Name: laser-induced brain injury in the motor cortex of rats
Uploaded: 2020-09-26T12:30:00.000+00:00
Duration: 7 min 5 s
Description: Watch this Scientific Journal Video about Laser-Induced Brain Injury in the Motor Cortex of Rats at JoVE.com

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 them adapt for least 3 days.</li>
</ol>
2. MCAO procedure
<ol>
	<li>Select 25 rats for MCAO allowing for 10&#8212;20% mortality associated with the procedure11.</li>
	<li>Perform MCAO using a standard technique, as previously described in detail12.</li>
</ol>
3. Laser-induced brain injury experimental procedure
<ol>
	<li>Assign 20 rats to a group marked as laser group and 20 rats to another control group (sham-operated).</li>
	<li>Subject the laser group rats to laser irradiation at 50J X 10 points&#160;in the following manner:
	<ol>
		<li>Anesthetize rat with a mixture of 2% isoflurane in oxygen allowing for the spontaneous ventilation. Check for sufficient anesthetic depth by pinching the tail with forceps to see the absence of the withdrawal reflex.</li>
		<li>Maintain the core body temperature of the rat at 37 &#730;C throughout the experimental procedure using a rectal temperature regulated heating pad.</li>
		<li>Remove local hair with a shaver and disinfect with 70% alcohol and 0.5% chlorhexidine gluconate. Repeat the disinfection step two more times. 
		NOTE: The size of the surgical incision should be approximately 3 cm. Remove hair&#160;at least 2 cm around the incision area.</li>
		<li>Place the rat on a stereotaxic head holder in a prone position and make a 3 cm incision to reflect the scalp laterally and to 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 at a 2 mm distance from the exposed area to produce a laser beam. 50J X 10 points was 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 second10. 
		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>
	</li>
	<li>Observe the rat for any signs of distress during post- anesthesia recovery. Prior to emergence from anesthesia, give 0.01mg/kg intramuscular buprenorphine for postoperative analgesia and continue with repeated doses every 12 h for at&#160;least 48 h.</li>
	<li>Subject control rats to the same conditions without subjecting them to the laser.</li>
</ol>
4. Neurological severity score (NSS)
<ol>
	<li>Evaluate the neurological severity score 24 h after the laser-induced brain injury using a 43-point score13. Test the animals for neurological deficits, behavior disturbances, beam-balancing task, and reflexes, assigning higher scores for more severe disabilities, as previously detailed13.</li>
</ol>
5. Post-injury manipulations
<ol>
	<li>After NSS evaluation, euthanize the rats by exposing them to 20% oxygen and 80% CO2 (via inspiration) and transcardially perfuse the rat with heparinized phosphate-buffered saline (PBS, 0.9% NaCl). 
	NOTE: Ensure that CO2 is delivered at a predetermined rate in accordance with the Institutional Animal Care and Use Committee guidelines. This step may also be performed under 5% isoflurane anesthesia.</li>
	<li>Harvest brains and prepare for further examination as described in an earlier protocol11.&#160;</li>
	<li>Evaluate for subarachnoid hemorrhage (SAH) through visual examination of the whole brain after its isolation from the skull. If necessary, a microscope or magnifying glasses may be used for this purpose.</li>
</ol>
6. Evaluation of the brain injury
<ol>
	<li>Determining the brain infarct volume and brain edema by TTC staining 
	NOTE: 2,3,5-Triphenyltetrazolium chloride (TTC) staining is a convenient procedure for brain infarct detection11.
	<ol>
		<li>Section the harvested brains into 6 coronal slices, each 2 mm thickness.</li>
		<li>Incubate the set of slices from each brain for 30 min at 37 &#176;C in 0.05% TTC.</li>
		<li>Following staining, scan the slices with an optical scanner with a resolution of 1600 X 1600 dpi.</li>
		<li>The unstained areas of the fixed brain slices are defined as infarcted12.</li>
		<li>Using an image processing software (e.g., freeware Image J) measure the unstained infarcted area, ipsi- and contralateral hemispheres for each of the 6 coronal slices.</li>
		<li>Calculate the infarcted volume as a percentage of the total brain: 
		<img alt="Equation 1" src="/files/ftp_upload/60928/60928eq1.jpg" /></li>
		<li>Calculate brain edema using Kaplan method: 
		<img alt="Equation 2" src="/files/ftp_upload/60928/60928eq2.jpg" /></li>
	</ol>
	</li>
	<li>Determining the extent of blood brain barrier (BBB) breakage 
	NOTE: Assess BBB breakage 24 h after the laser-induced brain injury as follows:
	<ol>
		<li>Administer 2% Evans Blue mixed with 4 mL/kg saline solution intravenously to rats via the cannulated tail vein and allow the solution to circulate for 1 h.</li>
		<li>Euthanize rats by exposing them to 20% oxygen and 80% CO2 (via inspiration) 24 h after the last NSS, as previously described13.</li>
		<li>Harvest the intravascularly localized dye as follows:
		<ol>
			<li>Open the chests of the rats with surgical pincettes and surgical scissors.</li>
			<li>Perfuse the animals with cooled 0.9% saline via the left ventricle using 110 mmHg until obtaining a colorless perfusion liquid from the right atrium.</li>
		</ol>
		</li>
		<li>Harvest the brains and slice them rostrocaudally into 2 mm slices.</li>
		<li>Separate the left brain slices from the right portions to evaluate injured and non-injured hemispheres separately.</li>
		<li>Weigh, homogenize using mortar and pestle, and then incubate the brain tissues in 50% trichloroacetic acid for 24 h.</li>
		<li>Centrifuge the homogenized brain slices at 10,000 &#215; g for 20 min.</li>
		<li>Mix 1 mL of the supernatant from the centrifuged brain with 1.5 mL of 96% ethanol at 1:3 and assess blood-brain barrier breakage using a fluorescence detector at 620 nm excitation wavelength (10 nm bandwidth) and 680 nm emission wavelength (10 nm bandwidth). 
		NOTE: Both groups of rats undergo the same protocol for determining BBB breakdown.</li>
	</ol>
	</li>
</ol>

<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>

The authors have nothing to disclose.

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 volume and brain edema also help minimize the risk of death. The use of lasers should be considered as a suitable technique for inducing brain injury with minimal adverse outcomes, given that the original MCAO techniques for triggering stroke (both transient and permanent) have been shown to produce deaths and SAH6.
Low body temperature findings in the laser group show that the laser technique does not occlude the hypothalamic artery that regulates body temperature, as the original MCAO does typically7, supporting the theory that the laser technique is more targeted. Low variability across the board of parameters investigated indicated consistency in the use of lasers to induce brain injury, but such fine results depend very much on the choice of power. Sufficient power provides desired outcomes, while little or surplus calibrations can cause under- or over-performance, which in either case is detrimental. Nevertheless, the ability to aim for the target still makes the technique less risky. Hence, correct handling makes it easier to obtain results with precision using the laser technique, as well as to regulate the method for desirable effects.
The precision and efficacy of the laser technique were evident in its ability to strike only the motor cortex without causing damage to the striatum, suggesting that the laser technique can produce localized injury that is almost impossible to achieve with MCAO10. This achievable outcome with the laser technique is due to the ability to regulate the laser beam and its power and makes the laser method a model technique for inducing smaller, peripheral, and deep and defined brain injury that cannot be obtained with MCAO. The simplicity of manipulating a laser machine makes it very desirable. Unlike MCAO techniques that demand arduous training and experts, using lasers is more simple, requiring no experts or expensive training. The use of the laser technique could boost research and help to uncover better outcomes more quickly than the MCAO method alone.
In terms of limitations of the laser technique, the use of laser beams does not produce brain injuries that are perfectly similar to acute vascular occlusive strokes. Specifically, lasers produce immediate tissue scars at the target site that are comparable to a vascular occlusive stroke that is several days old. The technique might, therefore, not be suitable for evaluating drugs that aim to prevent the spread of stroke but should be ideal in assessing isolated motor cortex stroke on prolonged motor, cognitive, and behavioral impairments. The use of a small number of rats for this research was also a limitation, with only half the number of rats (n = 10) in each group used for brain harvesting and examination of the size of the stroke, the extent of brain edema, BBB breakage, and SAH presence.
The lack of comparisons between our technique and other laser methods may also be deemed a limitation. We deliberated on performing comparative methods but decided not to do so because assessing the damage caused by these other laser methods is difficult. For example, the photothrombosis technique6 causes weak damage that makes it challenging to evaluate brain swelling and other conditions that may occur. Also, the use of craniotomy in the laser technique for ischemia is problematic because craniotomy is very invasive and can increase BBB&#8217;s permeability, causing additional brain injury that is not associated with stroke. Assessing such damage for comparison with our method is nearly impossible. The laser model induces stroke with radiation through the skull without craniotomy.
Like many models, the laser model has its benefits and limitations, with the most glaring drawback is its inability to mimic perfectly human stroke as precisely as other models. Nevertheless, the low variability in primary outcomes of most parameters, its precision, affordability, ability to induce smaller brain injuries, and its straightforward application makes it a suitable alternative technique for brain injury in rodents.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.

<table><tbody><tr><td>2,3,5-Triphenyltetrazolium chloride</td><td>SIGMA - ALDRICH</td><td>298-96-4</td><td></td></tr><tr><td>50% trichloroacetic acid</td><td>SIGMA - ALDRICH</td><td>76-03-9</td><td></td></tr><tr><td>Brain &amp;amp; Tissue Matrices</td><td>SIGMA - ALDRICH</td><td>15013</td><td></td></tr><tr><td>Cannula Venflon 22 G</td><td>KD-FIX</td><td>1.83604E+11</td><td></td></tr><tr><td>Centrifuge Sigma 2-16P</td><td>SIGMA - ALDRICH</td><td>Sigma 2-16P</td><td></td></tr><tr><td>Compact Analytical Balances</td><td>SIGMA - ALDRICH</td><td>HR-AZ/HR-A</td><td></td></tr><tr><td>Digital Weighing Scale</td><td>SIGMA - ALDRICH</td><td>Rs 4,000</td><td></td></tr><tr><td>Dissecting scissors</td><td>SIGMA - ALDRICH</td><td>Z265969</td><td></td></tr><tr><td>Eppendorf pipette</td><td>SIGMA - ALDRICH</td><td>Z683884</td><td></td></tr><tr><td>Eppendorf Tube</td><td>SIGMA - ALDRICH</td><td>EP0030119460</td><td></td></tr><tr><td>Ethanol 96 %</td><td>ROMICAL</td><td></td><td>Flammable Liquid</td></tr><tr><td>Evans Blue 2%</td><td>SIGMA - ALDRICH</td><td>314-13-6</td><td></td></tr><tr><td>Fluorescence detector</td><td>Tecan, M&amp;auml;nnedorf Switzerland</td><td>model Infinite 200 PRO multimode reader</td><td></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>Multiset</td><td>TEVA MEDICAL</td><td>998702</td><td></td></tr><tr><td>Olympus BX 40 microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Optical scanner</td><td>Canon</td><td>Cano Scan 4200F</td><td></td></tr><tr><td>Petri dishes</td><td>SIGMA - ALDRICH</td><td>P5606</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><tr><td>Sterile Syringe 2 ml</td><td>Braun</td><td>4606027V</td><td></td></tr><tr><td>Syringe-needle 27 G</td><td>Braun</td><td>305620</td><td></td></tr></tbody></table>

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 group (1 &#177; 0.3; Table 1; p&#60;0.01).
The laser-induced brain injury also caused a significant increase in infarct volume at the target hemisphere, compared to the sham-operated control group (2.4% &#177; 0.3 vs 0.5% &#177; 0.1; Table 2 and&#160; Figure 1A; p&#60;0.01), per the Mann-Whitney U test. However, the infarct volume of the laser model was smaller in comparison to the MCAO technique (2.4% &#177; 0.3 vs 9.9% &#177; 2.9).
Brain edema determined 24 h after brain injury are shown in Figure 1B and Table 2. There was no difference in brain edema between the laser-induced brain injury model and the sham-operated control group (3.4% &#177; 0.6 vs 0.7% &#177; 1.2). There was a significant difference in brain edema between the laser model and the MCAO technique (3.4 &#177; 0.6 vs 7 &#177; 2.6&#8224;). Data are presented as mean &#177; SEM.
Compared to the sham-operated control group, the laser-induced brain injury and MCAO technique both caused a significant increase in BBB breakage at the non-injured hemisphere (563 ng/g &#177; 66 and 1176 ng/g &#177; 168, respectively, vs 141 ng/g &#177; 14; Figure 2A and Table 2; p&#60;0.01) and target hemisphere (2204 ng/g &#177; 280 and 2764 ng/g &#177; 256, respectively, vs 134 ng/g &#177; 11; Figure 2B and Table 2; p&#60;0.01).
Histological examination of rats&#8217; brains are shown in Figure 3.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">NSS</td>
			<td colspan="2">Temperature, &#176;C</td>
			<td>SAH, %</td>
			<td>Mortality, %</td>
		</tr>
		<tr>
			<td>Groups</td>
			<td>mean &#177; SEM</td>
			<td>variability, %</td>
			<td>mean &#177; SEM</td>
			<td>variability, %</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sham-operated control</td>
			<td>1 &#177; 0.3</td>
			<td>97</td>
			<td>37.2 &#177; 0.1</td>
			<td>59</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Laser 50J x10</td>
			<td>16 &#177; 1.1*</td>
			<td>30</td>
			<td>37.4 &#177; 0.1</td>
			<td>84</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>p-MCAO</td>
			<td>20 &#177; 1.5*</td>
			<td>37</td>
			<td>38.3 &#177; 0.1*</td>
			<td>129</td>
			<td>20*</td>
			<td>20*</td>
		</tr>
	</tbody>
</table>
Table 1: Assessment of NSS, body temperature, subarachnoid hemorrhage, and mortality. * = p &#60; 0.01
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">BBB</td>
			<td colspan="2">Infarcted Volume</td>
			<td colspan="2">Brain Edema</td>
		</tr>
		<tr>
			<td>Groups</td>
			<td>mean &#177; SEM</td>
			<td>variability, %</td>
			<td>mean &#177; SEM</td>
			<td>variability, %</td>
			<td>mean &#177; SEM</td>
			<td>variability, %</td>
		</tr>
		<tr>
			<td>Sham-operated control</td>
			<td>134 &#177; 11</td>
			<td>25</td>
			<td>0.5 &#177; 0.1</td>
			<td>77</td>
			<td>0.7 &#177; 1.2</td>
			<td>573</td>
		</tr>
		<tr>
			<td>Laser 50J x10</td>
			<td>2204 &#177; 280*</td>
			<td>40</td>
			<td>2.4 &#177; 0.3*</td>
			<td>34</td>
			<td>3.4 &#177; 0.6</td>
			<td>58</td>
		</tr>
		<tr>
			<td>p-MCAO</td>
			<td>2764 &#177; 256*</td>
			<td>29</td>
			<td>9.9 &#177; 2.9*</td>
			<td>92</td>
			<td>7 &#177; 2.6*</td>
			<td>115</td>
		</tr>
	</tbody>
</table>
Table 2: Assessment of BBB breakdown, infarct zone, and brain edema. * = p &#60; 0.01
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60928/60928fig1.jpg" /> 
Figure 1: Assessment of brain injury in the laser model 24 h after the injury compared to the MCAO model and sham-operated control. (A) Assessment of infarct volume. There was an increase in infarct volume in the laser model compared to the sham-operated control (*p&#60;0.01). However, the infarct volume in the laser model was smaller compared to the MCAO model (*p&#60;0.01). (B) Assessment of total brain edema. There was an increase in brain edema in the MCAO model compared to either the laser model or sham-operated control. There was no difference in brain edema between the laser model and sham-operated control. The data are measured as % to the contralateral hemisphere and expressed as mean &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60928/60928fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60928/60928fig2.jpg" /> 
Figure 2: The extent of BBB breakdown compared to sham controls. (A) Contralateral (non-injured) hemisphere. Both&#160;the laser and MCAO models, led to a significant increase in BBB breakage at the non-injured hemisphere compared to the sham-operated control group (*p&#60;0.01). (B) Ipsilateral (injured) hemisphere. There was a difference in ipsilateral BBB breakdown in the laser and MCAO models compared to the sham-operated control (*p&#60;0.01). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60928/60928fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60928/60928fig3.jpg" /> 
Figure 3: Histological examination of rats&#8217; brains from sham, laser and MCAO groups. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60928/60928fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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) ...

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

5.2K Views.  Ben-Gurion University of the Negev. Ons protocol biedt een nieuwe methodologie voor het creëren van traumatisch hersenletsel bij ratten met behulp van laser bestraling om een focus impact in de motorische cortex te creëren. Het belangrijkste voordeel van deze techniek zijn de lage variabiliteit in infarctgebied, de lage sterftecijfers en de relatieve eenvoud van de procedure waarvoor geen deskundige handlers nodig zijn. Het aantonen van de procedure zal Dmitry Natanel, een onderzoeker van ons laboratorium.Om een laser-...

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

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular ...

Biology

Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain

The rat auditory cortex (AC) is becoming popular among auditory neuroscience investigators who are interested in experience-dependence plasticity, auditory perceptual processes, and cortical control of sound processing in the subcortical auditory nuclei. To address new challenges, a procedure to accurately locate and surgically expose the auditory cortex would expedite this research effort. Stereotactic neurosurgery is routinely used in pre-clinical research in animal models to engraft a needle or electrode at a pre-defined location within the auditory cortex. In the following protocol, we use stereotactic methods in a novel way. We identify four coordinate points over the surface of the temporal bone of the rat to define a window that, once opened, accurately exposes both the primary (A1) and secondary (Dorsal and Ventral) cortices of the AC. Using this method, we then perform a surgical ablation of the AC. After such a manipulation is performed, it is necessary to assess the localization, size, and extension of the lesions made in the cortex. Thus, we also describe a method to easily locate the AC ablation postmortem using a coordinate map constructed by transferring the cytoarchitectural limits of the AC to the surface of the brain.The combination of the stereotactically-guided location and ablation of the AC with the localization of the injured area in a coordinate map postmortem facilitates the validation of information obtained from the animal, and leads to a better analysis and comprehension of the data.

We describe a method for the stereotactically-guided location, exposure, and ablation of the auditory cortex in rats. The localization of the ablation is assessed using a coordinate map postmortem.

The rat auditory cortex (AC) is becoming popular among auditory neuroscience investigators who are interested in experience-dependence plasticity, ...

Behavior

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and human studies to investigate the cortical processing of nociceptive information. These laser-evoked brain potentials (LEPs) consist of several transient responses that are time-locked to the onset of laser stimuli. However, the functional properties of the LEP responses are still largely unknown, due to the lack of a sampling technique that can simultaneously record neural activities at the surface of the cortex (i.e., electrocorticogram [ECoG] and scalp electroencephalogram [scalp EEG]) and inside the brain (i.e., local field potential [LFP]). To address this issue, we present here an animal protocol using freely moving rats. This protocol is composed of three main procedures: (1) animal preparation and surgical procedures, (2) a simultaneous recording of ECoG and LFP in response to nociceptive laser stimuli, and (3) data analysis and feature extraction. Specifically, with the help of a 3D-printed protective shell, both ECoG and LFP electrodes implanted on the rat&#39;s skull were securely held together. During data collection, laser pulses were delivered on the rat&#39;s forepaws through gaps in the bottom of the chamber when the animal was in spontaneous stillness. Ongoing white noise was played to avoid the activation of the auditory system by the laser-generated ultrasounds. As a consequence, only nociceptive responses were selectively recorded. Using the standard analytical procedures (e.g., band-pass filtering, epoch extraction, and baseline correction) to extract stimulus-related brain responses, we obtained results showing that LEPs with a high signal-to-noise ratio were simultaneously recorded from ECoG and LFP electrodes. This methodology makes the simultaneous recording of ECoG and LFP activities possible, which provides a bridge of electrocortical signals at the mesoscopic and macroscopic levels, thereby facilitating the investigation of nociceptive information processing in the brain.

We developed a technique that simultaneously records both electrocorticography and local field potentials in response to nociceptive laser stimuli from freely moving rats. This technique helps establish a direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, which facilitates the investigation of nociceptive information processing in the brain.

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and ...

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.

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

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the United States. In fact, TBI is a contributing factor to over a third of all injury-related mortality. Nonetheless, the cellular and molecular mechanisms underlying the pathophysiology of TBI are poorly understood. Thus, preclinical models of TBI capable of replicating the injury mechanisms pertinent to TBI in human patients are a critical research need. The controlled cortical impact (CCI) model of TBI utilizes a mechanical device to directly impact the exposed cortex. While no model can full recapitulate the disparate injury patterns and heterogeneous nature of TBI in human patients, CCI is capable of inducing a wide range of clinically applicable TBI. Furthermore, CCI is easily standardized allowing investigators to compare results across experiments as well as across investigative groups. The following protocol is a detailed description of applying a severe CCI with a commercially available impacting device in a murine model of TBI.

Here we describe a protocol for the induction of murine traumatic brain injury via an open-head controlled cortical impact.

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the ...

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

rmTBI Induction in Mice via a Closed-Head Injury Method

Modified Mouse Model of Repetitive Mild Traumatic Brain Injury Incorporating Thinned-Skull Window and Fluid Percussion

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models with well-defined pathologies are urgently needed for studying the mechanisms of neuropathology after mild TBI and testing therapeutics. Replicating the entire sequelae of TBI in animal models has proven to be a challenge. Therefore, the availability of multiple animal models of TBI is necessary to account for the diverse aspects and severities seen in TBI patients. CHI is one of the most common methods for fabricating rodent models of rmTBI. However, this method is susceptible to many factors, including the impact method used, the thickness and shape of the skull bone, animal apnea, and the type of head support and immobilization utilized. The aim of this protocol is to demonstrate a combination of the thinned-skull window and fluid percussion injury (FPI) methods to produce a precise mouse model of CHI-associated rmTBI. The primary objective of this protocol is to minimize factors that could impact the accuracy and consistency of CHI and FPI modeling, including skull bone thickness, shape, and head support. By utilizing a thinned-skull window method, potential inflammation due to craniotomy and FPI is minimized, resulting in an improved mouse model that replicates the clinical features observed in patients with mild TBI. Results from behavior and histological analysis using hematoxylin and eosin (HE) staining suggest that rmTBI can lead to a cumulative injury that produces changes in both behavior and gross morphology of the brain. Overall, the modified CHI-associated rmTBI presents a useful tool for researchers to explore the underlying mechanisms that contribute to focal and diffuse pathophysiological changes in rmTBI.

Author Spotlight: Optimizing Rodent Models for Investigating the Mechanisms and Rehabilitation in Repeated Mild Traumatic Brain Injury

This protocol presents a modified mouse model of repetitive mild traumatic brain injury (rmTBI) induced via a closed-head injury (CHI) method. The approach features a thinned-skull window and fluid percussion to reduce the inflammation commonly caused by meninges exposure, along with improved reproducibility and accuracy in modeling rmTBI in rodents.

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models ...

Development of an Uncomplicated Mild Traumatic Brain Injury Model Modified by Weight-Drop Method and Evidenced by Magnetic Resonance Imaging

Mild traumatic brain injury (mTBI), known as concussion, accounts for more than 85% of brain injuries globally. Specifically, uncomplicated mTBI showing negative findings in routine clinical imaging in the acute phase hinders early and appropriate care in these patients. It has been acknowledged that different impact parameters may affect and even accelerate the progress of subsequent neuropsychological symptoms following mTBI. However, the association of impact parameters during concussion to the outcome has not been extensively examined. In the current study, an animal model with closed-head injury (CHI) modified from the weight-drop injury paradigm was described and demonstrated in detail. Adult male Sprague-Dawley rats (n = 20) were randomly assigned to CHI groups with different impact parameters (n = 4 per group). Longitudinal MR imaging studies, including T2-weighted imaging and diffusion tensor imaging, and sequential behavioral assessments, such as modified neurological severity score (mNSS) and the beam walk test, were conducted over a 50-day study period. Immunohistochemical staining for astrogliosis was performed on day 50 post-injury. Worse behavioral performance was observed in animals following repetitive CHI compared to the single injury and sham group. By using longitudinal magnetic resonance imaging (MRI), no significant brain contusion was observed at 24 h post-injury. Nevertheless, cortical atrophy and alteration of cortical fractional anisotropy (FA) were demonstrated on day 50 post-injury, suggesting the successful replication of clinical uncomplicated mTBI. Most importantly, changes in neurobehavioral outcomes and image features observed after mTBI were dependent on impact number, inter-injury intervals, and the selected impact site in the animals. This in vivo mTBI model combined with preclinical MRI provides a means to explore brain injury on a whole-brain scale. It also allows the investigation of imaging biomarkers sensitive to mTBI across varying impact parameters and severity levels.

Here, we present a protocol to establish a closed-head injury animal model replicating the neuroimage outcome of uncomplicated mild traumatic brain injury with the preserved brain structure in the acute phase and long-term brain atrophy. Longitudinal magnetic resonance imaging is the primary method used for evidence.

Mild traumatic brain injury (mTBI), known as concussion, accounts for more than 85% of brain injuries globally. Specifically, uncomplicated mTBI showing ...

Performing Laser-Induced Brain Injury in a Rat Model

Source: Kuts, R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/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. ...

JoVE Encyclopedia of Experiments

Neuropathology

Trauma

Induction of Traumatic Brain Injury in a Rat Model Using a Lateral Fluid Percussion Device

Source: McGuire, M. J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59637">Use of a Wireless Video-EEG System to Monitor Epileptiform Discharges Following Lateral Fluid-Percussion Induced Traumatic Brain Injury.</a> J. Vis. Exp. (2019)
This video demonstrates the induction of traumatic brain injury (TBI) in a rat using a lateral fluid percussion injury (FPI) device. The procedure involves creating a hole in the skull of an anesthetized rat to expose the dura mater, onto which a cannula is attached. The rat is then connected to the device, which generates a pressure wave that reaches the brain, mimicking TBI by causing neural cell death and vascular damage

Source: McGuire, M. J., et al. Use of a Wireless Video-EEG System to Monitor Epileptiform Discharges Following Lateral Fluid-Percussion Induced Traumatic ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

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