著者はマシュー・ケリー、ティツィアーナCogliati、ジェシカGumerson、ウーヴェウォルフルム、Rivkaルブロン、ヴィオラクレッチマーとゾーイマンに感謝し、原稿の彼らの重要な評価のためにしたいと思います。この作品は、Sofjaコワレフスカヤ賞（Humbodlt財団）とヨハネス・グーテンベルク大学マインツ、ドイツによって賄われていました。

<ol>
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The author declares no competing financial interests.

分析のための蝸牛組織を調製する場合、心に留めするためのいくつかの重要なポイントがあります。まず、遺伝的背景の違いは、それが必要なだけ同腹のコントロールを分析し、比較すること、蝸牛の表現型を変更することができます。第二に、被蓋膜の完全な除去は、免疫組織化学で最高の画像を得るために必要とSEMのために必須です。被蓋膜は不透明構造であり、イメージングがより困?...

一次繊毛は、ほとんどの哺乳動物細胞の表面から延びる長い微小管ベースの付属しています。一次繊毛常にセルごとに複数存在し、そのうちの多くの場合、運動性繊毛と混同しており、その目的は、膜の表面を横切って流体を移動させることです。一次繊毛は、対照的に、感覚的役割を採用し、その結果、また感覚繊毛と呼ばれます。いったん長い忘れて、この細胞小器官は、最近、ヒト遺伝病1の多数との会合の結果として「再発見」されました。理想的には、シグナリングオルガネラとして位置付け、一次繊毛は、組織の恒常性と疾患ではなく、開発の2の間だけでなく、重要であるそれらの多くは、多数のシグナル伝達経路を調節することが示されています。 繊毛機能不全に関連することが示され、最初のシグナル伝達経路の一つは、平面内細胞極性（PCP）経路としても知られている非標準Wntシグナル伝達経路、ました<sup<html> &gt; 3。最初にショウジョウバエで同定され、このシグナル伝達カスケードは、胚形成のために重要です。特に収束と拡張のプロセスのために上皮4の平面内での細胞の正しい向きのため。調節タンパク質のコアセットのシーケンシャルシグナル伝達は、最終的に細胞骨格の再編成につながると平面5における上皮細胞の協調分極が生じる方向性の手がかりを変換します。収束と拡張のプロセスは絶対に細長くする蝸 ​​牛管のために、正しい携帯パターニング6のために必要とされます。これはPCP経路の活性化を介して調節されるように、蝸牛のPCP変異体の最も印象的な表現型の一つが無秩序な感覚上皮7に短縮蝸牛管です。正確にこれを調節する方法が解明されないままでも同様に、繊毛を欠くマウス変異体は、また、そのような収束と拡張表現型8,9を示します 。 ve_content &quot;&gt;収束と拡張のプロセスは蝸牛管の伸長、および蝸牛管の中の感覚上皮の細胞パターニングのために重要であるため、開発蝸牛は脊椎動物の開発中にPCPシグナル伝達を検討する中で理想的な器官である。器官コルティ、その行蝸牛管を特殊な感覚上皮に与えられた用語は、一様に10を機能させるには蝸牛のために配向させる必要があり、非感覚支持細胞と機械刺激有毛細胞から構成されている。機械刺激有毛細胞がそのように理由で呼ばれていますクチクラ板から延びるstereociliary束それぞれの感覚有毛細胞11の（先端面）。mechanosensationのと不動としての命名法にもかかわらず、一次トランスデューサとしてこれらの行為は、実際に変更されたアクチンフィラメントベースの微絨毛で構成されている。各山形の髪の中でバンドルは、不動の3行は非常に注文し、定期的なPAで構成されています階段のケース状にttern。実微小管ベースの繊毛、kinociliaは、stereociliary 束 12の開発と方向のために必要とされていると呼ばれます。各有毛細胞の際に、一つの運動毛は、物理的に不動の一番高い行に中央に隣接して位置する、不動毛束に取り付けられています。運動毛の正確な機能は不明である、と1の仮説は、彼らが微絨毛12から成熟するにつれて運動毛が形に不動を「引っ張る」ということです。脊椎動物では、蝸牛内kinociliaは一過性のみ存在し、11,13,14を聞くの開始前にマウスでの有毛細胞から退避。 深刻な短縮蝸牛管、誤形成され、誤指向stereociliaryバンドルだけでなく、誤位置付け基礎体8,9の途上蝸牛結果の繊毛の完全な損失。機能的な繊毛はちょうど繊毛軸糸で構成されていません。繊毛に関連した多くのタンパク質機能は、基礎体温、移行帯、または繊毛軸糸15として繊毛関連のサブドメインに局在する複合体で起こります。中心体の母中心小体由来基礎体温は、また、細胞体に繊毛から離れて延びている微小管のための微小管組織化の中心であり、細胞内輸送、ならびに繊毛輸送を調節することができます。毛様体遷移ゾーンは、繊毛機能が毛様体化合物16のインポートおよびエクスポートを整理するのが規制されている別の領域です。 正確なメカニズムは、17不明であるものの、複数の研究は、繊毛および非標準Wnt（PCPシグナリング）との間の接続を確認しています。毛様体およびPCP遺伝子の冗長性と一般化された細胞異常に細胞極性の感度は、それが困難な直接PCP-特定の赤字に変異をリンクすることを可能にします。 PCPシグナル伝達の読み取りアウトの一つは、基礎体温とprimarの位置付けでありますしたがって、2次欠陥から、主なものを分離するのy繊毛は、困難です。ゼブラフィッシュとマウスの変異体におけるいくつかの研究は、18-20シグナリング繊毛およびWnt間の接続を示唆していません。データの矛盾は、種、組織、またはWntシグナル伝達に向かって毛様体の貢献の時間依存性の違いを反映しているのかもしれません。基礎体が機能している場合はさらに、正常なWnt応答性が保持されることがあります。細胞シグナル伝達経路中の毛様体タンパク質の役割をより深く理解し、他の生物学的現象は、細胞および発生生物学の我々の理解のためだけでなく、標的治療戦略の開発のために重要です。

<table>
	<tbody>
		<tr>
			<td>Tools/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone elastomere - Sylgard 184</td>
			<td>Sigma-Aldrich</td>
			<td>761028-5EA</td>
			<td>See Note 2</td>
		</tr>
		<tr>
			<td>Micro dissecting scissors-straight blade</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps (no. 5 and 55) and blunt forceps</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope.</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uncoated glass microscope slides</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slips (22 mm &#215; 40 mm &#215; 0.15 mm)</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Minutien pins</td>
			<td>&#160;Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM sample holder</td>
			<td>tousimis</td>
			<td>8762</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscopy studs</td>
			<td>TED PELLA</td>
			<td>16111</td>
			<td></td>
		</tr>
		<tr>
			<td>PELCO Tabs: Carbon adhesive</td>
			<td>TED PELLA</td>
			<td>16084-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Critical Point Dryer</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slides</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe Tissue&#160;</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Paint Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1&#215; Phosphate buffered saline (PBS)</td>
			<td>Gibco/Life Technologies</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160; (PFA) (EM Grade Required for EM)</td>
			<td>Various</td>
			<td></td>
			<td>Prepare a 4% solution in 1&#215; PBS made fresh each time. EM Grade Required for EM.</td>
		</tr>
		<tr>
			<td>2.5% Glutaraldehyde Grade1</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl (pH 7.5)</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl 2</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AffiniPure Fab Fragment Donkey Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-007-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G Mounting media</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>10&#215; Hanks&#8217; Balanced Salt Solution (HBSS)</td>
			<td>Gibco/Life Technologies</td>
			<td>14065</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Gibco/Life Technologies</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide (OsO4 )</td>
			<td>Sigma-Aldrich/Fluka Analytical</td>
			<td>75632</td>
			<td></td>
		</tr>
		<tr>
			<td>Tannic acid</td>
			<td>Sigma-Aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti Arl13b</td>
			<td>Protein Tech</td>
			<td>17711-1-AP</td>
			<td>Suggested concentration 1:1000</td>
		</tr>
		<tr>
			<td>anti acetylated tubulin (611-B1)</td>
			<td>Sigma-Aldrich</td>
			<td>T6793</td>
			<td>Suggested concentration 1:800</td>
		</tr>
		<tr>
			<td>anti gamma tubulin (GTU-88)</td>
			<td>Sigma-Aldrich</td>
			<td>T6557</td>
			<td>Suggested concentration 1:200</td>
		</tr>
		<tr>
			<td>anti Zo_1&#160;</td>
			<td>Invitrogen</td>
			<td>40-2300</td>
			<td>Suggested concentration 1:500</td>
		</tr>
		<tr>
			<td>Myosin VI</td>
			<td>Proteus Biosciences</td>
			<td>25-6791</td>
			<td>Suggested concentration 1:1000</td>
		</tr>
		<tr>
			<td>Myosin VIIa</td>
			<td>Proteus Biosciences</td>
			<td>25-6790</td>
			<td>Suggested concentration 1:1000</td>
		</tr>
		<tr>
			<td>anti Vangl2</td>
			<td>Merk Millipore</td>
			<td>ABN373</td>
			<td>Suggested concentration 1:250</td>
		</tr>
		<tr>
			<td>anti G&#945;i3</td>
			<td>Sigma-Aldrich</td>
			<td>G4040</td>
			<td>Suggested concentration 1:250</td>
		</tr>
		<tr>
			<td>Alexa Fluor&#174; 488 Phalloidin</td>
			<td>Invitrogen/Life Technologies</td>
			<td>A12379</td>
			<td>Suggested concentration 1:300-1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor&#174; 568 Phalloidin</td>
			<td>Invitrogen/Life Technologies</td>
			<td>A12380</td>
			<td>Suggested concentration 1:300-1000</td>
		</tr>
	</tbody>
</table>

evaluation of planar-cell-polarity phenotypes in ciliopathy mouse mutant cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

蝸牛解剖と組織調製 脳を除去した後、P0マウスヘッドの後正中線矢状切開、後ろから見骨迷路は、（ 図1A、白矢印）可視化し、除去することができる。 図1Bは、蝸牛有毛を有する単離骨迷路を示しています、腹、上向き（左）と後ろから、背側（右）。 1は、外側の軟骨の除去を開始...

Watch this Scientific Journal Video about Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea at JoVE.com

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

繊毛病マウス変異蝸牛における平面セル・-極性表現型の評価

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

evaluation-planar-cell-polarity-phenotypes-ciliopathy-mouse-mutant

Research

JoVE Journal

Medicine

10.3K ビュー.  Johannes Gutenberg-Universität Mainz. Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

ビデオ: Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

意識、無拘束マウスにおける高インスリン血症、正常血糖クランプ

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

医学

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

小説を使用して、脊髄損傷後のマウス後肢の骨の損失の経時的評価、 in vivoで、方法論

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

腔内MCAOのマウスモデル：クレシルバイオレット染色による脳梗塞の評価

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

マウス肺挿管の簡単な方法

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

定量的リアルタイムPCRによるマウス乳腺腫瘍モデルにおける肺転移の評価

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

外科用マウスモデルにおける変形性関節症の標準化組織形態評価

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

開放血管窓と生体内蛍光顕微鏡を用いたマウス蝸牛の心房血流測定

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. Dysregulated glucose metabolism is an important manifestation of the disease and is the key to its pathogenesis. Therefore, studies involving evaluation of glucose metabolism in PCOS are of utmost importance. Very few studies have quantified hepatic glucose production directly in PCOS models using non-radioactive glucose tracers. In this study, we discuss step-by-step instructions for the quantification of the rate of hepatic glucose production in a PCOS mouse model by measuring M+2 enrichment of [6,6-2H2]glucose, a stable isotopic glucose tracer, via gas chromatography - mass spectrometry (GCMS). This procedure involves creation of stable isotopic glucose tracer solution, use of tail vein catheter placement and infusion of the glucose tracer in both fasting and glucose-rich states in the same mouse in tandem. The enrichment of [6,6-2H2]glucose is measured using pentaacetate derivative in GCMS. This technique can be applied to a wide variety of studies involving direct measurement of the rate of hepatic glucose production.

This study describes the direct measurement of hepatic glucose production in a polycystic ovary syndrome mouse model by using a stable isotopic glucose tracer via tail vein in both fasting and glucose-rich states in tandem.

多嚢胞性卵巣症候群マウスモデルにおける肝グルコース産生の評価

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

ラットモデルにおける網膜病態生理学的評価

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

意識性ラットの鎖骨下静脈からの反復採血

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...