This work was funded by NIH grants HD043680, MH106392, DA013137, and NS100624.

All animal protocols were reviewed and approved by the Animal Care and Use Committee at the University of South Carolina (federal assurance number: D16-00028). Six adult male F344/N rat was pair housed in a controlled environment under a 12/12 light: dark cycle with ad libitum access to food and water. All animals were cared for using guidelines established by the National Institutes of Health in the Guide for the Care and Use of Laboratory Animals.
1. Virus packaging in 293 FT cells
<ol>
	<li>Culture the 293 FT cells (5&#215;105/mL) in gelatin coated 75 cm2 flasks with DMEM plus 10% FBS11. Grow cells at 37 &#176;C to be 50% confluent at transfection.</li>
	<li>Dilute 22.5 &#181;L of transfection reagent (e.g., Lipofectamine 3000) in 750 &#181;L of medium (e.g., Opti-MEM) in a 1.5 mL micro-centrifuge tube and vortex for 3 s.</li>
	<li>Dilute 20 &#181;g of EcoHIV plasmid DNA in 750 &#181;L of medium in a 1.5 mL micro-centrifuge tube and mix well.</li>
	<li>Add diluted DNA into the tube of diluted transfection reagent and mix gently. Incubate for 15 min at room temperature.</li>
	<li>Add the mixture to 10 mL of prewarmed DMEM medium in 75 cm2 flask. Incubate cells for 2 days at 37 &#176;C.</li>
	<li>Harvest and combine 24 mL of conditional medium from the two flasks that include the packaged lentivirus.</li>
	<li>Centrifuge all 24 mL of conditional medium at 500 x g for 10 minutes at 4 &#176;C. Transfer the clarified supernatant to a sterile 50 mL tube.</li>
	<li>Combine 8 mL of Lenti-x concentrator with 24 mL of clarified supernatant (1:3 ratio). Mix by gentle inversion. Incubate mixture at 4 &#176;C for two days.</li>
	<li>Centrifuge mixture at 1,500 x g for 45 minutes at 4 &#176;C. Carefully remove the supernatant.</li>
	<li>Gently re-suspend the pellet with 100 &#181;L of 100 mM PBS.</li>
	<li>Titer virus concentration with a p24 ELISA kit.</li>
</ol>
2. EcoHIV-EGFP virus stereotaxic surgeries
<ol>
	<li>Anesthetize rats using 3% sevoflurane. Proceed to step 2.2 when the rats are not responsive to noxious stimuli and reflexes are absent.</li>
	<li>Shave the hairs from the brain region and sterilize the skin twice with 70% ethanol and a chlorhexidine-based scrub. Secure the rat in a prone position in the stereotaxic apparatus.</li>
	<li>Make an incision (5-6 cm) through the skin along the scalp midline.</li>
	<li>Mark two drilling positions at 0.8 mm lateral, 1.2 mm rostral to bregma. Drill a hole (diameter 0.4 mm) in each skull position.</li>
	<li>Fill titered EcoHIV lentivirus solution (1.04 &#215; 106 TU/mL) in a 10 &#181;L injection syringe. Secure the syringe to the stereotaxic apparatus.</li>
	<li>Move down the needle close to the surface of drilling hole. Measure and move 2.5 mm in depth.</li>
	<li>Infuse 1 &#181;L of virus solution at a rate of 0.2 &#181;L/min. Keep the needle inside of the injection area for 5 min. Slowly move the needle up until it is outside of the rat skull.</li>
	<li>Suture the skin with a 4-0 silk thread.</li>
	<li>Sterilize the incision with 70% ethanol once. Subcutaneously inject butorphenol (Dorolex, 0.1 mg/kg body weight).</li>
	<li>Transfer the rat to a recovery chamber with a heating pad until it wakes up.</li>
</ol>
3. Visualization of brain sections
NOTE: Wait one to eight weeks after EcoHIV viral infusion.
<ol>
	<li>Anesthetize the rat with 5% sevoflurane. Continue to step 3.2 when the rats are not responsive to noxious stimuli and reflexes are absent.</li>
	<li>Fix the rat in a supine position inside a fume hood.</li>
	<li>Incise the skin along the thoracic midline. Cut the diaphragm and open the thoracic cavity.</li>
	<li>Insert a 20 G &#215; 25 mm needle into the left ventricle.</li>
	<li>Immediately open the right atrium with scissors.</li>
	<li>Perfuse 50 mL of prechilled 100 mM PBS at a rate of 5 mL/min.</li>
	<li>Perfuse 100 mL of cold 4% paraformaldehyde at a rate of 5 mL/min.</li>
	<li>Decapitate the rat, open the scalp and remove the brain.</li>
	<li>Postfix overnight with 4% paraformaldehyde.</li>
	<li>Transfer the brain to 40 mL of 30% sucrose in 100 mM PBS in 50 mL tube until the brain floats down to the bottom (about 3 days).</li>
	<li>Snap freeze the brain in methylbutanol for 2 min at - 80 &#176;C.</li>
	<li>Secure the brain tissue on a metal platform inside a -20 &#176;C cryostat.</li>
	<li>Cut 50 &#181;m thick coronal sections using the cryostat.</li>
	<li>Transfer the brain slices onto glass slides with a fine brush.</li>
	<li>Mount sections in 0.3 mL of antifade medium and cover with 22 mm 50 mm coverslips.</li>
	<li>Keep the glass slides in the dark at room temperature till dry.</li>
	<li>Image the targeted neurons with a confocal microscope using Z-stack based on brain region boundaries and morphological characteristics of neurons. 
	NOTE: The confocal microscope settings used were: magnification of 60 X (A/1.4, oil), and a Z-plane interval of 0.15 &#181;m (pinhole size 30 &#181;m; back-projected pinhole radius 167 nm) using a wavelength of 488 nm.</li>
</ol>

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None of the authors have conflicts of interest to declare.

In this protocol, we established an EcoHIV-induced HIV infection model in rats. Specifically, we described a bilateral stereotaxic injection of EcoHIV into the cortex which successfully induced active HIV infection in the rat brain 7 days post-injection. Futhermore, we demonstrate that EcoHIV infection in rats could be a good biological system to study key aspects of HAND. Eight weeks post- EcoHIV infection, rats exhibited significant neurocognitive impairments, which included the alterations in temporal processing and s...

Biological systems have enhanced our understanding of HIV-1 associated neurocognitive disorders (HAND) and their underlying neural mechanisms2. Determining which biological system is most appropriate for any given study is often dependent upon the question of interest2. The limitation of the range of host animal models challenges studies of HIV-1 disease development. To investigate HIV-1 viral replication and pathogenesis, Potash et al.3 created a mouse model of active HIV-1 infection, replacing the coding region of HIV surface envelope glycoprotein, gp120, with ecotropic MLV gp80, which led to successful viral replication in mice4. After tail vein injections in chimeric HIV (EcoHIV) mice, many characteristics were observed resembling those of HIV-1 seropositive individuals (e.g., infected lymphocytes and macrophages, targeted for antiviral immune responses, and inflammation3,5,6).
Although mice and rats are both members of the Muridae, fundamental species differences may influence their suitability for specific experimental questions7. Therefore, the extension of the EcoHIV infection model to rats (commonly used in studies of drug abuse and neurocognitive disorders) would be advantageous in the study of neuroHIV. For example, their larger size makes jugular catheter implantation for drug self-administration procedures more practical8. Drug self-administration techniques in rats have been utilized to evaluate motivation in HIV-19. Furthermore, many neurocognitive/behavioral tasks were initially designed for rats10. Here, we report the utilization of stereotaxic injections of EcoHIV in rats to extend the EcoHIV infection model and afford a key opportunity to address novel questions related to neuroHIV and HAND.

<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

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

The conditioned medium was collected from lentivirus of EcoHIV-EGFP infected 293FT cells. Next, it was concentrated and titered, then stereotaxically injected into the brain (cortical region) of F344/N rats. Seven days post-injection, rats were sacrificed and images were taken from coronal brain slices ranging from bregma 5.64 mm to bregma -4.68 mm. In Figure 1A, there are significant EcoHIV-EGFP signals throughout the brain, especially in the cortex and the hippocampal dentate gyrus. Furthe...

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

A Rat Model of EcoHIV Brain Infection

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

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

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Neuroscience

3.0K Views.  University of South Carolina. Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

Video: A Rat Model of EcoHIV Brain Infection

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.

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

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.

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.

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.

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).

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.

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.

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.

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.

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.

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

Validating Injury Response and Behavioral Assessments in an mTBI Rat Model

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.

Author Spotlight: Advancing Traumatic Brain Injury Research - A Closed-Head Model for Accurate Replication and Rapid Assessment 

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.

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