Name: generating a reproducible model of mid-gestational maternal immune activation using poly(i:c) to study susceptibility and resilience in offspring
Uploaded: 2022-08-17T13:40:00
Description: Watch this Scientific Journal Video about Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using PolyI:C to Study Susceptibility and Resilience in Offspring at JoVE.com

マウスMIAモデルの変動性に対処することに固執してくれたMyka Estes博士と、ここで説明するメソッドプロトコルの開発につながったEstesら8 のすべての貢献者に感謝します。ここで報告された研究は、NIMH 2P50 MH106438-06(A.K.M.)およびNIMH T32MH112507(K.P.)によってサポートされました。

すべてのプロトコルは、カリフォルニア大学デービス校の施設動物管理および使用委員会(IACUC)の承認の下で実行されます。
1.動物の準備
<ol><li>動物を取得するときは、最大の再現性を確保するために、次のパラメーターを一貫してください。<ol><li>ベンダーとベンダーの場所:以前に報告されたように、野生型C57BL / 6Jマウスは、ベンダーに応じて同じ用量の...

母体感染は、ヒトおよびげっ歯類および非ヒト霊長類の両方の脳発達の過程を変える4,5,7。ここでは、ポリ(I:C)を用いて妊娠中期の時点でマウスにMIAを誘導する手順を概説する。この方法には、妊娠前のBIRの評価が組み込まれているため、再現性が向上し、MIA8に対する子孫の回復力と感受性につながるメ?...

疫学的証拠は、母親の感染を、統合失調症(SZ)および自閉症スペクトラム障害(ASD)を含む精神医学的およびNDDのリスク増加に関連付けています1,2,3,4,5,6,7。MIAマウスモデルは、これらの障害の病因におけるMIAの因果関係と機構的役割をテストし、分子バイオマーカーを特定し、診断ツールと治療ツールの両方を開発するために開発されました4,6。このモデルの有用性と人気の高まりにもかかわらず、フィールド内のMIA誘導プロトコルにはかなりのばらつきがあり、研究間で結果を比較し、調査結果を再現することは困難です8,9。さらに、モデルのほとんどの反復は、MIAの2つの重要な翻訳的側面を調査していません:(i)多くの子孫はMIAに対して回復力があり、(ii)感受性子孫は表現型の異なる組み合わせを示す可能性があります8。
再現可能なMIAモデルを生成するために、研究者はダムで誘発されたMIAの大きさの少なくとも1つの定量的測定値を報告する必要があります。妊娠中にMIAを誘導するために、私たちの研究室では、ポリイノシティックを模倣した二本鎖RNAウイルスであるポリシチジル酸[ポリ(I:C)]の腹腔内(i.p.)注射を行います。 Poly(I:C)は、toll様受容体3(TLR3)10によって認識されるため、インフルエンザウイルスと同様の免疫カスケードを誘導します。その結果、ポリ(I:C)は急性期応答を活性化し、炎症誘発性サイトカインの急速な上昇をもたらします8,11,12。以前の研究では、MIA11,12,13の結果として、インターロイキン-6(IL-6)を含む炎症誘発性サイトカインの上昇が子孫に行動異常および神経病理学を引き起こすために必要であることが実証されています。したがって、ポリ(I:C)注射後2.5時間のピーク時に収集された母体血清中のIL-6のレベルは、フィールド内の検査室間で結果を比較するために使用できるMIAの説得力のある定量的尺度です。
単一の誘導プロトコル8,14で回復力と感受性の翻訳的に不可欠な要素に対処するMIAモデルを生成するために、研究者は典型的な誘導アプローチと妊娠前のダムのベースライン免疫反応性(BIR)の特性評価を組み合わせることができます8。最近、処女の雌C57BL/6マウスが、妊娠前のポリ(I:C)への低線量曝露に対して広範囲のIL-6応答を示すことが発見されました8。感受性の高い子孫を産み続けるのはこれらの雌のサブセットのみであり、BIRとポリ(I:C)用量8の組み合わせによって決定される特定の大きさの免疫活性化でのみ発生します。MIAは逆Uパターンで表現型を誘導します。子孫は、ダムが中程度の免疫反応性であり、母親の炎症の大きさが臨界範囲8に達するが、それを超えない場合に、最大の行動的および分子的異常を示します。ここでは、ポリ(I:C)の妊娠中期注射の結果として、異なる行動表現型を持つ回復力のある子孫と感受性のある子孫の両方を確実に作成する方法の詳細な方法が提供されています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% NaCl physiological endotoxin free saline</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td>Control and vehicle for Poly(I:C)</td>
		</tr>
		<tr>
			<td>35mm petri dish</td>
			<td>Thomas Scientific</td>
			<td>1219Z45</td>
			<td>Used to hold oil during tail bleed</td>
		</tr>
		<tr>
			<td>7.5% TGX gels</td>
			<td>Bio-rad</td>
			<td>4561084</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Ancare Nestlets</td>
			<td>Fisher Scientific</td>
			<td>NC9365966</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>anti-&#946;-tubulin</td>
			<td>Millipore</td>
			<td>MAB3408</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Bio-Plex Pro Mouse Cytokine Standards Group I</td>
			<td>Bio-rad</td>
			<td>171I50001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Plex Pro Reagent Kit with Flat Plate</td>
			<td>Bio-rad</td>
			<td>171304070M</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>ThermoFisher</td>
			<td>23209</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Covidien Monoject 1/2 mL Insulin Syringe with 28G x 1/2 in. Needle</td>
			<td>Spectrum</td>
			<td>552-58457-083</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D9779-10G</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Environmental enrichment</td>
			<td>Bio-serv</td>
			<td>K3327 and K3322</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Ethovision</td>
			<td>Noldus</td>
			<td>Ethovision</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Fluorsecent-tagged seondary ntibodies</td>
			<td>Li-cor</td>
			<td>925-32213 and 925-68072</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Food-grade edible oil (like olive, canola or grapeseed)</td>
			<td>Various vendors</td>
			<td></td>
			<td>Use to lubricate tail during tail bleeds</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>14060040</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>High molecular weight polyinositic:polycytidilic acid</td>
			<td>Invivogen</td>
			<td>#tlrl-pic-5</td>
			<td>Used to establish females&#39; BIR</td>
		</tr>
		<tr>
			<td>Humane Mouse Restrainer</td>
			<td>AIMS</td>
			<td>1000</td>
			<td>Used to restrain mouse during tail bleeds</td>
		</tr>
		<tr>
			<td>Image Studio Software</td>
			<td>Licor</td>
			<td>5.2</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Laemmli buffer</td>
			<td>Bio-rad</td>
			<td>1610737EDU</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Luminex200</td>
			<td>ThermoFisher</td>
			<td>APX10031</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvette CB300 300&#956;l Serum capillary tube</td>
			<td>Sarstedt</td>
			<td>16.440.100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixed molecular weight polyinositic:polycytidilic acid</td>
			<td>Sigma-Aldrich</td>
			<td>#P0913</td>
			<td>Gestational induction of MIA</td>
		</tr>
		<tr>
			<td>monoclonal anti-MEF2A</td>
			<td>AbCam</td>
			<td>ab76063</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>monoclonal anti-STAT3</td>
			<td>Cell signaling</td>
			<td>12640S</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Observer</td>
			<td>Noldus</td>
			<td>Observer</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Odyssey blocking buffer (TBS)</td>
			<td>Li-cor</td>
			<td>927-50003</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Odyssey CLx imaging system</td>
			<td>Li-cor</td>
			<td>9140</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Omnipure PBS</td>
			<td>Millipore</td>
			<td>65054L</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher</td>
			<td>23227</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>polyclonal anti_TH</td>
			<td>Pel-Freez</td>
			<td>P4101-150</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Bio-rad</td>
			<td>162-0177</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Qsonica Sonicator Q500</td>
			<td>Fisher Scientific</td>
			<td>15-338-282</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Quick blood stopper</td>
			<td>Petco</td>
			<td>17140</td>
			<td></td>
		</tr>
		<tr>
			<td>Seal-Rite 1.5 ml microcentrifuge tube, natural non-sterile</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Soldering stand</td>
			<td>Amazon</td>
			<td>B08Y12QC73</td>
			<td>Used to hold capillary tube during tail bleeds</td>
		</tr>
		<tr>
			<td>Sunflower seeds</td>
			<td>Bio-serv</td>
			<td>S5137-1</td>
			<td>Use to increase breeding efficiency</td>
		</tr>
		<tr>
			<td>The Bio-Plex Pro Mouse IL-6 set,</td>
			<td>Bio-rad</td>
			<td>171G5007M</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Bio-rad</td>
			<td>23209</td>
			<td>Optional</td>
		</tr>
	</tbody>
</table>

generating a reproducible model of mid-gestational maternal immune activation using poly(i:c) to study susceptibility and resilience in offspring

妊娠中の母親の免疫活性化(MIA)は、子孫の神経発達障害および神経精神障害のリスクの増加と一貫して関連しています。MIAの動物モデルは、因果関係をテストし、メカニズムを調査し、これらの障害の診断と治療法を開発するために使用されます。広く使用されているにもかかわらず、多くのMIAモデルは再現性の欠如に苦しんでおり、ほとんどすべてがこの危険因子の2つの重要な側面を無視しています:(i)多くの子孫はMIAに対して回復力があり、(ii)感受性の子孫は表現型の異なる組み合わせを示す可能性があります。再現性を高め、MIAに対する感受性と回復力の両方をモデル化するために、妊娠前の雌マウスのベースライン免疫反応性(BIR)を使用して、どの妊娠が回復力のある子孫またはMIAへの曝露後に定義された行動的および分子的異常を持つ子孫をもたらすかを予測します。ここでは、妊娠12.5日目に二本鎖RNA(dsRNA)ウイルス模倣ポリ(I:C)の腹腔内(i.p.)注射 を介して MIAを誘導する詳細な方法が提供される。この方法は、ダムで急性炎症反応を誘発し、その結果、マウスの脳発達に混乱が生じ、ヒトの精神障害および神経発達障害(NDD)の同様の影響を受けるドメインにマッピングされます。

E12.5で30 mg / kgのポリ(I:C)に曝露されたすべての動物が、一貫した行動異常を伴う子孫を産むわけではありません8,31。30 mg/kgと40 mg/kgのポリ(I:C)はどちらも、活動レベルの低下、低体温反応、体重減少など、ダムで確実に病気行動を引き起こし、IL-6の大幅な上昇を引き起こしますが、MIAに曝露された同腹仔のサブセットのみが、人間の精神医学的およ?...

Watch this Scientific Journal Video about Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using PolyI:C to Study Susceptibility and Resilience in Offspring at JoVE.com

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using PolyI:C to Study Susceptibility and Resilience in Offspring

母体感染は神経発達障害の危険因子です。母体免疫活性化(MIA)のマウスモデルは、感染が脳の発達と機能に与える影響を解明する可能性があります。ここでは、MIAにさらされた確実に回復力があり感受性のある子孫を産出するための一般的なガイドラインと手順が提供されています。

ポリ(I:C)を用いた妊娠中期の母体免疫活性化の再現モデルの生成による子孫の感受性と回復力の研究

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in ...

generating-reproducible-model-mid-gestational-maternal-immune

Research

JoVE Journal

Neuroscience

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using Poly(I:C) to Study Susceptibility and Resilience in Offspring

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

空間ナビゲーションで学習研究するために近赤外分光法をMazeSuiteおよび機能の使用

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

神経科学

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

ホールマウントマッスル分析による再現性のあるマウスの坐骨神経クラッシュと再生のその後の評価

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

モデルとしての嗅覚系は、軸索の成長パターンと形態を研究する<em&gt;インビボ</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

ゼブラフィッシュの再生モデルと網膜変性のレーザー誘起のミュラー グリア細胞の活性化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

嗅覚学習とマウスにおける差別の客観的かつ再現性のあるテスト

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

Here, a method for isolation of rat brain microvessels and for the preparation of membrane samples is described. This protocol has the clear advantage of producing enriched microvessel samples with acceptable protein yield from individual animals. Samples can then be used for robust protein analyses at the brain microvascular endothelium.

新鮮単離ラット脳微小循環から膜のサンプルを準備するシンプルで再現性のある方法

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

最小限の脊髄損傷のマウス モデルにおける内因性神経幹細胞の活性化を評価する Neurosphere アッセイ

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

軸索抑制と再生を研究するラットモデルにおける顔面神経外科

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

行動とEEGを組み合わせて、エピソード記憶に対するマインドフルネス瞑想の効果を研究する

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.