すべてのラットは、首都医科大学北京友好病院の動物実験委員会によって承認されたプロトコルに従って使用されました。
1.外科的準備、麻酔、および外科的技術
注:本研究では、体重200〜220 gの合計40匹の雌のWistarラットが商業的に入手されました。40匹のラットのうち、10匹を対照群として無作為に選択し、残りを試験群として治療した。すべての動物は、首都医科大学北京友好病院の動物施設の無菌環境に収容されました。
<ol><li>ペントバルビタールナトリウムを腹腔内投与して全身麻酔を行います(40 mg / kg)。.あるいは、3%〜4%のイソフルランを使用して麻酔を誘発し、1%〜3%(吸入)に維持する。乾燥を防ぐために眼科用軟膏を目に塗ります。次に、ラットを手術プラットフォームに置き、熱サポートを提供します。 注:手順の開始時に、ブプレノルフィン、0.05 mg / kg、SC、0.1〜0.2mLなどの鎮痛薬を投与します。</li>
	<li>つま先のつまみに対する反応の欠如によって麻酔の深さを確認してください。かみそりで背中全体から毛皮を剃ります。</li>
	<li>クロルヘキシジンやポビドンヨードなどの2段階のスクラブとそれに続くイソプロピルアルコールで少なくとも3サイクルの手術部位を滅菌します。手足をサージカルテープで固定し、手術用ハサミで背中の中央値約3cmを切開します。</li>
	<li>外科用ハサミを使用して皮下組織を通して切開を深くし、脊椎に付着した筋肉を切り落とします。</li>
	<li>13番目の 肋骨を視覚的に識別して露出させます(その肋骨に接続されている椎間腔は間隔T13\u2012L1です)。縫合糸を使用して13番目の 肋骨に印を付けます。</li>
	<li>識別後、脊椎に付着した筋肉を注意深く切除し、脊柱を露出させます。脊柱を正確に識別するために、棘上靭帯と棘間靭帯を切除します。外科用ハサミと鉗子でL4〜L5のレベルを公開します。 注:棘上靭帯は、薄い皮下組織が存在するため、簡単に識別できます。棘上靭帯の切除後、棘突起間の靭帯は棘間靭帯である。</li>
	<li>ケリー鉗子を使用して、L4〜5椎骨棘突起と横突起の一部を慎重に解剖し、脊髄を露出させます(図1)。</li>
	<li>円錐髄をL4〜L5レベルで完全に露出させ、円錐髄錐を虹彩切除ハサミで完全に横断します。脊髄の回復をブロックするために、組織パッキングを挿入します。</li>
	<li>4-0非吸収性縫合糸を使用して、外側の皮膚層の上に横たわる筋肉と皮膚を閉じます。</li>
	<li>コントロールグループについては、手順1.1〜7を実行し、円錐髄をそのまま残します。ステップ1.9に従って切開部を閉じます。</li>
</ol>2.動物の回復
<ol><li>術後最初の1時間はラットを温度管理されたインキュベーター(37°C)に入れ、胸骨または活発に動くまで監視します。 注:完全に回復するには約30分かかります。</li>
	<li>動物を十分な食物と水の入った清潔なケージに移します。ラットを別々のケージに入れてください。 注:切断の成功は、テストグループのラットが前足の助けを借りてのみ動くのに対し、コントロールグループのラットは正常に歩くことができた場合に示されます。</li>
</ol>3. 運用後管理
<ol><li>抗生物質であるペニシリンG(動物あたり50,000 U / mL)を腹腔内に注射します。.術後48時間、ブプレノルフィン、0.05 mg / kg、SC、0.1〜0.2 mLなどの鎮痛薬を6〜12時間ごとに投与します。.</li>
	<li>排尿を助けるために下腹部で膀胱を圧迫します。これを1日2回、同時に(午前8時と午後8時)6週間実行します。 注:排尿筋の正常な狭窄の喪失は、DUの象徴です。</li>
	<li>すべてのラットを代謝ケージに入れ、それぞれに尿収集漏斗を事前に計量した吸収紙の上に置き、排尿と失禁を監視します。</li>
	<li>排尿量(VV)を示す吸収紙の重量変化と残留尿量を別々に収集してメモします。</li>
</ol>4.尿流動態検査
<ol><li>術後6週間で、以下のように尿流動態測定装置を用いて膀胱鏡検査を行う。<ol><li>10%抱水クロラールを腹腔(3 mL / kg)に注射してラットを麻酔します。.</li>
		<li>排尿のために膀胱を圧縮し、次にテープを使用してラットを手術プラットフォームに固定します。</li>
		<li>硬膜外カテーテル(3F)を膀胱に挿入し、尿流動態測定装置、硬膜外カテーテル、輸液ポンプを三肢チューブで接続します。</li>
		<li>尿流動態測定のために生理食塩水を0.2 mL / minの速度でポンプで送ります( 材料の表を参照)。MCCとDOP、および膀胱のコンプライアンス(膀胱容積δ排尿筋の圧力で割って計算δ記録します)。</li>
	</ol></li>
</ol>5.統計分析
<ol><li>市販のソフトウェアを使用して統計分析を実行します。</li>
	<li>コルモゴロフ-スミルノフ検定を使用して、データの正規性を検定します。</li>
	<li>正規分布変数を標準偏差の平均値で表します。両側対応のスチューデント のt検定を使用して、両方のグループの膀胱メトログラムのパラメーターを比較します。 注: p < 0.05は、差に統計的有意性があることを示します。</li>
</ol>

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著者は開示するものは何もありません。

DUは、男性と女性の両方の下部尿路症状の一般的な原因です。これは、影響を受けた人々の生活の質(Qol)を大幅に低下させる可能性のある治療オプションがほとんどない複雑な症状の星座です18。DUは多因子性であると考えられているが、その病因の理解は初歩的なままである。研究によると、DUの病因は筋原性および神経原性の要因に関連している可能性があります。
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排尿筋活動低下症(DU)は、研究が続けられている典型的な下部尿路機能障害である。DUは国際排泄学会(ICS)1によって定義されていますが、この病気を指すために、「排尿筋不全」、「収縮性膀胱」、「排尿筋性反射症」2など、さまざまな用語が使用されています。2002年に国際禁制学会(ICS)によって定義されたDUは、強度と持続時間の低下の収縮であり、その結果、膀胱が排出される時間が長時間増加し、それによって通常の期間内に完全な膀胱排出を達成できなくなります。
DUは、男性の48%、女性の12%(>70歳)3 に、尿路症状が下がる可能性がある。それは多因子性のようであり、効果的な治療法は存在しません。DUは、多発性硬化症4、糖尿病5、パーキンソン病6、脳脳卒中7などの神経因性膀胱機能障害の患者に遍在していることが報告されています。DUは、腹腔鏡下子宮摘出術、前立腺全摘除術、または小骨盤8の他の外科的介入などの医原性神経損傷によっても引き起こされる可能性があります。DUの病態生理学の変化と利用可能な治療法は、研究に適した動物モデルがないため、依然として混乱しています。
排尿反射は、橋排尿中枢、仙骨副交感神経核、およびより上級の皮質中枢を組み合わせた脊髄球脊髄経路によって制御されています9。排尿反射の活性化と維持は、主に膀胱からより上級の皮質センターへの感覚信号の定期的な輸送に依存します。感覚機能障害がDUに寄与すると仮定することができます。
下部尿路機能障害に関連するほとんどの実験動物研究は、過活動膀胱(OAB)モデルに焦点を当てています10。これらのモデルは、OABの病態生理学および予後の合理的な理解を提供する。しかし、報告されているDUモデルはごくわずかであり、例えば、脊髄上損傷(局所病変、衰弱、および中大脳動脈閉塞)、脊髄切断または挫傷損傷、全身性(例:.、シクロホスファミド)または膀胱内投与刺激性または炎症性薬剤(例:.、酸、アクロレイン、およびリポ多糖)11,12,13,14。.これらの方法のうち、DU13の動物モデルを確立する際に使用できるのは、脊髄離断法または挫傷損傷法のみである。橋排尿センターと高次皮質センターの損傷を伴う試みは、重度の外傷のために放棄されました。そのため、排尿反射中枢の正確な位置を見つけて、最小限の副作用でDUを誘発することにますます注意が払われています。
前述のように、DUを誘導するメカニズムの1つは、脊髄を傷つけて排尿反射のシグナル伝達経路を損傷することです。アレンの体重減少法は、脊髄損傷を有する実験動物を確立するために開発された15。ただし、この方法に関するこれ以上の実験データはありません。さらに、動物の一部はDUなしで脳卒中後に脊髄機能を回復したので、DU動物モデル16を生成するための完全な方法とは考えられない。
1987年、ブレグマンはDU動物モデルを生成するために脊髄を切断するプロセスを解明し、実験データ17を取得しました。それにもかかわらず、この方法はDU動物モデルを確立するために適用されなかった。当時、研究者たちはまだDUの病因について混乱していました。OABまたはDUの誘導に関連する脊髄内の位置は互いに隣接しているため、DU17を誘導するための脊髄の損傷部位を正確に見つけることができませんでした。OABとDUは、この方法で一緒にまたは別々に導入されました。そのため、この方法はDUを導入しましたが、不正確であり、DUの発生と処理の理解には使用できませんでした。
上記のように、DUの適切な動物モデルの欠如は、DUの研究の主な障害の1つです。 研究者は、DUの病理をシミュレートできる正確で管理しやすいモデルを継続的に探しています。DUの治療選択肢でさえ、過去20年間に大幅に改善されていません。まとめると、DUの動物モデルを確立するための標準プロトコルを記述することが大いに必要とされている。
そこで本稿では、円錐髄郭清によりDUのラットモデルを首尾よく確立する方法について述べる。円錐髄質を分離するためにL4〜5レベルで切断を行った。最大膀胱測定容量(MCC)、排尿筋開放圧(DOP)、および膀胱のコンプライアンスを記録および分析して、プロトコルを検証しました。以下に述べるプロトコルは、実現可能性と信頼性の両方を標準化された方法で組み合わせて、DU動物モデルを確立し、DUの発生と処理をシミュレートします。このプロトコルは、DUのさらなる研究のための技術として使用することができる。

<table>
	<tbody>
		<tr>
			<td>0.9% saline</td>
			<td>Wuhan Prosai Company</td>
			<td>EY-C1178</td>
			<td>pump for urodynamic measurement</td>
		</tr>
		<tr>
			<td>10% chloral hydrate</td>
			<td>Shandong Yulong Co., Ltd</td>
			<td>H37022673</td>
			<td>3mL/kg&#65292;administered intraperitoneally</td>
		</tr>
		<tr>
			<td>Buprenorphine Hydrochloride Injection</td>
			<td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co. LTD</td>
			<td>H12020275</td>
			<td>0.05mg/kg subcutaneously 24h and 48h postoperation</td>
		</tr>
		<tr>
			<td>Epidural Catheter</td>
			<td>Shandong Xinghua Co, Ltd</td>
			<td>VABR3L</td>
			<td>for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>Alta Technology Co., Ltd</td>
			<td>1ST5637</td>
			<td>50,000 unit/ml per animal</td>
		</tr>
		<tr>
			<td>pentobarbital</td>
			<td>Beijing solabo Technology Co., Ltd</td>
			<td>NK-WF0001</td>
			<td>40 mg/kg, administered intraperitoneally</td>
		</tr>
		<tr>
			<td>Suture line(4-0)</td>
			<td>ETHICON</td>
			<td>VCP422H</td>
			<td>suture the injury</td>
		</tr>
		<tr>
			<td>Three-limb tube</td>
			<td>Shandong Xinghua Co, Ltd</td>
			<td>VAB3T</td>
			<td>for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Trace infusion pump</td>
			<td>Zhejiang Smith Medical Instrument Co., Ltd</td>
			<td>20162540335</td>
			<td>Pump the saline at a speed of 0.2ml/min for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Urodynamic measurement equipment</td>
			<td>Medical Measurement SystemsB.V.</td>
			<td>08-0467</td>
			<td>urodynamic measurement equipment can not only help the diagnosis of dysuria, but also provide objective materials for treatment and therapeutic effect. It is the most commonly used examination method in clinical diagnosis and treatment of lower urinary tract functional diseases</td>
		</tr>
		<tr>
			<td>Wistar Rats</td>
			<td>HFK Biotechnology Co.Ltd,Beijing ,China</td>
			<td>SCXK2012-0023</td>
			<td>200-220g</td>
		</tr>
	</tbody>
</table>

detrusor underactivity model in rats by conus medullaris transection

提示されたプロトコルの目標は、円錐髄膜剥離を通じてラットの排尿筋活動低下(DU)モデルを確立することでした。体重200〜220 gの雌Wistarラット40匹(対照群:10匹、試験群:30匹)で椎弓切除術を行い、試験群では髄膜円錐をL4〜5レベルで切除しました。全てのラットを同じ環境条件下で6週間飼育し、給餌した。試験群では、排尿を1日2回6週間行い、平均残尿量を記録した。膀胱メトログラムは両方のグループで実施されました。最大膀胱測定容量(MCC)、排尿筋開口圧(DOP)、および膀胱のコンプライアンスが記録され、計算されました。試験群は、脊髄ショック中および手術後の両方で、手術後に有意な尿閉を示した。しかし、対照群に異常は認められなかった。対照群と比較した場合、試験群のMCCおよび膀胱のコンプライアンスは試験群よりも有意に高かった(3.24 ± 2.261 mL 対 1.04 ± 0.571 mL;0.43 ± 0.578 mL/cmH 2 O 対 0.032 ± 0.016 mL/cmH 2 O)、試験群のDOPは対照群よりも低かった(20.28 ± 14.022 cmH 2 O vs 35 ± 13.258 cmH2円錐髄膜解離によってDUの動物モデルを確立するこの方法は、DUの病態生理学をよりよく理解する絶好の機会を提供します。

円錐髄膜切開の全手順は、経験豊富な外科医によって45分以内に完了することができます。当研究室では、100例以上の円錐髄膜切開手術を行ってきました。成功率は、ラットの生存率とDUの誘導の成功によって定義されるように、95%以上です。尿流動態検査によりDUの誘導が確認された。
我々の経験に基づいて、DUの誘導は、残留尿量によって事前に評価することができま...

Watch this Scientific Journal Video about Detrusor Underactivity Model in Rats by Conus Medullaris Transection at JoVE.com

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

ラットにおける円錐髄膜剥離による排尿筋活動低下モデルを確立する方法を提示する。排尿筋活動低下は、これらの動物において首尾よく刺激された。このモデルは、尿路機能の研究に使用できます。

円錐髄質断面によるラットの排尿筋活動不足モデル

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was ...

detrusor-underactivity-model-in-rats-by-conus-medullaris-transection

Research

JoVE Journal

Neuroscience

2.0K ビュー.  Capital Medical University. ラットにおける円錐髄膜剥離による排尿筋活動低下モデルを確立する方法を提示する。排尿筋活動低下は、これらの動物において首尾よく刺激された。このモデルは、尿路機能の研究に使用できます。

ビデオ: Detrusor Underactivity Model in Rats by Conus Medullaris Transection

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

視神経切断：中枢神経系における成人ニューロンのアポトーシスのモデル

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

神経科学

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

哺乳類の中枢神経系における視神経切断後の実験操作のための方法

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

ラットのショ糖の渇望のインキュベーションを評価するための一般的な方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

局所トランスジェニックラットの発生により<em&gt;子宮内</em&gt;エレクトロポレーションおよび<em&gt;インビボ</em&gt;生物発光スクリーニング

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

モーター神経切断とライブゼブラフィッシュにおけるグリア細胞の挙動のタイムラプスイメージング

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

幼虫のゼブラフィッシュにおける脊髄切断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

神経回路基盤記憶形成を解剖するための新生児ラットの左右で非対称匂い学習モデル

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

ラットの視神経の部分断裂: 網膜神経節細胞の二次変性を評価する新しい手術アプローチとの確立モデル

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, and neuroinflammation is often observed as a co-occurrence. Here, we describe a sub-acute CMHs rat model induced by lipopolysaccharide (LPS) injection, as well as a method for detecting CMHs. Systemic LPS injection is relatively simple, economical, and cost-effective. One major advantage of LPS injection is its stability to induce inflammation. CMHs caused by LPS injection could be detected by gross observation, hematoxylin and eosin (HE) staining, Perl&#39;s Prussian staining, Evans blue (EB) double-labeling, and magnetic resonance imaging-susceptibility weighted imaging (MRI-SWI) technology. Finally, other methods of developing CMHs animal models, including their advantages and/or disadvantages, are also discussed in this report.

We present a protocol to induce and detect CMHs caused by LPS injection in Sprague-Dawley rats, which may be utilized in future research investigations on the pathogenesis of CMHs.

ラットにおけるリポ多糖投与による亜急性の脳 Microhemorrhages

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, ...