performing laser-induced brain injury in a rat model

Performing Laser-Induced Brain Injury in a Rat Model

To perform a laser-induced brain injury, first, assign twenty 300- to 350-gram Sprague Dawley rats into the laser group. After confirming a lack of response to withdrawal reflex, place one rat on a rectal temperature-regulated heating pad, and use a shaver to remove enough hair from the injury site, with an approximately two-centimeter hair-free margin around the incision. Disinfect the exposed skin with 70% ethanol, and place the rat in the prone position in a stereotaxic head holder.
Make a three-centimeter incision to allow lateral reflection of the scalp to expose the area between bregma and lambda. Holding an Nd:YAG laser with peak wavelength of 1,064 nanometers at a two-millimeter distance from the skull, administer 50 joules times 10 points, with a one-second pulse duration, to the exposed area of the skull above the right hemisphere. After the laser injury has been delivered, remove the rat from the device, and close the scalp with 3-0 silk surgical sutures. Then, place the rat in its cage with monitoring until full recumbency.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Laser-induced brain injury experimental procedure
<ol>
	<li>Anesthetize the rat with a mixture of 2% isoflurane in oxygen, allowing for spontaneous ventilation. Check for sufficient anesthetic depth by pinching the tail with forceps to see if the withdrawal reflex is absent.</li>
	<li>Using a rectal temperature-regulated heating pad, maintain the rat&#39;s core body temperature at 37 &#176;C throughout the experimental procedure.</li>
	<li>Remove local hair with a shaver and disinfect it with 70% alcohol and 0.5% chlorhexidine gluconate. Repeat the disinfection step two more times. 
	NOTE: The surgical incision should be approximately 3 cm wide. Remove hair at least 2 cm around the incision area.</li>
	<li>Place the rat on a stereotaxic head holder in a prone position. Make a 3 cm incision to reflect the scalp laterally and expose the area between Bregma and Lambda.</li>
	<li>Maintain anesthesia through the nose cone.</li>
	<li>Use Neodymium-YAG (Nd-YAG) laser (peak wavelength 1064 nm) to administer 50J X 10 points, with 1 s pulse duration, to the exposed area of the skull above the right hemisphere.</li>
	<li>Ensure that the laser-generating part of the apparatus is 2 mm away from the exposed area to produce a laser beam. 50J X 10 points were selected after careful evaluation of different energy/surface combinations. This combination is efficient and does not cause bone destruction of the skull after administration for less than a second. 
	NOTE: 2 mm is the distance between the terminal of the laser beam (from the optical cable it is passed through) and the skull bone. In case a focusing lens is used, the distance should be calculated, taking into account the angle of inclination of the lens to focus the beam in the desired area of damage. Ensure proper safety when using a laser device, including appropriate training and eye protection.</li>
	<li>Remove the rat from the device and close the scalp with 3-0 silk surgical sutures.</li>
	<li>Discontinue anesthesia and return the rat to its cage for recovery. Administer 0.1 mL of 0.25% bupivacaine locally to reduce the postoperative pain immediately after surgery. 
	NOTE: The entire procedure should last less than 5 min if performed correctly.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Brain &#38; Tissue Matrices</td>
			<td>SIGMA - ALDRICH</td>
			<td>15013</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Weighing Scale</td>
			<td>SIGMA - ALDRICH</td>
			<td>RS 4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96 %</td>
			<td>ROMICAL</td>
			<td></td>
			<td>Flammable Liquid</td>
		</tr>
		<tr>
			<td>Heater with thermometer</td>
			<td>Heatingpad-1</td>
			<td>Model: HEATINGPAD-1/2</td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion Cuff</td>
			<td>ABN</td>
			<td>IC-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluran, USP 100%</td>
			<td>Piramamal Critical Care, Inc</td>
			<td>NDC 66794-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades 11</td>
			<td>SIGMA - ALDRICH</td>
			<td>S2771</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharplan 3000 Nd:YAG (neodymium-doped yttrium aluminum garnet) laser machine</td>
			<td>Laser Industries Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic head holder</td>
			<td>KOPF</td>
			<td>900LS</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Kuts, R., et al. <a href="https://app.jove.com/v/60928">Laser-Induced Brain Injury in the Motor Cortex of Rats.</a> J. Vis. Exp. (2020)
This video demonstrates a procedure to generate precise, controlled brain injury in the rat motor cortex using a laser.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take an anesthetized rat placed on a rectal temperature-regulated heating pad to maintain its body temperature.
Remove the fur and disinfect the surgical site.
Next, secure the rat in a prone position on a stereotaxic unit.
Make a midline surgical incision. Then, expose the area between the skull reference points.
Position the laser appropriately above the right hemisphere to target the motor cortex.
Administer longer wavelength, high-energy light pulses to the exposed area of the skull, penetrating deeply into the motor cortex.
The tissue absorbs these pulses, causing localized heating and cellular damage, inducing brain injury.
After laser exposure, remove the rat from the stereotaxic unit.
Suture the surgical incision and return the rat to its cage.
Allow it to recover. The laser-induced brain injury rat model can be used for neurological analysis.

10 Görüntüleme. Bir Sıçan Modelinde Lazer Kaynaklı Beyin Hasarı Gerçekleştirme

Video: Bir Sıçan Modelinde Lazer Kaynaklı Beyin Hasarı Gerçekleştirme - Deney

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Insan kaynaklı pluripotent kök hücre türetilen nöral hücrelerin terapötik transplantasyonu ile fare beyin yaralanması kontrollü Kortiik darbe modeli

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

JoVE Journal

Gelişim Biyolojisi

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

Stereotaksik Atlas güdümlü lazer yakalama mikrodiseksiyon travma yaralanma tarafından etkilenen beyin bölgeleri

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Nörobilim

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Akut miyokard infarktüsünde farelerde gerçek zamanlı basınç-hacim Analizi

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Performing Laser-Induced Brain Injury in a Rat Model

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Performing Laser-Induced Brain Injury in a Rat Model