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 Table of Materials.
1. Implantation of EEG electrodes
<ol>
	<li>Inject 0.05mg/kg of atropine subcutaneously 15 min before anesthesia to prevent respiratory obstruction and asphyxia caused by secretions. Administer an intramuscular injection of 20 mg/kg zoletil and 5 mg/kg xylazine to anesthetize the rats14. Use tweezers to clamp the toes of the rat to confirm deep anesthesia.</li>
	<li>Remove hair from the rat&#39;s head with a hair shaver. Fix the rat&#39;s head on a brain stereotaxic device using non-penetrating ear bars. Use sterile cotton balls to apply ethanol and povidone iodine three times to the surgical site to disinfect the skin.</li>
	<li>Cut the skin of the rat&#39;s head along the decapitated suture with a surgical blade. Remove the muscle covering the skull and completely expose the skull. Use sterile cotton swabs to stop bleeding throughout the process.</li>
	<li>Blow dry the surface of the skull with an ear wash ball to help dental cement adhere tightly to the skull. Mark the installation position of the skull nail (Diameter 1.2 mm, Length 3 mm) with a black marker (Figure 1, step 1). The specific positions are the anterior fontanelle point and four other sites.</li>
	<li>Use the needle of a 10 mL syringe to rotate and drill through four areas in sequence. Insert four skull nails into the skull in sequence, ensuring contact with the cerebral cortex (Figure 1, steps 2-3). 
	NOTE: Use sterile cotton swabs to absorb blood in case of bleeding to prevent rusting of the bone nails.</li>
	<li>Wrap the silver wire of the EEG electrode around the skull nail. Embed the electromyographic electrode into the muscle and fix it with a 6-0 suture.</li>
	<li>Mix the denture base resin with self-setting denture powder and fix the electrode to the skull. Use an ear wash ball to blow air onto the surface of the dental cement to accelerate curing.</li>
	<li>Inject 10,000 units of penicillin to prevent infection. House each rat in a separate cage to prevent mutual tearing and damage to the electrodes. Subcutaneously injected 0.2 mg/kg meloxicam for three consecutive days to alleviate postoperative pain. Allow 3 days for recovery of rat wounds and electrode fixation (Figure 1, step 4).</li>
</ol>
2. Surgical process of cerebral ischemic coma model
<ol>
	<li>Three days later, re-anesthetize the rats and place them in a supine position. Use sterile cotton balls to&#160;disinfect the surgical site via three repeated applications of iodine followed by an&#160;alcohol rinse/wipe. Make an incision about 2-3 cm long with a scalpel from the upper margin of the sternum lengthwise along the middle of the neck (Figure 1, step 5).</li>
	<li>Bluntly separate the subcutaneous tissue and sternohyoid muscle, fully exposing the trachea and the longus colli muscles on both sides of the trachea15. 
	NOTE: Avoid stimulating the trachea throughout the entire procedure.</li>
	<li>Bluntly separate the longus colli muscles from the level of the thyroid gland downward, exposing the first and second cervical vertebrae. Expand the neck area with a rat tissue dilator, fully exposing the surgical site.</li>
	<li>Use fine forceps to carefully separate the muscles and tissues visible in the cervical intervertebral space, exposing the characteristic location where the vertebral artery enters the first cervical vertebra. It can be observed that the vertebral artery passes through the first cervical vertebra (Figure 1, step 6). 
	NOTE: Placing a 1 mL syringe under the neck provides a clearer surgical manipulation space.</li>
	<li>Preheat the electrocoagulation pen and insert it into the area for 3-5 s to ensure that the vertebral artery is electrocoagulated and severed. Separate the muscles and fascia along the inner edge of the sternocleidomastoid muscle, expose and free the bilateral CCAs, and tie a loose knot. 
	NOTE: The electrocoagulation pen must be preheated; otherwise, it cannot quickly coagulate the vertebral artery, leading to bleeding. Both vertebral arteries are electrocoagulated and loose knots are tied around the CCAs.</li>
	<li>Quickly tighten the first loose knot to block blood flow in the CCA when the rats regain consciousness and exhibit limb twitching, while the EEG detector detects active EEG and EMG signals. The rats will struggle for a few seconds and then gradually lose consciousness (Figure 1, step 7).</li>
	<li>After releasing the fixation, observe that the limbs of the rat are stiff, the righting reflex disappears, but breathing is maintained. At this point, the electromyogram (EMG) presents a straight line, and the EEG is rapidly suppressed, indicating that the 4-VO induced cerebral ischemia model was successful16. 
	NOTE: Respiratory depression occurs in some rats during bilateral CCA ligation. Rapid mechanical stimulation can restore spontaneous respiration in some rats.</li>
	<li>According to the &#34;needle control tie method16,&#34; bind the CCA with a 0.5 mm diameter syringe needle using 6-0 nylon thread about 1.5 cm away from the bifurcation of the CCA, ICA, and ECA. Carefully pull out the needle; this second knot will subsequently cause the carotid artery to narrow (Figure 1, step 8). 
	NOTE: The ligature used for CCA stenosis needs to be made of nylon material, which is stable. Nylon thread is not affected by blood and does not thicken; otherwise, it can cause extreme stenosis of the CCA in rats and increase the mortality rate.</li>
	<li>After 30 min of ischemia, untie the first knot, and the CCA will undergo reperfusion, but the second knot will result in CCA stenosis, inducing a sustained coma (Figure 1, step 9). Close the subcutaneous tissue with absorbable monofilament sutures and the skin with non-absorbable monofilament sutures.</li>
</ol>
3. Animal recovery
<ol>
	<li>Place the rats on insulation pads and inject 10,000 units of penicillin to prevent infection.&#160;Subcutaneously inject&#160;0.2 mg/kg meloxicam for three consecutive days to alleviate postoperative pain.</li>
	<li>After 60 min of reperfusion, ensure the rats gradually recover.</li>
</ol>

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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coma+after+global+ischemic+brain+injury:+Pathophysiology+and+emerging+therapies.">Coma after global ischemic brain injury: Pathophysiology and emerging therapies.</a> Crit Care Clin. 24 (1), 25-44 (2008).</li><li>You, 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=Amplitude-integrated+electroencephalography+predicts+outcome+in+patients+with+coma+after+acute+brain+injury.">Amplitude-integrated electroencephalography predicts outcome in patients with coma after acute brain injury.</a> Neurosc Bull. 34 (4), 639-646 (2018).</li><li>He, F., Jiang, Y., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+naloxone+treatment+on+opioidinduced+side+effects+A+meta-analysis+of+randomized+and+controlled+trails.">The effect of naloxone treatment on opioidinduced side effects A meta-analysis of randomized and controlled trails.</a> Medicine (United States). 95 (37), e4729 (2016).</li><li>Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+of+coma+and+coma+arousal+therapy+in+Caraka+Sahit&#257;+and+its+corollary+in+modern+medicine.">Description of coma and coma arousal therapy in Caraka Sahit&#257; and its corollary in modern medicine.</a> Neurol India. 65 (2), 250-252 (2017).</li><li>Cossu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+options+to+enhance+coma+arousal+after+traumatic+brain+injury:+State+of+the+art+of+current+treatments+to+improve+coma+recovery.">Therapeutic options to enhance coma arousal after traumatic brain injury: State of the art of current treatments to improve coma recovery.</a> Brit J Neurosurg. 28 (2), 187-198 (2014).</li><li>Li, J., Cheng, Q., Liu, F. K., Huang, Z., Feng, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+stimulation+to+improve+arousal+in+comatose+patients+after+traumatic+brain+injury:+a+systematic+review+of+the+literature.">Sensory stimulation to improve arousal in comatose patients after traumatic brain injury: a systematic review of the literature.</a> Neurol Sci. 41 (9), 2367-2376 (2020).</li><li>Pulsinelli, W. A., Levy, D. E., Duffy, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+blood+flow+in+the+four-vessel+occlusion+rat+model.">Cerebral blood flow in the four-vessel occlusion rat model.</a> Stroke. 14 (5), 832-834 (1983).</li><li>Pulsinelli, W. A., Brierley, J. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reperfusion+after+cerebral+ischemia:+Influence+of+duration+of+ischemia.">Reperfusion after cerebral ischemia: Influence of duration of ischemia.</a> Stroke. 17 (3), 460-466 (1986).</li><li>Toda, 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=Highly+reproducible+rat+model+of+reversible+forebrain+ischemia+-+Modified+four-vessel+occlusion+model+and+its+metabolic+feature.">Highly reproducible rat model of reversible forebrain ischemia - Modified four-vessel occlusion model and its metabolic feature.</a> Acta Neurochir (Wien). 144 (12), 1297-1304 (2002).</li><li>Ma, R., Lu, D., Wang, J., Xie, Q., Guo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+pharmacological+activity+and+safety+of+different+stereochemical+configurations+of+borneol:+L-borneol,+D-borneol,+and+synthetic+borneol.">Comparison of pharmacological activity and safety of different stereochemical configurations of borneol: L-borneol, D-borneol, and synthetic borneol.</a> Biomed Pharmacother. 164, 114668 (2023).</li><li>Ji, X. 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>

The authors have nothing to disclose.

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><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.

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...

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.

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 ...

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

Research

JoVE Journal

Medicine

429 Views.  South China University of Technology. This protocol describes the process for inducing a cerebral ischemic coma model using a modified four-vessel occlusion method.

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

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

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

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 ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

A Model of Transient Cerebral Ischemia by Occlusion of Middle Cerebral Artery

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Author Spotlight: Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...

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 ...

Modified MCAO/R Model with Preserved ECA for Enhanced Reperfusion and Stability

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion

The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug development.However, among the commonly used modeling methods, the Koizumi method often faces scrutiny due to its ligation of the common carotid artery (CCA) and its inability to achieve adequate reperfusion. Similarly, the Longa method has been criticized for disconnecting and ligating the external carotid artery (ECA). This study aims to introduce a modified model preparation method that preserves the integrity of the ECA, involves inserting a monofilament nylon suture through the CCA, repairing the ligated CCA incision, and maintaining reperfusion from the CCA. Reperfusion of blood flow was confirmed using laser speckle flow imaging. Evaluation methods such as the Longa scale, Modified Neurological Severity Score, triphenyltetrazolium chloride (TTC) staining, and immunofluorescence labeling of neurons demonstrated that this approach could induce stable ischemic nerve damage. This modified MCAO/R model protocol is simple and stable, providing valuable guidance for practitioners in the field of cerebral ischemia.

Author Spotlight: A Novel Approach to Cerebral Ischemia Modeling &#8211; Enhancing Reperfusion and Simplifying Procedure

This protocol describes the process of preparing for middle cerebral artery occlusion reperfusion via the common carotid artery.

Cerebral Ischemic Coma Model Induced by Modified Four-Vessel Occlusion

Summary

Explore More Videos

Chapters in this video

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

Mouse Model of Middle Cerebral Artery Occlusion

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

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

Transient Middle Cerebral Artery Occlusion Model of Stroke

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

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion