We would like to thank the Department of Anesthesiology of Soroka University Medical Center and the laboratory staff of Ben-Gurion University of the Negev for their help in the performance of this experiment.

The following procedure was conducted according to the Guidelines of the Use of Experimental Animals of the European Community. The experiments were also approved by the Animal Care Committee at the Ben-Gurion University of the Negev.
1. Animal selection and preparation
<ol>
	<li>Select 65 male Sprague-Dawley rats weighing 300 to 350 g with no overt pathology for this procedure. The smaller size poses technical difficulties for the MCAO procedure.</li>
	<li>Assign 3 rats per cage and let the...

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+health+estimates:+deaths+by+cause,+age,+sex+and+country,+2000-2012.">Global health estimates: deaths by cause, age, sex and country, 2000-2012.</a> World Health Organization. 9, (2014).</li><li>Meadows, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+focal+and+multifocal+cerebral+ischemia:+a+review.">Experimental models of focal and multifocal cerebral ischemia: a review.</a> Reviews in the Neurosciences. 29, 661-674 (2018).</li><li>Durukan, A., Strbian, D., Tatlisumak, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+ischemic+stroke:+a+useful+tool+for+stroke+drug+development.">Rodent models of ischemic stroke: a useful tool for stroke drug development.</a> Current Pharmaceutical Designs. 14, 359-370 (2008).</li><li>Fluri, F., Schuhmann, M. K., Kleinschnitz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ischemic+stroke+and+their+application+in+clinical+research.">Animal models of ischemic stroke and their application in clinical research.</a> Drug Design, Development and Therapy. 9, 3445-3454 (2015).</li><li>Li, F., Omae, T., Fisher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+hyperthermia+and+its+mechanism+in+the+intraluminal+suture+middle+cerebral+artery+occlusion+model+of+rats.">Spontaneous hyperthermia and its mechanism in the intraluminal suture middle cerebral artery occlusion model of rats.</a> Stroke. 30, 2464-2470 (1999).</li><li>Boyko, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+of+focal+ischemia+using+an+internal+carotid+artery+approach.">An experimental model of focal ischemia using an internal carotid artery approach.</a> Journal of Neuroscience Methods. 193, 246-253 (2010).</li><li>Zhao, Q., Memezawa, H., Smith, M. L., Siesjo, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperthermia+complicates+middle+cerebral+artery+occlusion+induced+by+an+intraluminal+filament.">Hyperthermia complicates middle cerebral artery occlusion induced by an intraluminal filament.</a> Brain Research. 649, 253-259 (1994).</li><li>Braeuninger, S., Kleinschnitz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+cerebral+ischemia:+procedural+pitfalls+and+translational+problems.">Rodent models of focal cerebral ischemia: procedural pitfalls and translational problems.</a> Experimental and Translational Stroke Medicine. 1, 8 (2009).</li><li>Choi, B. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobehavioural+deficits+correlate+with+the+cerebral+infarction+volume+of+stroke+animals:+a+comparative+study+on+ischaemia-reperfusion+and+photothrombosis+models.">Neurobehavioural deficits correlate with the cerebral infarction volume of stroke animals: a comparative study on ischaemia-reperfusion and photothrombosis models.</a> Environmental Toxicology and Pharmacology. 33, 60-69 (2012).</li><li>Boyko, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Alternative+Model+of+Laser-Induced+Stroke+in+the+Motor+Cortex+of+Rats.">An Alternative Model of Laser-Induced Stroke in the Motor Cortex of Rats.</a> Biological Procedure Online. 21, 9 (2019).</li><li>Bleilevens, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+anesthesia+and+cerebral+blood+flow+on+neuronal+injury+in+a+rat+middle+cerebral+artery+occlusion+(MCAO)+model.">Effect of anesthesia and cerebral blood flow on neuronal injury in a rat middle cerebral artery occlusion (MCAO) model.</a> Experimental Brain Research. 224, 155-164 (2013).</li><li>Kuts, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Middle+Cerebral+Artery+Occlusion+Technique+for+Inducing+Post-stroke+Depression+in+Rats.">A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats.</a> Journal of Visualized Experiments. (147), e58875 (2019).</li><li>Boyko, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+neuro-behavioral+parallels+in+the+rat+model+of+stroke.">Morphological and neuro-behavioral parallels in the rat model of stroke.</a> Behavioural Brain Research. 223, 17-23 (2011).</li></ol>

It is fair to assume that the laser technique is minimally invasive, given that no deaths or SAH occurred in the laser group. The primary cause of death and SAH is the damage to blood vessels that leads to an elevation of intracranial pressure (ICP), as shown in the original MCAO techniques10. The absence of death and SAH in the laser group is likely due to the specific effects of lasers: they do not have direct impact on blood vessels and can induce coagulation in case of leakage. Low infarct vol...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60928">Laser-Induced Brain Injury in the Motor Cortex of Rats</a>. The Authors section was updated.
One of the author names was updated from:
Dmitri Frank
to
Dmitry Frank

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60928">Laser-Induced Brain Injury in the Motor Cortex of Rats</a>. The Authors section was updated.

Erratum: Laser-Induced Brain Injury in the Motor Cortex of Rats

Globally, stroke is the second leading cause of death and the third leading cause of disability1. Stroke also leads to severe disability, often requiring extra care from medical staff and relatives. There is, therefore, a need to understand the complications associated with the disorder and improve the potential for more positive outcomes.&#160;
The use of animal models is the initial step to understanding diseases. To ensure the best research outcomes, a typical model would include a simple technique, affordability, high reproducibility, and minimal variability. The determinants in ischemic stroke models include brain edema volume, infarct size, the extent of the blood-brain barrier (BBB) breakdown, and functional impairment generally evaluated via neurological severity score2.
The most widely used stroke induction technique in rodent models occludes the middle cerebral artery (MCA) transiently or permanently3. This technique produces a stroke model similar to the ones in humans: it has a penumbra surrounding the stroked area, is highly reproducible, and regulates ischemia duration and reperfusion4. Nevertheless, the MCAO method has some complications. The technique is prone to intracranial hemorrhage and injury to the ipsilateral retina with a dysfunction of the visual cortex and common hyperthermia that often lead to additional outcomes5,6,7. Other limitations include high variations in induced stroke (arising from the probable extension of the ischemia to unintended regions, like the external carotid artery region), insufficient occlusion of the MCA, and premature reperfusion. Also, rats of different strains and sizes exhibit various infarct volumes8. In addition to all the disadvantages mentioned, MCAO model cannot induce small isolated strokes in deep brain areas, because it is limited technically in terms of its requirement of minimum vessel size for catheterization. This makes the need for an alternative model all the more critical. Another method, photothrombosis, provides a possible alternative to MCAO procedures but does not improve on the efficiency9. This technique targets stroke with light and offers some improvements on the previous models. However, photothrombosis requires an invasive craniotomy that is associated with secondary compications9.
In the light of outlined shortcomings, the protocol presented here provides a capable alternative laser technique for inducing brain injury in rodents. The mechanism of action of the laser technique is based on the laser&#8217;s photothermal effects imparted on living tissues, which leads to the absorption of light beams by body tissues and their conversion into heat. The advantages of using a laser technique are its safety and ease of manipulation. A laser&#8217;s ability to produce heat to stop bleeding makes it very important in medicine, while its ability to amplify different beams at a given meet point ensures that lasers avoid destroying healthy tissues that stands in the&#160;way of&#160;the&#160;target point10. The laser beam used in this protocol can pass through a low liquid medium, such as bone, without emitting its energy and/or causing any destruction. Once it reaches a high liquid medium, such as brain tissues, it uses up its energy to destroy the target tissues. The technique, therefore, can induce brain injury only in the appropriate area of the brain.
The technique presented here showed a tremendous amount of ability to regulate its levels of irradiation, producing the chosen variations of brain injury intended from the start. Unlike the original MCAO that impacts both the cortex and striatum, the laser technique was able to regulate the impact of brain injury, inducing injury only on the intended motor cortex. Herein, the laser-induced brain injury protocol and a summary of representative results for the procedure performed on the cerebral cortex of rats are provided.

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			<td height="42" style="height:43px;width:216px;">2,3,5-Triphenyltetrazolium chloride</td>
			<td style="width:123px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">298-96-4</td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:43px;width:216px;">50% trichloroacetic acid</td>
			<td style="width:123px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">76-03-9</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Brain &#38; Tissue Matrices</td>
			<td style="width:123px;">SIGMA - ALDRICH</td>
			<td>15013</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Cannula Venflon 22 G</td>
			<td style="width:123px;">KD-FIX</td>
			<td style="width:161px;">1.83604E+11</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Centrifuge Sigma 2-16P</td>
			<td style="width:123px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">Sigma 2-16P</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Compact Analytical Balances</td>
			<td style="width:123px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">HR-AZ/HR-A</td>
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			<td height="42" style="height:43px;width:216px;">Digital Weighing Scale</td>
			<td style="width:123px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">Rs 4,000</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Dissecting scissors</td>
			<td style="width:123px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">Z265969</td>
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			<td height="42" style="height:43px;width:216px;">Eppendorf pipette</td>
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			<td height="42" style="height:43px;width:216px;">Eppendorf Tube</td>
			<td style="width:123px;">SIGMA - ALDRICH</td>
			<td>EP0030119460</td>
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		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Ethanol 96 %</td>
			<td style="width:123px;">ROMICAL</td>
			<td style="width:161px;"></td>
			<td>Flammable Liquid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Evans Blue 2%</td>
			<td style="width:123px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">314-13-6</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Fluorescence detector</td>
			<td style="width:123px;">Tecan, M&#228;nnedorf Switzerland</td>
			<td style="width:161px;">model Infinite 200 PRO multimode reader</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Heater with thermometer</td>
			<td>Heatingpad-1</td>
			<td>Model: HEATINGPAD-1/2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Infusion Cuff</td>
			<td style="width:123px;">ABN</td>
			<td style="width:161px;">IC-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Isofluran, USP 100%</td>
			<td style="width:123px;">Piramamal Critical Care, Inc</td>
			<td style="width:161px;">NDC 66794-017</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Multiset</td>
			<td style="width:123px;">TEVA MEDICAL</td>
			<td style="width:161px;">998702</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Olympus BX 40 microscope</td>
			<td style="width:123px;">Olympus</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Optical scanner</td>
			<td style="width:123px;">Canon</td>
			<td style="width:161px;">Cano Scan 4200F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Petri dishes</td>
			<td style="width:123px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">P5606</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Scalpel blades 11</td>
			<td style="width:123px;">SIGMA - ALDRICH</td>
			<td style="width:161px;">S2771</td>
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		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Sharplan 3000 Nd:YAG (neodymium-doped yttrium aluminum garnet) laser machine</td>
			<td style="width:123px;">Laser Industries Ltd</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Stereotaxic head holder</td>
			<td style="width:123px;">KOPF</td>
			<td style="width:161px;">900LS</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">Sterile Syringe 2 ml</td>
			<td style="width:123px;">Braun</td>
			<td>4606027V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe-needle 27 G</td>
			<td style="width:123px;">Braun</td>
			<td style="width:161px;">305620</td>
			<td></td>
		</tr>
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laser-induced brain injury in the motor cortex of rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

No deaths or SAH were registered in either the control or experimental groups (Table 1). The MCAO group had a 20% rate of both mortality and SAH.
The relative body temperature changes in the rats of both groups were also similar, despite a difference in the variability of both groups (Table 1).
There was a significantly worse NSS in both the laser (16 &#177; 1.1) and MCAO (20 &#177; 1.5) models, compared to the sham-operated control...

Watch this Scientific Journal Video about Laser-Induced Brain Injury in the Motor Cortex of Rats at JoVE.com

Laser-Induced Brain Injury in the Motor Cortex of Rats

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Our protocol provides a novel methodology for creating traumatic brain injury in rats using laser irradiation to create a focus impact in the motor cortex. The main advantage of this technique are the low variability in infarct area, the low mortality rates, and the relative simplicity of the procedure which doesn't require expert handlers. Demonstrating the procedure will be Dmitry Natanel, a researcher from our laboratory.To perform a laser-induced brain injury, first assign 20, 300 to 350 gram Sprague Dawley rats into the laser group and 20 to the control Sham-operated 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 1064 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. 24 hours after the procedure, use a 43 point scoring system to evaluate the neurological severity score. Testing the animals for neurological deficits, behavior disturbances, beam balancing task, and reflexes, and assigning higher scores for more severe disabilities.After the assessment, harvest the brains from each experimental and control animal according to standard protocols. To assess the brain infarct volume, section the harvested brains into six two millimeter thick coronal slices and incubate each slice in 0.05%TTC for 30 minutes at 37 degrees Celsius. Following staining, scan this slices with an optical scanner with a 1600 by 1600 dots per inch resolution.The unstained areas of the fixed brain slices are defined as infarcted. To evaluate the incidence of blood brain barrier breakage, 24 hours after the laser induced injury load a syringe with 2%Evans blue dye diluted in four milliliters per kilogram of saline solution and deliver the solution intravenously to the injured and control rats via the cannulated tail vein. Allow the solution to circulate for one hour before using surgical pincets and scissors to open the chest of the first animal.Perfuse the exposed heart with cooled 0.9%saline via the left ventricle at 110 millimeters of mercury of pressure until a colorless profusion liquid is observed from the right atrium. Next, harvest the brain and obtain two millimeter rostral caudal slices. Separate the left brain slices from the right brain slices to allow evaluation of the injured and non-injured hemispheres separately, and weigh the samples.Homogenize the samples using a mortar and pestle and incubate the tissue samples in 50%trichloroacetic acid for 24 hours. The next day, centrifuge the homogenized brain tissue samples for 20 minutes at 10, 000 times G and room temperature, and mix one milliliter of the supernatant with 1.5 milliliters of 96%ethanol at one to three ratio. Then, use a fluorescence detector at a 620 nanometer excitation and the 680 nanometer emission wavelength to assess the blood brain barrier breakage.As observed by histological analysis, Evans blue staining can be used to reveal the incidence of brain injury at laser and MCAO model animals. In this representative analysis no deaths or subarachnoid hemorrhages were registered in either the control or experimental groups, and the MCAO group had a 20%rate of both mortality and subarachnoid hemorrhage. The relative body temperature changes in the rats from both groups were also similar, despite a difference in the variability of both groups.The neurological severity scores were significantly worse in both the laser and MCAO models compared to the Sham-operated control group. Laser-induced to brain injury also caused a significant increase in infarct volume at the target hemisphere compared to the Sham-operated control group. However, the infarct volume of the laser model was smaller compared to the animals injured by the MCAO technique.No difference in brain edema was observed between the laser-induced brain injury model and the Sham-operated control group. However, there was a significant difference in brain edema between the laser model and MCAO injured groups. Compared to the Sham-operated control group, the laser-induced brain injury and MCAO techniques both caused a significant increase in blood brain barrier breakage at the non-injured and target hemispheres.While attempting this model, remember to precisely target the motor cortex area and to standardize the energy, the number of points being irradiated, and the total exposition time. Following this procedure, a wide plethora of behavioral tests, such as beam walking, Froedert, and other evaluations can be performed.

laser-induced-brain-injury-in-the-motor-cortex-of-rats

Research

JoVE Journal

Neuroscience

5.2K vistas.  Ben-Gurion University of the Negev. Nuestro protocolo proporciona una metodología novedosa para crear lesiones cerebrales traumáticas en ratas que utilizan irradiación láser para crear un impacto de enfoque en la corteza motora. La principal ventaja de esta técnica son la baja variabilidad en el área de infarto, las bajas tasas de mortalidad y la relativa simplicidad del procedimiento que no requiere manejadores expertos. Demostrando el procedimiento estará Dmitry Natanel, un investigador de nuestro laboratorio.Pa...