This work was supported by the National Natural Science Foundation of China (82173781 and 82373835), Postdoctoral research project (BKS212055), Science and Technology Innovation Project of Foshan Science and Technology Bureau (2320001007331), Guangdong Basic and Applied Basic Research Foundation (2019A1515010806), Key Field Projects (Intelligent Manufacturing) of General Universities in Guangdong Province (2020ZDZX2057), and the Scientific Research Projects (Characteristic Innovation) of General Universities in Guangdong Province (2019KTSCX195).

The experimental protocol was conducted in accordance with the requirements of the Use of Laboratory Animals and Institutional Animal Care and Use Committee at Foshan University (Record number: 2023-643656). Male Sprague Dawley (SD) rats (200 g &#177; 20 g, 6-8 weeks old) were used for this study. All animal research data have been written up in accordance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. The details of the reagents and equipment used in the study are listed in the Tabl...

Single-Step Surgery for Inducing Cerebral Ischemic Coma in Rats

<ol>
	<li>Hou, Y., 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=Rhodiola+crenulata+alleviates+hypobaric+hypoxia-induced+brain+injury+by+maintaining+BBB+integrity+and+balancing+energy+metabolism+dysfunction.">Rhodiola crenulata alleviates hypobaric hypoxia-induced brain injury by maintaining BBB integrity and balancing energy metabolism dysfunction.</a> Phytomedicine. 128, 155529 (2024).</li><li>Rong, 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=A+modified+model+preparation+for+middle+cerebral+artery+occlusion+reperfusion.">A modified model preparation for middle cerebral artery occlusion reperfusion.</a> J Vis Exp. 207, e67060 (2024).</li><li>Hoesch, R. E., Koenig, M. A., Geocadin, R. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+bilateral+hemispheric+ischemia+in+the+unanesthetized+rat.">A new model of bilateral hemispheric ischemia in the unanesthetized rat.</a> Stroke. 10 (3), 267-272 (1979).</li><li>Plaschke, 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=Pronounced+arterial+collateralization+was+induced+after+permanent+rat+cerebral+four-vessel+occlusion.+Relation+to+neuropathology+and+capillary+ultrastructure.">Pronounced arterial collateralization was induced after permanent rat cerebral four-vessel occlusion. Relation to neuropathology and capillary ultrastructure.</a> J Neural Transm. 110 (7), 719-732 (2003).</li><li>Sugio, K., Horigome, N., Sakaguchi, T., Goto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+bilateral+hemispheric+ischemia-+modified+four-vessel+occlusion+in+rats:+To+the+editor.">A model of bilateral hemispheric ischemia- modified four-vessel occlusion in rats: To the editor.</a> Stroke. 19 (7), 922 (1988).</li><li>Sadelli, 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=Global+cerebral+ischemia+in+rats+leads+to+amnesia+due+to+selective+neuronal+death+followed+by+astroglial+scar+formation+in+the+CA1+layer.">Global cerebral ischemia in rats leads to amnesia due to selective neuronal death followed by astroglial scar formation in the CA1 layer.</a> Neurobiol Learn Mem. 141, 168-178 (2017).</li><li>Song, B. W., 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=1H-pyrrole-2,5-dione-based+small+molecule-induced+generation+of+mesenchymal+stem+cell-derived+functional+endothelial+cells+that+facilitate+rapid+endothelialization+after+vascular+injury.">1H-pyrrole-2,5-dione-based small molecule-induced generation of mesenchymal stem cell-derived functional endothelial cells that facilitate rapid endothelialization after vascular injury.</a> Stem Cell Res Ther. 6 (1), 174 (2015).</li><li>Sun, W., 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+modified+four+vessel+occlusion+model+of+global+cerebral+ischemia+in+rats.">A modified four vessel occlusion model of global cerebral ischemia in rats.</a> J Neurosci Methods. 352, 109090 (2021).</li><li>Xiaobing, J., 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=Modifying+the+four+vessel+occlusion+to+establish+ischemic+brain+injury+coma+model+in+rats.">Modifying the four vessel occlusion to establish ischemic brain injury coma model in rats.</a> Sichuan Med J. 29 (4), 384-386 (2008).</li><li>Wang, W., 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=Levodopa+improves+cognitive+function+and+the+deficits+of+structural+synaptic+plasticity+in+hippocampus+induced+by+global+cerebral+ischemia/reperfusion+injury+in+rats.">Levodopa improves cognitive function and the deficits of structural synaptic plasticity in hippocampus induced by global cerebral ischemia/reperfusion injury in rats.</a> Front Neurosci. 14, 586321 (2020).</li><li>Zhan, L., Lu, X., Xu, W., Sun, W., Xu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+MLKL-dependent+necroptosis+via+downregulating+interleukin-1R1+contributes+to+neuroprotection+of+hypoxic+preconditioning+in+transient+global+cerebral+ischemic+rats.">Inhibition of MLKL-dependent necroptosis via downregulating interleukin-1R1 contributes to neuroprotection of hypoxic preconditioning in transient global cerebral ischemic rats.</a> J Neuroinflammation. 18 (1), 97 (2021).</li><li>Konaka, K., Miyashita, K., Ishibashi-Ueda, H., Naritomi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+hyperthermia+caused+by+four-vessel+occlusion+of+main+cerebral+arteries.">Severe hyperthermia caused by four-vessel occlusion of main cerebral arteries.</a> Internal Med. 48 (24), 2137-2140 (2009).</li><li>Ferreira, E. D. F., Romanini, C. V., Mori, M. A., de Oliveira, R. M. W., Milani, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Middle-aged,+but+not+young,+rats+develop+cognitive+impairment+and+cortical+neurodegeneration+following+the+four-vessel+occlusion/internal+carotid+artery+model+of+chronic+cerebral+hypoperfusion.">Middle-aged, but not young, rats develop cognitive impairment and cortical neurodegeneration following the four-vessel occlusion/internal carotid artery model of chronic cerebral hypoperfusion.</a> Eur J Neurosci. 34 (7), 1131-1140 (2011).</li><li>Ma, B., 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=Protective+effects+of+extract+of+Coeloglossum+viride+var.+bracteatum+on+ischemia-induced+neuronal+death+and+cognitive+impairment+in+rats.">Protective effects of extract of Coeloglossum viride var. bracteatum on ischemia-induced neuronal death and cognitive impairment in rats.</a> Behav Pharmacol. 19 (4), 325-333 (2008).</li><li>Song, Y. Y., Chen, Y. H., Li, J. F., Sun, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+animal+models+of+vascular+dementia.">A review of animal models of vascular dementia.</a> Chin J Comp Med. 33 (12), 75-85 (2023).</li><li>Liu, B., 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=Autophagy+activation+aggravates+neuronal+injury+in+the+hippocampus+of+vascular+dementia+rats.">Autophagy activation aggravates neuronal injury in the hippocampus of vascular dementia rats.</a> Neural Regen Res. 9 (13), 1288-1296 (2014).</li><li>Li, J., Takeda, Y., Hirakawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threshold+of+ischemic+depolarization+for+neuronal+injury+following+four-vessel+occlusion+in+the+rat+cortex.">Threshold of ischemic depolarization for neuronal injury following four-vessel occlusion in the rat cortex.</a> J Neurosurg Anesthesiol. 12 (3), 247-254 (2000).</li><li>Rishitha, N., Muthuraman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ameliorative+potential+of+thymoquinone+in+four+vessel+occlusion+induced+vascular+dementia+in+rats.">Ameliorative potential of thymoquinone in four vessel occlusion induced vascular dementia in rats.</a> Alzheimer Dement. 19 (S13), e71053 (2023).</li><li>Oruc, S., 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+antioxidant+and+antiapoptotic+effects+of+crocin+pretreatment+on+global+cerebral+ischemia+reperfusion+injury+induced+by+four+vessels+occlusion+in+rats.">The antioxidant and antiapoptotic effects of crocin pretreatment on global cerebral ischemia reperfusion injury induced by four vessels occlusion in rats.</a> Life Sci. 154, 79-86 (2016).</li><li>Lu, D., 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+modified+method+to+reduce+variable+outcomes+in+a+rat+model+of+four-vessel+arterial+occlusion.">A modified method to reduce variable outcomes in a rat model of four-vessel arterial occlusion.</a> Neurol Res. 38 (12), 1102-1110 (2016).</li><li>Idt-Kastner, R. S., Paschen, W., Ophoff, B. G., Hossmann, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+four-vessel+occlusion+model+for+inducing+incomplete+forebrain+ischemia+in+rats.">A modified four-vessel occlusion model for inducing incomplete forebrain ischemia in rats.</a> Stroke. 20 (7), 938-946 (1989).</li><li>McBean, D. E., Kelly, P. A. 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+global+cerebral+ischemia:+A+comparison+of+two-vessel+occlusion+and+four-vessel+occlusion.">Rodent models of global cerebral ischemia: A comparison of two-vessel occlusion and four-vessel occlusion.</a> Gen Pharmacol. 30 (4), 431-434 (1998).</li><li>Todd, N. V., Picozzi, P., Alan Crockard, H., Russell, A. R. 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Y., 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=Potential+targets+for+protecting+against+hippocampal+cell+apoptosis+after+transient+cerebral+ischemia-reperfusion+injury+in+aged+rats.">Potential targets for protecting against hippocampal cell apoptosis after transient cerebral ischemia-reperfusion injury in aged rats.</a> Neural Regen Res. 9 (11), 1122-1128 (2014).</li><li>Mart&#237;nez-Alonso, E., 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=Differential+association+of+4E-BP2-interacting+proteins+is+related+to+selective+delayed+neuronal+death+after+ischemia.">Differential association of 4E-BP2-interacting proteins is related to selective delayed neuronal death after ischemia.</a> Int J Mol Sci. 22 (19), 10327 (2021).</li></ol>

Four-vessel occlusion induces global ischemic and hypoxic brain injury, which can simulate acute coma, cardiac arrest, asphyxia, shock, severe arrhythmia, and other critical clinical conditions caused by cerebral ischemia in clinical practice. Meanwhile, four-vessel occlusion can lead to damage mainly in the hippocampus17,18, which is the primary functional brain area responsible for cognitive memory19,20...

Ischemic brain injury is the most common brain injury in clinical practice, accounting for approximately 75% of cerebrovascular disease cases. Ischemia can lead to severe secondary brain injuries and diseases1,2, and coma is the most severe symptom caused by ischemic hypoxic brain injury. It is also the final pathway for many critical conditions3. Coma is a critical and severe illness in clinical practice that is difficult to manage4. The longer the coma lasts, the greater the potential danger. Prompt awakening is the primary goal in preventing the deterioration and progression of the condition. Although naloxone injection has a wide range of clinical applications in promoting wakefulness, it still has some side effects5. Therefore, the development of safe and effective wakefulness-promoting drugs is an urgent problem that needs to be addressed. Establishing a simple and easy-to-operate brain ischemic coma model is essential for elucidating the pathogenesis of ischemic coma and for drug development6,7,8.
The purpose of this study is to introduce a model of inducing global ischemic coma through electrocoagulation of bilateral vertebral arteries (VA) and temporary ligation of bilateral common carotid arteries (CCA), which is simple and user-friendly for novices. The previous protocol involved exposing the bilateral pterygoid foramen of the first posterior cervical vertebra during the first operation and electrically burning the pterygoid foramen to block the bilateral VAs. A second operation was performed 24 h later to induce total ischemic coma by ligation of the bilateral CCAs9,10,11,12. However, due to invisibility, there is a risk of incomplete electrocoagulation, bleeding, brainstem, and spinal cord injury, as well as a prolonged experimental period. Therefore, it is necessary to address these issues.
Here, we present an improved method for modeling ischemic coma. The main procedure involves making a median anterior neck incision, performing electrical resection of the bilateral VAs under visual conditions, and briefly ligating the bilateral CCAs during a single operation to block the blood supply to the entire brain, causing rapid electroencephalogram (EEG) inhibition and leading to coma. This method also induces a brief continuous coma after reperfusion. This procedure is easy to perform, novice-friendly, and reduces the risk of secondary trauma infection in animals, thereby shortening the experimental period.
The protocol is suitable for the study of global ischemic coma caused by cardiac arrest. It is also ideal for the study of ischemic dementia, mainly because the hippocampal brain area is extremely sensitive to ischemia; thus, transient cerebral ischemia can lead to damage or even loss of hippocampal neurons13, resulting in cognitive dysfunction. Therefore, the protocol can provide a reference for practitioners studying cerebral ischemia, ischemic coma, and ischemic dementia.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16 channel microfiber photoelectrode array</td>
			<td>Jiangsu Yige Biotechnology Co., Ltd</td>
			<td>2605</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Surgical suture</td>
			<td>Nantong Holycon Medical Devices Co.,Ltd.</td>
			<td>B-104</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 Surgical suture</td>
			<td>Ningbo MEDICAL Needle Co., Ltd.</td>
			<td>JM1216-742417</td>
			<td></td>
		</tr>
		<tr>
			<td>EEG electrode</td>
			<td>Kedou Brain machine Technology Co., LTD</td>
			<td>KD-EEGEMG</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrocoagulation pen</td>
			<td>CONPUVON Company</td>
			<td>465</td>
			<td></td>
		</tr>
		<tr>
			<td>Lunion Stage Automatic Sleep Staging System</td>
			<td>Shanghai Lulian Intelligent Technology Co., Ltd.</td>
			<td>1336</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniature hand-held skull drill</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>87001</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin sodium</td>
			<td>Chengdu Kelong Chemical Co., Ltd.</td>
			<td>17121709-2</td>
			<td></td>
		</tr>
		<tr>
			<td>SD rats</td>
			<td>SPF ( Beijing ) Biotechnology Co.,Ltd.</td>
			<td>180-220g</td>
			<td></td>
		</tr>
		<tr>
			<td>Skull nail</td>
			<td>GLOBALEBIO,LTD</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic instrument</td>
			<td>Rayward Life Technology Co., Ltd</td>
			<td>68801</td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil 50</td>
			<td>Vic Trading (Shanghai) Co., LTD</td>
			<td>BN 88SHA</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

Cerebral Ischemic Coma Model Induced by Modified Four-Vessel Occlusion

The scope of this research is to introduce a model of global ischemic coma, induced by electrocoagulation of the vertebral artery and the temporary ligation of the common carotid artery simultaneously, which is simple and useful, friendly for novices. We present a improved method for the modeling ischemic coma. This procedure is easy to perform, novice-friendly, and reduce the risk of secondary trauma effects in animals, shortening the experimental period.Our protocol enable novice to learn the methods of global ischemic coma more quickly, contributing to exploration of a pathologic mechanism and the development of new drugs.

Due to inflammation and other stimulation caused by the implantation of electrodes, the EEG may be unstable, so the rats need to recover for 3 days. Rats with normal EEG and EMG after 3 days could be included for coma model preparation. When the rats were anesthetized, EEG and EMG activity was slightly suppressed but proceeded smoothly. There was no significant change in EEG and EMG activity after electrocoagulation blocking the bilateral VAs. After about 30 min, the drug was metabolized, the rats gradually regained cons...

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 ...

cerebral-ischemic-coma-model-induced-by-modified-four-vessel-occlusion