We would like to acknowledge Beth Levine for critically reviewing the manuscript, Valerie Tassinari for developing the genotyping protocol, and Matt Ferola and Carlos Maya for their exceptional animal care.

すべての実験動物の管理と使用のための健康ガイドの国立研究所によって記載されたプロトコルに従って実施されており、ALS TDIの制度的動物のケアと使用委員会（IACUC）によって承認されました。 1.動物、住宅、および研究デザイン注意：SOD1-G93Aマウスコロニーは、もともとガーニーら7によって生産高コピーB6SJLTgN（SOD1G93A）1Gur株に由来するものでした。コロニーは、現在、野生型C57BL / 6-SJL F1の雌とトランスジェニックC57BL / 6-SJL男性を交配することによって維持されています。 F1動物をB6雌とSJL男性を交配することによって生成されます。マウスは、生​​後35〜45日でALSのTDI毎週に出荷されます。 <ol><li>マウス少なくとも1週間は研究に割り当てる前（18〜23°Cと40〜60％の湿度の温度で、12時間の明/暗サイクル）動物施設に順応できるようにします。 </li><li>ハウスオスSOD1-G93Aマイクグループを収容するので、ケージごとに電子1マウスは、男性は戦うために傾向があります。ケージ当たり最大2つの家の女性SOD1-G93Aマウス。プラスチック小屋の形で、環境エンリッチメントを提供します。マウスは、水/食品飲料に薬物投与を必要とする研究の一部である場合を除き、食料および水を自由に摂取させてください。これらのケースでは、配給、レコード食料と水。 </li><li>年齢50日目の周りの薬剤有効性試験を開始し、64ゴミをマッチさせたジェンダーバランスのとれたマウス（コホートごとに32）が挙げられます。最後に、研究を行う研究者が実験条件を知らされていることを確認。 注：これは、典型的な薬剤の有効性の研究は、しかし、それは前または後の研究を開始することが望ましいかもしれない治療的介入の種類に基づいて、年齢50日に開始することをお勧めします。 注意：コホートあたり32匹のマウスを用いた研究は、性特異的薬剤の効果を検出するために供給されています。何の性別固有の比較が必要とされない場合は、コホートあたり24匹のマウスは、全体的な薬剤の効果を検出するために十分なはずです。</li></ol> 2.体重<ol><li>グラムの10分の1の精度で日常の研究におけるすべてのマウスのレコードの体重。注意：体重は疾患進行の慢性薬物治療から生じる可能性の倦怠感の敏感な指標です。 </li><li>マウスの体重は、このように、朝に高いこと、日中かなり変動毎日同じ時期にそれらを記録。 </li></ol> 3.神経学的スコアリングシステム（NeuroScore） <ol><li> A）マウスがその尾によって中断され、b）のマウスは、歩くことが許可され、麻痺cの発症後）：順次実行次の3つの条件の下でマウスを観察することで、毎日、各マウスのための神経学的スコア（NS）を評価マウスは、その側面に配置されています。 <ol><li>尾懸垂試験では、OBSながら、離れた食品流域から、1〜2秒のための彼らのホームケージのワイヤー上から約1.5 &quot;尾の付け根からマウスを開催後肢をアービング。懸濁試験を3回繰り返し、最も一貫性のある結果を記録します。 </li><li>歩行テストでは、いくつかのトラクション25センチ歩行距離を提供してきれいな表面上でマウスを置く（ 例えば 、ペーパータオルは、滑り防止のためにダウンしてテープで固定しました）。その歩行を観察しながらマウスを75センチ合計（ペーパータオルの3倍の長さ）を歩くことを許可します。 </li><li>その左側または右側にマウスを置き、テスト「立ち直り反射」とストップウォッチを使用するために、それが両側から自力自体を右にかかる時間を測定します。注：このテストの唯一の試験が必要です。 </li></ol></li><li> 0〜4注に独立規模で（左または右）各後肢のNSを決定します。右後肢NSが1である間例えば0のスコアは、1は、左後肢NSが0であることを示しています。 </li><li> （ 表1に提供要約）観測の次のセットに対応する神経学的スコア（0-4）を割り当てます。<ol><li>以下が観察されている場合は、NS 0（発症前）を割り当てます、マウスを尾によって中断されると、後肢は通常のスプレイつまりを提示し 、それが完全に離れて横正中線から延長され、それが2秒間、この位置に留まりますか長いです。マウスが歩いさせると、通常の歩行が観察されます。 </li><li>以下が観察されている場合は、NS 1（最初の症状）を割り当て：マウスを尾によって中断されると、後肢は異常な広がりを示し、 すなわち、それが崩壊している、または部分的に崩壊し、横方向の正中線に向かってか、尾懸垂中に震えるか、後退させます/握りしめ。マウスが歩いて許可された場合、通常のまたはわずかに遅い歩行が観察されます。 </li><li>以下が観察されている場合は、NS 2（麻痺の発症）を割り当てます。マウスを尾によって中断されると、後肢はあまり伸びるない、部分的または完全に崩壊しています。 （まだ関節の動きがあるかもしれません）。マウスが歩いさせると、後肢を前方MOTのために使用されますイオンは、しかし、つま先を90センチ歩いたり、足の一部がケージの底/テーブルに沿ってドラッグしている間に少なくとも二度下向きにカール。マウスは、その左右側に配置されている場合には、両側から10秒以内に自身を右することができます。 </li><li>以下が観察されている場合は、NS 3（麻痺）を割り当て：マウスを尾によって中断されると、後肢または最小限の関節の動きの剛性麻痺があります。マウスを歩くことを許可されている場合には、前方が後肢は前進運動のために使用されていない動きがあります。マウスは、その左右側に配置されている場合には、両側から10秒以内に自身を右することができます。注：まれに、麻痺の発症後、尿の水分が後肢に表示されない場合があります。左未処理の尿の水分が尿に「バーン」と皮膚病変を引き起こす可能性があります。髪をクリッピングすることによって尿の水分を扱い、暖かい水が尿を除去するために浸す適用され、静かに乾燥ブロット。皮膚病変が存在する場合antibio適用チック軟膏。 </li><li>以下が観察されている場合は、NS 4（動物愛護エンドポイント）を割り当てます。マウスを尾によって中断されると、後肢で剛性麻痺があります。マウスを歩くことを許可された場合、何の前進運動はありません。マウスは、その左右に配置されているとき、それはどちらの側から10秒以内に自分自身を右することはできない。 すなわち、立ち直り反射の欠如。 </li></ol></li></ol> 4.データ入力と分析<ol><li>薬効の研究の過程で、研究室情報管理システム（LIMS）に各マウスについて、市販の表計算ソフトに収集されたデータを入力します。 注意：ALS TDI-開発LIMSソフトウェアは、ALSのTDI研究者のための時間をかけて、これらのデータは便利なアクセスできるように、内部の研究から収集したすべてのデータの中央リポジトリです。 LIMSは、すべての試験日に戻ってその系統、体重または神経学的スコアに、そのユニークなIDで、任意の個々のマウスを追跡し、その有効期間中に注意し、他の観測値が含まれています。 LIMSは、最終的にはその後の分析のために使用されているスプレッドシートを生成します。 </li><li>薬効試験の完了時、 すなわち、全てのマウスは、左と右の後肢NSを平均化することによって毎日のために、各マウスのための組み合わせNSを計算するために人道的エンドポイント用表計算ソフト（材料の表を参照）に達したとき。次に、各マウスそれぞれのマウスは、特定のNSに費やした日数の間、それぞれのNSで年齢の中央値を計算します。 </li><li>統計ソフトウェアに表計算ソフトからデータをインポートすることで、時間をかけて、NSの全体的な変化を分析（材料の表を参照のこと）。中央値NS時の年齢と回帰における効果などの薬物治療と、期待NSを推定順序ロジスティック回帰モデルを使用してこれらのデータを合わせます。ログランクカイ二乗検定を用いて効果尤度確率を推定し、NS進行に対する薬物治療の効果は統計的であるかどうかを判断LY有意な（ステップバイステップの手順については、補足コードファイルを参照してください）​​。 注：p値&lt;0.05は、統計学的に有意であると考えられます。 注：統計分析するこの方法は、NSデータと個々のマウスのNS軌跡の可変性の序性質を尊重します。個々のNS軌跡がNS応答の十分に反映していないNS時間に関連するグループの平均値が得られる著しく異なるものとすることができるので、好ましいです。加えて、いくつかのケースでは、特にNSときゼロから1つの範囲で変動することができます。 </li><li>最後に、薬剤処理動物と対照動物のためのグラフ（;補足コードは、ステップバイステップの手順については、ファイルの図 2を参照）、「序数はNS時の年齢中央値対NSを期待」を生成する（材料の表を参照してください）ソフトウェアのグラフを使用します。 私は （便利なy軸から疾患の発症年齢の中央値を補間することにより、薬物治療群と対照群との間のシフトを推定するためにこれらのグラフを使用してください。Eは、x軸上のNS 2）。 注：二つのグループ間の年齢の中央値の差は、薬剤誘発性のシフトを表しており、効果の大きさの実用的な尺度として扱われます。 注意：予想NS対年齢の中央値曲線が右へシフトが遅く症候性疾患の進行を示しています。逆に、予想NS対年齢の中央値曲線の左方シフトが加速症候性疾患の進行を示しています。 </li></ol>

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The authors have nothing to disclose.

本稿では、適切に適用された場合、迅速かつ簡単にビデオプロトコルを記述し、確実にSOD1-G93Aマウスにおける疾患の進行を評価し、薬物誘発性の変化を識別することができます。別のグループは、SOD1-G93Aマウス8〜10のための表現型のスコアリングシステムを開発してきたが、それらは多くの場合、手続きに十分な詳細を提供し、レプリケーションのために不十分であるしないでくだ?...

1990年代半ばにおけるその開発SOD1-G93Aトランスジェニックマウスモデルは、筋萎縮性側索硬化症（ALS）1の最も広く使用される動物モデルとなっている以来。このトランスジェニックマウスモデルは、遺伝的に、アミノ酸93（G93A）のアラニンへの変異ALS関連グリシンを保有する人のCu / Znのスーパーオキシドジスムターゼ1（SOD1）遺伝子の変異型を過剰発現するように設計されました。 G93A変異を一括メイク家族性ALS症例2の約​​1/5 SOD1遺伝子の多くの変異の一つです。 ALSとの明らかな遺伝的リンクに加えて、このような運動ニューロンの損失、進行性の筋力低下や萎縮などのヒトでALSの病理学的特徴の多くを再現するため、SOD1-G93Aモデルは、最終的に、ALS創薬研究の主力となっています麻痺と死3。多くの場合、トランスジェニック動物モデルの場合と同様にかかわらず、固有の双方向性がありますSOD1-G93Aマウスに関連する学的変動。スコットら 。少なくとも24ごみをマッチさせた男女バランスのマウスのコホートは、4生物学的変動によって作成されたノイズを克服するために、薬剤の有効性の研究のデザインに必要とされていることを確認しました。攻撃的な疾患の進行、毎日モニタリングの必要性と組み合わせたこれらの研究に必要な動物の多くは、神経筋強度および機能を測定するための精巧な、時間がかかり、潜在的にストレスの多い技術の使用を禁止する（例えば、筋電、握力、回転棒、等）。代わりに、それは確実に迅速な方法でSOD1-G93Aマウスの神経筋機能を評価するin vivoでのスクリーニングツールの必要性を強調しています。 ALS TDIでプロトコルが平均でマウスあたり30秒未満でSOD1-G93Aマウスの神経筋機能の信頼性の評価が可能になるように開発されました。このプロトコルのencompaプレスで、ビデオでは、この報告書に記載されている単純な神経学的スコアリングシステム（NeuroScore）を使用して、後肢機能のSSES毎日評価。後肢の赤字が最も早く、SOD1-G93Aマウス5,6における疾患の神経学的兆候を報告しているためNeuroScoreは、後肢の機能に焦点を当てています。さらに、ALS TDIが開発したプロトコルは、不全麻痺（わずかなまたは部分的な麻痺）の発症の公平な評価を提供する麻痺の進行および重症度、それが疾患の進行における薬剤誘発性の変化を識別するのに十分な感度です。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>B6SJLTgN(SOD1G93A)1Gur strain</td>
			<td>The Jackson Laboratory, Bar Harbor, ME</td>
			<td>002726</td>
			<td>Strain of mice used in this study</td>
		</tr>
		<tr>
			<td>Breeders</td>
			<td>Biomedical Research Models, Inc., Worcester, MA</td>
			<td></td>
			<td>Colony maintainance</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Navigator OHAUS, Parsippany, NJ</td>
			<td>Model N14120</td>
			<td>Used to weigh mice</td>
		</tr>
		<tr>
			<td>Nutra-GelH</td>
			<td>Bio-Serv, Flemington, NJ</td>
			<td>#S4798</td>
			<td>Wet food provided to sick mice</td>
		</tr>
		<tr>
			<td>Irradiated Lab Animal Diet</td>
			<td>Harlan, Indianapolis, IN</td>
			<td>2918-111914M</td>
			<td>Chow diet</td>
		</tr>
		<tr>
			<td>Lab-grade Sani-chips</td>
			<td>Harlan Teklad, Indianapolis, IN</td>
			<td>7090</td>
			<td>Bedding</td>
		</tr>
		<tr>
			<td>JMPH&#160;</td>
			<td>SAS Institute, Inc., SAS Campus Drive, Cary, NC</td>
			<td>&#160;v10.0.2</td>
			<td>Statistical software</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft, One Microsoft Way, Redmond, WA</td>
			<td>Excel 2013 (v 15.0)</td>
			<td>Spreadsheet software</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad software Inc., La Jolla, CA</td>
			<td>v5.4</td>
			<td>Graphing software</td>
		</tr>
		<tr>
			<td>Mouse Huts</td>
			<td>Bio-Serv, Flemington, NJ</td>
			<td>K3272</td>
			<td>For environmental enrichment</td>
		</tr>
	</tbody>
</table>

a quick phenotypic neurological scoring system for evaluating disease progression in the sod1-g93a mouse model of als

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening protocol, demonstrated in video herein, which reliably assesses the neuromuscular function of SOD1-G93A mice in a quick manner. This protocol encompasses a simple neurological scoring system (NeuroScore) designed to assess hindlimb function. NeuroScore is focused on hindlimb function because hindlimb deficits are the earliest reported neurological sign of disease in SOD1-G93A mice. The protocol developed by ALS TDI provides an unbiased assessment of onset of paresis (slight or partial paralysis), progression and severity of paralysis and it is sensitive enough to identify drug-induced changes in disease progression. In this report, the combination of a detailed manuscript with video minimizes scoring ambiguities and inter-experimenter variability thus allowing for the protocol to be adopted by other laboratories and enabling comparisons between studies taking place at different settings. We believe that this video protocol can serve as an excellent training tool for present and future ALS researchers.

90男性と94女性の非処置SOD1-G93Aマウスのデータは2014年の間に、ALS TDIで学ん神経学的スコアを評価しました。結果は、女性に比べNS 1におけるその寿命の大部分によって証明されるように、雄マウスは、典型的に、雌のマウスよりもより攻撃的な疾患進行を有することを示しています。 NS 2、NS 3は、男女全体で事実上等しい頻度で発生します。それは「通常の」表現型と考えられているので、NS...

Watch this Scientific Journal Video about A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS at JoVE.com

A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

This video protocol describes a sensitive, reliable, and quick method for evaluating the neuromuscular deficits in a transgenic mouse model of amyotrophic lateral sclerosis.

ALSのSOD1-G93Aマウスモデルにおける疾患進行を評価するためのクイック表現型の神経学的スコアリングシステム

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening ...

a-quick-phenotypic-neurological-scoring-system-for-evaluating-disease

Research

JoVE Journal

Medicine

19.2K ビュー.  ALS Therapy Development Institute. This video protocol describes a sensitive, reliable, and quick method for evaluating the neuromuscular deficits in a transgenic mouse model of amyotrophic lateral sclerosis.

ビデオ: A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

パーキンソン病の単方向、病変6-OHDAモデルマウスの開発

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

医学

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

人間の神経筋システムを研究するために経頭蓋磁気刺激の活用

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

新しいハーブ治療法を評価するために敗血症の動物モデルの使用

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

臨床試験および筋萎縮性側索硬化症（ALS）のマウスモデルにおける脊髄の除去

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

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  ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

膵臓癌の進行の生物発光同所性モデル

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

検証済みの内視鏡スコアリングシステムを使用して、大腸炎および結腸直腸腫瘍の重症度を評価するためにマウスでの柔軟な大腸内視鏡検査

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

マウスモデルにおけるUTIとCAUTIの樹立とその性状

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

マウスの弁膜症の包括的表現型の特徴のための心エコーアプローチとプロトコル

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

マウスモデルにおける覚醒時膀胱内圧測定の実施手順の評価

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...