, China). The levels of IL-10 and β-endorphin were measured using commercial IL-10 ELISA kit (Invitrogen) and β-endorphin ELISA kit (Phoenix Pharmaceuticals) according to the operation manuals. The concentrations of IL-10 and β-endorphin were calculated by a calibration curve performed at the same time. The linear range was 1–500 and 1–100 pg/ml for the IL-10 and β-endorphin assay, respectively.

Experimental design and statistical analyses

For the behavioral study, 5–6 male rats were randomly assigned to each group, and the behavioral measurements were carried out at different time points. For the analysis of dose-response curve, the percentage of maximal possible effect (% MPE) was calculated using the following formula: (post-drug threshold in ipsilateral hindlimb – pre-drug threshold in ipsilateral hindlimb) × 100%/(post-drug threshold in contralateral hindlimb – pre-drug threshold in ipsilateral hindlimb). The % MPE values approximated to 100 indicate normal mechanical thresholds or thermal latency (i.e., near contralateral thresholds), while values approximated to 0 indicate mechanical allodynia or thermal hyperalgesia. Half-effective dose (ED50) were calculated by using GraphPad Prism (Version 7.0, GraphPad Software, Inc., San Diego, CA, USA) by fitting nonlinear least squares curves to the relation Y = a + bx, where x = [D]n/(ED50n + [D]n). The values of ED50 and b (Emax) were projected by yielding a minimum anime studio pro crack sum of squares of deviations from the theoretical curve [50].

Results were summarized as means ± SEM or 95% confidence intervals. The analysis of variance was performed by unpaired and two-tailed Student’s t test and one-way or repeated measures two-way ANOVA using GraphPad Prism to determine the significant difference. The post hoc Student-Newman-Keuls test was conducted when the effect of the drug (dose) (for one-way ANOVA, the factor was drug [dose]; for two-way ANOVA, the factors were drug [dose], time, and their interaction) was statistically significant. Values of P < 0.05 were considered statistical significance.

Results

Intrathecal GW9508 exerted mechanical antiallodynia and thermal antihyperalgesia in neuropathic pain via GPR40 activation

The antinociceptive effects of the GPR40 agonist GW9508 were assessed in neuropathic rats induced by L5/L6 spinal nerve ligation approximately 2 weeks after surgery. Six groups of neuropathic rats received an intrathecal injection of either normal saline (10 μl) or GW9508 (1, 3, 10, 30, or 100 μg). The hindpaw withdrawal thresholds to the von Frey monofilament stimulus and paw withdrawal latencies to the thermal stimulus were subsequently (in a 10-min interval) measured before and 0.5, 1, 2 and 4 h post-intrathecal injection. In normal saline-treated rats, withdrawal thresholds in either contralateral or ipsilateral hindpaws remained unchanged during the observation period of 4 h. In contrast, the intrathecal injection of GW9508 attenuated mechanical allodynia in ipsilateral hindpaws in a dose- and time-dependent manner, without significantly affecting withdrawal thresholds in contralateral hindpaws (Fig. 1a). Mechanical thresholds at 1 h after injection were used for % MPE calculation and subsequent dose-response analysis. The projected Emax and ED50 values for mechanical antiallodynia inhibition were 80% MPE and 6.7 μg (95% confidence intervals 4.1–10.8 μg) (Fig. 1b). Furthermore, the intrathecal injection of GW9508 also dose- and time-dependently reduced the thermal hyperalgesia in ipsilateral hindpaws (Fig. 1c), with the projected Emax value of 100% MPE and ED50 value of 5.4 μg (95% confidence intervals 3.14–9.30 μg) (Fig. 1d).

Inhibitory effects of GW9508 given intrathecally on mechanical allodynia (a, b) and thermal hyperalgesia (c, d) in neuropathic rats induced by spinal nerve ligation. Dose-response analyses of GW9508 on mechanical allodynia (b) and thermal hyperalgesia (d) were calculated using the thresholds measured 1 h after injection, best projected by the nonlinear least squares method. Effects of the selective GPR40 antagonist GW1100 (e) and GPR120 antagonist AH7614 (f) on GW9508-induced mechanical antiallodynia. Data are presented as means ± SEM (N = 6~7 per group). *P < 0.05 vs. the vehicle control group; analyzed by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test

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GW9508 is also an agonist of GPR120, although with the affinity is 100-fold less than that of GPR40 [51]. The selective GPR40 antagonist GW1100 and GPR120 antagonist AH7614 were used to confirm whether or not GW9508 attenuated neuropathic pain through the activation of GPR40. Three groups of neuropathic rats received intrathecal injection of the vehicle (30% DMSO, 70% PEG400, 10 μl) and GW1100 (100 and 300 μg), respectively, followed by the second injection of GW9508 (30 μg) 30 min later. Paw withdrawal thresholds were measured before and 0.5, 1, 2, and 4 h post-GW9508 administration. Intrathecal injection of GW9508 exerted the time-dependent mechanical antiallodynia. Although GW1100 did not significantly alter baseline withdrawal responses in either hindpaws, it dose-dependently reversed GW9508-induced mechanical antiallodynia (P < 0.05, by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test; Fig. 1e). In addition, four groups of neuropathic rats were intrathecally administrated with the vehicle (10 μl) + saline (10 μl), vehicle (10 μl) + GW9508 (30 μg), AH7614 (30 μg) + saline (10 μl), and AH7614 (30 μg) + GW9508 (30 μg). The first intrathecal injection was followed by the second injection 30 min later. Intrathecal injection of GW9508 produced mechanical antiallodynia time-dependently (P < 0.05, by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test), which was not significantly altered by pretreatment with intrathecal AH7614 (Fig. 1f).

Intrathecal GW9508 stimulated IL-10 and β-endorphin expression in the spinal dorsal horn

GPR40 shows diversified distributions in the brain, pancreatic islets, spinal cord, pituitary, liver, heart, and skeletal muscle [2,3,4, 52]. We first examined its expression in the spinal cords of neuropathic rats by utilizing double immunofluorescence staining. Frozen sections were obtained from the spinal lumbar enlargements of neuropathic rats induced by spinal nerve ligation approximately 2 weeks after surgery. Immunofluorescence was stained with antibodies against GPR40 with cellular biomarkers for microglia, astrocytes, and neurons (Iba-1, GFAP, or NeuN, respectively). Photomicrographs were taken from graphpad prism tutorial - Free Activators entire spinal cord (× 10 magnification) and dorsal horn laminae I–V (× 30 magnification). As shown in Additional file 1: Figure S1A, GPR40-positive cells were mainly expressed in the contralateral and ipsilateral dorsal horns, as well as in the ventral horns. In addition, double immunofluorescence staining of GPR40 with Iba-1, GFAP, and NeuN revealed that GPR40 was colocalized with microglia, astrocytes, and neurons in the contralateral and ipsilateral dorsal horn I–V laminae. Interestingly, the ipsilateral dorsal horn exhibited much higher expression of GPR40 immunostaining on all of microglia, astrocytes, and neurons, compared to the contralateral side (Fig. 2a–i). The ImageJ program was used to quantify immunofluorescence intensity (immunolabeled surface area) in the dorsal horn (× 10 magnification). As shown in Additional file 1: Figure S1B, the GPR40-immunolabeled surface areas in the ipsilateral spinal dorsal horn were significantly increased by 102%, compared with the contralateral side (P < 0.05, by unpaired and two-tailed Student’s t test). Double immunostaining of GPR40/Iba-1, GPR40/GFAP, and GPR40/NeuN in the ipsilateral dorsal horn was significantly increased by 165%, 203%, and 219%, respectively (P < 0.05, by unpaired and two-tailed Student’s t test; Fig. 2j–l).

Expression of GPR40 in microglia (ac), astrocytes (df), and neurons (gi) in the spinal dorsal horn of neuropathic rats. Frozen sections of the spinal lumbar enlargements were obtained approximately 2 weeks after spinal nerve ligation. Immunofluorescence was double stained with GPR40/Iba-1, GPR40/GFAP, and GPR40/NeuN, and photomicrographs were taken from the spinal cord section (a, d, g; 500 μm) and amplified dorsal horn laminae I–V (b, c, e, f, h, i; 50 μm). Arrows indicate double immunostaining of GPR40 with each cellular biomarker. Double immunolabeled surface areas of GPR40/Iba-1 (j), GPR40/GFAP (k), and GPR40/NeuN (l) from the spinal dorsal horn laminae I–V indicated in white lines were quantified using the ImageJ program. Data are presented as means ± SEM (N = 5–6 per group). *P < 0.05 vs. contralateral side; analyzed by unpaired and two-tailed Student’s t test

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We then assessed the stimulatory effects of GW9508 given intrathecally on the spinal level of IL-10 and its cell localization. Spinal enlargements were isolated from neuropathic rats 1 h after intrathecal normal saline (10 μl) or GW9508 (30 μg) treatment for IL-10 measurement. As shown in Additional file 2: Figure S2A and S2B, both gene and protein levels of IL-10 were significantly increased by the intrathecal injection of GW9508 (P < 0.05, by unpaired and two-tailed Student’s t test). Moreover, frozen sections were obtained 1 h after intrathecal normal saline (10 μl) or GW9508 (30 μg) treatment. Immunofluorescence was stained with antibodies against GPR40 with Iba-1, GFAP, and NeuN. In neuropathic rats treated with intrathecal normal saline, IL-10 was widely spread in the gray matter of both contralateral and ipsilateral spinal dorsal horns and in the ventral horns, which was markedly enhanced by the intrathecal administration of GW9508 (30 μg) in both contralateral and ipsilateral sides (Additional file 2: Figure S2C and S2D). Double immunofluorescence staining further revealed Antivirus - Crack All Windows/Mac OS Software Full Version IL-10 was colocalized with Iba-1 in the dorsal horn of saline-treated rats (Fig. 3a–c). Intrathecal injection of GW9508 significantly enhanced the co-immunostaining of IL-10/Iba-1 in both contralateral and ipsilateral dorsal horns (Fig. 3d–f). In addition, IL-10 was also colocalized with GFAP (Fig. 3g–i) and the intrathecal injection of GW9508 significantly enhanced the double immunostaining of IL-10/GFAP in both contralateral and ipsilateral dorsal horns (Fig. 3j–l). In contrast, although IL-10 was colocalized with NeuN (Fig. 3m–o), the intrathecal GW9508 failed to significantly increase the immunostaining of IL-10/NeuN either in the contralateral or ipsilateral dorsal horn (Fig. 3p–r).

Effect of intrathecal GW9508 on spinal IL-10 expression on spinal microglia (af), astrocytes (gl), and neurons (mr) in neuropathic rats induced by spinal nerve ligation. Frozen sections of the spinal lumbar enlargements were obtained 1 h after intrathecal saline (10 μl) or GW9508 (30 μg) treatment. Immunofluorescence was double stained with IL-10/Iba-1, IL-10/GFAP, and IL-10/NeuN, and photomicrographs were taken from the entire spinal cord section (a, d, g, j, m, p; 500 μm) and amplified dorsal horn laminae I–V (b, c, e, f, h, i, k, l, n, o, q, r; 50 μm). Arrows indicate double immunostaining of IL-10 and each cellular biomarker. Double immunolabeled surface areas of IL-10/Iba-1 (s), IL-10/GFAP (t), and IL-10/NeuN (u) from the spinal dorsal horn graphpad prism tutorial - Free Activators I-V indicated in white lines were quantified using the ImageJ program. Data are presented as means ± SEM (N = 5~7 per group). *P < 0.05 vs. saline group; analyzed by unpaired and two-tailed Student’s t test

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The ImageJ program was used graphpad prism tutorial - Free Activators quantify immunofluorescence intensity in the dorsal horn. As shown in Additional file 2: Figure S2E, the intrathecal GW9508 significantly increased the IL-10-immunolabeled surface areas by 520% and 380% in the contralateral and ipsilateral dorsal horns, compared with saline group (P < 0.05, by unpaired and two-tailed Student’s t test). Furthermore, GW9508 significantly increased the double immunostaining of IL-10/Iba-1 by 290% and 200%, respectively, in the contralateral and ipsilateral dorsal horns (P < 0.05, by unpaired and two-tailed Student’s t test; Fig. 3s). Although the baseline double immunostaining intensity of IL-10/GFAP was much lower than that of IL-10/Iba-1, it was significantly increased by GW9508 injection by 280% and 230% in contralateral and ipsilateral spinal dorsal horns, respectively (P < 0.05, by unpaired and two-tailed Student’s t test; Fig. 3t). However, GW9508 failed to significantly increase the double immunostaining intensity of IL-10/NeuN in either contralateral or ipsilateral spinal dorsal horn (Fig. 3u).

We further assessed the stimulatory effect of intrathecal GW9508 treatment on β-endorphin expression in the above same spinal enlargement samples and spinal slides. β-Endorphin was markedly enhanced by the intrathecal GW9508 in both gene and protein level as well (Additional file 3: Figure S3A and S3B). β-Endorphin was also widely spread in the gray matter of both contralateral and ipsilateral spinal dorsal horns and ventral horns from the control neuropathic rats, and the intrathecal GW9508 injection significantly enhanced the spinal β-endorphin expression (Additional file 3: Figure S3C and S3D). β-Endorphin was colocalized with Iba-1 in the dorsal horns of control rats (Fig. 4a–c). Intrathecal injection of GW9508 significantly increased the double immunostaining of β-endorphin/Iba-1 both in the contralateral and ipsilateral spinal dorsal horns (Fig. 4d–f). In addition, β-endorphin was also colocalized with GFAP (Fig. 4g–i) and the intrathecal injection of GW9508 significantly enhanced its co-immunostaining in both contralateral and ipsilateral dorsal horns (Fig. 4j–l). In contrast, although β-endorphin was also colocalized with NeuN (Fig. 4m–o), the intrathecal GW9508 failed to significantly increase its double immunostaining in either the contralateral or ipsilateral dorsal horn (Fig. 4p–r). The ImageJ quantification in the dorsal horn showed that the intrathecal GW9508 significantly increased the immunofluorescence intensity of β-endorphin by 700% and 800% (P < 0.05, by unpaired and two-tailed Student’s t test; Additional file 3: Figure S3E), double immunofluorescence intensity of β-endorphin/Iba-1 by 380% and 430% (P < 0.05, by unpaired and two-tailed Student’s t test; Fig. 4s), and β-endorphin/GFAP by 360% and 260% (P = 0.06 and 0.13, by unpaired and two-tailed Student’s t test; Fig. 4t) in contralateral and ipsilateral spinal dorsal horn, respectively, compared with the saline group. In contrast, the intrathecal GW9508 failed to stimulate the double immunostaining of β-endorphin/NeuN in either contralateral or ipsilateral spinal dorsal horn (Fig. 4u).

Effect of GW9508 given intrathecally on spinal β-endorphin expression on spinal microglia (af), astrocytes (gl), and neurons (mr) in neuropathic rats induced by spinal nerve ligation. Frozen sections of the spinal lumbar enlargements were obtained 1 h after intrathecal saline (10 μl) or GW9508 (30 μg) treatment. Immunofluorescence was double stained with β-endorphin/Iba-1, β-endorphin/GFAP, and β-endorphin/NeuN, and photomicrographs were taken from the entire spinal cord section (a, d, g, j, m, p: 500 μm) and amplified dorsal horn laminae I–V (b, c, e, f, h, i, k, l, n, o, q, r: 50 μm). Arrows indicate double immunostaining of β-endorphin with each cellular biomarker. Double immunolabeled surface areas of β-endorphin/Iba-1 (s), β-endorphin/GFAP (t), and β-endorphin/NeuN (u) from the spinal dorsal horn laminae I-V indicated in white lines were quantified using the ImageJ program. Data are presented as means ± SEM (N = 5~7 per group). *P < 0.05 vs. saline group; analyzed by unpaired and two-tailed Student’s t test

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GW9508 stimulated IL-10 and β-endorphin expression in cultured primary microglia and astrocytes

To further assess the cell expression of GPR40, microglia, astrocytes, and neurons were isolated from the spinal cords of neonatal rats and were co-immunostained with GPR40/Iba-1, GPR40/GFAP, and GPR40/NeuN, respectively, in addition to the nucleus staining with DAPI. As shown in Fig. 5a–c, GPR40 was expressed on microglia, astrocytes, and neurons, with 100% positive in each cell population.

Expression of GPR40 (ac) and stimulatory effects of GW9508 on mRNA expression of IL-10, the β-endorphin precursor proopiomelanocortin (POMC) and dynorphin A precursor prodynorphin (PDYN) (dg), and secretion of IL-10 and β-endorphin (h, i) in cultured primary microglia, astrocytes, and neurons originated from the spinal cords of 1-day-old neonatal rats. For the immunostaining study, immunofluorescence was double stained with GPR40/Iba-1, GPR40/GFAP, and GPR40/NeuN. For the stimulatory study, cultured cells and cultural medium were collected 2 h after GW9508 incubation, and the mRNA expression of IL-10, POMC, and PDYN and the peptide concentrations of IL-10 and β-endorphin were measured using quantitative real-time PCR and commercial ELISA kits, respectively. Data are presented as means ± SEM (N = 4~7 per group). *P < 0.05 vs. control group; analyzed by unpaired and two-tailed Student’s t test

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We then tested the stimulatory effect of GPR40 activation on IL-10 and β-endorphin expression. Primary microglia, astrocytes, and neurons from the spinal cords of neonatal rats were incubated with 100 μM GW9508 for 2 h, and the cellular mRNA expression of IL-10, POMC, and PDYN was measured by using quantitative real-time PCR. Incubation with GW9508 for 2 h significantly increased the expression of IL-10 mRNA in cultured primary microglia and astrocytes (P < 0.05, by unpaired and two-tailed Student’s t test), but not neurons (Fig. 5d). Further analysis showed that the GW9508 treatment concentration dependently increased the POMC expression in microglia, with an EC50 value of 13.4 μM (Fig. 5e). In addition, the treatment with GW9508 for 2 h significantly increased the mRNA expression of POMC in cultured primary microglia and astrocytes (P < 0.05, by unpaired and two-tailed Student’s t test), but not in neurons (Fig. 5f). In contrast, GW9508 failed to affect the PDYN mRNA expression in microglia, astrocytes, or neurons (Fig. 5g).

The secretory effects of GW9508 on IL-10 and β-endorphin were also investigated. Primary cultures of microglia, astrocytes, and neurons were incubated with 100 μM GW9508 for 2 h, and the concentration of IL-10 and β-endorphin in the cultural medium were measured using commercial ELISA kits. As exhibited in Fig. 5h and i, the incubation with GW9508 for 2 h significantly increased the IL-10 and β-endorphin secretion from microglia and astrocytes (P < 0.05, by unpaired and two-tailed Student’s t test). However, the treatment with GW9508 did not affect the IL-10 or β-endorphin secretion from neurons.

To confirm the stimulatory effect of GW9508 on IL-10 and β-endorphin expression through GPR40, the selective GPR40 antagonist GW1100 was utilized. As shown in Fig. 6a and b, the pretreatment with GW1100 (50 μM) for 30 min did not significantly the change basal expression of IL-10 or POMC mRNA in the cultured primary microglia, but totally blocked the GW9508 (100 μM)-induced elevation of IL-10 and POMC mRNA (P < 0.05, by one-way ANOVA followed by the post hoc Student-Newman-Keuls test).

Effects of the specific GPR40 antagonist GW1100 (a, b), glial inhibitor minocycline (cf), IL-10 (g, h), and β-endorphin (i, j) neutralizing antibodies on the GW9508-enhanced mRNA expression of IL-10 and the β-endorphin precursor proopiomelanocortin (POMC) in microglia and astrocytes. Cultured primary microglial cells (ad, gj) and astrocytes (e, f), originated from the spinal cords of neonatal rats, were collected 2 h after drug treatment, and the mRNA expression of IL-10 and POMC was determined using quantitative real-time PCR. Data are presented as means ± SEM (N = 4~8 per group). Asterisk (*) and number sign (#) indicate P < 0.05 vs. the vehicle control group and GW9508 group, respectively; analyzed by one-way ANOVA followed by the post hoc Student-Newman-Keuls test

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Minocycline is generally considered as a microglia inhibitor [53,54,55], but may inhibit astrocyte activation as well [56,57,58]. To confirm its glial inhibitory effect, minocycline was used to block the GW9508-induced mRNA expression of IL-10 and POMC in microglia and astrocytes. Pretreatment with minocycline (60 μM) in the cultured primary microglia for 1 h did not change the baseline expression of IL-10 and POMC mRNA, but entirely blocked the GW9508 (100 μM)-elevated IL-10 and POMC mRNA expression (P < 0.05, by one-way ANOVA followed by the post hoc Student-Newman-Keuls test; Fig. 6c, d). Moreover, as exhibited in Fig. 6e and f, GW9508 also markedly increased the IL-10 and POMC mRNA expression in the cultured primary astrocytes, and the pretreatment with minocycline significantly inhibited the GW9508-stimulated POMC mRNA expression (P < 0.05, by one-way ANOVA followed by the post hoc Student-Newman-Keuls test). Minocycline also appeared to inhibit the GW9508-induced IL-10 expression, although it did not reach statistical significance (P = 0.12).

We have recently discovered that exogenous IL-10 and endogenous IL-10 after GLP-1 receptor activation induced the β-endorphin expression in microglia [49]. Following GPR40 activation, the IL-10 and β-endorphin neutralizing antibodies were used to illustrate IL-10 to induce β-endorphin and vice versa. As shown above, the treatment with GW9085 (100 μM) significantly increased the IL-10 and POMC mRNA expression in the cultured primary microglia, whereas the treatment with either the IL-10 antibody (4 μg/ml) or β-endorphin antibody (1:300 dilution) had no effects on the baseline expression of both IL-10 and POMC mRNA. The IL-10 neutralizing antibody completely inhibited the GW9508-stimulated mRNA expression of POMC (P < 0.05, by one-way ANOVA followed by the post hoc Student-Newman-Keuls test) but not IL-10 (Fig. 6g, h). In contrast, the β-endorphin neutralizing antibody did not exhibit any inhibitory effects on the GW9508-stimulated mRNA expression of either IL-10 or POMC (Fig. 6i, j).

Mitogen-activated protein kinases (MAPKs), including p38, ERK, and JNK, are evolutionarily conserved enzymes that play crucial roles in various cellular processes [59, 60]. We assessed whether GW9508 stimulated IL-10 and POMC mRNA expression through MAPK activation. In cultured primary microglia, the treatment with GW9508 (100 μM) for 30 min significantly increased the p38 phosphorylation by 1.7-fold (P < 0.05, by unpaired and two-tailed Student’s t test; Fig. 7a). In addition, pretreatment with the selective p38 inhibitor SB203580 (30 μM) significantly inhibited the GW9508-stimulated IL-10 and POMC mRNA expression (P < 0.05, by one-way ANOVA followed by followed by the post hoc Student-Newman-Keuls test), although it did not significantly affect their baseline expression (Fig. 7b, c). As shown in Fig. 7d–i, the treatment with GW9508 also stimulated both JNK and ERK1/2 phosphorylation by 1.4- and 1.5-fold, respectively (P < 0.05, by unpaired and two-tailed Student’s t test), and pretreatment with the selective JNK inhibitor SP600125 (30 μM) and ERK1/2 inhibitor U0126 (30 μM) significantly attenuated the GW9508-increased IL-10 and POMC mRNA expression (P < 0.05, by one-way ANOVA followed by the post hoc Student-Newman-Keuls test), without affecting their baseline expression.

Effects of the phosphorylation of p38 (ac), JNK (df), and ERK1/2 mitogen-activated protein kinase (MAPK) (gi) on GW9508-enhanced mRNA expression of IL-10 and β-endorphin in microglia. Cultured primary microglial cells were originated from the spinal cords of neonatal rats. For the phosphorylation immunoblotting study, microglia were collected 30 min after the GW9508 treatment, and their lysates were immunoblotted with the antibodies of anti-phospho-p38, anti-phospho-ERK1/2, and anti-phospho-JNK, respectively. Immunoblots of each experiment are placed on the upper panels, and densitometric analyses are shown in the lower panels. For the MAPK inhibitor study, microglia were collected 2 h after drug treatment, and the mRNA expression of IL-10 and the β-endorphin precursor proopiomelanocortin (POMC) was determined using quantitative real-time PCR. Data are presented as means ± SEM (N = 5~8 per group). Asterisk (*) and number sign (#) indicate P < 0.05 vs. the vehicle control group and GW9508 group, respectively; analyzed by unpaired and two-tailed Student’s t test and one-way ANOVA followed by the post hoc Student-Newman-Keuls test

Full size image

GW9508 exerted antinociception via spinal glial IL-10/β-endorphin pathway

Minocycline was used to block the GW9508-induced mechanical antiallodynia. Two groups of neuropathic rats were pretreated with intrathecal saline (10 μl) or minocycline (100 μg) for 4 h followed by the intrathecal injection of GW9508 (30 μg). Paw withdrawal thresholds were measured before and 0, 0.5, 1, 2, and 4 h post-GW9508 administration. The intrathecal injection of GW9508 exerted profound mechanical antiallodynia in a time-dependent manner, which was reversed by the pretreatment with minocycline (P < 0.05, by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test), although it did not alter the baseline withdrawal response in either paw (Fig. 8a).

Blockade effects of the glial inhibitor minocycline (a), specific IL-10 antibody (b), β-endorphin antiserum (c), and μ-opioid receptor-preferred antagonist naloxone (d), given intrathecally, on the spinal GW9508-induced mechanical antiallodynia in neuropathic rats. Neuropathic rats, 2 weeks after spinal nerve ligation, received a single injection of the vehicle (10 μl) or each blocking agent followed by a single intrathecal injection of GW9508 (30 μg) 4 h (for minocycline) or 30 min (for the rest drugs) later for subsequent assessment of withdrawal responses to mechanical stimuli. Data are presented as means ± SEM (N = 5~8 per group). *P < 0.05, vs. vehicle + GW9508 group; analyzed by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test

Full size image

In order to confirm the causal role of spinal IL-10/β-endorphin in the GW9508-induced mechanical antiallodynia, the IL-10 neutralizing antibody was first applied. Two groups of neuropathic rats received an intrathecal injection of normal saline (10 μl) or the IL-10 antibody (2 μg) for 30 min followed by an intrathecal injection of GW9508 (30 μg). Paw withdrawal thresholds were measured before and 0, 0.5, 1, 2, and 4 h post-GW9508 administration. Pretreatment with intrathecal IL-10 neutralizing antibody completely reversed the mechanical antiallodynia produced by intrathecal GW9508 (P < 0.05, by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test), although it did not significantly alter the baseline withdrawal responses in either contralateral or ipsilateral hindpaws (Fig. 8b).

In addition, two groups of neuropathic rats received an intrathecal injection of the blank serum (1:10 dilution, 10 μl) or the β-endorphin antiserum (1:10 dilution, 10 μl) for 30 min followed by an intrathecal injection of GW9508 (30 μg). Paw withdrawal thresholds were measured before and 0, 0.5, 1, 2, and 4 h post-GW9508 administration. The intrathecal injection of GW9508 exerted profound mechanical antiallodynia in a time-dependent manner, which was completely reversed by the pretreatment with intrathecal β-endorphin antiserum (P < 0.05, by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test), although it did not significantly alter baseline withdrawal responses in either hindpaws (Fig. 8c).

Furthermore, as β-endorphin is an endogenous ligand of μ-opioid receptors [61], the μ-opioid receptor-preferred antagonist naloxone was utilized to illustrate the pivotal role of β-endorphin in the GW9508-induced mechanical antiallodynia. Two groups of neuropathic rats received an intrathecal injection of normal saline (10 μl) or naloxone (20 μg) for 30 min, followed by an intrathecal injection of GW9508 (30 μg). Pretreatment with intrathecal naloxone did not significantly affect baseline withdrawal responses in either hindpaws, graphpad prism tutorial - Free Activators completely blocked the GW9508-exerted mechanical antiallodynia in the ipsilateral paws (P < 0.05, by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test; Fig. 8d).

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Our present study demonstrated that the GPR40 agonist GW9508, given intrathecally, was effective in attenuating mechanical allodynia and thermal hyperalgesia in ipsilateral hindpaws of neuropathic rats in a dose-dependent manner, with Emax values of graphpad prism tutorial - Free Activators and 100% MPE and ED50 values of 6.7 and 5.4 μg, respectively. It did not significantly affect the normal reflex nociceptive pain states in contralateral hindpaws. We also confirmed that the GW9508-induced mechanical antiallodynia was through GPR40 activation, as the selective GPR40 antagonist GW1100 (but not selective GPR120 antagonist AH7614) dose-dependently and nearly completely suppressed the GW9508-induced antinociception and as the GPR120 was not expressed in the central nervous system including the spinal cord [18]. Our results were in accordance with the previous findings, which showed graphpad prism tutorial - Free Activators the GPR40 agonists DHA, GW9508, and MEDICA16, given orally, intracerebroventricularly, or intrathecally, attenuated pain behaviors in the rodent models of inflammatory pain, visceral pain, and neuropathic pain [14, 18, 23, 24]. Taken together, all the results indicated that GPR40 agonists were specifically antinociceptive in painful hypersensitivity states.

The more striking finding from the current study is that GPR40 activation-induced antinociception is mediated by the spinal glial expression of IL-10 and subsequent β-endorphin. The notion is supported by the following findings: (1) Intrathecal injection of GW9508 stimulated the expression of IL-10 and β-endorphin in microglia and astrocytes, but not in neurons, in both contralateral and ipsilateral spinal cords of neuropathic rats, directly identified by double immunofluorescence staining of IL-10 and β-endorphin with cellular biomarkers, i.e., Iba-1, GFAP, and NeuN. (2) Treatment with GW9508 stimulated the gene and protein expression of IL-10 and β-endorphin in primary cultures of microglia and astrocytes, but not of neurons, in a GW1100-reversible manner. The specific IL-10 neutralizing antibody in primary microglia completely inhibited the GW9508-stimulated mRNA expression of POMC but not IL-10, whereas the β-endorphin antibody did not affect the GW9508-stimulated IL-10 or POMC gene expression. The results are in agreement with our previous findings that the IL-10 neutralizing antibody completely inhibited the exenatide-stimulated expression of POMC [39] and that IL-10 stimulated the β-endorphin expression but not vice versa [46]. (3) Intrathecal injection of the IL-10 neutralizing antibody, β-endorphin antiserum, and μ-opioid receptor-preferred antagonist naloxone entirely eliminated the spinal GW9508-induced mechanical antiallodynia in neuropathic rats. (4) Minocycline is generally deemed as a microglial inhibitor [53,54,55], but it also inhibited astrocyte activation as well [56,57,58]. Treatment with minocycline in the current study inhibited the GW9508-stimulated IL-10 and POMC gene expression in the cultured primary microglia and astrocytes. Moreover, the intrathecal injection of minocycline completely blocked the spinal GW9508-induced mechanical antiallodynia in neuropathic rats. These results further highlight the broad significance of the newly discovered spinal glial IL-10/β-endorphin pathway in pain process and modulation. It was recently discovered that IL-10 and IL-24, a member of the IL-10 family, were effective in relieving neuropathic pain and bone cancer pain in rats by the promotion of β-endorphin synthesis [62]. Moreover, GLP-1 receptor agonist exenatide induced the antinociception through spinal microglial IL-10 and β-endorphin pathway via the cAMP/PKA/p38β/CREB and JAK1/STAT3 signal transduction mechanisms [49].

β-Endorphin is an endogenous opioid neuropeptide that is known to be produced in neurons within the central nervous system including the hypothalamus and modulates pain transmission and transduction by the activation of μ-opioid receptors [63,64,65,66,67]. However, recent findings indicate that endogenous opioid peptides (including endorphins and dynorphins) are also present in astrocytes and microglia [29, 33, 39, 68, 69]. Particularly, β-endorphin and dynorphin A secreted by GLP-1 receptor agonists, herbal cynandione A, and aconitines effectively attenuated painful hypersensitivity [29,30,31

Источник: https://jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-019-1457-9

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    SolisWorkspace

    By using SolisWorkspace, you can access software that you need for your studies, such as SPSS, Mathematica, MatLab, NVivo or Stella. You can use the software using anytime, any place, using devices with Windows, Apple or any other operating system. For more information, visit the page SolisWorkspace.

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    Other Software

    Utrecht University also offers some applications and suites which you may install locally (and free of charge) on your own laptop or computer. See below for specific information:

    Adobe Creative Cloud is available for all students of Utrecht University on the public student computers on campus. Applications covered by this version include Photoshop, Indesign and Illustrator. 

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    With ArcGIS Pro, you can explore, visualize, and analyze data; create 2D maps and 3D scenes; and share your work to your ArcGIS Online. 

    ArcGIS Pro is available in MyWorkplace. When starting ArcGIS Pro choose 'enterprise login', type uni-utrecht (so now it reads: uni-utrecht.maps.arcgis.com), then type your Solis-id and password.

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    The ArcGIS license of Utrecht University allows students, faculty and staff to install ArcGIS Pro software on their personally-owned windows computer. Summary of Steps:

    1. Download the installers. You can find the installers on Blackboard under Communities (top right) - Gis Community - Content - ESRI Software - ArcGisPro. (If the Gis Community is not available in Blackboard, please contact Maarten Zeylmans Van Emmichoven.)
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    4. Execute the second installation by double clicking ArcGIS_Data_Interop<version>.exe
    5. Start ArcGIS Pro, choose 'enterprise login', type uni-utrecht (so now it reads: uni-utrecht.maps.arcgis.com), then type your Solis-id and password.

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    Mathematica is a software application focused on mathematics. It uses symbolic language (mathematical formulas).

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    1. First, create a Wolfram ID, using your @students.uu.nl or @ucr.nl email address. Other email accounts do not work.
    2. Then you can request an activation code on the portal of Wolfram. You will receive the code and download links directly on the screen via email.

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    MATLAB (developed by MathWorks) is a multi-paradigm numerical computing environment and fourth-generation programming language. MATLAB allows matrix manipulations, plotting of functions and data, implementation of algorithms, creation of user interfaces.

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    The MATLAB license of Utrecht University allows students (and faculty and staff) to install MathWorks software on their personally-owned computers.

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    1. Log in to the MathWorks Portal, using your @students.uu.nl email address. snapgene crack windows download - Activators Patch you don't have a MathWorks account, you may need to create one first.)
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    MATLAB Online and Simulink Online

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    Office 365 is a collaboration platform developed by Microsoft. Every UU employee and -student has a license and can use it for free. This includes Word, Excel, Access, Teams and PowerPoint, with a licence that allows you to use it on 5 different devices.

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    Students can download Windows 10 Education for free. Windows 10 Education contains more options that the standard Windows 10 Home operating system.

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    • Yes, it’s an Apple MacBook with Windows - you can order the free upgrade on SURFspot.nl.
    • Yes, it’s an Apple MacBook without Windows - first, order the cheapest Windows license, on Microsoft.com or elsewhere. Next, you can order the free upgrade on SURFspot.nl.

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    • You plan to buy a laptop with Windows – in most cases, Windows is installed on the laptop, with an ‘OEM license’. Order the cheapest Windows license. Next, you can order the free upgrade on SURFspot.nl.
    • You plan to buy an Apple MacBook – in most cases, it is possible to obtain an ‘OEM license’ with your laptop. Order the cheapest Windows license. Next, you can order the free upgrade on SURFspot.nl.

    The Microsoft Azure DevTools for Teaching (formerly called Dreamspark/Imagine) offers free Microsoft software with a valid Windows licence. Log in with your Solis-id and password.

    Please note: this software can only be used by students and teachers of STEM (Science, Technology, Engineering and Mathematics) to learn/teach design, development and testing of software applications, and researchers STEM (Science, Technology, Engineering and Mathematics) to perform non-commercial research.

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    QSR NVIVO is a software package that can be used for the qualitative analysis of primarily textual information.

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    • Go to https://portal.mynvivo.com e and create an account with your UU email address. You'll have to accept FIFA 17 3DM Crack PC Free Download Full Version Is Here privacy terms of NVivo, which are accepted by Utrecht University with reservation.
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    • After logging on to the website, you will see advertisements for other QSR products, such as NVivo Transcription*. The use of those products is not allowed under the terms of the Utrecht University license.

    *ITS is working on a transcription service for Utrecht University. More information about graphpad prism tutorial - Free Activators will be published in due time.

    Qualtrics is a powerful online survey tool that enables UU students and staff to create complex surveys that meet a variety of research needs. You can use this tool to design, distribute and analyze surveys. Log in with your Solis-id, password and two-factor-authentication at https://survey.uu.nl.

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    Visit the Qualtrics page.

    Reference Management tools are used to systematically collect, organise and use references to sources. How to do this in the most efficient way depends on the type of sources you use most frequently and also on your personal workflow and preferences. Read more about Reference Management in the LibGuide by Utrecht University Library. Whatever tool you choose, it is always relatively easy to switch and import your references in another tool.

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    ZoteroCorporation for Digital Scholarship (an independent nonprofit organization)Open Source and free of charge
    • MyWorkPlace
    • Private device (please install this yourself, see Wikipedia-link)
    MendeleyElsevierFree of charge
    • MyWorkPlace
    • Private device (please install this yourself, see Wikipedia-link)
    RefWorksEx Libris Group / ProQuestCommercial. There is a university wide campus license, so you can use this without extra chargeWeb-based, no installation necessary.

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    EndNoteClarivate Analytics (previously Thomson Reuters)Commercial. There is a university wide campus license, so you can use this without extra charge
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    To install Endnote on a private device (pc/laptop):

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    Employees and students of Utrecht University can use this software for free. Go to this website, log in using your Solis-id and password and graphpad prism tutorial - Free Activators the License Code.

    You will find a direct link to Van Dale Online in the start menu of your Solis workspace. You do not have to log in to the site. You have access to the full range of Van Dale dictionaries, including the complete Dutch dictionary and other language dictionaries.

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    Are you outside the campus? Start the software via MyWorkplace or browse to www.vandale.nl and log in under ‘Inloggen Onderwijs / SURFconext’ using your Solis-id. 

    Do you spend a lot of time behind your laptop or PC? Check out if WORK & MOVE is something for you. With this software, short breaks in your work provide mental and physical moments of exercise. This way you stay focused and energetic! We recommend that you install WORK & MOVE if you regularly work at a computer for more than two hours. WORK & MOVE is a Windows application. Follow these steps to install this application:

    1. Download WORK & MOVE from WorkAndMove.com (or go to this website and choose "Software installation (Click Once)"), and install it by double clicking the downloaded file and following the steps in the installation wizard. Make sure you have an active internet connection during installation.
    2. Go to this website, log in with your Solis ID and password and download the license file License.json from the WORK and MOVE folder.
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    Activation of GPR40 produces mechanical antiallodynia via the spinal glial interleukin-10/β-endorphin pathway

    Journal of Neuroinflammationvolume 16, Article number: 84 (2019) Cite this article

    • 2164 Accesses

    • 18 Citations

    • Metrics details

    Abstract

    Background

    The G protein-coupled receptor 40 (GPR40), broadly expressed in various tissues such as the spinal cord, exerts multiple physiological functions including pain regulation. This study aimed to elucidate the mechanisms underlying GPR40 activation-induced antinociception in neuropathic pain, particularly related to the spinal glial expression of IL-10 and subsequent β-endorphin.

    Methods

    Spinal nerve ligation-induced neuropathic pain model was used in this study. β-Endorphin and IL-10 levels were measured in the spinal cord and cultured primary microglia, astrocytes, and neurons. Double immunofluorescence staining of β-endorphin with glial and neuronal cellular biomarkers was also detected in the spinal cord and cultured primary microglia, astrocytes, and neurons.

    Results

    GPR40 was expressed on microglia, astrocytes, and neurons in the spinal cords and upregulated by spinal nerve ligation. Intrathecal injection of the GPR40 agonist GW9508 dose-dependently attenuated mechanical allodynia and thermal hyperalgesia in neuropathic rats, with Emax values of 80% and 100% MPE and ED50 values of 6.7 and 5.4 μg, respectively. Its mechanical antiallodynia was blocked by the selective GPR40 antagonist GW1100 but not GPR120 antagonist AH7614. Intrathecal GW9508 significantly enhanced IL-10 and β-endorphin immunostaining in spinal microglia and astrocytes but not in neurons. GW9508 also markedly stimulated gene and protein expression of IL-10 and β-endorphin in cultured primary spinal microglia and astrocytes but not in neurons, originated from 1-day-old neonatal rats. The IL-10 antibody inhibited GW9508-stimulated gene expression of the β-endorphin precursor proopiomelanocortin (POMC) but not IL-10, whereas the β-endorphin antibody did not affect GW9508-stimulated IL-10 or POMC gene expression. GW9508 increased phosphorylation of mitogen-activated protein kinases (MAPKs) including p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), and its stimulatory effects on IL-10 and POMC expression were blocked by each MAPK isoform inhibitor. Spinal GW9508-induced mechanical antiallodynia was completely blocked by intrathecal minocycline, IL-10 neutralizing antibody, β-endorphin antiserum, and μ-opioid receptor-preferred antagonist naloxone.

    Conclusions

    Our results illustrate that GPR40 activation produces antinociception via the spinal glial IL-10/β-endorphin antinociceptive pathway.

    Introduction

    The orphan G protein-coupled receptor 40 (GPR40), also known as free fatty acid (FFA) receptor 1, was first discovered in 1997 [1] and deorphanized in 2003 [2]. Belonging to the family A of the G protein-coupled receptor, it is activated by saturated/unsaturated medium- and long-chain FFA [2, 3]. GPR40 is widely expressed in various tissues, such as the brain, pancreas islet, heart, and spinal cord, with preferential expression in pancreatic β-cells [2,3,4,5]. GPR40 activation exerts multiple physiological functions in glucose metabolism, including regulation of the secretion of insulin [3], incretin [6], and glucagon [7]. Accumulative studies have proved GPR40 to be a promising target molecule for the treatment of type 2 diabetes mellitus, and its ligands are under clinical investigations as antidiabetic drugs [8,9,10,11]. In addition, GPR40 produces anti-neuroinflammation [12] and neuroprotection [13, 14]. GPR40 activation directly led to ERK activation, which subsequently performed profound functions in neuronal plasticity and long-term memory [15, 16]. The GPR40 agonist GW9508 significantly improved amyloid β-impaired cognitive performance in a mouse model of Alzheimer’s disease [17].

    GPR40 has been extensively demonstrated to be implicated with pain regulation. The GPR40 endogenous agonist docosahexaenoic acid (DHA) or exogenous agonist GW9508, given orally, intraperitoneally, or intracerebroventricularly, remarkably reduced pain behaviors in the mouse models of inflammatory pain or visceral pain induced by acetic acid, formalin, complete Freund’s adjuvant (CFA), carrageenan, or cyclophosphamide [14, 18,19,20,21,22]. Given intracerebroventricularly, they also significantly attenuated mechanical hyperalgesia induced by bilateral carotid artery occlusion in the mouse model of central post-stroke pain, which was reversed by the GPR40 antagonist GW1100 [23]. Furthermore, intrathecal injection of GW9508, DHA, and MEDICA16 significantly reduced mechanical allodynia and thermal hyperalgesia in the mouse models of neuropathic pain induced by spinal nerve injury and inflammatory pain induced by CFA and carrageenan [24], although a study reported these agents, given intrathecally, were ineffective in reducing formalin-induced pain [18].

    The mechanisms underlying GPR40 activation-induced antinociception remain unclear. It was reported that the descending inhibitory serotonergic and noradrenergic systems contributed to supraspinal GPR40 antinociception since intracerebroventricular injection of GW9508 facilitated the release of noradrenaline and 5-HT in the spinal cord and that the serotonergic and noradrenergic depleting and blocking agents 6-hydroxydopamine, dl-p-chlorophenylalanine, yohimbine, and WAY100635 inhibited GW9508-induced antinociception in the mouse formalin test [25]. DHA-induced antinociception also possibly occurred through the inhibition of microglia and astrocyte activation-induced spinal neuroinflammation and the secretion of inflammatory cytokines [23, 26]. On the other hand, the secretion of β-endorphin has been assumed to be associated with GPR40 activation-induced antinociception, as the GPR40 agonists accelerated β-endorphin release from the hypothalamus, and pretreatment with the β-endorphin antiserum, opioid receptor antagonist naloxone, and μ-opioid receptor antagonists β-funaltrexamine and CTOP blocked DHA- and GW9508-induced antinociception in the mouse inflammatory pain models of acetic acid, formalin, and CFA [18, 20, 21, 27, 28].

    We recently uncovered that various glucagon-like peptide-1 (GLP-1) receptor agonists (exenatide, WB4–24, and shanzhiside methylester), Cynanchi-derived cynandione A, and Aconitum-derived bulleyaconitine A, bullatine A, and lappaconitine effectively relieved chronic pain through the microglial secretion of β-endorphin and dynorphin A [29,30,31,32,33,34,35]. In addition, interleukin-10 (IL-10) is a well-known anti-inflammatory and immunosuppressive cytokine, expressed in astrocytes [36, 37] and microglia [38, 39], and its antinociception has been markedly confirmed in various pain models [40,41,42,43,44,45]. Intrathecal injection of exogenous IL-10-induced mechanical antiallodynia was mediated by the spinal microglial expression of β-endorphin [39]. Moreover, the GLP-1 receptor agonist exenatide stimulated the spinal expression of β-endorphin and produced mechanical antiallodynia in neuropathic pain through the autocrine microglial secretion of IL-10, via a cAMP/PKA/p38β/CREB signal pathway [46]. Thus, it is possible that GPR40 activation-induced β-endorphin secretion, likewise GLP-1 receptor activation, is mediated by IL-10 expression.

    The present study aimed to investigate the mechanisms underlying GPR40 activation-induced antinociception in neuropathic pain, especially related to the spinal glial IL-10/β-endorphin antinociceptive pathway. We first tested the effect of GW9508, given intrathecally, on mechanical allodynia and thermal hyperalgesia in neuropathic rats. Furthermore, we studied the glial expression of IL-10 and β-endorphin in the spinal cord and cultured primary cells and the signal transduction pathways. Lastly, we explored the association between the spinal glial expression of IL-10/β-endorphin and GW9508-induced mechanical antiallodynia. Our study, for the first time, reveals that GPR40 activation produces antinociception in neuropathic pain through the spinal glial expression of IL-10 and subsequent β-endorphin, highlighting the role of the newly discovered glial IL-10/β-endorphin pathway in pain transmission and transduction.

    Materials and methods

    Drugs and reagents

    GW9508, naloxone, GW1100, and AH7614 were purchased from Target Mol (Shanghai, China), Sigma-Aldrich (St. Louis, MO, USA), Cayman Chemical Company (Ann Arbor, Michigan, USA), and ApexBio (Houston, TX, USA), respectively. SP600125, SB203580, and U0126 were from Selleck Chemicals (Houston, TX, USA). Polyclonal antiserum against β-endorphin was purchased from Abcam (Cambridge, UK), and polyclonal antibodies against IL-10 and β-endorphin were obtained from Thermo Fisher Scientific (Vienna, Austria) and Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA), respectively. GW1100 or AH7614 was dissolved in 30% dimethyl sulfoxide (DMSO) and 70% polyethylene glycol (PEG) 400 or 50% PEG 400 in 0.9% normal saline. All other drugs or reagents were dissolved or diluted in normal saline.

    Experimental animals

    Male or female 1-day-old neonatal and male adult (160–180 g) Wistar rats were from the Shanghai Experimental Animal Institute for Biological Sciences (Shanghai, China). The adult rats were kept in the Laboratory Animal Center of Shanghai Jiao Tong University in a temperature- and humidity-controlled environment with an automatic 12-h light/dark cycle and access to food and water ad libitum. The animals were under 3–5 days’ adaptation before being used for any experiments. All the research protocols were approved by the Animal Care and Welfare Committee of the Shanghai Jiao Tong University (Shanghai, China) and carried out in accordance with the animal care guidelines of the US National Institutes of Health.

    Primary cell culture

    Primary microglia, astrocytes, and neurons were isolated from the spinal cords of 1-day-old neonatal rats. The isolated spinal cords were minced and incubated with 0.05% trypsin-EDTA (Invitrogen, Carlsbad, CA, USA) for 7 min. The dissociated cells were suspended in DMEM supplemented with 10% (v/v) fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). The cell suspension was filtered by a 40-μm cell strainer and then plated into 75-cm2 tissue cultural flasks (1 × 107 cells/flask) pre-coated with poly-l-lysine (0.1 mg/ml) and maintained at 37 °C in a 5% carbon dioxide incubator. For microglial cell preparation, the aliquots were collected after shaking the flasks at 260 rpm, 37 °C for 1.5–2 h after 10 days of culture. The cell suspension was then transferred to new plates, and unattached cells were removed by washing with phosphate-buffered saline (PBS) before drug treatments. Harvested microglial cells exhibited a purity > 95%, as determined by the microglial cellular marker ionized calcium binding adaptor molecule-1 (Iba-1) immunoreactivity [29, 39].

    For the astrocyte preparation, the mixed cells were incubated with 4 ml of 0.05% trypsin-EDTA in the cell incubator for 3 min to separate the oligodendrocytes. After neutralization, the floating cell suspension was discarded. The remaining monolayer of astrocytes was then trypsinized and sub-cultured in new plates for further experiments. Harvested astrocytes exhibited a purity > 90%, as determined by the astrocytic cellular marker glial fibrillary acid protein (GFAP) immunoreactivity [29].

    For neuronal cells, the cell suspension was plated into a 10-cm dish after being filtered by 40 μm for about 30 min to remove non-neural cells, and then, the cell suspension was plated into poly-l-lysine pre-coated plates. After incubation with complete DMEM for 1–2 h, the medium was changed to the Neurobasal (Invitrogen) containing 2% B27 supplement and 0.5 mM glutamine. The following experiments were performed 3–4 days later. Harvested neurons exhibited a purity of 85%, as determined by the neuronal cellular marker neuronal specific nuclear protein (NeuN) immunoreactivity [29].

    Intrathecal catheterization and injection in rats

    An 18-cm PE-10 catheter (OD 0.55 mm, ID 0.30 mm; AniLab Software & Instruments Co., Ningbo, China) was inserted into the lumbar level of the spinal cord under inhaled isoflurane anesthesia (4% for induction and 2% for maintenance) run by a gas anesthesia system (Ugo Basile, Comerio, Italy). The intrathecal catheterization was checked after 1-week recovery from surgery by intrathecal administration of 10 μl 4% lidocaine with 15 μl normal saline flush. Intrathecally catheterized rats that had no motor impairments after surgery and developed instant bilateral hindlimb paralysis after lidocaine injection were included in further research. For intrathecal injection, 10 μl of the drug solution was administered in a 50-μl syringe (Shanghai Anting Micro-Injector Factory, Shanghai, China) followed by a 15-μl saline flush.

    Rat model of neuropathic pain and behavior assessment

    After environmental adaption, L5/L6 spinal nerve ligation surgery was performed on rats under inhaled isoflurane anesthesia as described previously [47, 48]. Briefly, the left L5 and L6 spinal nerves were isolated and tightly ligated with 6–0 silk thread. After ligation, the wound was sutured after giving antibiotic and the rats were allowed to recover for about 2 weeks. As intrathecal injection was needed in the research, intrathecal catheterization was performed at the same time just before nerve ligation. After recovery, only rats with significant unilateral allodynia to mechanical stimulation (hindlimb withdrawal thresholds in the operated side < 8 g) and with no motor deficits were included in the further experiments.

    To evaluate mechanical allodynia, the animals were acclimatized to transparent plexiglass boxes on a metal grid for about 30 min. The hindlimb withdrawal threshold was measured by using a 2450 CE Electronic Von Frey hair (IITC Life Science Inc., Woodland Hill, CA, USA). The electronic handheld transducer with a no. 15 monofilament (with forces ranging from 0.1 to 90 g) was applied perpendicularly to the base of the third and fourth toe of the hindpaws with increasing force until a sudden withdrawal. The lowest force to induce the withdrawal response was considered as the threshold. The average of three repeated measurements at a 2-min interval was recorded as each hindlimb for each time point.

    To assess thermal hyperalgesia, the rats were placed in plexiglass boxes on a heated glass surface (about 37 °C) for about 30 min for adaption. After that, a radiant heat source (setting at a low density of 50) was applied to the plantar medial surface of each hindpaw. The hindpaw withdrawal latency was measured by a 390G Plantar Test Analgesia Meter (IITC Life Science Inc.). The paw withdrawal latency was defined as the time from the onset of radiant heat application to the withdrawal of the hindpaw. The cutoff latency was set at 30 s to avoid tissue damage. Each value at a time point was the mean value of three repeated measurements at a 2-min interval between tests.

    Neuropathic rats, starting the drug testing 2–3 weeks after the spinal nerve ligation, were randomly assigned to experimental groups, and the behavior observations were performed in a manner blind to the experimental conditions.

    RNA extraction and quantitative real-time PCR measurement

    For tissue samples, spinal lumbar enlargements (L3–5) from rats were isolated and homogenized using an electronic homogenizer at 4000 rpm for 15 s in TRIzol (Invitrogen) on ice. For cell samples, cells were collected after drug treatment and lysed in TRIzol for 5 min. Total RNAs extracted from the spinal cords and cells were reversely transcribed into cDNA using a ReverTra Ace qPCR RT kit (Toyobo Co., Osaka, Japan) according to the manufacturer’s protocols. Quantitative real-time PCR was then carried out by using the Realmaster Mix (SYBR Green I) (Toyobo Co.) in a Mastercycler ep realplex (Eppendorf, Hamburg, Germany). The fold change was calculated using the 2−ΔΔCt method after normalization to GAPDH. The primers were 5′-CCA AGG TCA TCC ATG ACA AC-3′ and 5′-TCC ACA GTC TTC TGA GTG GC-3′ for GAPDH [29], 5′-GGC TCA GCA CTG CTA TGT TGC C-3′ and 5′-AGC ATG TGG GTC TGG CTG ACT G-3′ for IL-10 [49], 5′-CTT TCC GCG ACA GAG CCT-3′ and 5′-CCA GCT CCA CAC GTC TAT GG-3′ for the β-endorphin precursor proopiomelanocortin (POMC) (NM_139326.2), and 5′-CCT GTC CTT GTG TTC CCT GT-3′ and 5′-AGA GGC AGT CAG GGT GAG AA-3′ for the dynorphin precursor prodynorphin (PDYN) [32]. All the primers were synthesized by Synbio Technologies (Suzhou, China).

    Protein extraction and Western blotting

    Cultured primary microglia were seeded in 6-well plates. The cells were treated with GW9508 (100 μM) for about 30 min on the next day, and then harvested with cold PBS and lysed in the radioimmunoprecipitation assay (RIPA) lysis buffer with the addition of the phosphates inhibitor cocktail and the protease inhibitor cocktail (Biotool, Houston, USA) after being centrifuged. Cell lysates were denatured at 100 °C for 10 min and separated in 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk in 1 × TBS containing 0.1% Tween-20 (TBST) and incubated with different primary antibodies against phospho-p38, phospho-ERK1/2, phospho-JNK (1:1000, rabbit polyclonal, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:2500, mouse monoclonal, Proteintech Group, Rosemont, IL, USA) diluted in the antibody diluent (Beyotime Biotechnology, Shanghai, China) at 4 °C overnight. On the following day, the membrane was thoroughly washed in TBST for 4 times (10 min each time) and further incubated with the Dylight 680-conjugated anti-mouse IgG and Dylight 800-conjugated anti-rabbit IgG (H + L) (Cell Signaling Technology) at room temperature for 1 h. The fluorescent density of each protein band was scanned by using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA) after being washed 4 times in TBST over 40 min. The relative expression of each protein was analyzed and quantified by using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) for the band ratios of phospho-p38/GAPDH, phospho-ERK1/2/GAPDH, and phospho-JNK/GAPDH.

    Immunofluorescence staining

    Double immunofluorescence staining of GPR40, β-endorphin or IL-10, and cellular biomarkers of microglia, astrocytes, and neurons was undertaken and observed using a TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) in cultured primary cells and the spinal cord sections using modified protocols [29].

    For immunocytochemistry, cultured primary microglia, astrocytes, and neurons were seeded on poly-l-lysine pre-coated coverslips placed at the bottom of 12-well plates (5 × 104 cells/well). After culture overnight, cells were washed with PBS and fixed in cool 4% paraformaldehyde for 1 h and were further blocked in the PBS containing 10% goat serum and 0.5% Triton X-100 for 1 h after being washed three times for 15 min in total with PBS. The cell flasks were then incubated with various primary antibodies (Table 1) in blocking buffer at 4 °C overnight, followed by incubation with Alexa 555-conjugated goat anti-rabbit secondary antibody (1:200; Life Technologies, Carlsbad, CA, USA) and Alexa 488-conjugated goat anti-mouse secondary antibody (1:200; Life Technologies) for 1 h at 37 °C. Nucleic dye reagent 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, 0.1 μg/ml; Beyotime, Shanghai, China) was used to stain cell nuclei. The expression of GPR40, Iba-1, GFAP, and NeuN was visualized under a laser scanning confocal microscope. Colocalization analysis was carried out by utilizing a computer-assisted image analysis program (ImageJ Software, National Institutes of Health, USA) equipped with a colocalization finder, under which colocalized pixels appeared as white points [29].

    Full size table

    For immunohistochemistry, rats were anesthetized by intraperitoneal pentobarbital sodium (40 mg/kg) and then intracardially perfused with 100 ml normal saline, followed by 60 ml of 4% paraformaldehyde. The spinal lumbar enlargements (L3–5) were isolated and fixed in paraformaldehyde for 12 h and dehydrated in gradient sucrose solutions (10%, 20%, and 30% in PBS) at 4 °C until precipitated at the bottom. Tissue sections were embedded in the OCT embedding agent (Leica Microsystems) and cut into 30-μm slices. The tissue sections were then incubated with 10% goat serum (v/v) and 0.5% Triton X-100 (v/v) in PBS for 1 h. The following steps were the same as the immunocytochemistry, with various primary antibodies (Table 1) and secondary antibodies.

    To quantify the relative intensity of GPR40, IL-10, and β-endorphin or their co-expression in microglia, astrocytes, and neurons in the spinal cord, photomicrographs of the medial threefourths of the dorsal horn (laminas I–V) were captured under × 10 or × 30 magnification. The positively stained surface area was measured by using the ImageJ Software after the low and high thresholds were set to exclude the background fluorescence. Only immunofluorescent intensity measurements from positively stained areas were included. The same setup configurations were used to measure all the surface areas in each experimental group at the same time. The averaged value of the positive immunofluorescence area fraction from three non-adjacent sections of each spinal cord was recorded as the relative expression or co-expression.

    IL-10 and β-endorphin measurement by ELISA kits

    IL-10 and β-endorphin were measured both in primary cells and spinal enlargements of neuropathic rats. For primary cells, cell culture supernatants were collected after treatment with GW9508 for 2 h and then centrifuged at 5000 rpm at 4 °C for 5 min. The supernatants after being centrifuged were prepared as the protein sample for ELISA. For the spinal enlargements, they were isolated from neuropathic rats after drug administrations, homogenized at 4000 rpm for 15 s with a homogenizer (Fluko Equipment) in 10 mM Tris-HCl (1 g of tissue/5 ml), and centrifuged at 4000 rpm at 4 °C for 15 min. The total protein concentration was measured by using the standard BCA assay (Beyotime Institute of Biotechnology, Shanghai, China). The levels of IL-10 and β-endorphin were measured using commercial IL-10 ELISA kit (Invitrogen) and β-endorphin ELISA kit (Phoenix Pharmaceuticals) according to the operation manuals. The concentrations of IL-10 and β-endorphin were calculated by a calibration curve performed at the same time. The linear range was 1–500 and 1–100 pg/ml for the IL-10 and β-endorphin assay, respectively.

    Experimental design and statistical analyses

    For the behavioral study, 5–6 male rats were randomly assigned to each group, and the behavioral measurements were carried out at different time points. For the analysis of dose-response curve, the percentage of maximal possible effect (% MPE) was calculated using the following formula: (post-drug threshold in ipsilateral hindlimb – pre-drug threshold in ipsilateral hindlimb) × 100%/(post-drug threshold in contralateral hindlimb – pre-drug threshold in ipsilateral hindlimb). The % MPE values approximated to 100 indicate normal mechanical thresholds or thermal latency (i.e., near contralateral thresholds), while values approximated to 0 indicate mechanical allodynia or thermal hyperalgesia. Half-effective dose (ED50) were calculated by using GraphPad Prism (Version 7.0, GraphPad Software, Inc., San Diego, CA, USA) by fitting nonlinear least squares curves to the relation Y = a + bx, where x = [D]n/(ED50n + [D]n). The values of ED50 and b (Emax) were projected by yielding a minimum residual sum of squares of deviations from the theoretical curve [50].

    Results were summarized as means ± SEM or 95% confidence intervals. The analysis of variance was performed by unpaired and two-tailed Student’s t test and one-way or repeated measures two-way ANOVA using GraphPad Prism to determine the significant difference. The post hoc Student-Newman-Keuls test was conducted when the effect of the drug (dose) (for one-way ANOVA, the factor was drug [dose]; for two-way ANOVA, the factors were drug [dose], time, and their interaction) was statistically significant. Values of P < 0.05 were considered statistical significance.

    Results

    Intrathecal GW9508 exerted mechanical antiallodynia and thermal antihyperalgesia in neuropathic pain via GPR40 activation

    The antinociceptive effects of the GPR40 agonist GW9508 were assessed in neuropathic rats induced by L5/L6 spinal nerve ligation approximately 2 weeks after surgery. Six groups of neuropathic rats received an intrathecal injection of either normal saline (10 μl) or GW9508 (1, 3, 10, 30, or 100 μg). The hindpaw withdrawal thresholds to the von Frey monofilament stimulus and paw withdrawal latencies to the thermal stimulus were subsequently (in a 10-min interval) measured before and 0.5, 1, 2 and 4 h post-intrathecal injection. In normal saline-treated rats, withdrawal thresholds in either contralateral or ipsilateral hindpaws remained unchanged during the observation period of 4 h. In contrast, the intrathecal injection of GW9508 attenuated mechanical allodynia in ipsilateral hindpaws in a dose- and time-dependent manner, without significantly affecting withdrawal thresholds in contralateral hindpaws (Fig. 1a). Mechanical thresholds at 1 h after injection were used for % MPE calculation and subsequent dose-response analysis. The projected Emax and ED50 values for mechanical antiallodynia inhibition were 80% MPE and 6.7 μg (95% confidence intervals 4.1–10.8 μg) (Fig. 1b). Furthermore, the intrathecal injection of GW9508 also dose- and time-dependently reduced the thermal hyperalgesia in ipsilateral hindpaws (Fig. 1c), with the projected Emax value of 100% MPE and ED50 value of 5.4 μg (95% confidence intervals 3.14–9.30 μg) (Fig. 1d).

    Inhibitory effects of GW9508 given intrathecally on mechanical allodynia (a, b) and thermal hyperalgesia (c, d) in neuropathic rats induced by spinal nerve ligation. Dose-response analyses of GW9508 on mechanical allodynia (b) and thermal hyperalgesia (d) were calculated using the thresholds measured 1 h after injection, best projected by the nonlinear least squares method. Effects of the selective GPR40 antagonist GW1100 (e) and GPR120 antagonist AH7614 (f) on GW9508-induced mechanical antiallodynia. Data are presented as means ± SEM (N = 6~7 per group). *P < 0.05 vs. the vehicle control group; analyzed by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test

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    GW9508 is also an agonist of GPR120, although with the affinity is 100-fold less than that of GPR40 [51]. The selective GPR40 antagonist GW1100 and GPR120 antagonist AH7614 were used to confirm whether or not GW9508 attenuated neuropathic pain through the activation of GPR40. Three groups of neuropathic rats received intrathecal injection of the vehicle (30% DMSO, 70% PEG400, 10 μl) and GW1100 (100 and 300 μg), respectively, followed by the second injection of GW9508 (30 μg) 30 min later. Paw withdrawal thresholds were measured before and 0.5, 1, 2, and 4 h post-GW9508 administration. Intrathecal injection of GW9508 exerted the time-dependent mechanical antiallodynia. Although GW1100 did not significantly alter baseline withdrawal responses in either hindpaws, it dose-dependently reversed GW9508-induced mechanical antiallodynia (P < 0.05, by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test; Fig. 1e). In addition, four groups of neuropathic rats were intrathecally administrated with the vehicle (10 μl) + saline (10 μl), vehicle (10 μl) + GW9508 (30 μg), AH7614 (30 μg) + saline (10 μl), and AH7614 (30 μg) + GW9508 (30 μg). The first intrathecal injection was followed by the second injection 30 min later. Intrathecal injection of GW9508 produced mechanical antiallodynia time-dependently (P < 0.05, by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test), which was not significantly altered by pretreatment with intrathecal AH7614 (Fig. 1f).

    Intrathecal GW9508 stimulated IL-10 and β-endorphin expression in the spinal dorsal horn

    GPR40 shows diversified distributions in the brain, pancreatic islets, spinal cord, pituitary, liver, heart, and skeletal muscle [2,3,4, 52]. We first examined its expression in the spinal cords of neuropathic rats by utilizing double immunofluorescence staining. Frozen sections were obtained from the spinal lumbar enlargements of neuropathic rats induced by spinal nerve ligation approximately 2 weeks after surgery. Immunofluorescence was stained with antibodies against GPR40 with cellular biomarkers for microglia, astrocytes, and neurons (Iba-1, GFAP, or NeuN, respectively). Photomicrographs were taken from the entire spinal cord (× 10 magnification) and dorsal horn laminae I–V (× 30 magnification). As shown in Additional file 1: Figure S1A, GPR40-positive cells were mainly expressed in the contralateral and ipsilateral dorsal horns, as well as in the ventral horns. In addition, double immunofluorescence staining of GPR40 with Iba-1, GFAP, and NeuN revealed that GPR40 was colocalized with microglia, astrocytes, and neurons in the contralateral and ipsilateral dorsal horn I–V laminae. Interestingly, the ipsilateral dorsal horn exhibited much higher expression of GPR40 immunostaining on all of microglia, astrocytes, and neurons, compared to the contralateral side (Fig. 2a–i). The ImageJ program was used to quantify immunofluorescence intensity (immunolabeled surface area) in the dorsal horn (× 10 magnification). As shown in Additional file 1: Figure S1B, the GPR40-immunolabeled surface areas in the ipsilateral spinal dorsal horn were significantly increased by 102%, compared with the contralateral side (P < 0.05, by unpaired and two-tailed Student’s t test). Double immunostaining of GPR40/Iba-1, GPR40/GFAP, and GPR40/NeuN in the ipsilateral dorsal horn was significantly increased by 165%, 203%, and 219%, respectively (P < 0.05, by unpaired and two-tailed Student’s t test; Fig. 2j–l).

    Expression of GPR40 in microglia (ac), astrocytes (df), and neurons (gi) in the spinal dorsal horn of neuropathic rats. Frozen sections of the spinal lumbar enlargements were obtained approximately 2 weeks after spinal nerve ligation. Immunofluorescence was double stained with GPR40/Iba-1, GPR40/GFAP, and GPR40/NeuN, and photomicrographs were taken from the spinal cord section (a, d, g; 500 μm) and amplified dorsal horn laminae I–V (b, c, e, f, h, i; 50 μm). Arrows indicate double immunostaining of GPR40 with each cellular biomarker. Double immunolabeled surface areas of GPR40/Iba-1 (j), GPR40/GFAP (k), and GPR40/NeuN (l) from the spinal dorsal horn laminae I–V indicated in white lines were quantified using the ImageJ program. Data are presented as means ± SEM (N = 5–6 per group). *P < 0.05 vs. contralateral side; analyzed by unpaired and two-tailed Student’s t test

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    We then assessed the stimulatory effects of GW9508 given intrathecally on the spinal level of IL-10 and its cell localization. Spinal enlargements were isolated from neuropathic rats 1 h after intrathecal normal saline (10 μl) or GW9508 (30 μg) treatment for IL-10 measurement. As shown in Additional file 2: Figure S2A and S2B, both gene and protein levels of IL-10 were significantly increased by the intrathecal injection of GW9508 (P < 0.05, by unpaired and two-tailed Student’s t test). Moreover, frozen sections were obtained 1 h after intrathecal normal saline (10 μl) or GW9508 (30 μg) treatment. Immunofluorescence was stained with antibodies against GPR40 with Iba-1, GFAP, and NeuN. In neuropathic rats treated with intrathecal normal saline, IL-10 was widely spread in the gray matter of both contralateral and ipsilateral spinal dorsal horns and in the ventral horns, which was markedly enhanced by the intrathecal administration of GW9508 (30 μg) in both contralateral and ipsilateral sides (Additional file 2: Figure S2C and S2D). Double immunofluorescence staining further revealed that IL-10 was colocalized with Iba-1 in the dorsal horn of saline-treated rats (Fig. 3a–c). Intrathecal injection of GW9508 significantly enhanced the co-immunostaining of IL-10/Iba-1 in both contralateral and ipsilateral dorsal horns (Fig. 3d–f). In addition, IL-10 was also colocalized with GFAP (Fig. 3g–i) and the intrathecal injection of GW9508 significantly enhanced the double immunostaining of IL-10/GFAP in both contralateral and ipsilateral dorsal horns (Fig. 3j–l). In contrast, although IL-10 was colocalized with NeuN (Fig. 3m–o), the intrathecal GW9508 failed to significantly increase the immunostaining of IL-10/NeuN either in the contralateral or ipsilateral dorsal horn (Fig. 3p–r).

    Effect of intrathecal GW9508 on spinal IL-10 expression on spinal microglia (af), astrocytes (gl), and neurons (mr) in neuropathic rats induced by spinal nerve ligation. Frozen sections of the spinal lumbar enlargements were obtained 1 h after intrathecal saline (10 μl) or GW9508 (30 μg) treatment. Immunofluorescence was double stained with IL-10/Iba-1, IL-10/GFAP, and IL-10/NeuN, and photomicrographs were taken from the entire spinal cord section (a, d, g, j, m, p; 500 μm) and amplified dorsal horn laminae I–V (b, c, e, f, h, i, k, l, n, o, q, r; 50 μm). Arrows indicate double immunostaining of IL-10 and each cellular biomarker. Double immunolabeled surface areas of IL-10/Iba-1 (s), IL-10/GFAP (t), and IL-10/NeuN (u) from the spinal dorsal horn laminae I-V indicated in white lines were quantified using the ImageJ program. Data are presented as means ± SEM (N = 5~7 per group). *P < 0.05 vs. saline group; analyzed by unpaired and two-tailed Student’s t test

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    The ImageJ program was used to quantify immunofluorescence intensity in the dorsal horn. As shown in Additional file 2: Figure S2E, the intrathecal GW9508 significantly increased the IL-10-immunolabeled surface areas by 520% and 380% in the contralateral and ipsilateral dorsal horns, compared with saline group (P < 0.05, by unpaired and two-tailed Student’s t test). Furthermore, GW9508 significantly increased the double immunostaining of IL-10/Iba-1 by 290% and 200%, respectively, in the contralateral and ipsilateral dorsal horns (P < 0.05, by unpaired and two-tailed Student’s t test; Fig. 3s). Although the baseline double immunostaining intensity of IL-10/GFAP was much lower than that of IL-10/Iba-1, it was significantly increased by GW9508 injection by 280% and 230% in contralateral and ipsilateral spinal dorsal horns, respectively (P < 0.05, by unpaired and two-tailed Student’s t test; Fig. 3t). However, GW9508 failed to significantly increase the double immunostaining intensity of IL-10/NeuN in either contralateral or ipsilateral spinal dorsal horn (Fig. 3u).

    We further assessed the stimulatory effect of intrathecal GW9508 treatment on β-endorphin expression in the above same spinal enlargement samples and spinal slides. β-Endorphin was markedly enhanced by the intrathecal GW9508 in both gene and protein level as well (Additional file 3: Figure S3A and S3B). β-Endorphin was also widely spread in the gray matter of both contralateral and ipsilateral spinal dorsal horns and ventral horns from the control neuropathic rats, and the intrathecal GW9508 injection significantly enhanced the spinal β-endorphin expression (Additional file 3: Figure S3C and S3D). β-Endorphin was colocalized with Iba-1 in the dorsal horns of control rats (Fig. 4a–c). Intrathecal injection of GW9508 significantly increased the double immunostaining of β-endorphin/Iba-1 both in the contralateral and ipsilateral spinal dorsal horns (Fig. 4d–f). In addition, β-endorphin was also colocalized with GFAP (Fig. 4g–i) and the intrathecal injection of GW9508 significantly enhanced its co-immunostaining in both contralateral and ipsilateral dorsal horns (Fig. 4j–l). In contrast, although β-endorphin was also colocalized with NeuN (Fig. 4m–o), the intrathecal GW9508 failed to significantly increase its double immunostaining in either the contralateral or ipsilateral dorsal horn (Fig. 4p–r). The ImageJ quantification in the dorsal horn showed that the intrathecal GW9508 significantly increased the immunofluorescence intensity of β-endorphin by 700% and 800% (P < 0.05, by unpaired and two-tailed Student’s t test; Additional file 3: Figure S3E), double immunofluorescence intensity of β-endorphin/Iba-1 by 380% and 430% (P < 0.05, by unpaired and two-tailed Student’s t test; Fig. 4s), and β-endorphin/GFAP by 360% and 260% (P = 0.06 and 0.13, by unpaired and two-tailed Student’s t test; Fig. 4t) in contralateral and ipsilateral spinal dorsal horn, respectively, compared with the saline group. In contrast, the intrathecal GW9508 failed to stimulate the double immunostaining of β-endorphin/NeuN in either contralateral or ipsilateral spinal dorsal horn (Fig. 4u).

    Effect of GW9508 given intrathecally on spinal β-endorphin expression on spinal microglia (af), astrocytes (gl), and neurons (mr) in neuropathic rats induced by spinal nerve ligation. Frozen sections of the spinal lumbar enlargements were obtained 1 h after intrathecal saline (10 μl) or GW9508 (30 μg) treatment. Immunofluorescence was double stained with β-endorphin/Iba-1, β-endorphin/GFAP, and β-endorphin/NeuN, and photomicrographs were taken from the entire spinal cord section (a, d, g, j, m, p: 500 μm) and amplified dorsal horn laminae I–V (b, c, e, f, h, i, k, l, n, o, q, r: 50 μm). Arrows indicate double immunostaining of β-endorphin with each cellular biomarker. Double immunolabeled surface areas of β-endorphin/Iba-1 (s), β-endorphin/GFAP (t), and β-endorphin/NeuN (u) from the spinal dorsal horn laminae I-V indicated in white lines were quantified using the ImageJ program. Data are presented as means ± SEM (N = 5~7 per group). *P < 0.05 vs. saline group; analyzed by unpaired and two-tailed Student’s t test

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    GW9508 stimulated IL-10 and β-endorphin expression in cultured primary microglia and astrocytes

    To further assess the cell expression of GPR40, microglia, astrocytes, and neurons were isolated from the spinal cords of neonatal rats and were co-immunostained with GPR40/Iba-1, GPR40/GFAP, and GPR40/NeuN, respectively, in addition to the nucleus staining with DAPI. As shown in Fig. 5a–c, GPR40 was expressed on microglia, astrocytes, and neurons, with 100% positive in each cell population.

    Expression of GPR40 (ac) and stimulatory effects of GW9508 on mRNA expression of IL-10, the β-endorphin precursor proopiomelanocortin (POMC) and dynorphin A precursor prodynorphin (PDYN) (dg), and secretion of IL-10 and β-endorphin (h, i) in cultured primary microglia, astrocytes, and neurons originated from the spinal cords of 1-day-old neonatal rats. For the immunostaining study, immunofluorescence was double stained with GPR40/Iba-1, GPR40/GFAP, and GPR40/NeuN. For the stimulatory study, cultured cells and cultural medium were collected 2 h after GW9508 incubation, and the mRNA expression of IL-10, POMC, and PDYN and the peptide concentrations of IL-10 and β-endorphin were measured using quantitative real-time PCR and commercial ELISA kits, respectively. Data are presented as means ± SEM (N = 4~7 per group). *P < 0.05 vs. control group; analyzed by unpaired and two-tailed Student’s t test

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    We then tested the stimulatory effect of GPR40 activation on IL-10 and β-endorphin expression. Primary microglia, astrocytes, and neurons from the spinal cords of neonatal rats were incubated with 100 μM GW9508 for 2 h, and the cellular mRNA expression of IL-10, POMC, and PDYN was measured by using quantitative real-time PCR. Incubation with GW9508 for 2 h significantly increased the expression of IL-10 mRNA in cultured primary microglia and astrocytes (P < 0.05, by unpaired and two-tailed Student’s t test), but not neurons (Fig. 5d). Further analysis showed that the GW9508 treatment concentration dependently increased the POMC expression in microglia, with an EC50 value of 13.4 μM (Fig. 5e). In addition, the treatment with GW9508 for 2 h significantly increased the mRNA expression of POMC in cultured primary microglia and astrocytes (P < 0.05, by unpaired and two-tailed Student’s t test), but not in neurons (Fig. 5f). In contrast, GW9508 failed to affect the PDYN mRNA expression in microglia, astrocytes, or neurons (Fig. 5g).

    The secretory effects of GW9508 on IL-10 and β-endorphin were also investigated. Primary cultures of microglia, astrocytes, and neurons were incubated with 100 μM GW9508 for 2 h, and the concentration of IL-10 and β-endorphin in the cultural medium were measured using commercial ELISA kits. As exhibited in Fig. 5h and i, the incubation with GW9508 for 2 h significantly increased the IL-10 and β-endorphin secretion from microglia and astrocytes (P < 0.05, by unpaired and two-tailed Student’s t test). However, the treatment with GW9508 did not affect the IL-10 or β-endorphin secretion from neurons.

    To confirm the stimulatory effect of GW9508 on IL-10 and β-endorphin expression through GPR40, the selective GPR40 antagonist GW1100 was utilized. As shown in Fig. 6a and b, the pretreatment with GW1100 (50 μM) for 30 min did not significantly the change basal expression of IL-10 or POMC mRNA in the cultured primary microglia, but totally blocked the GW9508 (100 μM)-induced elevation of IL-10 and POMC mRNA (P < 0.05, by one-way ANOVA followed by the post hoc Student-Newman-Keuls test).

    Effects of the specific GPR40 antagonist GW1100 (a, b), glial inhibitor minocycline (cf), IL-10 (g, h), and β-endorphin (i, j) neutralizing antibodies on the GW9508-enhanced mRNA expression of IL-10 and the β-endorphin precursor proopiomelanocortin (POMC) in microglia and astrocytes. Cultured primary microglial cells (ad, gj) and astrocytes (e, f), originated from the spinal cords of neonatal rats, were collected 2 h after drug treatment, and the mRNA expression of IL-10 and POMC was determined using quantitative real-time PCR. Data are presented as means ± SEM (N = 4~8 per group). Asterisk (*) and number sign (#) indicate P < 0.05 vs. the vehicle control group and GW9508 group, respectively; analyzed by one-way ANOVA followed by the post hoc Student-Newman-Keuls test

    Full size image

    Minocycline is generally considered as a microglia inhibitor [53,54,55], but may inhibit astrocyte activation as well [56,57,58]. To confirm its glial inhibitory effect, minocycline was used to block the GW9508-induced mRNA expression of IL-10 and POMC in microglia and astrocytes. Pretreatment with minocycline (60 μM) in the cultured primary microglia for 1 h did not change the baseline expression of IL-10 and POMC mRNA, but entirely blocked the GW9508 (100 μM)-elevated IL-10 and POMC mRNA expression (P < 0.05, by one-way ANOVA followed by the post hoc Student-Newman-Keuls test; Fig. 6c, d). Moreover, as exhibited in Fig. 6e and f, GW9508 also markedly increased the IL-10 and POMC mRNA expression in the cultured primary astrocytes, and the pretreatment with minocycline significantly inhibited the GW9508-stimulated POMC mRNA expression (P < 0.05, by one-way ANOVA followed by the post hoc Student-Newman-Keuls test). Minocycline also appeared to inhibit the GW9508-induced IL-10 expression, although it did not reach statistical significance (P = 0.12).

    We have recently discovered that exogenous IL-10 and endogenous IL-10 after GLP-1 receptor activation induced the β-endorphin expression in microglia [49]. Following GPR40 activation, the IL-10 and β-endorphin neutralizing antibodies were used to illustrate IL-10 to induce β-endorphin and vice versa. As shown above, the treatment with GW9085 (100 μM) significantly increased the IL-10 and POMC mRNA expression in the cultured primary microglia, whereas the treatment with either the IL-10 antibody (4 μg/ml) or β-endorphin antibody (1:300 dilution) had no effects on the baseline expression of both IL-10 and POMC mRNA. The IL-10 neutralizing antibody completely inhibited the GW9508-stimulated mRNA expression of POMC (P < 0.05, by one-way ANOVA followed by the post hoc Student-Newman-Keuls test) but not IL-10 (Fig. 6g, h). In contrast, the β-endorphin neutralizing antibody did not exhibit any inhibitory effects on the GW9508-stimulated mRNA expression of either IL-10 or POMC (Fig. 6i, j).

    Mitogen-activated protein kinases (MAPKs), including p38, ERK, and JNK, are evolutionarily conserved enzymes that play crucial roles in various cellular processes [59, 60]. We assessed whether GW9508 stimulated IL-10 and POMC mRNA expression through MAPK activation. In cultured primary microglia, the treatment with GW9508 (100 μM) for 30 min significantly increased the p38 phosphorylation by 1.7-fold (P < 0.05, by unpaired and two-tailed Student’s t test; Fig. 7a). In addition, pretreatment with the selective p38 inhibitor SB203580 (30 μM) significantly inhibited the GW9508-stimulated IL-10 and POMC mRNA expression (P < 0.05, by one-way ANOVA followed by followed by the post hoc Student-Newman-Keuls test), although it did not significantly affect their baseline expression (Fig. 7b, c). As shown in Fig. 7d–i, the treatment with GW9508 also stimulated both JNK and ERK1/2 phosphorylation by 1.4- and 1.5-fold, respectively (P < 0.05, by unpaired and two-tailed Student’s t test), and pretreatment with the selective JNK inhibitor SP600125 (30 μM) and ERK1/2 inhibitor U0126 (30 μM) significantly attenuated the GW9508-increased IL-10 and POMC mRNA expression (P < 0.05, by one-way ANOVA followed by the post hoc Student-Newman-Keuls test), without affecting their baseline expression.

    Effects of the phosphorylation of p38 (ac), JNK (df), and ERK1/2 mitogen-activated protein kinase (MAPK) (gi) on GW9508-enhanced mRNA expression of IL-10 and β-endorphin in microglia. Cultured primary microglial cells were originated from the spinal cords of neonatal rats. For the phosphorylation immunoblotting study, microglia were collected 30 min after the GW9508 treatment, and their lysates were immunoblotted with the antibodies of anti-phospho-p38, anti-phospho-ERK1/2, and anti-phospho-JNK, respectively. Immunoblots of each experiment are placed on the upper panels, and densitometric analyses are shown in the lower panels. For the MAPK inhibitor study, microglia were collected 2 h after drug treatment, and the mRNA expression of IL-10 and the β-endorphin precursor proopiomelanocortin (POMC) was determined using quantitative real-time PCR. Data are presented as means ± SEM (N = 5~8 per group). Asterisk (*) and number sign (#) indicate P < 0.05 vs. the vehicle control group and GW9508 group, respectively; analyzed by unpaired and two-tailed Student’s t test and one-way ANOVA followed by the post hoc Student-Newman-Keuls test

    Full size image

    GW9508 exerted antinociception via spinal glial IL-10/β-endorphin pathway

    Minocycline was used to block the GW9508-induced mechanical antiallodynia. Two groups of neuropathic rats were pretreated with intrathecal saline (10 μl) or minocycline (100 μg) for 4 h followed by the intrathecal injection of GW9508 (30 μg). Paw withdrawal thresholds were measured before and 0, 0.5, 1, 2, and 4 h post-GW9508 administration. The intrathecal injection of GW9508 exerted profound mechanical antiallodynia in a time-dependent manner, which was reversed by the pretreatment with minocycline (P < 0.05, by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test), although it did not alter the baseline withdrawal response in either paw (Fig. 8a).

    Blockade effects of the glial inhibitor minocycline (a), specific IL-10 antibody (b), β-endorphin antiserum (c), and μ-opioid receptor-preferred antagonist naloxone (d), given intrathecally, on the spinal GW9508-induced mechanical antiallodynia in neuropathic rats. Neuropathic rats, 2 weeks after spinal nerve ligation, received a single injection of the vehicle (10 μl) or each blocking agent followed by a single intrathecal injection of GW9508 (30 μg) 4 h (for minocycline) or 30 min (for the rest drugs) later for subsequent assessment of withdrawal responses to mechanical stimuli. Data are presented as means ± SEM (N = 5~8 per group). *P < 0.05, vs. vehicle + GW9508 group; analyzed by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test

    Full size image

    In order to confirm the causal role of spinal IL-10/β-endorphin in the GW9508-induced mechanical antiallodynia, the IL-10 neutralizing antibody was first applied. Two groups of neuropathic rats received an intrathecal injection of normal saline (10 μl) or the IL-10 antibody (2 μg) for 30 min followed by an intrathecal injection of GW9508 (30 μg). Paw withdrawal thresholds were measured before and 0, 0.5, 1, 2, and 4 h post-GW9508 administration. Pretreatment with intrathecal IL-10 neutralizing antibody completely reversed the mechanical antiallodynia produced by intrathecal GW9508 (P < 0.05, by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test), although it did not significantly alter the baseline withdrawal responses in either contralateral or ipsilateral hindpaws (Fig. 8b).

    In addition, two groups of neuropathic rats received an intrathecal injection of the blank serum (1:10 dilution, 10 μl) or the β-endorphin antiserum (1:10 dilution, 10 μl) for 30 min followed by an intrathecal injection of GW9508 (30 μg). Paw withdrawal thresholds were measured before and 0, 0.5, 1, 2, and 4 h post-GW9508 administration. The intrathecal injection of GW9508 exerted profound mechanical antiallodynia in a time-dependent manner, which was completely reversed by the pretreatment with intrathecal β-endorphin antiserum (P < 0.05, by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test), although it did not significantly alter baseline withdrawal responses in either hindpaws (Fig. 8c).

    Furthermore, as β-endorphin is an endogenous ligand of μ-opioid receptors [61], the μ-opioid receptor-preferred antagonist naloxone was utilized to illustrate the pivotal role of β-endorphin in the GW9508-induced mechanical antiallodynia. Two groups of neuropathic rats received an intrathecal injection of normal saline (10 μl) or naloxone (20 μg) for 30 min, followed by an intrathecal injection of GW9508 (30 μg). Pretreatment with intrathecal naloxone did not significantly affect baseline withdrawal responses in either hindpaws, but completely blocked the GW9508-exerted mechanical antiallodynia in the ipsilateral paws (P < 0.05, by repeated measures two-way ANOVA followed by the post hoc Student-Newman-Keuls test; Fig. 8d).

    Discussion

    Our present study demonstrated that the GPR40 agonist GW9508, given intrathecally, was effective in attenuating mechanical allodynia and thermal hyperalgesia in ipsilateral hindpaws of neuropathic rats in a dose-dependent manner, with Emax values of 80% and 100% MPE and ED50 values of 6.7 and 5.4 μg, respectively. It did not significantly affect the normal reflex nociceptive pain states in contralateral hindpaws. We also confirmed that the GW9508-induced mechanical antiallodynia was through GPR40 activation, as the selective GPR40 antagonist GW1100 (but not selective GPR120 antagonist AH7614) dose-dependently and nearly completely suppressed the GW9508-induced antinociception and as the GPR120 was not expressed in the central nervous system including the spinal cord [18]. Our results were in accordance with the previous findings, which showed that the GPR40 agonists DHA, GW9508, and MEDICA16, given orally, intracerebroventricularly, or intrathecally, attenuated pain behaviors in the rodent models of inflammatory pain, visceral pain, and neuropathic pain [14, 18, 23, 24]. Taken together, all the results indicated that GPR40 agonists were specifically antinociceptive in painful hypersensitivity states.

    The more striking finding from the current study is that GPR40 activation-induced antinociception is mediated by the spinal glial expression of IL-10 and subsequent β-endorphin. The notion is supported by the following findings: (1) Intrathecal injection of GW9508 stimulated the expression of IL-10 and β-endorphin in microglia and astrocytes, but not in neurons, in both contralateral and ipsilateral spinal cords of neuropathic rats, directly identified by double immunofluorescence staining of IL-10 and β-endorphin with cellular biomarkers, i.e., Iba-1, GFAP, and NeuN. (2) Treatment with GW9508 stimulated the gene and protein expression of IL-10 and β-endorphin in primary cultures of microglia and astrocytes, but not of neurons, in a GW1100-reversible manner. The specific IL-10 neutralizing antibody in primary microglia completely inhibited the GW9508-stimulated mRNA expression of POMC but not IL-10, whereas the β-endorphin antibody did not affect the GW9508-stimulated IL-10 or POMC gene expression. The results are in agreement with our previous findings that the IL-10 neutralizing antibody completely inhibited the exenatide-stimulated expression of POMC [39] and that IL-10 stimulated the β-endorphin expression but not vice versa [46]. (3) Intrathecal injection of the IL-10 neutralizing antibody, β-endorphin antiserum, and μ-opioid receptor-preferred antagonist naloxone entirely eliminated the spinal GW9508-induced mechanical antiallodynia in neuropathic rats. (4) Minocycline is generally deemed as a microglial inhibitor [53,54,55], but it also inhibited astrocyte activation as well [56,57,58]. Treatment with minocycline in the current study inhibited the GW9508-stimulated IL-10 and POMC gene expression in the cultured primary microglia and astrocytes. Moreover, the intrathecal injection of minocycline completely blocked the spinal GW9508-induced mechanical antiallodynia in neuropathic rats. These results further highlight the broad significance of the newly discovered spinal glial IL-10/β-endorphin pathway in pain process and modulation. It was recently discovered that IL-10 and IL-24, a member of the IL-10 family, were effective in relieving neuropathic pain and bone cancer pain in rats by the promotion of β-endorphin synthesis [62]. Moreover, GLP-1 receptor agonist exenatide induced the antinociception through spinal microglial IL-10 and β-endorphin pathway via the cAMP/PKA/p38β/CREB and JAK1/STAT3 signal transduction mechanisms [49].

    β-Endorphin is an endogenous opioid neuropeptide that is known to be produced in neurons within the central nervous system including the hypothalamus and modulates pain transmission and transduction by the activation of μ-opioid receptors [63,64,65,66,67]. However, recent findings indicate that endogenous opioid peptides (including endorphins and dynorphins) are also present in astrocytes and microglia [29, 33, 39, 68, 69]. Particularly, β-endorphin and dynorphin A secreted by GLP-1 receptor agonists, herbal cynandione A, and aconitines effectively attenuated painful hypersensitivity [29,30,31

    Источник: https://jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-019-1457-9

    How do I access Prism Academy from a Group Subscription?

    What is Prism Academy?

    Prism Academy is an online learning portal, accessed via the Prism application and included with valid, paid Prism subscriptions. In Prism Academy, users can access a wealth of product tutorials and educational content. Prism Academy includes over 125 video tutorials taught by experts and exclusive online access to the leading biostatistics resource for working scientists, Intuitive Biostatistics.

    Why should my users use Prism Academy? 

    Designed for beginners and advanced users, your users will learn how to master unique tools and features within Prism. It is a self-driven learning platform for users of all levels.

    Your users will get the most out of Prism by

    • Becoming more efficient and productive using the Prism features

    • Improving accuracy in interpreting and presenting their research

    • Testing their knowledge with interactive quizzes and certifications

    Group Subscription Access to Prism Academy


    Who has access to Prism Academy? 

    The users that have activated Prism under your group subscription have access to Prism Academy, included with the valid subscription, on the computer on which they have activated Prism

    To access the online learning materials, each individual user that has activated under the group subscription will need to register for their own customer account when they access Prism Academy, preferably, using their institutional email address. They are required to register for individual customer accounts (rather than one shared account) because Prism Academy will provide each user with a personalized view of their training progress.


    When can my users access Prism Academy?

    Users can access Prism Academy throughout the entire duration of their valid subscription. If a subscription is canceled or expired, Prism Academy will no longer be accessible.  

     

    Where is Prism Academy located? 

    The resources are available through an online portal, accessed via the Prism software itself. The users of the group subscription can access Prism Academy directly through the installed  Prism application on the computer that has been activated under your group license serial number. Just launch Prism and click the Prism Academy button on the bottom left of the Prism welcome screen. There is also a Prism Academy button in the toolbar. 

     

    How do my users access Prism Academy under our group subscription? 

    Your users can access Prism Academy directly through their Prism application on the machine that is licensed under your serial number. Your users must be using Prism v9.1 or higher to access Prism Academy.

    To access, they simply click the Prism Academy button on the bottom left of the Prism welcome screen. They can also access Prism Academy from the Help option in the Prism toolbar.

    Users will then be directed to a browser window and prompted to log in to access Prism Academy. If a user is signing in for the first time, they will need to create a new customer account using their institutional email address. 

     

    How can I tell if my users have accessed  Prism Academy from our set of activated computers under the license?

    When you sign in to your Prism admin account page at graphpad.com/myaccount, you can click the “Activations” tab on your dashboard to view the list of computers activated under your group license. If a user has accessed Prism Academy from a Prism-activated computer, you will see an additional line beneath the Prism activation information that says “Academy” with the associated email address.

    Note: This email address has nothing to do with the allowed user email addresses under your Prism license. This is simply the email address/customer account used to sign into the Prism Academy training portal on that particular computer.

     

    Can I change the Prism Academy email associated with a specific Prism activation? 

    Yes, administrators of Prism group subscriptions have the ability to clear the email associated with a Prism Academy account from their online admin account page. To do this, you will need to log in to https://www.graphpad.com/myaccount/  and select the “Activations” tab. There, you will see a list of activated machines and their associated email addresses. Once Prism Academy has been accessed on a machine, you will see a line under the activation entry that is labeled “Academy”. There will be a blue “(x)” next to the email address associated with the Prism Academy account. Clearing this association does not affect the Prism software activation in any way. 

    The next time someone tries to access the Prism Academy from the Prism app on that particular computer, they will be prompted to sign in to Prism Academy, rather than automatically launching under the previous Prism Academy email address associated with that machine. This function essentially resets the computer’s link to a particular Prism academy account. This action of clearing the associated email does not block a user or clock the actual Prism license in any way.

     

    Why are some of my users unable to access Prism Academy?

    Be sure that all of your users are running at least Prism v9.1.0, which is when we added Prism Academy access to the software itself. If users are running older versions of Prism, they do not have access to Prism Academy. 

    Once they update their software, they will see the Prism Academy option available from the software itself, both at the welcome screen when they first launch the app and in the toolbar at the top of the app.

    We are also happy to troubleshoot and assist further if needed. Please reach out to us at help.graphpad.com.

    Источник: https://www.graphpad.com/support/faq/access-prism-academy-from-a-group-subscription/

    googleのランキングの要因は伝説のものです。

    そこには、googleのランキングを知らせる200の以上のシグナルと噂(この統計は、2006年に始まったが、物事がそれ以来少しも変わってきたと言うことはおそらく安全だ)されており、正確なリストを作る要因だけでなく、彼らの重要な順序は、多年生の議論の対象です。

    で、私たちますが検索エンジン・ウォッチは、決してグーグルの完全なリストを主張することができますランキング要因(と彼らはあなたに嘘をついていることができますと言う誰も-そう、彼らはgoogleからの方にも、おそらく)、我々は掘り下げました主題はかなり少しです。

    去年私たちの勇敢な編集者christopher ratcliffは、いくつかのgoogleの重要なランク付け要素の詳細を調べる10回シリーズの記事を書きました。このガイドはあなたの参照の便宜のためにそのシリーズからの主要な洞察を要約します、それぞれのランキング要因の完全な説明へのリンク。

    コンテンツの鮮度からコンテンツの品質、内部リンクからバックリンクまで、googleの堅実なランキングを達成するために必要な主なポイント、およびそれらのヒット方法について説明しました。

    それでさらに面倒なことなしに始めましょう。

    にジャンプします。

    パート1:ページ要因
    パート2:キーワード
    パート3:品質の高い内容
    パート4:コンテンツの鮮度
    パート5:重複するコンテンツとシンジケーション
    パート6:信頼、権威および専門知識
    パート7:サイトレベルの信号
    パート8:内部リンク
    パート9:アウトバウンドリンク
    パート10:バックリンク
    ボーナス:ランクブレイン

    パート1:ページ要因
    googleのランク付け要因に関するガイドの最初の部分では、googleがページのランク付けに使用する単純で技術的な要素(タイトルタグ、h1タグ、メタ説明)について説明します。

    これらはすべてあなたが完全にコントロールできる要素であり、googleがあなたのサイトをランク付けする方法とあなたのサイトがserpに現れる方法の両方に重要な影響を与えます。したがって、それらを適切に最適化する方法を学ぶことは非常に重要です。

    タイトルタグ、h1タグ、および検索用のメタ説明を最適化する方法に関するいくつかの重要なポイント:

    ランク付けしたいキーワードをタイトルタグに含めます。キーワードがタグの先頭に近いほど、そのキーワードでページがランク付けされる可能性が高くなります。
    そうは言っても、あなたのタイトルタグが人間のために書かれていることを確認してください – それは彼らがまだ論理的な意味を成す必要があり、キーワードだけでいっぱいに詰め込まれることを意味します
    あなたのウェブサイトでタイトルタグを重複させないでください。
    ターゲットキーワードもh1タグに含める必要がありますが、h1はタイトルタグと異なる場合があります
    通常は1ページに1つのh1タグしか使用できませんが、h2タグとh3タグを使用してコンテンツをさらに分割することができます。
    メタディスクリプションは厳密にはランキングのシグナルではありませんが、優れたメタディスクリプションはクリック率を大幅に向上させることができるため、賢明に使用してください。
    seoのタイトルタグとメタ説明の書き方についてさらに深く知りたい場合は、2つの別々のガイドをチェックしてください。

    seoのメタタイトルタグの書き方
    seoのためのメタ説明を書く方法

    パート2:キーワード
    ランキング要因ガイドのパート2では、seoに関する議論の永遠の主題であるキーワードについて説明しています。

    seoにおけるキーワードの役割は、検索の初期から大きく変化してきましたが、ロングテールキーワードと自然言語検索の進化により、控え目なキーワードは依然として検索最適化の重要な基本要素の1つであり、googleの重要なランキングです。信号。

    しかし、前のセクションで説明したように、キーワードが重要であるからといって、それらを狂ったように詰め込むべきではありません。ここでは、キーワードの上手な使い方についてのgoogleのランキング要因に関するガイドからのハイライトをいくつか紹介します。

    キーワードの関連性と掲載順位は、頻度よりもはるかに重要です。最初の文ではない場合、あなたのキーワードまたはキーフレーズはあなたのページの最初の100単語に現れるべきです
    グーグルは最初にメタ情報とヘッダーを優先し、次にボディーコピー、そして最後にサイドバーとフッターを優先する。
    キーフレーズは、検索者が検索エンジンに入力する内容と完全に一致するようにしてください。これは、自然言語検索クエリに最適化したい場合は、キーワードを会話形式で表現することを意味します。
    過度のキーワードの繰り返し、および他のコンテンツに関係のないキーワードの使用は、ペナルティを受ける可能性があります。
    ドメインのurlにキーワードを含めると、 seoが少し向上します。

    パート3:品質の高い内容
    あなたのブログやサイトをグーグルで高く評価する方法として「品質の高いコンテンツ」という言葉が使われているのは間違いありません。「品質の高いコンテンツを制作する」。

    まあ、それはすべて非常によくて良いです、しかしそれは実際的にはどういう意味ですか?制作しているコンテンツがgoogleにとって十分に高品質であるかどうかをどうやって知ることができますか。

    googleのランキング要因に関するガイドの第3部では、スペルや文法から読みやすさ、フォーマット、長さまで、コンテンツの品質を評価するための14のヒントを紹介します。これが私たちのポインタのいくつかです:

    前に説明したように、コンテンツは、アルゴリズムだけでなく人間に訴えるように書かれていることを確認し、キーワードで飽和させないでください。
    fleschの読みやすさテストでコンテンツの読みやすさのスコアを確認し、60%以上になることを目指します
    文章や段落を短くして、改行(モバイルでは読みやすくするための余白が多くなります)や小見出しでそれらを分割してください。
    文章や段落を短くしたいのですが、全体的な内容は空想のままにすることができます。詳細な内容は品質の大きな指標です。

    パート4:コンテンツの鮮度
    ページ上のコンテンツのシグナルを続けていくと、webページが最近公開された時期もランク付けのシグナルです。ただし、最近のイベントの検索、話題のトピック、定期的な定期的なイベントなど、検索の種類は異なります。

    googleのアルゴリズムは、検索を最も関連性があり最新の結果と一致させるときに、これらすべてを考慮に入れようとします。

    昨年、mozは、コンテンツの鮮度がgoogleのランキングにどのように影響するかについて包括的な調査結果を発表しました。これは、googleの順位要素に関するガイドの第4部にある洞察の基盤となります。主な要点は次のとおりです。

    webページには、その発行日に基づいて、コンテンツが古くなるにつれて時間の経過とともに減衰する即時の「鮮度スコア」を付与できます。コンテンツを定期的に更新すると、そのスコアを維持するのに役立ちます。
    コンテンツにリンクしている外部サイトの数の増加は、関連性と鮮度の指標と見なすことができます。
    「新鮮な」サイトからのリンクはあなたのコンテンツにその新鮮さを伝えるのを助けることができます
    最新の結果が常に最良というわけではありません。ニュース価値の低いトピックでは、詳細で信頼性の高い結果が新しいコンテンツよりも細いコンテンツを上回る可能性があります。

    パート5:重複するコンテンツとシンジケーション
    あなたのウェブサイトで他人のコンテンツを再公開すること、またはあなた自身のコンテンツを社内で再利用することが許容されるのはいつですか?seoコミュニティは、偶然にも「重複コンテンツペナルティ」を犯してしまうという恐怖を共有しており、それを回避する方法についてアドバイスがたくさんあります。

    確かに、許可なく他人のコンテンツを盗んで再公開することはひどいやり方であり、これを頻繁に行うことはスパムで質の低いwebサイトの明らかな兆候です。ただし、ann smartyがfaqで重複コンテンツについて説明しているように、「重複コンテンツのペナルティ」というようなことはありません。グーグルの誰もそのようなペナルティの存在を確認したことはなく、また「重複した内容」のアルゴリズムの更新もなかった。

    それでは、重複したコンテンツを公開することの危険性は何ですか?つまり、オンラインで同じ投稿に複数のバージョンがある場合、googleはどの記事をランク付けするかについて電話をかけ、最初に公開された記事とは無関係に検索を行います。最高権威。

    同様に、ランキングをめぐって競合する同じ内部コンテンツの複数のバージョンがある場合(これには同じサイトの別々のデスクトップバージョンとモバイルバージョンが含まれます)、足を踏み入れて自分を撮影することができます。

    どうすればこれをすべて回避できますか。googleのランク付け要素の記事の第5部では、重複して配信されるコンテンツを管理してgoogleが優先urlのみをインデックスに登録するようにする方法について説明しています。いくつかのポイントが含まれます:

    自分のサイトに重複したコンテンツがある場合は、301リダイレクトを設定して、googleが自分の優先ページにインデックスを付けるようにする
    別の携帯サイトの代わりにレスポンシブwebサイトを使用する
    シンジケートコンテンツにrel = canonicalタグまたはmeta noindexタグを使用して、どの記事がオリジナルであるかをgoogleに伝えます。

    パート6:信頼、権威および専門知識
    特にリンク構築キャンペーンに関しては、高いレベルの権限を持つwebサイトがより大きな重みを持っていることを私たちは知っています。しかし、グーグルはあなたのウェブサイトの信頼、権威そして専門性のレベルをどのように評価しているのでしょうか?

    googleのランキングの要因に関するガイドの第6部では、googleがどのように権限、信頼、専門知識を計算するのかと同様に、あなたのサイトのページ品質評価を構成する要因を調べます。主なポイントは次のとおりです。

    コンテンツの品質、コンテンツの量、およびwebサイト情報は、すべてページ品質評価の要素です。
    論理サイトアーキテクチャは、より高いレベルの権限を手助けすることができます。
    ブログを始めることはあなたのビジネスが関連していて信頼できることを示すのを助けることができます – そしてまた、コンテンツの新鮮さを手助けすること
    特に総レビュー数が多い場合は、マイナスの顧客レビューがページの品質評価に影響を与えることはありません。googleは実際の評価よりも、コンテンツのレビューを確認する傾向があります。

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    関連記事
    2019年のモバイル検索とビデオ:あなたはどのくらい目に見えますか? facebookはローカル検索エンジンです。あなたはそれを一つのように扱っていますか? seo用の17のトッププラグインと拡張 seoにおけるサイバーセキュリティ:webサイトのセキュリティがseoのパフォーマンスに与える影響
    パート7:サイトレベルの信号
    ページ上のコンテンツから離れて、googleのランキング要因に関するガイドの第7部ではサイトレベルのシグナルに注目しています。

    googleは、サイトレベルであなたのランキングに影響を与える可能性のある要因をどのように考慮していますか?ここにいくつかあります…

    https:googleは2014年に、httpsを「非常に軽量な信号」として使用し始めたと発表しました。それ以来それが強化されたかどうかはわからないが、特にあなたのウェブサイトが金融取引を扱うならば、httpsの使用も一般的にちょうど良い習慣です
    携帯性:2015年の最初の「mobilegeddon」更新以降、携帯性はgoogleの検索結果における重要な要素となっています。それ以来、信号は強化されてきました
    サイトの速度:特に携帯電話でサイトの速度を評価して最適化することに時間をかけてください。そうすれば、検索のランキングが向上することに気付くでしょう。

    パート8:内部リンク
    私たちのランキングの要因のパート8、9、10はすべて、インターネットの神経系を扱うものです:リンク。さまざまな種類のリンクがどのようにあなたのサイトが検索でうまくランク付けするのを助けますか?

    まず、内部リンクです。starcomのjason mcgovernによると、内部リンクは、特定のページのコンテンツが重要であることをgoogle(および訪問者)に伝えるために使用できる数少ない方法の1つです。それでは、どのようにあなたはあなたのウェブサイトの他のページに内部的にリンクすることについて行くべきですか?

    パート8では、「ハブページ」やその構築方法など、内部リンクを使用してサイトの指標やユーザーエクスペリエンスを向上させる方法について説明します。

    重要なポイントを理解したら、seoのための内部リンクの完全ガイド:例とベストプラクティスを必ずチェックしてください。

    パート9:アウトバウンドリンク
    アウトバウンドリンク、つまり外部リンクは、あなたのサイトから別のウェブサイトへと外側を向いているリンクです。それらはあなたがリンクしているサイトにあなた自身のサイトのランキング力の一部を(リンクがスーパースパムのウェブサイトに行かない限りあなたに何の害もなしに)伝えます。

    しかし、これはどのようにあなたに利益をもたらしますか?なぜあなたは他のサイトへのリンクジュースの景品をリンクしているのでしょうか?

    実際、googleのgoogleのランキング要因ガイドの第9部で明らかにされているように、関連性のある権威のあるサイトへの外部リンクはあなたのランキングに役立ちます。アウトバウンドリンクとseoに関するその他の重要なポイントは次のとおりです。

    pagerankの保持は神話です – あなたのサイトが内部リンクより外部リンクを持つことによってリンクジュースを「漏らす」ことは不可能です
    実際、アウトバウンドリンクは信頼のシグナルとしてカウントされます – あなたがあなたのデータと研究をバックアップするために参照にリンクしているならば、あなたは明らかにあなたの仕事をきちんと終えて信頼できます。
    アフィリエイトリンクも問題ありませんが、必ずgoogleのベストプラクティスに従ってnofollowメタタグを使用してください。

    パート10:バックリンク
    なぜバックリンク(第三者からあなたのサイトに戻るリンク)がseoにとって重要なのですか?先ほど説明したように、自分のサイトから別のwebサイトへの外部リンクは、ランク付けの力の一部を通ります – その逆もまた当てはまります。

    オンラインで他の場所からあなたのサイトにリンクを張ることはあなたの検索ランキングを改善するための重要な方法です。実際、昨年google irelandのsearch quality senior strategistであるandrey lipattsevによって明らかにされたように、あなたのウェブサイトへのリンクは上位3つのランク付け要因の1つです。

    それで、seoでのリンク構築の周りに急成長している取引があります – リンクを構築する方法についてのアドバイスとリンク自体を売買することの両方において。ただし、有料リンクの構築はブラックハットseoと見なされ、ペナルティを受ける可能性があります。

    グーグルは、無料ギフトと交換されるブログ上のリンクなど、さまざまな種類の有料リンクをさまざまな時期に締め付けてきた。これにより、多くのseo がリンク構築の実行に慎重になりました。しかし、グーグルは原則的にリンク構築に対して何もしていない – それどころか、グーグルはリンクに頼ってどんなウェブサイトがすべてについてであるか、そして特定の検索においてそれらをどれくらい優先するかを知っている。

    では、どのようにして正しい方法でバックリンクを獲得することができるでしょうか。googleランキング要因ガイドの第10部からのヒントをいくつか紹介します。

    言うまでもなく、あなたのウェブサイトを参照している個々のドメインの数は、googleのアルゴリズムにおける重要な要素です – しかし、それらの権威もそうです。(グレッグギフォードがあなたに言うように、ローカルseoを除いて)少ない、権威あるバックリンクを持つことは、低品質のリンクをたくさん持つことよりもseo価値の観点からもっと価値があります。
    あなたのニッチの関連サイトからのバックリンクもまた、無関係なサイトやページよりもはるかに価値があります。
    同じドメインからのリンクが多すぎるとスパムと見なされる可能性があるため、さまざまなwebサイトからのリンクが適しています。
    長文の常緑樹コンテンツ内のリンクも、短いニュースベースの投稿内のリンクよりも価値があります。

    ボーナス:ランクブレインとseo
    googleのランキング要因に私たちのガイドの公式な一部ではありませんが、私はgoogleが正式の一つとしてrankbrain名付けられたように、このラウンドアップの一環としてrankbrainとseoにダン・テイラーの優れたガイドが含まれるだろうと思って最も重要な3つの信号ことウェブサイトのランキングに貢献する。

    彼のガイドの中で、dan taylorはrankbrainがどのように機能するのか、またどのような機械学習があるのか​​、そしてassociation rule learning(arl)の基礎となる概念を分解して解き明かします。
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    ワードプレスは、基本的にはブログを構築するソフトではありますが、ワードプレスの中で使用されているテーマというデザインを構成する概念があるのですがそのテーマのデザインやcssなどを駆使する事で、一般企業や、中小企業、商店のホームページとして作る事も可能です。

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    Students

    SolisWorkspace

    By using SolisWorkspace, you can access software that you need for your studies, such as SPSS, Mathematica, MatLab, NVivo or Stella. You can use the software using anytime, any place, using devices with Windows, Apple or any other operating system. For more information, visit the page SolisWorkspace.

    SURFspot

    You can also buy software and hardware with a discount on SURFspot.nl. (Click on "English" at the upper right corner on the website.) Log on using your Solis-id and password.

    Other Software

    Utrecht University also offers some applications and suites which you may install locally (and free of charge) on your own laptop or computer. See below for specific information:

    Adobe Creative Cloud is available for all students of Utrecht University on the public student computers on campus. Applications covered by this version include Photoshop, Indesign and Illustrator. 

    In order to use the applications, it is necessary to create a Adobe ID. You'll have to accept the privacy terms of Adobe. These terms are not a part of the contract between Utrecht University and Adobe (via SURF); sharing information with Adobe is your own responsibility.

    How to create an Adobe ID is described in this manual. Once you have completed the registration, you can use these Adobe applications. You only need to create an Adobe ID once.

    If you want to use Adobe Creative Cloud on your privately owned computer, you can buy a license on SURFspot.nl, with a considerable discount.

    With ArcGIS Pro, you can explore, visualize, and analyze data; create 2D maps and 3D scenes; and share your work to your ArcGIS Online. 

    ArcGIS Pro is available in MyWorkplace. When starting ArcGIS Pro choose 'enterprise login', type uni-utrecht (so now it reads: uni-utrecht.maps.arcgis.com), then type your Solis-id and password.

    Local installation on your Windows computer/laptop

    The ArcGIS license of Utrecht University allows students, faculty and staff to install ArcGIS Pro software on their personally-owned windows computer. Summary of Steps:

    1. Download the installers. You can find the installers on Blackboard under Communities (top right) - Gis Community - Content - ESRI Software - ArcGisPro. (If the Gis Community is not available in Blackboard, please contact Maarten Zeylmans Van Emmichoven.)
    2. Unpack the 7z file.
    3. Start the installation by double clicking ArcGISPro<version>.exe - confirm licenses and folder destinations etcetera - click Finish.
    4. Execute the second installation by double clicking ArcGIS_Data_Interop<version>.exe
    5. Start ArcGIS Pro, choose 'enterprise login', type uni-utrecht (so now it reads: uni-utrecht.maps.arcgis.com), then type your Solis-id and password.

    ArcGIS Pro regularly and automatically checks if updates are available

    ArcGIS Online

    ArcGIS Online is a cloud-based mapping and analysis solution. Use it to make maps, analyze data, share and collaborate. Get access to workflow-specific apps, maps and data from around the globe, and tools for being mobile in the field. Log in with your Solis-id and password on ArcGIS Online.

    Mathematica is a software application focused on mathematics. It uses symbolic language (mathematical formulas).

    Use the software via SolisWorkspace (free of charge)

    Myworkplace is the easiest way to use the software for free.

    Local installation on your PC

    You may install Mathematica on your own PC. There are versions available for Windows, Linux and Mac OS.

    1. First, create a Wolfram ID, using your @students.uu.nl or @ucr.nl email address. Other email accounts do not work.
    2. Then you can request an activation code on the portal of Wolfram. You will receive the code and download links directly on the screen via email.

    Every year you will receive an email to confirm you are still a student; this prolongs your license for another year.

    Need help with the installation? Visit the support page of the supplier. 

    MATLAB (developed by MathWorks) is a multi-paradigm numerical computing environment and fourth-generation programming language. MATLAB allows matrix manipulations, plotting of functions and data, implementation of algorithms, creation of user interfaces.

    Use the software via SolisWorkspace (free of charge)

    Myworkplace is the easiest way to use the software for free.

    Local installation on your PC

    The MATLAB license of Utrecht University allows students (and faculty and staff) to install MathWorks software on their personally-owned computers.

    Summary of Steps:

    1. Log in to the MathWorks Portal, using your @students.uu.nl email address. (If you don't have a MathWorks account, you may need to create one first.)
    2. Download the installer for your selected version of MATLAB and run it.
      1. Windows users: double click on the Installer named MATLAB_<version number>_win64.exe in your download folder.
      2. Mac users: double click on the Installer named MATLAB_<version number>_maci64.zip - which will extract the files in the folder named MATLAB_<version number>_maci64. Start the Installer by double clicking InstallForMacOSX.
    3. In the installer, select Sign in with a MathWorks Account and follow the instructions.
    4. When prompted, select the Academic – Total Headcount license labeled MATLAB (Individual).
    5. In the Product Selection screen, select the products you want to download and then click Begin Install.

    When the end date of the license is near, you'll see a pop-up. Start the application as Administrator (Start - click with the alternative (usually right) mouse button - More - Run as Administrator) - Click Help – Licensing – Activate Software.

    MATLAB and Simulink Training

    All students (and faculty and staff) also have free access to MATLAB and Simulink Training Online. Access through matlabacademy.mathworks.com. (If you don't have a MathWorks account, you may need to create one first on the MathWorks Portal.)

    MATLAB Online and Simulink Online

    All students (and faculty and staff) also have free access to MATLAB Online and Simulink Online. With MATLAB Online and Simulink Online you can access MATLAB and Simulink in your webbrowser without installation. You can also share files with others or across different applications. Access MATLAB Online and Simulink Online throught matlab.mathworks.com.

    MATLAB Mobile

    You can also find free MATLAB Mobile apps in the Apple App Store or Google Play.

    Office 365 is a collaboration platform developed by Microsoft. Every UU employee and -student has a license and can use it for free. This includes Word, Excel, Access, Teams and PowerPoint, with a licence that allows you to use it on 5 different devices.

    Office 365 can be accessed via office.com. You can log on with your email address that ends with @uu.nl or @students.uu.nl and your Solis password, after which you’ll see the Office 365 homepage. From this page you have access to all the online Office 365 applications.

    Please note: If you're asked to enter a product key, click on 'sign in to an existing office 365 subscription'.

    Students can download Windows 10 Education for free. Windows 10 Education contains more options that the standard Windows 10 Home operating system.

    Do you already own a laptop?

    • Yes, it’s a laptop with Windows – you can order the free upgrade on SURFspot.nl.
    • Yes, it’s an Apple MacBook with Windows - you can order the free upgrade on SURFspot.nl.
    • Yes, it’s an Apple MacBook without Windows - first, order the cheapest Windows license, on Microsoft.com or elsewhere. Next, you can order the free upgrade on SURFspot.nl.

    You don’t own a laptop, and plan to buy one?

    • You plan to buy a laptop with Windows – in most cases, Windows is installed on the laptop, with an ‘OEM license’. Order the cheapest Windows license. Next, you can order the free upgrade on SURFspot.nl.
    • You plan to buy an Apple MacBook – in most cases, it is possible to obtain an ‘OEM license’ with your laptop. Order the cheapest Windows license. Next, you can order the free upgrade on SURFspot.nl.

    The Microsoft Azure DevTools for Teaching (formerly called Dreamspark/Imagine) offers free Microsoft software with a valid Windows licence. Log in with your Solis-id and password.

    Please note: this software can only be used by students and teachers of STEM (Science, Technology, Engineering and Mathematics) to learn/teach design, development and testing of software applications, and researchers STEM (Science, Technology, Engineering and Mathematics) to perform non-commercial research.

    Access is limited to employees and students of the Faculty of Science.

    QSR NVIVO is a software package that can be used for the qualitative analysis of primarily textual information.

    Use the software via SolisWorkspace (free of charge)

    Myworkplace is the easiest way to use the software for free.

    Local install on your own PC

    • Go to https://portal.mynvivo.com e and create an account with your UU email address. You'll have to accept the privacy terms of NVivo, which are accepted by Utrecht University with reservation.
    • After logging on to the website, you can download and install NVivo.
    • The first time you start the application, you will be asked to activate NVivo. Click “provide enterprise key to activate” and use the license code that you'll find on this website (log in using your Solis-id and password)

    Please note:

    • It is NOT necessary to share your personal financial/payment information in order to use NVivo.
    • After logging on to the website, you will see advertisements for other QSR products, such as NVivo Transcription*. The use of those products is not allowed under the terms of the Utrecht University license.

    *ITS is working on a transcription service for Utrecht University. More information about this will be published in due time.

    Qualtrics is a powerful online survey tool that enables UU students and staff to create complex surveys that meet a variety of research needs. You can use this tool to design, distribute and analyze surveys. Log in with your Solis-id, password and two-factor-authentication at https://survey.uu.nl.

    More information

    Visit the Qualtrics page.

    Reference Management tools are used to systematically collect, organise and use references to sources. How to do this in the most efficient way depends on the type of sources you use most frequently and also on your personal workflow and preferences. Read more about Reference Management in the LibGuide by Utrecht University Library. Whatever tool you choose, it is always relatively easy to switch and import your references in another tool.

    Name and Wikipedia-linkProducerLicenceAvailable on
    ZoteroCorporation for Digital Scholarship (an independent nonprofit organization)Open Source and free of charge
    • MyWorkPlace
    • Private device (please install this yourself, see Wikipedia-link)
    MendeleyElsevierFree of charge
    • MyWorkPlace
    • Private device (please install this yourself, see Wikipedia-link)
    RefWorksEx Libris Group / ProQuestCommercial. There is a university wide campus license, so you can use this without extra chargeWeb-based, no installation necessary.

    Plugin for Microsoft Word is available on

    • MyWorkPlace
    • Private device (please install this yourself, see Wikipedia-link)
    EndNoteClarivate Analytics (previously Thomson Reuters)Commercial. There is a university wide campus license, so you can use this without extra charge
    • MyWorkPlace
    • Private device (please install this yourself, see below)

    To install Endnote on a private device (pc/laptop):

    MacOS:

    • Go to this website, log in using your Solis-id and password.
    • Download the installation file and double click to install Endnote.

    Windows:

    • Go to this website, log in using your Solis-id and password.
    • Download the msi-file and License.dat and save them in the same folder/directory.
    • Double click the msi-file to install Endnote.
    • When the installation is completed: start Endnote and click ‘I accept the licence agreement’. Endnote will ask you to create an account; this is not necessary.
    • You can exit Endnote now, and delete the two files you downloaded.

    Think-cell facilitates the creation of charts on Microsoft PowerPoint presentation slides from Microsoft Excel data sheets, e.g., bar charts, waterfall charts, Marimekko charts and Gantt charts. It consists of a Microsoft Office add-in for PowerPoint and Excel, which integrates seamlessly with these applications. Some features overlap with those provided by built-in charts in Office 2016. See the Think-cell website.

    Employees and students of Utrecht University can use this software for free. Go to this website, log in using your Solis-id and password and download the License Code.

    You will find a direct link to Van Dale Online in the start menu of your Solis workspace. You do not have to log in to the site. You have access to the full range of Van Dale dictionaries, including the complete Dutch dictionary and other language dictionaries.

    Use the software via SolisWorkspace (free of charge)

    Myworkplace is the easiest way to use the software for free.

    On campus

    On campus, but not using an UU computer? Start the software via MyWorkplace or visit the website uu.vandale.nl.

    Access off campus

    Are you outside the campus? Start the software via MyWorkplace or browse to www.vandale.nl and log in under ‘Inloggen Onderwijs / SURFconext’ using your Solis-id. 

    Do you spend a lot of time behind your laptop or PC? Check out if WORK & MOVE is something for you. With this software, short breaks in your work provide mental and physical moments of exercise. This way you stay focused and energetic! We recommend that you install WORK & MOVE if you regularly work at a computer for more than two hours. WORK & MOVE is a Windows application. Follow these steps to install this application:

    1. Download WORK & MOVE from WorkAndMove.com (or go to this website and choose "Software installation (Click Once)"), and install it by double clicking the downloaded file and following the steps in the installation wizard. Make sure you have an active internet connection during installation.
    2. Go to this website, log in with your Solis ID and password and download the license file License.json from the WORK and MOVE folder.
    3. Start WORK & MOVE, click Help - Show license page (toon licentiepagina) - Import license file (importeer het licentiebestand). Choose the json file there.
    Источник: https://students.uu.nl/en/free-software

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