Name: a rat model of ecohiv brain infection
Uploaded: 2021-01-21T14:30:00
Description: Watch this Scientific Journal Video about A Rat Model of EcoHIV Brain Infection at JoVE.com

Este trabajo fue financiado por las subvenciones HD043680, MH106392, DA013137 y NS100624 de los NIH.

Todos los protocolos de animales fueron revisados y aprobados por el Comité de Cuidado y Uso de Animales de la Universidad de Carolina del Sur (número de garantía federal: D16-00028). La rata masculina adulta de seises F344/N era par alojada en un ambiente controlado bajo luz 12/12: ciclo oscuro con el acceso del ad libitum al alimento y al agua. Todos los animales fueron atendidos utilizando las directrices establecidas por los Institutos Nacionales de Salud en la Guía para el Cuidado y Uso de Animales de Laboratori...

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En este protocolo, establecimos un modelo de infección por VIH inducida por EcoHIV en ratas. Específicamente, describimos una inyección stereotaxic bilateral de EcoHIV en la corteza que indujo con éxito la infección vih activa en el cerebro de la rata 7 días de poste-inyección. Además, demostramos que la infección por EcoHIV en ratas podría ser un buen sistema biológico para estudiar aspectos clave de HAND. Ocho semanas de infección post-EcoHIV, ratas exhibieron deterioros neurocognitivos significativos, que ...

Los sistemas biológicos han mejorado nuestra comprensión de los trastornos neurocognitivos asociados al VIH-1 (HAND) y sus mecanismos neuronales subyacentes2. La determinación del sistema biológico más apropiado para un estudio determinado depende a menudo de la cuestión de interés2. La limitación de la gama de modelos animales huésped desafía los estudios de desarrollo de la enfermedad VIH-1. Para investigar la replicación viral y la patogénesis del VIH-1, Potash et al.3 crearon un modelo de ratón de infección activa por VIH-1, reemplazando la región codificante de la glicoproteína de la envoltura superficial del VIH, gp120, con mlv ecotrópica gp80, lo que llevó a la replicación viral exitosa en ratones4. Después de las inyecciones de venas de cola en ratones quiméricos de VIH (EcoHIV), se observaron muchas características que se asemejaban a las de los individuos seropositivos del VIH-1 (por ejemplo, linfocitos y macrófagos infectados, dirigidos a respuestas inmunitarias antivirales, e inflamación3,5,6).
Aunque los ratones y las ratas son miembros de muridos, las diferencias fundamentales de las especies pueden influir en su idoneidad para cuestiones experimentales específicas7. Por lo tanto, la extensión del modelo de infección por EcoHIV a ratas (comúnmente utilizado en estudios de abuso de drogas y trastornos neurocognitivos) sería ventajosa en el estudio de neuroHIV. Por ejemplo, su mayor tamaño hace que la implantación de catéteres yugulares para procedimientos de autoadministración de fármacos sea más práctica8. Las técnicas de autoadministración de fármacos en ratas se han utilizado para evaluar la motivación en el VIH-19. Además, muchas tareas neurocognitivas/conductuales fueron diseñadas inicialmente para ratas10. Aquí, divulgamos la utilización de inyecciones stereotaxic de EcoHIV en ratas para extender el modelo de la infección de EcoHIV y para permitir una oportunidad dominante de dirigir las preguntas nuevas relacionadas con neuroHIV y la MANO.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>293FT cells</td>
			<td>ThermoFisher Scientific</td>
			<td>R70007</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic solution</td>
			<td>Cellgro</td>
			<td>30004CI</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Corning BioCoatGelatin 75cm&#178; Rectangular Canted Neck Cell Culture Flask with Vented Cap</td>
			<td>Life Technologies</td>
			<td>354488</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning DMEM with L-Glutamine, 4.5 g/L Glucose and Sodium Pyruvate</td>
			<td>Life Technologies</td>
			<td>10013CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>637-137</td>
			<td></td>
		</tr>
		<tr>
			<td>drill</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14095</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #7 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14097</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Snap-Cap Microcentrifuge Biopur Safe-Lock Tubes</td>
			<td>Life Technologies</td>
			<td>22600028</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethicon Vicryl Plus Antibacterial, 4-0 Polyglactin 910 Suture, 27in. FS-2</td>
			<td>Med Vet International</td>
			<td>VCP422H</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>1701</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Lipofectamine 3000 Transfection Reagent</td>
			<td>Life Technologies</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Forceps</td>
			<td>World Precision Instruments</td>
			<td>15914</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>World Precision Instruments</td>
			<td>500216</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentivirus-Associated p24 ELISA Kit</td>
			<td>Cell Biolabs, inc.</td>
			<td>VPK-107-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Lenti-X Concentrator</td>
			<td>Takara</td>
			<td>PT4421-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold</td>
			<td>Fisher Scientific</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Merritt Veterinary Supply</td>
			<td>347075</td>
			<td></td>
		</tr>
		<tr>
			<td>stereotaxic apparatus</td>
			<td>Kopf Instruments</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-154%</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors</td>
			<td>World Precision Instruments</td>
			<td>500086</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rat model of ecohiv brain infection

Se ha estudiado bien que el modelo de ratón infectado ecoHIV es de gran utilidad en la investigación de las complicaciones neurológicas asociadas al VIH. El establecimiento del modelo de rata infectada EcoHIV para estudios de abuso de drogas y trastornos neurocognitivos, sería beneficioso en el estudio de neuroHIV y trastornos neurocognitivos asociados al VIH-1 (HAND). En el actual estudio, demostramos la creación acertada de un modelo de la rata de la infección vih activa usando el VIH quimérico (EcoHIV). En primer lugar, la construcción lentiviral de EcoHIV se envasaba en células cultivadas de 293 FT durante 48 horas. Entonces, el medio condicional fue concentrado y ºteado. Después, realizamos inyecciones stereotaxic bilaterales del EcoHIV-EGFP en el tejido cerebral de la rata de F344/N. Una semana después de la infección, se detectaron señales de fluorescencia de EGFP en el tejido cerebral infectado, lo que indica que EcoHIV induce con éxito una infección activa por VIH en ratas. Además, immunostaining para el marcador microglial de la célula, Iba1, fue realizado. Los resultados indicaron que la microglía era el tipo de célula predominante que albergaba EcoHIV. Además, las ratas de EcoHIV exhibieron alteraciones en el proceso temporal, un mecanismo neurobehavioral subyacente potencial de la MANO así como la disfunción sináptica ocho semanas después de la infección. En conjunto, el presente estudio extiende el modelo EcoHIV de la infección por VIH-1 a la rata que ofrece un valioso sistema biológico para estudiar los reservorios virales del VIH-1 en el cerebro, así como la MANO y las comorbilidades asociadas, como el abuso de drogas.

El medio condicionado fue recogido del lentivirus de las células infectadas EcoHIV-EGFP 293FT. Después, fue concentrado y ºdado, entonces stereotaxically inyectado en el cerebro (región cortical) de las ratas de F344/N. Siete días después de la inyección, las ratas fueron sacrificadas y las imágenes fueron tomadas de las rebanadas coronales del cerebro que se extendían de bregma 5.64 milímetros a bregma -4.68 milímetros. En la Figura 1A,hay señales significativas de EcoHIV-EGFP e...

Watch this Scientific Journal Video about A Rat Model of EcoHIV Brain Infection at JoVE.com

A Rat Model of EcoHIV Brain Infection

Aquí, presentamos un protocolo para establecer un nuevo modelo de rata de infección activa por VIH usando VIH quimérico (EcoHIV), que es fundamental para mejorar nuestra comprensión de los reservorios virales del VIH-1 en el cerebro y ofrecer un sistema para estudiar los trastornos neurocognitivos asociados al VIH y las comorbilidades asociadas (es decir, el abuso de drogas).

Un modelo de rata de la infección cerebral EcoHIV

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Usando ratas, este protocolo se puede utilizar para establecer un nuevo modelo de la infección vih activa usando el VIH quimérico. con ratas, el establecimiento del modelo de infección por EcoHIV para estudios de abuso de drogas y trastornos neurocognitivos podría ser beneficioso para el estudio del neuro VIH y los trastornos neurocognitivos asociados al VIH-1. La demostración del procedimiento será Hailong Li, un investigador asociado de mi laboratorio.Para el envasado del virus en 293 células T, sembra de cinco veces 10 a la quinta 293 células T por mililitro de DMEM suplementado con 10% de FBS en cada uno de los dos frascos de 75 centímetros cuadrados recubiertos de gelatina e incubar las células a 37 grados centígrados hasta que los cultivos alcancen el 50% de confluencia. El día de la transfección, diluir 22,5 microlitros de reactivo de transfección en 750 microlitros de medio por matraz en un tubo de microcentrífuga de 1,5 mililitros y vórtice la solución durante tres segundos. Diluir 20 microgramos del ADN quimérico del plásmido EcoHIV en 750 microlitros de medio en un segundo tubo de microcentrífuga de 1,5 mililitros por matraz con mezcla exhaustiva.Combine el ADN diluido con el reactivo de transfección diluido con una mezcla suave e incube la solución resultante durante 15 minutos a temperatura ambiente. Al final de la incubación, añadir cada suspensión de virus a 10 mililitros de medio DMEM de preguerra y añadir cada solución complementada por virus a un matraz de 293 células T. Después de dos días de cultivo celular, acoge el sobrenadante de los matraces en un solo tubo cónico de 50 mililitros para la centrifugación y transfiera el sobrenadante clarificado a un nuevo tubo de 50 mililitros.Agregue ocho mililitros de Lenti-X Concentrator al sobrenadante clarificado y mezcle con una inversión suave. Después de una incubación de dos días a cuatro grados Centígrados, centrífuga la mezcla y retire cuidadosamente el sobrenadante, luego resuspiente suavemente el pellet con 100 microlitros de PBS de 100 milimolares y use un kit P24 ELISA para diezmar la concentración del virus. Para la inyección ecoHIV-EGFP, después de confirmar una falta de respuesta al reflejo del pedal, afeite el cabello de la región cerebral y esterilice la piel expuesta dos veces con etanol al 70% y un exfoliante a base de clorhexidina.Asegure la rata en una posición propensa en un aparato esteretáxico y haga una incisión de cinco a seis centímetros a través de la piel a lo largo de la línea media del cuero cabelludo. Marque una posición de perforación a 0,8 milímetros de lateral y una rostral de 1,2 milímetros a bregma y perfore un agujero de 0,4 milímetros de diámetro en cada posición del cráneo. Cargue de 1,04 veces de 10 a las sextas unidades de transducción por mililitro de solución de Lentivirus EcoHIV titulada en una jeringa de inyección de 10 microlitros y asegure la jeringa al aparato estereotáxico.Mueva la aguja cerca de la superficie de un orificio de perforación e inserte la aguja 2,5 mililitros en el orificio. Infunda un microlitro de solución de virus a una velocidad de 0,2 microlitros de virus por minuto. Cuando se haya inyectado todo el virus, deje la aguja dentro del área de inyección durante cinco minutos antes de retraer lentamente la aguja hasta que esté fuera del cráneo de la rata.Cierre la incisión de la piel con una sutura de hilo de seda 4-0 y esterilice la solución con etanol al 70%, luego coloque a la rata en una cámara de recuperación con una almohadilla térmica con monitoreo hasta que recoste. De una a ocho semanas después de la infusión viral, después de confirmar una falta de respuesta al reflejo del pedal, fije la rata en una posición supina dentro de una campana extractora e incise la piel a lo largo de la línea media torácica. Corte el diafragma para abrir la cavidad torácica e inserte una aguja calibre 20 por 25 milímetros en el ventrículo izquierdo.Abra inmediatamente la aurícula derecha con tijeras y perfunda 50 mililitros de PBS preenfriado de 100 milimolares a una velocidad de flujo de cinco mililitros por minuto. Cuando se haya entregado todo el PBS, perfundir con 100 mililitros de paraformadehído frío al 4%. Cuando se haya entregado todo el fijador, extraiga el cerebro del cráneo y fije el tejido durante la noche en paraformacionaldehído fresco al 4%.A la mañana siguiente, coloque la muestra en 40 mililitros de sacarosa al 30% en PBS de 100 milimolares en un tubo de 50 mililitros durante aproximadamente tres días hasta que el cerebro se asiente en la parte inferior del tubo antes de congelar el tejido cerebral en butanol de metilo durante dos minutos a menos 80 grados Celsius. Después de congelar, use un criostato de menos 20 grados Celsius y un pincel fino para adquirir secciones coronales de 50 micras de espesor. Una vez obtenidas todas las secciones, cubra las muestras con 300 microlitros de medio antidesvanecimiento y montelas con 22 cubrebocas de 50 mililitros.Cuando el medio se ha secado, imagen de las secciones por microscopía confocal. Siete días después de la inyección de Lentivirus, las imágenes de rebanadas cerebrales coronales de rata revelan una presencia significativa de señales ecoHIV-EGFP en todo el tejido cerebral, especialmente dentro de la corteza y la circunvolución dentada del hipocampo. El etiquetado dual con las sondas Iba1 y EcoHIV-EGFP proporciona una fuerte evidencia de que la microglía es el tipo de célula predominante que alberga la expresión de EcoHIV en el cerebro.Ocho semanas después de la infección, EcoHIV inyectó animales exhiben una insensibilidad relativa a la manipulación del intervalo interestímulo como lo demuestra una función de intervalo relativamente más plana en comparación con los controles salinos. Además, las ratas inyectadas EcoHIV muestran alteraciones profundas en su morfología dendrítica de la espina dorsal según lo evidenciado por una frecuencia relativa creciente de espinas dendríticas más cortas con diámetros crecientes de la cabeza y del cuello en relación con animales de control. La concentración y titulación del medio de condición antes de la inyección estereotáxica es fundamental para garantizar los resultados consistentes y replicables en todos los experimentos.Esta inyección estereotáxica regional específica de EcoHIV proporciona una infección eficiente por el VIH dentro del cerebro y produce déficits de procesamiento temporal en ratas. La utilización de inyecciones estereotáxicas contra el VIH en ratas para extender el modelo de infección por EcoHIV ofrece una oportunidad clave para abordar nuevas preguntas relacionadas con el neuro VIH en cuestión.

Research

JoVE Journal

Neuroscience

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Gen de entrega de cerebro de rata postnatal por la inyección no ventricular plásmido y electroporación

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Investigación

Neurociencias

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

La cirugía estereotáxica para la lesión excitotóxica de áreas específicas del cerebro en la rata adulta

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Un modelo de rata de hipoperfusión leve intrauterina con microcoil de transcatheter estenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Una evaluación In Vivo de la interrupción de la Barrera Blood - Brain en un modelo de rata de ictus isquémico

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

Modelo de una rata de la fatiga Central mediante la método de plataforma múltiple

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Un enfoque alternativo para estudiar acontecimientos primarios en neurodegeneración con rodajas de cerebro de rata Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

Un enfoque de sobremesa para la ubicación específica de la barrera hematoencefálica que se abre con ultrasonido enfocado en un modelo de rata

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Un método de limpieza de tejido hidrófobo para el tejido cerebral de rata

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor.&#160;It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10&#8211;15 years of treatment. Understanding the mechanisms of this malady and developing both novel and effective anti-dyskinesia treatments requires consistent and accurate modeling for pre-clinical testing of therapeutic interventions. A detailed method for reliable induction and comprehensive rating of LIDs following 6-OHDA-induced nigral lesioning in a rat model of PD is presented here. Dependable LID assessment in rats provides a powerful tool that can be readily utilized across laboratories to test emerging therapies focused on reducing or eliminating this common treatment-induced burden for individuals with PD.

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson&#039;s Disease

This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson&#39;s disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

Inducción y evaluación de discinesias inducidas por levodopa en un modelo de rata de la enfermedad de Parkinson

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements ...

Validación de la respuesta a las lesiones y las evaluaciones conductuales en un modelo de rata mTBI

Rat Model of Closed-Head Mild Traumatic Injury and its Validation

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful insights, developing a stable and reproducible animal model is essential. In this study, we report a detailed description of a closed-head mTBI model and a representative validation method using Sprague-Dawley rats to verify the modeling effect. The model involves dropping a 550 g mass weight from a height of 100 cm directly onto the head of a rat on a destructible surface, followed by a 180-degree turn. To assess the injury, rats underwent a series of neurobehavioral assessments 10 min post-injury, including time of loss of consciousness, first seeking-behavior time, escape ability, and beam balance ability test. During the acute and subacute stages following the injury, behavioral tests were conducted to assess motor coordination ability (Beam task), anxiety (Open Field test), and learning and memory abilities (Morris Water Maze test). The closed-head mTBI model produced a consistent injury response with minimal mortality and replicated real-life situations. The validation method effectively verified the model development and ensured the stability and consistency of the model.

Autor destacado: Avance en la investigación de lesiones cerebrales traumáticas: un modelo de cabeza cerrada para una replicación precisa y una evaluación rápida 

Here, we present a closed-head mild traumatic brain injury (mTBI) rat model and its validation exhibiting remarkable similarity to human mTBI concerning behavioral manifestations during the acute and subacute stages.

Modelo de Rata de Lesión Traumática Leve de Cabeza Cerrada y su Validación

Animal models are crucial for advancing our understanding of mild traumatic brain injury (mTBI) and guiding clinical research. To achieve meaningful ...