我々の研究は、NIH、国立眼研究所の学内研究プログラムによってサポートされています。

使用されるすべての機器や試薬は無菌である。プロトコルは、アニマルケアによって承認され、（動物実験のプロトコールNEI - 553）NEI / NIHで委員会（ACUC）を使用して、NIHのガイドラインと規則にしたがって実施した。 1。血管新生因子を含有するペレットを生産この部分では、関心の血管新生因子を含むペレットを用意しています。 <ol><li> 10％（w / v）のPBSとスクラルファートのソリューションとなります。室温下で保管してください。 </li><li>無水エタノールで12％（w / v）のポリ- HEMAを行います。室温下で保管してください。 </li><li>の1μg/μlのbFGFのソリューションとなります。 -80の℃で保存</li><li> 50ペレットを作るために、スクラルファートとbFGFのストック溶液4μlの1μlのポリ- HEMAの5μlを、、徹底的にボルテックスして混合する。 </li><li>ペトリ皿の中できれいなパラフィルムの断片を配置する、組織培養フードで15分間UV光の下でそれを公開します。 oneペレット用パラフィルム上の混合溶液0.2μlをドロップします。このように、混合溶液の10μlを、50ペレットを生成する必要があります。 1〜2時間室温でペレットを乾燥させて。乾燥ペレットを数日間4℃に保存、または数ヶ月-80 ° Cすることができます。 </li><li>使用時には、てことピンセットの先端でペレットを拾う。それらを壊したり、それらを離れて跳ぶよう特に注意してください。 </li></ol> 2。マウスの角膜のポケットアッセイを実行するこのパートでは、我々は、マウスの角膜でポケットを作る方法をデモンストレーションします、そしてポケットにペレットを挿入する方法。ペレットに含まれる血管新生因子は、角膜の周辺地域（図1）に徐々に放出されます。このデモでは、8週メスC57/BL6マウスを使用してください。 <ol><li>マウスは、ケタミン（75 mg / kg体重）及びキシラジン（5mg/kg、ip注射）の混合物と深く体系的に麻酔です。これは、30〜60分間麻酔の状態でマウスを保持します。局所麻酔薬（0.5％プロパラカイン塩酸）が角膜に適用されます。 5分後、解剖顕微鏡下で片目を修正。 </li><li>非常に穏やかなカットは、フォングレーフェ白内障ナイフで角膜輪部（図1に示す）から角膜1.2 -1.4 mmの途中で作られています。角膜を通して切らないように、特に注意してください。 </li><li>ポケットは、水平に角膜の中央にナイフを挿入し、角膜輪部慎重に（図1）に向かってナイフを拡張することにより、角膜の上皮層の下に行われます。ポケットのサイズは1血管新生因子を含有するペレットを収容するために十分な大きさでなければなりません。角膜を介してではない穿刺には十分に注意してください。 </li><li>ゆっくりポケットにピンセットでペレットを挿入します。可能な限りフラットとしてペレットを作ることを試みて下さい。 </li><li>ペレットの注入後、局所眼科用抗生物質の軟膏を眼に適用されます。インストゥルメントは動物の間に70％のアルコールパッドで拭いています。 </li><li>それは直立姿勢を維持し、そのホームケージにそれを返すことができるようになるまで、暖かく乾燥した領域にマウスを移動し、それを監視する。全身鎮痛薬（例えば、ブプレノルフィン、2 mg / kg投与、腹腔内注射）と局所抗生物質軟膏（例、ゲンタマイシン）手術後2日間1日2回与えられているとマウスが密接に3日間監視されています。ヒーリングは最初の48時間後に手術以内に完了する予定です。ポケット付きの目は、ペレットの移植後4日から10日間の細隙灯biomicroスコープで検査されるでしょう。 </li></ol> 3。代表的な結果<ol><li>ペレットを移植7日後、マウスに麻酔を全身および局所的に2.1で説明されているようです。 </li><li>解剖顕微鏡下で血管新生因子によって誘導される角膜の血管を観察します。車で治療角膜では、ない血管の成長は、（図2A、ビヒクル処理角膜）を観察することはできません。 bFGFで処理された角膜では、堅牢な血管新生は、（図2B）見ることができます。血管の成長は、血管（新しい血管の先頭に通常の輪部血管から）、および全血中の血管領域（図2Bは、bFGFの赤い破線で区切らの最大長を用いて評価することができる処理角膜）。他の定量法でも2,5を記載されている。 </li></ol><img src="/files/ftp_upload/3077/3077fig1.jpg" alt="図1" /> 図1。 H＆E染色と。正常な角膜。いいえ血管は正常な角膜には存在しません。B。角膜の上皮層の下にポケットを作る。C。ポケットにペレットを埋め込 ​​みます。D。血管新生因子は、ペレットから放出され、新しい血管は、通常の輪部血管から成長しています。 <img src="/files/ftp_upload/3077/3077fig2.jpg" alt="図2" /> 五日のIM後の図2。代表結果ペレットのプランテーション。。唯一の車両を含むペレットと角膜。角膜内に新しい血管の形成しません。唯一の既存の通常の輪部血管が表示されます。B。ペレットを含むbFGFと角膜。堅牢な新しい血管は、（ドット裏地）既存の通常の輪部血管から育った。

<ol>
	<li>Cao, Y., Linden, P., Shima, D., Browne, F., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+angiogenic+activity+and+hypoxia+induction+of+heterodimers+of+placenta+growth+factor/vascular+endothelial+growth+factor.">In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor.</a> The Journal of clinical investigation. 98, 2507-2511 (1996).</li><li>Rogers, M. S., Birsner, A. E., D'Amato, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cornea+micropocket+angiogenesis+assay.">The mouse cornea micropocket angiogenesis assay.</a> Nature. 2, 2545-2550 (2007).</li><li>Poche, R. A., Saik, J. E., West, J. L., Dickinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cornea+as+a+transplantation+site+for+live+imaging+of+engineered+tissue+constructs.">The mouse cornea as a transplantation site for live imaging of engineered tissue constructs.</a> Cold Spring Harbor protocols. , 5416-54 (2010).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-B+is+dispensable+for+blood+vessel+growth+but+critical+for+their+survival,+and+VEGF-B+targeting+inhibits+pathological+angiogenesis.">VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis.</a> Proceedings of the National Academy of Sciences of the United States of America. , 106-6152 (2009).</li><li>Tong, S., Yuan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose+response+of+angiogenesis+to+basic+fibroblast+growth+factor+in+rat+corneal+pocket+assay:+I.+Experimental+characterizations.">Dose response of angiogenesis to basic fibroblast growth factor in rat corneal pocket assay: I. Experimental characterizations.</a> Microvascular research. 75, 10-15 (2008).</li><li>Muthukkaruppan, V., Auerbach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+the+mouse+cornea.">Angiogenesis in the mouse cornea.</a> Science. 205, 1416-1418 (1979).</li><li>Ziche, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+assay+for+angiogenesis.">Corneal assay for angiogenesis.</a> Methods in molecular medicine. 46, 131-142 (2001).</li><li>Ziche, M., Morbidelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+corneal+pocket+assay.">The corneal pocket assay.</a> Methods in molecular biology. 467, 319-329 (2009).</li></ol>

利害の衝突は宣言されません。

マウスの角膜のポケットアッセイのモデルは、最初Muthukkaruppan VRとアウエルバッハR 6が1979年に記述されていた。詳細なプロトコルは、異なる研究室2,3,7,8で公開されている。当研究室では長年4に変更され、我々の研究でこのメソッドを使用しています。マウスの角膜のポケットアッセイは非常に再現性を持つ比較的単純なモデルです。このモデルのもう一つの利点は?...

<table border="1"><tbody><tr><th>機器</th><th>会社</th><th>カタログ番号</th></tr><tr><td>解剖顕微鏡</td><td>世界の精密機器</td><td> PZMIV - BS </td></tr><tr><td>フォングレーフェ白内障のナイフ（1.5x25mmブレード、フラット） </td><td>アンブラー外科</td><td> 3401E </td></tr><tr><td>デュモン＃5鉗子</td><td>ファイン科学ツール</td><td> 11252〜50 </td></tr><tr><td>細隙灯</td><td>ツァイス</td><td> 30 SL - M </td></tr></tbody></table> 表1。機器<table border="1"><tbody><tr><th>試薬</th><th>会社</th><th>カタログ＃ </th><th>コメント</th></tr><tr><td> bFGFの（塩基性線維芽細胞増殖因子） </td><td>ぺプロ株式会社</td><td> 100 - 18B </td><td></td></tr><tr><td>スクラルファート（ショ糖octasulfate - アルミニウム錯体） </td><td>シグマ</td><td> S0652 </td><td> PBS中10％ </td></tr><tr><td>ポリ- HEMA（ポリ（2 - ヒドロキシエチルメタクリレート）） </td><td>シグマ</td><td> P3932 </td><td>エタノールで12％ </td></tr></tbody></table> 表2。試薬および材料

a mouse model of the cornea pocket assay for angiogenesis study

正常な角膜は、血管組織の明らかである。しかし、血管は、強力な血管新生因子が1を投与している角膜に成長し、生き残るために誘導することができる。この一意性は、角膜のポケットアッセイ血管新生の研究によく使われるモデルのひとつです。角膜は、セルの複数の層を構成する。それは、その血管新生効果2,3を調査するために角膜への関心の血管新生因子を含むペレットを埋め込 ​​むことが可能である。ここで、我々は（私が）血管新生因子を含有するペレット（II）の（III）興味の血管新生因子によって誘導される血管新生を解析角膜にペレットを埋め込むを生成する方法のステップのデモンストレーションでステップを提供しています。塩基性線維芽細胞増殖因子（bFGF）は最も強力な血管新生因子4の一つとして知られているので、角膜の血管新生を誘導するために使用されています。

Watch this Scientific Journal Video about A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study at JoVE.com

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

角膜は、血管組織を欠いている点でユニークです。しかし、堅牢な血管の成長と生存が強力な血管新生因子によって角膜に誘導することができる。そのため、角膜は私達と血管新生の研究のための貴重なツールを提供することができます。このプロトコルは、角膜のポケットアッセイのマウスモデルを実行する方法と、このモデルを用いて血管新生因子によって誘導される血管新生を評価する方法を示しています。

血管新生研究のための角膜ポケットアッセイのマウスモデル

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

a-mouse-model-of-the-cornea-pocket-assay-for-angiogenesis-study

Research

JoVE Journal

Medicine

15.9K ビュー.  National Eye Institute. 角膜は、血管組織を欠いている点でユニークです。しかし、堅牢な血管の成長と生存が強力な血管新生因子によって角膜に誘導することができる。そのため、角膜は私達と血管新生の研究のための貴重なツールを提供することができます。このプロトコルは、角膜のポケットアッセイのマウスモデルを実行する方法と、このモデルを用いて血管新生因子?...

ビデオ: A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSDの、ニューラル血液学的および行動的相関関係を明らかにするための動物モデルにおけるバイオマーカー

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

医学

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人間の神経内分泌腫瘍療法の研究のための三次元モデルとしての人間の神経内分泌腫瘍細胞株

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

再狭窄の研究のための頸動脈内ステント留置のマウスモデル

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

ヒアルロン酸ナトリウムの網膜下注入によるげっ歯類における網膜剥離モデル

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

肺特異的血管形成を研究するためにマウスの肺にフィブリンゲルの移植

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

腫瘍の増殖、浸潤及び血管新生を研究するために、ニワトリでのヒト多発性骨髄腫異種移植モデルの確立

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

整形外科術後の高次脳機能障害と組織再生を研究するマウス モデル

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

角膜上皮摩耗眼はりのモデルとして角膜創傷治癒

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

ヒンドリム虚血のマウスモデルにおける治療血管新生の評価

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...