The authors have no acknowledgements.

This protocol was approved by the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital. Animal care and experiments were conducted according to the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals.
1. Surgical Preparation
<ol>
	<li>Prior to surgery, flush the catheters with sterile saline to ensure patency.</li>
	<li>Put a heating blanket on the operating table, and cover the table with a sterile drape.</li>
	<li>Place Male Sprague&#8722;Dawley rats (270-330 g) in an acrylic box with 3.0%-4.0% of isoflurane in 100% oxygen.</li>
	<li>Apply lubricant to the rat&#39;s eyes.</li>
	<li>Place the rat on the operating table in the supine position, and maintain anesthesia using a facial mask with continuous administration of inhaled isoflurane (1.0%-2.5 vol.%).</li>
	<li>Place a rectal probe to monitor and maintain body temperature between 37.0-38.0 &#176;C.</li>
</ol>
2. Femoral Artery Catheterization
<ol>
	<li>Gently scrub the right inguinal area using Betadine and 70% ethanol.</li>
	<li>Make a 2-cm horizontal skin incision using scissors on the right inguinal area.</li>
	<li>Using a retractor, expose the surgical field.</li>
	<li>Dissect the femoral artery from the surrounding vein and nerve. Isolate a 1-cm section of the artery using curved forceps and blunted forceps.</li>
	<li>Using a 4.0 black silk suture, place a loose tie on both the proximal and distal ends of the artery to maximize the exposure.</li>
	<li>Make an incision on the femoral artery using micro-scissors.</li>
	<li>Insert a 2-Fr. balloon-tipped catheter into the femoral artery using micro-forceps. Secure the catheter to the vessel about 1 cm from the catheter head with the proximal ligature, and then tie the distal ligature.</li>
</ol>
3. Carotid Artery Catheterization
<ol>
	<li>Scrub the right anterior neck using betadine and 70% ethanol, and then make a skin incision.</li>
	<li>Using a retractor, expose the surgical field. Isolate a 1-cm section of the artery, and tie it loosely using a silk suture on both the proximal and distal ends of the artery to maximize the exposure.</li>
	<li>Puncture the carotid artery using a 24 G intravenous catheter, and advance the proximal 1 cm of the catheter towards the heart.</li>
	<li>Secure the catheter with the proximal ligature, and then tie the distal ligature.</li>
	<li>Using a 3-way stopcock, connect the distal end of the catheter to the saline-filled micro-tube and external reservoir.</li>
</ol>
4. Tail Artery Catheterization
<ol>
	<li>Scrub the ventral area of the tail using betadine and 70% ethanol, and make a 2-cm skin incision.</li>
	<li>Dissect the tail artery from the surrounding structures and isolate a 1 cm section of the artery using curved forceps and blunted forceps.</li>
	<li>Using a 4.0 black silk suture, place a loose tie on both the proximal and distal ends of the artery to maximize the exposure.</li>
	<li>Puncture the tail artery using a 24 G intravenous catheter, and advance the catheter into the artery.</li>
	<li>Secure the catheter to the vessel (about 1 cm from the catheter head) with the proximal ligature, and tie the distal ligature.</li>
	<li>Connect the distal part of the catheter to the arterial pressure monitoring device.</li>
</ol>
5. Induction of Spinal Cord Ischemia
<ol>
	<li>After catheterization is complete, advance the 2 Fr. balloon-tipped catheter into the descending thoracic aorta so that the tip of the catheter reaches the left subclavian artery.</li>
	<li>Insert to a depth of 10 cm from the insertion site.</li>
	<li>Inject 150 units of heparin at a concentration of 100 units/mL into the carotid catheter.</li>
	<li>Inflate the catheter balloon with 0.05 mL of saline.</li>
	<li>Simultaneously, drain the blood into the external blood container from the carotid artery to regulate the proximal arterial pressure to 80 mmHg.</li>
	<li>Connect the arterial pressure monitoring system to the remaining lumen of the 3-way stop cock, and control the amount of blood drainage with monitoring the arterial pressure.</li>
	<li>Confirm the success of aortic occlusion by an abrupt decrease and continuous loss of distal arterial pressure.</li>
	<li>After aortic occlusion of 9, 10, or 11 min, deflate the Fogarty catheter balloon, and reinfuse the drained blood.</li>
</ol>
6. Post-surgical Care and Neurologic Assessment
<ol>
	<li>While monitoring the arterial pressure through the tail artery, remove the catheters from the femoral and carotid arteries and close the wounds with silk sutures.</li>
	<li>After confirming that the arterial pressure is recovered to the normal range, remove the catheter from the tail artery and close the wound.</li>
	<li>After recovery from the anesthesia, return the rat to its cage.</li>
	<li>Assess hind limb motor function at 24 h after surgery using the motor deficit index (Table 1). The motor deficit index is defined as the sum of the ambulation score and the placing/stepping reflex score. A motor deficit index of 6 indicates the maximum deficit.</li>
</ol>
7. Histopathologic Evaluation
<ol>
	<li>After the neurologic assessment, anesthetize rats with mask-delivered isoflurane, sacrifice them by transcardial perfusion with 100 mL of heparinized saline under anesthesia, and extract the spinal cord.8</li>
	<li>Embed the cord sections from the level of lumbar vertebrae 3-5 in paraffin.</li>
	<li>Stain transverse sections with hematoxylin and eosin (H &#38; E).</li>
	<li>Observe the motor neuron injury under a microscope.9</li>
</ol>
8. Statistical Analysis
<ol>
	<li>Perform statistical analysis using Statistical Package for the Social Sciences version 20. The motor deficit index of the three groups was compared using the Kruskal-Wallis test, followed by a Mann-Whitney U-test. A P-value &#60;0.05 was considered statistically significant.</li>
</ol>

<ol>
	<li>Greenberg, R. K., 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=Contemporary+analysis+of+descending+thoracic+and+thoracoabdominal+aneurysm+repair:+a+comparison+of+endovascular+and+open+techniques.">Contemporary analysis of descending thoracic and thoracoabdominal aneurysm repair: a comparison of endovascular and open techniques.</a> Circulation. 118 (8), 808-817 (2008).</li><li>Okita, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fighting+spinal+cord+complication+during+surgery+for+thoracoabdominal+aortic+disease.">Fighting spinal cord complication during surgery for thoracoabdominal aortic disease.</a> Gen Thorac Cardiovasc Surg. 59 (2), 79-90 (2011).</li><li>Fleck, T. 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=Improved+outcome+in+thoracoabdominal+aortic+aneurysm+repair:+the+role+of+cerebrospinal+fluid+drainage.">Improved outcome in thoracoabdominal aortic aneurysm repair: the role of cerebrospinal fluid drainage.</a> Neurocrit Care. 2 (1), 11-16 (2005).</li><li>Kouchoukos, N. T., 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=Hypothermic+bypass+and+circulatory+arrest+for+operations+on+the+descending+thoracic+and+thoracoabdominal+aorta.">Hypothermic bypass and circulatory arrest for operations on the descending thoracic and thoracoabdominal aorta.</a> Ann Thorac Surg. 60 (1), 67-76 (1995).</li><li>Taira, Y., Marsala, M. <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+proximal+arterial+perfusion+pressure+on+function,+spinal+cord+blood+flow,+and+histopathologic+changes+after+increasing+intervals+of+aortic+occlusion+in+the+rat.">Effect of proximal arterial perfusion pressure on function, spinal cord blood flow, and histopathologic changes after increasing intervals of aortic occlusion in the rat.</a> Stroke. 27 (10), 1850-1858 (1996).</li><li>Tveten, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+vascularity.+III.+The+spinal+cord+arteries+in+man.">Spinal cord vascularity. 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B., Leusen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemispheric+blood+flow+in+the+rat+after+unilateral+common+carotid+occlusion:+evolution+with+time.">Hemispheric blood flow in the rat after unilateral common carotid occlusion: evolution with time.</a> Stroke. 16 (1), 69-73 (1985).</li><li>Coyle, P., Panzenbeck, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collateral+development+after+carotid+artery+occlusion+in+Fischer+344+rats.">Collateral development after carotid artery occlusion in Fischer 344 rats.</a> Stroke. 21 (2), 316-321 (1990).</li><li>Levine, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoxic-ischemic+encephalopathy+in+rats.">Anoxic-ischemic encephalopathy in rats.</a> Am J Pathol. 36, 1-17 (1960).</li><li>Prior, B. 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=Time+course+of+changes+in+collateral+blood+flow+and+isolated+vessel+size+and+gene+expression+after+femoral+artery+occlusion+in+rats.">Time course of changes in collateral blood flow and isolated vessel size and gene expression after femoral artery occlusion in rats.</a> Am J Physiol Heart Circ Physiol. 287 (6), H2434-H2447 (2004).</li><li>Yang, H. T., Feng, Y., Allen, L. A., Protter, A., Terjung, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+specificity+of+bFGFincreased+collateral+flow+in+experimental+peripheral+arterial+insufficiency.">Efficacy and specificity of bFGFincreased collateral flow in experimental peripheral arterial insufficiency.</a> Am J Physiol Heart Circ Physiol. 278 (6), H1966-H1973 (2000).</li><li>Kakinohana, M., Fuchigami, T., Nakamura, S., Sasara, T., Kawabata, T., Sugahara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrathecal+administration+of+morphine,+but+not+small+dose,+induced+spastic+paraparesis+after+a+noninjurious+interval+of+aortic+occlusion+in+rats.">Intrathecal administration of morphine, but not small dose, induced spastic paraparesis after a noninjurious interval of aortic occlusion in rats.</a> Anesth Analg. 96 (3), 769-775 (2003).</li><li>Horiuchi, T., 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=The+effects+of+the+delta-opioid+agonist+SNC80+on+hind-limb+motor+function+and+neuronal+injury+after+spinal+cord+ischemia+in+rats.">The effects of the delta-opioid agonist SNC80 on hind-limb motor function and neuronal injury after spinal cord ischemia in rats.</a> Anesth Analg. 99 (1), 235-240 (2004).</li><li>Griepp, R. B., Griepp, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+perfusion+and+protection+during+descending+thoracic+and+thoracoabdominal+aortic+surgery:+the+collateral+network+concept.">Spinal cord perfusion and protection during descending thoracic and thoracoabdominal aortic surgery: the collateral network concept.</a> Ann Thorac Surg. 83 (2), S865-S869 (2007).</li></ol>

The authors have no competing financial interests. This work was supported by grant 2012R1A1A3014010 from the National Research Foundation of Korean Government.

In the current study, we demonstrated a rat model of spinal cord ischemia based on Taira and Marsala&#39;s method5 that induces variable degrees of motor deficit in the hind limb depending on the aortic occlusion time.
The length of aortic occlusion can affect the degree of motor deficit. If the aortic occlusion time is longer, the motor deficit becomes more severe. Thus, researchers can achieve a certain degree of motor deficit by controlling the aortic occlusion time in this model.
Our model involves the ligation of the common carotid artery and the femoral artery, and the possibility of neurologic deficits resulting from the ligation of these arteries is a potential concern. However, rats have efficient collateral network systems. Thus, when the carotid artery or femoral artery is ligated, sufficient blood flow can be provided by the extensive collateral network. Unilateral carotid artery occlusion is reported to produce only minor effects on cerebral blood flow.10,11 Although unilateral carotid artery occlusion can produce stroke in rats, this occurs only when in combination with severe systemic hypoxia.12 During our experiment, no systemic hypoxia occurred and none of the rats presented with neurological deficits suggestive of cerebral infarction. Furthermore, when the femoral artery of rats is ligated, the collateral circulation provides sufficient blood flow to the hind limb muscles.13 This collateral circulation provides sufficient blood flow to the hind limb muscles when they are at rest, but does not provide sufficient flow during exercise.14
Furthermore, the proximal arterial pressure during spinal cord ischemia affects the development of motor deficits. According to a previous study,5 the collateral blood supply almost disappeared at the proximal arterial pressure of 40 mmHg during aortic clamping in a rat spinal ischemia model. Thus, subsequent studies maintained the proximal arterial pressure at 40 mmHg during spinal cord ischemia in their models of rat spinal cord ischemia.9,15,16 However, in this protocol, we maintained the proximal arterial pressure at 80 mmHg during aortic occlusion because it is recommended to maintain the mean arterial pressure at 80 mmHg or greater for preserving adequate spinal cord perfusion during spinal cord ischemia in the clinical practice,17 although there might be a difference in what constitutes an adequate proximal arterial pressure between humans and rodents.
The spinal cord vasculature and collateral system of rats and humans are similar,6,7 which makes rats an appropriate choice for an experimental spinal cord ischemia model. However, it should not be discounted that the results may be different according to the species in which spinal cord ischemia occurs.
In conclusion, researchers can easily adopt this rat model of spinal cord ischemia and achieve highly reproducible findings. Furthermore, they can modify the aortic occlusion time to vary the degree of motor deficit produced. As such, this model can facilitate further studies examining the underlying pathophysiology of neurologic complications following thoracoabdominal aortic aneurysm, and allow the development of neuroprotective strategies against these complications.

Paraplegia is a fatal complication of thoracoabdominal aortic aneurysm surgery. It results from spinal cord ischemia-reperfusion injury that occurs during cross-clamping and unclamping of the aorta.1 Several strategies including systemic hypothermia and cerebrospinal drainage have been introduced to protect the spinal cord,2,3,4 but many patients remain affected by the injury.
Several animal spinal cord ischemia models have been introduced to investigate its pathogenesis and devise protective strategies against the injury. In the current study, we outline a rat model of spinal cord ischemia based on Taira and Marsala&#39;s method.5 The spinal circulation system in rats is very similar to the spinal cord vascular and collateral system in humans, although there are some differences in the size and location.6,7 Thus, a rat is an anatomically suitable animal to utilize for an experimental model investigating the pathogenesis, complications, and treatment of spinal cord ischemia. Moreover, this spinal cord ischemia model produces reliable aortic occlusion with minimal intervention by utilizing an intravascular balloon occlusion of the thoracic aorta.
In this study, we demonstrated that this rat model of spinal cord ischemia induces reproducible motor deficits in the hind limbs that vary in severity depending on the aortic occlusion time.

<table><tbody><tr><td>Fogarty Arterial Embolectomy catheter</td><td>Edward Life Sciences</td><td>120602F</td><td>a balloon-tipped catheter inserted into the femoral artery</td></tr><tr><td>BD Insyte-N Autoguard Shielded IV catheter&amp;nbsp;</td><td>BD&amp;nbsp;</td><td>381411</td><td>24-gauge intravenous catheter</td></tr><tr><td>50 mL syringe</td><td>KOREA VACCINE&amp;nbsp;</td><td>KOVAX-SYRINGE 50mL</td><td>Facial mask</td></tr><tr><td>1 mL syringe</td><td>KOREA VACCINE</td><td>KOVAX-SYRINGE 1ml</td><td></td></tr><tr><td>Recal probe</td><td>HARVARD APPARATUS</td><td>50-7221F</td><td>Rectal probe for temperature monitoring</td></tr><tr><td>Micro dissecting spring scissor</td><td>Jeung do bio &amp;amp; Plant co.LTD.</td><td>JD-S-10</td><td>Micro-scissor</td></tr><tr><td>SCISSOR (SHARP-SHARP)</td><td>Jeung do bio &amp;amp; Plant co.LTD.</td><td>S-51-12-S</td><td>Scissors</td></tr><tr><td>Retractor</td><td>Jeung do bio &amp;amp; Plant co.LTD.</td><td>JD-S-74A</td><td>Retractor</td></tr><tr><td>Micro forcep&amp;nbsp;</td><td>Jeung do bio &amp;amp; Plant co.LTD.</td><td>JD-S-29</td><td>Micro-forceps</td></tr><tr><td>MOSQUITO FORCEP (Curved)</td><td>Jeung do bio &amp;amp; Plant co.LTD.</td><td>S-44-CPK</td><td>Curved forceps</td></tr><tr><td>DRESSING FORCEP&amp;nbsp;</td><td>Jeung do bio &amp;amp; Plant co.LTD.</td><td>S-37-16S</td><td>Blunted forceps</td></tr><tr><td>4/0 black silk&amp;nbsp;</td><td>Woori Medical</td><td>S431</td><td>4.0 black silk suture</td></tr><tr><td>3-WAY STOCK</td><td>Seonwon Medcal</td><td>D-98-01</td><td>3-way stopcock</td></tr><tr><td>Patient monitor</td><td>PHILIPS</td><td>MP20</td><td>The arterial pressure monitoring device.&amp;nbsp;</td></tr><tr><td>Heating blanket</td><td>Self production</td><td></td><td>Heating blanket</td></tr><tr><td>Microtube and external reservoir</td><td>Self production</td><td></td><td>Microtube and external reservoir</td></tr><tr><td>Heparin</td><td>JW Pharmaceutical</td><td></td><td>Heparin</td></tr><tr><td>0.9% NS 1000ml</td><td>JW Pharmaceutical</td><td></td><td>Normal saline</td></tr><tr><td>Isoflurane</td><td>Hana Med</td><td></td><td>Isoflurane</td></tr></tbody></table>

reproducible motor deficit following aortic occlusion in a rat model of spinal cord ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

During a period of spinal cord ischemia, aortic occlusion was performed for 9 min (n=3), 10 min (n=3), or 11 min (n=3). The motor deficit index in rats is presented in Table 2. Rats that underwent 9 min of aortic occlusion showed a mild and reversible motor impairment in the hind-limb. Rats subjected to 10 min of aortic occlusion presented with moderate motor deficit, but not complete paralysis. Rats that underwent 11 min of occlusion time displayed complete and persistent paralysis.
Representative photographs of spinal cord sections stained with H &#38; E are shown in Figure 1. The motor neurons in the spinal cord sections were more preserved in rats subjected to a shorter duration of aortic occlusion.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55814/55814fig1.jpg" /> 
Figure 1. Histologic Examination of Spinal Cord Sections 
The motor neurons were more preserved in rats subjected to a shorter duration of aortic occlusion (original magnification, 200X). Scale bar in all images = 50 &#181;m. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55814/55814fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ambulation (walking with lower extremities)</td>
			<td>Placing/stepping reflex</td>
		</tr>
		<tr>
			<td>0: normal (symmetrical and coordinated ambulation)</td>
			<td>0: normal</td>
		</tr>
		<tr>
			<td>1: toes flat under body when walking but ataxia present</td>
			<td>1: weak</td>
		</tr>
		<tr>
			<td>2: knuckle-walking</td>
			<td>2: no stepping</td>
		</tr>
		<tr>
			<td>3: movement in lower extremities but unable to knuckle-walk</td>
			<td></td>
		</tr>
		<tr>
			<td>4: no movement, drags lower extremities</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Evaluation of Ambulation and the Placing/stepping Reflex.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aortic occlusion time&#160;</td>
			<td>24 h after surgery&#160;</td>
		</tr>
		<tr>
			<td>9 min (n = 3)</td>
			<td>2 (2 &#8722; 3)</td>
		</tr>
		<tr>
			<td>&#160; 10 min (n = 3)</td>
			<td>4 (4 &#8722; 4)</td>
		</tr>
		<tr>
			<td>&#160; 11 min (n = 3)</td>
			<td>5 (5 &#8722; 6)</td>
		</tr>
	</tbody>
</table>
Table 2. Motor Deficit Index. Values are presented as median (interquartile range).

Watch this Scientific Journal Video about Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia at JoVE.com

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

reproducible-motor-deficit-following-aortic-occlusion-rat-model

Nanyang Technological University

Research

JoVE Journal

Medicine

7.1K Views.  SMG-SNU Boramae Medical Center, Seoul National University College of Medicine, Seoul. This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

Video: Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Neuroscience

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.

Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains ...

Middle Cerebral Artery Occlusion in Rats with Preserved Vascular Anatomy

A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have significantly contributed to our understanding of its pathological mechanisms and the development of potential treatments. Presently, there are two common methods involving filament (endovascular suture) techniques to induce animal models of cerebral ischemia. However, these methods have inherent limitations, such as reduced blood perfusion to the brain, damage to the external carotid artery system, impaired food and/or water intake, and sensory dysfunction of the face. This article introduces a new method for inducing a rat ischemic stroke model without compromising the cerebral vascular anatomy. In this study, the common carotid artery (CCA) of Sprague-Dawley rats was exposed, and an incision was made. A filament was then inserted through the incision into the internal carotid artery to occlude the middle cerebral artery. After 1.5 h of induced ischemia, the occluding filament was fully removed from both the internal carotid artery and the CCA. The incision in the CCA was subsequently sutured using 11-0 microsurgical sutures under a microscope (magnification 4x). Through the utilization of microsurgical techniques to repair the CCA, this study successfully developed a unique method to induce an ischemic stroke model in rats while preserving the anatomical integrity of cerebral blood vessels.

The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have ...

Immunofluorescence Staining to Study Cell Death in Rat MCAO Stroke Models

Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery (MCA). Current pharmacological approaches for the treatment of ischemic stroke are limited; therefore, novel therapies providing effective neuroprotection against ischemic injury following stroke are urgently needed. In the brain tissue following ischemic stroke, the area of the ischemic penumbra is salvageable but at risk of progressing to irreversible damage. The penumbral area surrounding the infarcted core is a conceptual target for neuroprotection. Since the blood flow is partially maintained in the penumbral area, neuronal and non-neuronal cells in this area transiently survive after stroke, and these cells that are still viable may be rescued by appropriate medical interventions. Understanding the pathophysiology of the penumbra is important in the development of neuroprotective therapies because the cell death pathways activated in the ischemic penumbra may indicate therapeutic targets, such as RUNX1 and cathepsins. These protein targets functioning as mediators of programmed cell death can be further exploited in translational research. With moderate size and similarity to the human brain, the in vivo rat model of MCA occlusion (MCAO) mimics the human ischemic stroke and offers an applicable tool to investigate the penumbral pathology, examine the cell death signaling, and evaluate the effects of potential targets in the context of MCAO. Here, we describe how to induce MCAO in rats and how to perform immunofluorescence staining for the detection of cell death signaling in the rat brain following MCAO.

Rat in vivo models are indispensable tools to investigate the pathological mechanisms and therapeutic targets for ischemic stroke. This work will describe performing immunofluorescence staining in infarcted brain slices following middle cerebral artery occlusion (MCAO) in rats.

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery ...

Single-Step Surgery for Inducing Cerebral Ischemic Coma in Rats

Cerebral Ischemic Coma Model Induced by Modified Four-Vessel Occlusion

Coma caused by cerebral ischemia is the most serious complication of cerebral ischemia. Four-vessel occlusion can establish a cerebral ischemic coma model for disease research and drug development. However, the commonly used four-vessel occlusion method mainly involves inserting an electrocoagulation pen into the bilateral pterygoid foramen of the first cervical vertebra behind the neck to electrocoagulate the vertebral arteries. This process carries the risk of incomplete electrocoagulation, bleeding, and damage to the brainstem and spinal cord. Twenty-four hours after surgery, re-anesthetized rats undergo carotid artery ligation in front of the neck. Two surgeries expose the rats to a higher risk of infection and increase the experimental period. In this study, during a single surgical procedure, an anterior cervical incision was used to locate the key site where the vertebral artery penetrates the first cervical vertebra. The bilateral vertebral arteries were electrocauterized under visual conditions, while the bilateral common carotid arteries were separated to place loose knots. When the limbs of the rats began twitching, the bilateral common carotid arteries were quickly ligated to induce ischemic coma. This method can avoid the risk of infection caused by two surgical operations and is easy to perform with a high success rate, providing a useful reference for relevant practitioners.

Author Spotlight: Enhancing Cerebral Ischemia Research with a Simplified Rat Model

This protocol describes the process for inducing a cerebral ischemic coma model using a modified four-vessel occlusion method.

Coma caused by cerebral ischemia is the most serious complication of cerebral ischemia. Four-vessel occlusion can establish a cerebral ischemic coma model ...