私たちは、資金調達源ゲノムカナダとゲノムケベック州、およびモントリオール大学と同様に（S2D）プロジェクト&quot;病にシナプス&quot;私たちに資金を供給するためのイノベーションのためのカナダの財団からの資金に感謝。

1。 de novoの突然変異によって引き起こされるかもしれない疾患の選定以下の基準に対応する疾患は、de novo突然変異の仮説と合うことができる。 <ol><li>生殖適応度が減少します。 </li><li>病気の頻度は、広く多様な環境にもかかわらず、比較的高いと一定です。 </li><li>病気は、より高い父親の年齢に関連付けられています。 </li><li>古典的な連携と関連研究は、疾患の遺伝のかなりの部分を説明するために失敗しました。 </li><li>双子のコンコーダンスデータは、de novoのモデルをサポートしています。 </li></ol> のde novo変異が一部の遺伝的根拠を説明するかもしれない共通の病気が重要な第一歩である可能性の分析。 2。ケースとDNAサンプルの選択適切なサンプルの選択は 、de novo変異の同定の成功にとって重要です。 de novoの変異を見つけることのチャンスを最大にするために、我々は、以下を推奨します。 <ol><li>影響を受けない両親、古い父とし、病気の拡大家族の歴史を持つ、発症、重症の表現型の早い年齢でケースを選択してください。 </li><li>その利用可能なDNAの研究を行うのに十分な患者を選択してください。特に重要な培養に供されていない主な細胞源（例えば血液のDNAまたは唾液のDNA）からのDNAの可用性は、です。 </li><li>両親のDNAの可用性は、突然変異の送信ステータスを（対デノボを継承）を決定するために重要です。候補遺伝子が同定されれば、追加の影響を受けるのコホートと正常対照の可用性は、遺伝学的検証研究が必要である。 </li><li>突然変異率は、スクリーニングする遺伝子の量とのde novo突然変異に起因するかもしれない例割合の推定値に基づいて、サンプルのサイズを見積もります。 </li></ol> 3。遺伝子再配列、2つの主要な方法<ol><li>高品質、低スループットシーケンシング このアプローチは、候補遺伝子アプローチに基づいています。 <ol><li>候補遺伝子の選択（複数可） 6つの主要な基準に基づいて構築されたスコアリングシステムに基づいて最適な候補遺伝子を選択してください。次に、表1に示す6つの基準を使用して起因するすべてのポイントの合計に相当する合計を計算する。選択と選択されていない遺伝子の分布の図1における私たちのプロジェクトからの例を参照してください。 <img src="/files/ftp_upload/2534/2534table1.jpg" alt="表1"/> 表1。候補遺伝子の選択に使用される基準 <img src="/files/ftp_upload/2534/2534fig1.jpg" alt="図1"/> 図1我々のプロジェクトで順序付けのために選択し、選択されていない遺伝子の分布を示すグラフ。我々は彼らのスコアの値に応じて遺伝子をソートすることで、候補のプロパティによって選ばれた遺伝子の分布を得た。例えば、SHANK3とNRXN1遺伝子、我々は新生突然変異を発見した二つの遺伝子は、（最大値は12です）、それぞれスコア7と6を持っていた。 </li><li> Exonprimerを通じてPrimers3ソフトウェアを使用して設計したプライマー。唯一のコード領域、スプライスジャンクショ​​ンは、エクソンの各側に余分な50塩基対を含むカバーされるべきである。 </li><li> Taqポリメラーゼ、反応容積の選択のためのPCR条件等を最適化する</li><li>すべてのPCR断片を最適化する</li><li>標準的な手順にしたがって血液サンプルから抽出したゲノムDNAの5 ngを増幅する</li><li>シークエンシングのために送信する前に、2％アガロースゲルをロードすることによってあなたのPCR産物の品質管理を行います。無作為にサンプルを選択する。 </li><li>一方の鎖上のDNA AnalyzerのシーケンスPCR産物を。トレースの90％以上の分析が可能な場合はフラグメントは正常塩基配列とみなされます。これは、大規模なスクリーニングに適用されます。 </li><li>バリアントの検出<ol><li>このようなPolyPhred、Polyscanと突然変異のサーベイヤーとしてゲノム変異の検出と遺伝子型のためのツールを使用してください。 2つ以上の検出ツールの組み合わせは理想的です。例えば、V.5をPolyPhredとデフォルトの設定でv.6をPolyPhred同じ変化を検出しません。 V.3 Polyscanはindelsの（93％）のためのSNP（96％）と少ないため高い偽陽性突然変異率を持っています。 PolyPhred V.6は真indelsの大多数を検出しましたが、偽陽性の突然変異率（indelsのための）（90％）Polyscan V.3全体のより低いを持っていませんでした。我々はPolyscan V.3のためのSNP検出のオプションを削除して、両方PolyPhred V.5を保持し、バリアントの検出のためv.6をPolyPhredてください。突然変異のサーベイとPolyscanはindelsの検出に優れています。注：Polyscanを使用するときにSNP検出のオプションが適用されるべきではない。のみ&quot;INDEL&quot;オプションをオンにする必要があります。 Polyscanは、SNP検出のための多くの偽陽性に生成されます。 </li><li>検出されたそれぞれのユニークな小説エクソンバリアントの場合は、フラグメントとresequをreamplifyingして手動で確認する発端者と技術的なアーティファクトを除去するためにリバースとフォワードプライマーを使用して両方の両親をencing。 </li></ol></li></ol></li><li>全体exomeシークエンシング このアプローチは、人間のゲノムのコード領域の大部分をターゲットにハイスループットシーケンシングです。我々は現在、潜在的な候補遺伝子の検出を加速する、私たちの研究室ではこの新しいアプローチを使用しています。 <ol><li>ロシュのような他の人により、Agilentまたはその他の同様の製品によって設計された16,000以上の遺伝子（ゲノムの50 MB）のコード領域を標的とする&quot;SureSelect人間のすべてのエクソンを&quot;命ずる。キャプチャキットを注文する前に、シーケンシングプラットフォームは、（イルミナ対SOLID）を決定する必要があります。 </li><li> Agilentのプロトコルに応じてキャプチャを実行してください</li><li>あなたのそれぞれの利用可能な次世代のプラットフォーム上でシーケンスの製品を</li><li>全体exomeシーケンシングからバリアントの検出：次世代シーケンシングプラットフォームからのゲノム変異の検出と遺伝子型のいくつかのバイオインフォマティクスのツールは、BWA、Bfast、アライメントを実行するビオスコープとして利用可能です。そのあとで追加の自由に利用できる下流のツールは、（たとえば、SAMツール、Varscan、Annovar）それは変種を呼び出して、注釈を付けるために必要になります。配列アラインメントとバリアントの呼び出しと注釈を組み込んだ商用ソフトウェアはまた、ネクストジェン（Softgenetics）、CLCバイオ、など、など、用意されています</li></ol></li></ol> 4。ゲノムバリアントの優先順位付け同定された変異体は、その後のde novoおよびタンパク質またはmRNAの機能及び/または構造体への有害さの彼らの確率に応じてフォローアップのために優先順位付けされます。バリアントは、以下のようになるはずのde novo変異の検出のための優先事項をフォローアップ： <ol><li>ユニークなバリエーション（一つのケースで一度に観察） </li><li>両親には存在しないバリエーション</li><li>タンパク質-切り捨てるバリエーション：ナンセンス、フレームシフト、スプライシング変異につながるindelsの。 </li><li>ミスセンスとサイレント変異（例えば、mRNAのスプライシングに影響を与える）機能的に破壊的であると予測。タンパク質上の機能予測の効果のためのPolyphen、SIFTとPantherを使用してください。 </li></ol>全体exomeシーケンシングを使用している場合、候補遺伝子の選択は、さらなる研究のための優先順位を付ける変種のための戦略として使用することができます。 5。遺伝学的検証<ol><li>追加の患者のケースで遺伝子全体を再シーケンス（他の原因となる変異を識別するため）およびコントロールで。対照試料は、優先順位付けの亜種の対立遺伝子頻度を評価するために使用されます。別の患者（ただし、コントロールサンプルまたは公共のデータベース内）にある少なくとも二つの異なるde novoの有害な突然変異を（ナンセンス、スプライシング、フレームシフト、および有害なミスセンスを予測）を含む任意の遺伝子は高度にさらに検証研究のために優先されるべき。患者由来リンパ芽球様細胞株における潜在的なスプライシングの欠陥のための1）のテストを：これが含まれています。我々の経験では、ほとんどの遺伝子は、リンパ芽球様細胞株からのRT - PCR産物を得、2）さらに、テストの機能レベルでの変異/遺伝子）リンパ芽球様細胞株および3からのタンパク質抽出物の定量的なウェスタンブロット分析により改変されたタンパク質の発現レベルを調べる動物（C虫、ゼブラフィッシュ）と、以前に私たちのグループ（例：5,6）によって行われたとして、細胞株モデル。 </li></ol> 6。代表的な結果： このプロトコルに続いて、我々は統合失調症と自閉症のための新しい遺伝子を同定することができた。一例として、私たちの最近SHANK3遺伝子の探索（図2）です。 SHANK3遺伝子内の異なる2つのde novo変異、三の影響を受ける弟一、影響を受けた女性の1つのミスセンス変異で見つかったものナンセンス突然変異。 <img src="/files/ftp_upload/2534/2534fig2.jpg" alt="図2"/> 図2。家族PED 419の三の影響を受ける人の兄弟にR1117Xナンセンス突然変異の（）分離。発端者は矢印で示されます。PED 56の（B）発端者のR536Wのミスセンス変異の分離ではなく、彼女の非罹患兄弟。

<ol>
	<li>Bassett, A. S., Bury, A., Hodgkinson, K. A., Honer, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproductive+fitness+in+familial+schizophrenia.">Reproductive fitness in familial schizophrenia.</a> Schizophr Res. 21, 151-160 (1996).</li><li>Jablensky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schizophrenia:+manifestations,+incidence+and+course+in+different+cultures.+A+World+Health+Organization+ten-country+study.">Schizophrenia: manifestations, incidence and course in different cultures. A World Health Organization ten-country study.</a> Psychol Med Monogr. , 1-97 (1992).</li><li>Malaspina, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schizophrenia+risk+and+paternal+age:+a+potential+role+for+de+novo+mutations+in+schizophrenia+vulnerability+genes.">Schizophrenia risk and paternal age: a potential role for de novo mutations in schizophrenia vulnerability genes.</a> CNS Spectr. 7, 26-29 (2002).</li><li>DeLisi, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+scan+for+linkage+to+chromosomal+regions+in+382+sibling+pairs+with+schizophrenia+or+schizoaffective+disorder.">A genome-wide scan for linkage to chromosomal regions in 382 sibling pairs with schizophrenia or schizoaffective disorder.</a> Am J Psychiatry. 159, 803-812 (2002).</li><li>Gauthier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+Novo+SHANK3+Mutations+in+Patients+Ascertained+for+Schizophrenia.">De Novo SHANK3 Mutations in Patients Ascertained for Schizophrenia.</a> Proc Natl Acad Sci U S A. , (2010).</li><li>Piton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+calcium-related+gene+IL1RAPL1+are+associated+with+autism.">Mutations in the calcium-related gene IL1RAPL1 are associated with autism.</a> Hum Mol Genet. 17, 3965-3974 (2008).</li><li>Krawczak, M., Ball, E. V., Cooper, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neighboring-nucleotide+effects+on+the+rates+of+germ-line+single-base-pair+substitution+in+human+genes.">Neighboring-nucleotide effects on the rates of germ-line single-base-pair substitution in human genes.</a> Am J Hum Genet. 63, 474-488 (1998).</li></ol>

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ここで説明する手順は、de novo変異から、一部では、可能性の高い結果という特定の一般的な疾患を識別するために、そしてこの仮説を証明することを目指しています。 de novoの変異は、例えば、疾患の数、遺伝性がん症候群の開発のための十分に確立されたメカニズムですが、不十分な一般的な疾患で検討されている。ごく最近、次世代シーケンシングの出現との効果的なコストになっているDNAの大量のシーケンシングを必要とするのde novo変異、、の同定に関わる技術的な課題から一部の結果でこの。さらに、ヒトの新生突然変異率は、ごく最近まで、唯一の見積もりでした。ごく最近では、そこにレポートが直接ヒトでの突然変異率を決定している。これらの測定に先立ち、それはこの種の研究に必要なサンプルサイズを予測し、観測された新生突然変異率が基準率より大きいかどうかを確認することは困難であった。全ゲノム対の候補遺伝子をシーケンシング？報告された疾患の変異の大部分はミスセンス/ナンセンス変異であると私たちのスクリーニング戦略は、既知の変異の68％以上を特定するというスプライス部位の変異（HGMDウェブサイトによると）ですので。アミノ酸置換の重症度と臨床表現型の可能性との間の明確な関係もあります。保存的アミノ酸置換と比較して、ナンセンスの変化は臨床的に7を提示する可能性が9.0倍です。従って、この時点で、シーケンスの候補遺伝子は、最も費用対効果の高い戦略です。 概説する手順の成功は、詳細に概説し、二つの例、自閉症や統合失調症を使用して示されているいくつかの重要なステップ、に依存する。その画面、DNAの源、そして効率的にde novoの変異を識別する方法の詳細に患者の病気が選択するかなどの回避する必要がある多くの落とし穴が、、があります。我々は、最も効率的にそのような自然突然変異に起因するいかなる疾患の症例の割合を決定するための方法を提供する。

a strategy to identify de novo mutations in common disorders such as autism and schizophrenia

そのような自閉症や統合失調症などの一般的な疾患のメカニズム、などのde novo変異の役割を支持する証拠のいくつかの行があります。最初に、ヒトの新生突然変異率が比較的高い、新たな突然変異が集団で高い頻度で生成されるので。しかし、de novoの変異が最も一般的な疾患で報告されていない。このような胚の段階で致死性または減少生殖適応度などの表現型に対する強い否定的な選択がある重篤な疾患につながる遺伝子の突然変異は、複数の家族に送信されず、したがって、連鎖遺伝子のマッピングまたは協会によって検出されません。の研究。一卵性双生児で非常に高い一致と二卵性双生児の非常に低い一致の観察にも強く例かなりの部分は、新しい変異に起因するかもしれないという仮説をサポートしています。このように、自閉症や統合失調症などの疾患のためのケースです。第二に、削減生殖適応度1と非常にさまざまな環境要因にもかかわらず、いくつかの疾患の発生率は比較的高いと一定の速度で世界的に維持されます。これは全世界で約1％の発生率で、自閉症や統合失調症の場合です。突然変異荷重は新生突然変異による有害な突然変異とその製造または反対選択のバランスとして考えることができます。再現率の低下は、最終的に減少した疾患の有病率につながる、人口の突然変異対立遺伝子の数を減らす必要ネガティブ選択の要因となる。これらの選択圧が異なる環境で異なる強度の傾向にあります。それにもかかわらず、これらの重度の精神障害は、それら2に対して強い負の選択にもかかわらず、文化や国の幅広い世界的な人口の一定の比較的高い有病率で維持されています。これは高い新しい突然変異率があった場合を除き1つが低下生殖適応度と病気に予測するものではありません。最後に、父方の年齢の影響：父方のde novo変異の年齢関連の増加から生じる可能性が増加する父方の年齢と病気のリスクの有意な増加は、あります。これは、自閉症や統合失調症3のケースです。突然変異率の男女比は、おそらく男性では年齢とともに生殖細胞の分裂の高い数のために、約4-6:1と推定されている。したがって、一つは 、de novo変異がより頻繁に男性、特に高齢男性4から来ることを予測する。遺伝学的研究はこれまで、自閉症や統合失調症などの複合体の疾患遺伝子の素因となる多くの遺伝子を、同定に失敗した、となぜ病気がヒトゲノム中の遺伝子のわずか3％に同定されている理由は新しい突然変異の頻度が高い場合は、部分的に説明することがあります。病気の原因としてのde novo変異の同定は、勉強の両親と影響を受けた科目を含む標的分子のアプローチが必要です。疾患の遺伝的根拠はde novoの突然変異から一部と、このリンクを確立するために分子的なアプローチにつながる可能性がある場合を決定するためのプロセスは、例として、自閉症や統合失調症を用いて、例示される。

Watch this Scientific Journal Video about A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia at JoVE.com

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

自閉症や統合失調症などの一般的な疾患を引き起こすのde novo変異を見つけるための分子遺伝学的戦略。

そのような自閉症や統合失調症のような一般的な疾患でのde novo変異を同定するための戦略

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

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25.6K ビュー.  Universite de Montreal. 自閉症や統合失調症などの一般的な疾患を引き起こすのde novo変異を見つけるための分子遺伝学的戦略。

ビデオ: A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

局所脳虚血への早期虚血耐性を誘導するために適切な前処理刺激として二国間の総頚動脈閉塞

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

医学

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

ザ<em&gt;卵で</em肉腫のためXenograftmodelとして&gt; CAMアッセイ

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

下部尿路薬理学を評価するための方法として膀胱平滑筋収縮ストリップ

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

疫学研究では心血管疾患の危険因子への微小血管の応答を研究するための便利なツールとしての眼底写真

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

代替として舌下免疫療法は、急性呼吸器感染症に対する保護を誘導するために

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

病における網膜障壁を研究するツールとしてアデノ随伴ウイルスを使用して、

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

内分泌と外分泌リプログラミングおよび増殖のためのモデルとしての膵管の連結を介して、マウスの膵臓への外科的損傷

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

生体内でウサギの一般的な頚動脈内皮細胞への遺伝子導入

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits. Thanks to animal models, basic research can investigate the mechanisms underlying pathological processes such as MetS. Here, we describe the methods used to develop an experimental rabbit model of diet-induced MetS and its assessment. After a period of acclimation, animals are fed a high-fat (10% hydrogenated coconut oil and 5% lard), high-sucrose (15% sucrose dissolved in water) diet for 28 weeks. During this period, several experimental procedures were performed to evaluate the different components of MetS: morphological and blood pressure measurements, glucose tolerance determination, and the analysis of several plasma markers. At the end of the experimental period, animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia with low HDL, high LDL, and an increase of triglyceride (TG) levels, thus reproducing the main components of human MetS. This chronic model allows new perspectives for understanding the underlying mechanisms in the progression of the disease, the detection of preclinical and clinical markers that allow the identification of patients at risk, or even the testing of new therapeutic approaches for the treatment of this complex pathology.

We describe methods to develop an experimental model of diet-induced metabolic syndrome (MetS) in rabbits using a high-fat, high-sucrose diet. Animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia, thus reproducing the main components of human MetS. This chronic model will allow acquisition of knowledge underlying mechanisms of disease progression.

ウサギの食餌によるメタボリック シンドロームの実験的モデル: 方法論的検討・開発・評価

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

耐久性のある遺伝子発現により一般的な頚動脈移植に頸静脈の家兎モデル

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...