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

Agradecemos a la Dra. Myka Estes por su persistencia en abordar la variabilidad en el modelo MIA de ratón y a todos los colaboradores en Estes et al.8 por su trabajo que condujo al desarrollo del protocolo de métodos descrito aquí. La investigación reportada aquí fue apoyada por NIMH 2P50 MH106438-06 (A.K.M.) y NIMH T32MH112507 (K.P.).

Todos los protocolos se realizan bajo la aprobación del Comité Institucional de Cuidado y Uso de Animales de la Universidad de California-Davis (IACUC).
1. Preparación de animales
<ol><li>Al adquirir animales, mantenga los siguientes parámetros consistentes para garantizar la máxima reproducibilidad.<ol><li>Ubicación del proveedor y del proveedor: como se informó anteriormente, los ratones salvajes tipo C57BL / 6J exhiben diferentes respuestas a la misma dosis de poli...

La infección materna altera el curso del desarrollo cerebral en humanos y en roedores y primates no humanos 4,5,7. Aquí, se describe un procedimiento para inducir MIA en ratones en un punto de tiempo gestacional medio usando poli (I: C). Este método incorpora la evaluación del BIR antes del embarazo, lo que aumenta la reproducibilidad y ofrece la oportunidad de investigar mecánicamente los mecanismos que conducen a la resil...

La evidencia epidemiológica vincula la infección materna con un mayor riesgo de NDD psiquiátrica, incluida la esquizofrenia (SZ) y el trastorno del espectro autista (TEA)1,2,3,4,5,6,7. El modelo de ratón MIA fue desarrollado para probar la causalidad y el papel mecanicista de MIA en la etiología de estos trastornos, así como para identificar biomarcadores moleculares y desarrollar herramientas diagnósticas y terapéuticas 4,6. A pesar de la utilidad de este modelo y su creciente popularidad, existe una variabilidad considerable en los protocolos de inducción de MIA dentro del campo, lo que dificulta la comparación de los resultados entre los estudios y la replicación de los hallazgos 8,9. Además, la mayoría de las iteraciones del modelo no investigan dos aspectos traslacionales importantes de la MIA: (i) muchos descendientes son resistentes a la MIA, y (ii) los descendientes susceptibles pueden exhibir distintas combinaciones de fenotipos8.
Para generar un modelo de MIA reproducible, los investigadores deben informar al menos una medida cuantitativa de la magnitud de MIA inducida en presas. Para inducir MIA durante la gestación, nuestro laboratorio realiza inyecciones intraperitoneales (i.p.) del poliinositítico imitador viral de ARN bicatenario: ácido policitidílico [poli(I:C)]. Poly(I:C) induce una cascada inmune similar a los virus de la influenza, ya que es reconocido por el receptor tipo toll 3 (TLR3)10. Como resultado, poli(I:C) activa la respuesta de fase aguda que resulta en una rápida elevación de citoquinas proinflamatorias 8,11,12. Estudios previos han demostrado que la elevación de citoquinas proinflamatorias, incluyendo la interleucina-6 (IL-6), es necesaria para producir anomalías conductuales y neuropatología en la descendencia como resultado de MIA11,12,13. Por lo tanto, el nivel de IL-6 en el suero materno recolectado durante su pico a las 2,5 h después de la inyección de poli(I: C) es una medida cuantitativa convincente de MIA que se puede utilizar para comparar los resultados entre laboratorios dentro del campo.
Con el fin de generar un modelo MIA que aborde los elementos traslacionalmente esenciales de resiliencia y susceptibilidad con un solo protocolo de inducción 8,14, los investigadores pueden combinar enfoques típicos de inducción con la caracterización de la inmunorreactividad basal (BIR) de la madre antes del embarazo8. Recientemente, se descubrió que los ratones hembra vírgenes C57BL / 6 muestran una amplia gama de respuestas IL-6 a una exposición a dosis bajas a poli(I: C) antes del embarazo8. Es sólo un subconjunto de estas hembras que llegan a producir descendencia susceptible, y sólo a ciertas magnitudes de activación inmune según lo dictado por la combinación de BIR y poli(I:C) dosis8. MIA induce fenotipos en un patrón de U invertida; La descendencia muestra las mayores aberraciones conductuales y moleculares cuando las madres son moderadamente inmunorreactivas, y la magnitud de la inflamación materna alcanza, pero no supera, un rango crítico8. Aquí, se proporciona un método detallado de cómo crear de manera confiable descendientes resistentes y susceptibles con fenotipos conductuales divergentes como resultado de la inyección gestacional media de poli (I: C).

<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

La activación inmune materna (MIA) durante el embarazo está constantemente relacionada con un mayor riesgo de trastornos del desarrollo neurológico y neuropsiquiátricos en la descendencia. Los modelos animales de MIA se utilizan para probar la causalidad, investigar mecanismos y desarrollar diagnósticos y tratamientos para estos trastornos. A pesar de su uso generalizado, muchos modelos MIA sufren de una falta de reproducibilidad y casi todos ignoran dos aspectos importantes de este factor de riesgo: (i) muchos descendientes son resistentes a MIA, y (ii) los descendientes susceptibles pueden exhibir distintas combinaciones de fenotipos. Para aumentar la reproducibilidad y modelar tanto la susceptibilidad como la resistencia a la MIA, la inmunorreactividad basal (BIR) de ratones hembra antes del embarazo se utiliza para predecir qué embarazos darán lugar a una descendencia resistente o descendencia con anomalías moleculares y de comportamiento definidas después de la exposición a la MIA. Aquí, se proporciona un método detallado para inducir MIA a través de la inyección intraperitoneal (i.p.) del poli (I: C) imitador viral de ARN bicatenario (dsRNA) a los 12,5 días de gestación. Este método induce una respuesta inflamatoria aguda en la madre, lo que resulta en perturbaciones en el desarrollo del cerebro en ratones que se asignan a dominios impactados de manera similar en los trastornos psiquiátricos y del neurodesarrollo humano (NDD).

No todos los animales expuestos a 30 mg/kg de poli(I:C) en E12.5 producen descendencia con anomalías de comportamiento consistentes 8,31. Aunque tanto 30 mg/kg como 40 mg/kg de poli(I:C) producen de manera confiable comportamientos de enfermedad en las presas, incluyendo disminución de los niveles de actividad, respuestas hipotérmicas y pérdida de peso, y también causan elevaciones significativas en IL-6, solo un subconjunto de camadas expuestas a MIA desarr...

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

La infección materna es un factor de riesgo para los trastornos del desarrollo neurológico. Los modelos de ratón de activación inmune materna (MIA) pueden dilucidar el impacto de la infección en el desarrollo y la función del cerebro. Aquí, se proporcionan pautas generales y un procedimiento para producir crías confiablemente resistentes y susceptibles expuestas a MIA.

Generación de un modelo reproducible de activación inmune materna a mediados de la gestación utilizando Poly(I: C) para estudiar la susceptibilidad y la resiliencia en la descendencia

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.

Utilizando MazeSuite y funcional espectroscopia cercana al infrarrojo para estudiar el aprendizaje de la navegación espacial

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

Investigación

Neurociencias

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.

Ratón reproducible aplastamiento del nervio ciático y posterior evaluación de la regeneración mediante el análisis de músculo entero Monte

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.

El sistema olfativo como un modelo para estudiar los patrones de crecimiento axonal y Morfología<em&gt; En Vivo</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.

Activación de la célula de Müller glía en un modelo de regeneración en el pez cebra y degeneración retiniana inducida por láser

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.

Una prueba objetiva y Reproducible de aprendizaje olfativo y discriminación en ratones

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.

Un método Simple y Reproducible para preparar muestras de membrana de microvasos de cerebro recién aislado de rata

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.

Un ensayo desarrolló para evaluar la activación de células neuronales endógenos en un modelo murino de lesión medular mínima

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.

Cirugía de nervios faciales en el modelo de rata para estudiar la inhibición y regeneración axonal

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.

Combinación de comportamiento y EEG para estudiar los efectos de la meditación de atención plena en la memoria episódica

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.