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A novel embolic middle cerebral artery occlusion model induced by thrombus formed in common carotid artery in rat
Journal of the Neurological Sciences 359 (2015) 275–279
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
A novel embolic middle cerebral artery occlusion model induced by thrombus formed in common carotid artery in rat Yin-Zhong Ma a, Li Li a, Jun-Ke Song a, Zi-Ran Niu a, Hai-Feng Liu b,c, Xiang-Shan Zhou c, Fu-Sheng Xie b, Guan-Hua Du a,⁎ a b c
Beijing Key Laboratory of Drug Target and Screening Research, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China Shandong Ehua Biopharmaceutical Co., Ltd., Shandong 252201, China Shandong Dong-e E-Jiao Co., Ltd., Shandong 252201, China
a r t i c l e
i n f o
Article history: Received 23 June 2015 Received in revised form 31 August 2015 Accepted 21 September 2015 Available online 5 October 2015 Keywords: Stroke t-PA Thrombolytic Cerebral embolism Therapeutic time window Disease model
a b s t r a c t Stroke is a major cause of death and disability worldwide. However, treatment options to date are very limited. To meet the need for validating the novel therapeutic approaches and understanding the physiopathology of the ischemic brain injury, experimental stroke models were critical for preclinical research. However, commonly used embolic stroke models are reluctant to mimic the clinical situation and not suitable for thrombolytic timing studies. In this paper, we established a standard method for producing a rat embolic stroke model with autologous thrombus formed within the common carotid artery (CCA) by constant galvanic stimulation. Then the thrombus was shattered and channeled into the origin of the MCA and small (lacunar) artery. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured at 2, 4 and 6 h postischemia. This model developed a predictable infarct volume (38.37 ± 2.88%) and gradually reduced blood flow (20% of preischemic baselines) within the middle cerebral artery (MCA) territory. The thrombus occluded in the MCA was able to be lysed by a tissue-type plasminogen activator (t-PA) within 4 h postischemia. The techniques presented in this paper would help investigators to overcome technical problems for stroke research. © 2015 Published by Elsevier B.V.
1. Introduction Stroke is one of the leading causes of death and disability worldwide, but until now the treatment options for acute stroke remain limited [10]. To develop the novel therapeutic approaches and understanding the physiopathology of the ischemic brain injury, experimental strokes were valuable tools. An ideal experimental stroke model needs to have two features: occluded by autologous blood clots which can be lysed by thrombolytics, such as t-PA [1]; the thrombosis and infarction closely simulate human ischemic stroke. At present, two major types of ischemic stroke models have been developed: intraluminal suture middle cerebral artery occlusion (MCAO) and embolic MCAO. The intraluminal MCAO was developed by Koizumi in 1986 to simulate this impactful human pathology in rats. A modification of the MCAO method was later presented by Longa (Longa et al., 1989). Both techniques have been widely used to identify molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities. However, it does not reliably reproduce the
inhomogeneous vascular findings at the microvascular and macrovascular levels in acute territorial stroke [2]. Furthermore, the therapeutic window for therapeutic options may be substantially shorter in this model than in humans [5,9]. Embolic MCAO used blood drawn from the animal to form autologous clots of specific size and composition in vitro and are subsequently (e.g. 24 h later) introduced into the cerebral circulation via a cannula inserted and advanced along the internal carotid artery. Embolic MCAO causes most human strokes and therefore models that simulate this type of occlusion are useful for testing new thrombolytic agents. However, the current embolic model failed to mimic the thrombosis and thrombusaging since the thrombus was formed from dried blood clots in vitro or other artificial emboli. In this article, we demonstrated a standard method for producing embolic stroke with thrombus formed by galvanic stimulation in CCA in rats. This model could develop a predictable infarct volume within the MCA territory. The technique presented in this article was suitable for the study of thrombolytic time window. 2. Materials and methods
Abbreviations: t-PA, tissue-type plasminogen activator; CCA, common carotid artery; MCAO, middle cerebral artery occlusion; ECA, external carotid artery; ICA, internal carotid artery; PPA, pterygopalatine artery; PU, perfusion unit; CBF, cerebral focal perfusion. ⁎ Corresponding author. E-mail address: [email protected] (G.-H. Du).
http://dx.doi.org/10.1016/j.jns.2015.09.362 0022-510X/© 2015 Published by Elsevier B.V.
2.1. Experimental animals Male Sprague–Dawley rats (250–290 g) were purchased from Vital River Laboratory Animal Technology Co., Beijing. All animal experiments
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were approved by the Institutional Animal Care and Use Committee of the Peking Union Medical College and in accordance with the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals. All animals were acclimatized for 2 d or more prior to surgery.
2.2. Experimental embolic stroke models and treatments Sterilize all surgical tools by autoclaving (minimum 121°°C, 15 psi, for 20 min). Sanitize the surgery table and associated surgical equipment using 75% ethanol. Anesthetize the rat with isoflurane (5% for induction, 2–3% for maintenance) in 70% N2O and 30% O2 by a face mask. Confirm anesthesia by a toe pinch. Shave the fur on the ventral neck and head regions with electric clippers to expose the skin areas. Temperature of the rat was maintained at 36.5–37.5°°C using a homeothermic blanket control unit. Disinfect the shaved skin and surrounding fur with 10% povidone– iodine followed by 75% ethanol. Make a 2-cm-long midline incision on the neck. Use retractors to expose the surgical field and dissect the right CCA, external carotid artery (ECA), internal carotid artery (ICA), and pterygopalatine artery (PPA). Dissect the CCA free from the surrounding nerves and fascia (without harming the vagal nerve). Wipe dry the CCA with sterile cotton-wool mops. As was shown in Fig. 1A, three artery clamps were used to close PPA, ECA and the right CCA (the distal end). Put the CCA into the electric clamp from the YLS-14B thrombus formation tester (Jinan Yiyan Science & Technology Development Co., Ltd) and initiated the galvanic stimulation (1.00 mA) for 225 s. The thrombus could be observed (about 5 mm-long, dark black) after the clamp was removed. The thrombus was smashed 5–10 times by an ophthalmic forceps with serrated soft tip. Arteriopalmus was re-emerging when the thrombus pieces were
smashed into homogeneous pieces. The artery clamp on the right CCA was relieved for 1–3 s to flush the comminuted thrombus into the ICA and the CCA was reclipped for another 10–15 min to channel the thrombus into the MCA/lacunar artery by Willis circulation. Recheck the recanalization of CCA after relief of all the artery clamps. Closed the midline incision by suture. Laser-Doppler flowmetry (2 mm posterior, 5 mm lateral to bregma) was used to monitor cerebral perfusion throughout the surgery. Only rats that showed sustained ischemia to less than 20% of pre-embolic baselines were included. Animals with spontaneous revascularization before thrombolysis were excluded (6 of a total of 67 rats). Exclusion took place before assignment into the various treatment groups: vehicle saline injected at 2–6 h after ischemia (n = 10); t-PA infused as a 10% bolus, and the remainder was infused continuously over a 30 min interval at 2–6 h after ischemia (n = 10).
2.3. Monitoring on cerebral focal perfusion (CBF) Focal CBF was monitored by means of laser-Doppler flowmetry (Perimed, Sweden) throughout the surgery. Prior to thrombus occlusion, a 1.2 cm long midline incision was made in the scalp to expose the skull bone. The tissues on the skull bone were removed with a dental scraper and sterile cotton-wool mops. The laser-Doppler probe was sticked at 2 mm posterior and 5 mm lateral to the bregma above the intact dura. The perfusion unit (PU) value was recorded for 5 min as the baselines. The results were calculated as the percentage change of PU values at the endpoint compared with each baseline. The scalp incision was closed by suture after the measurement. Special attention should be paid to avoid the confounding effects of superfluous light, heterogeneous distribution of superficial blood vessels, and movement artifacts on laser-Doppler flowmetry.
Fig. 1. The CBF and mortality rate was measured in thromboembolic stroke model after the thrombolytic therapy. (A) Schematic diagram of the surgery. (B) Focal CBF was measured 0.25–6 h after the infarction continuously. t-PA treatment significantly restores the (C) focal CBF and lowers the (D) mortality rate. Data represent CBF as percentage change from postischemia baseline and values are means ± SEM of 10 experiments.
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2.4. Neurological deficit tests
3. Result
Neurological deficits in the animals were assessed 24 h after the surgery using a modified 5-point Bederson scale, as described in detail previously [6]. The scoring was as follows: 0, no deficit; 1, forelimb flexion deficit on contralateral side; 2, decreased resistance to lateral push and torso turning to the ipsilateral side when held by the tail; 3, very significant circling to the affected side and reduced capability to bear weight on the affected side; 4, animal rarely moves spontaneously and prefers to lay down or stay at rest.
3.1. General features of the embolic stroke model
2.5. Rotating rod test A rotating rod apparatus was modified and used to assess motor performance (diameter increased from 10 cm to 13 cm and lined with 1.5 mm thick rubber) [12]. Rats were placed on the elevated rotating rod, which was started at 22 rpm over 60 s. Timing started after hindpaw movement was observed. The scoring was as follows: 0 (walked on rod for more than 60 s), 1 (walked on rod for less than 60 s), 2 (fell off right after the rotation started), or 3 (fell off before the rotation started).
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The physiological parameters remained within the normal range in all groups. In the vehicle-treated group, there were 3 rats that died 1 d later of modeling and 4 in 10 rats died in 5 d. The overall mortality was 40% in vehicle-treated stroke-only rats. By contrast, the t-PA-treated animals had a significantly lower mortality (2 of 10 died in 5 d) as compared with the vehicle group. As was shown in Fig. 1B, once after the occlusion, cerebral perfusion in the middle cerebral artery territory dropped below 80% of preischemic baselines gradually. t-PA treatment significantly elevated the focal CBF and decreased the mortality to 20% (Fig. 1C–D). 3.2. Success rate of model In all stroke-only rats (67 rats), 11 rats died of intracranial hemorrhage and edema in 24 h later of modeling, and 6 rats were excluded from the success rate account as these rats were found to have spontaneous revascularization before thrombolysis. The success rate of our experimental embolic stroke model is 74.6%.
2.6. Forelimb function test 3.3. Analysis of brain lesion A grip strength meter was modified and used to assess the hemiparesis and strength of the forelimb [8]. A rope of 0.5 mm diameter and 20 cm long was fastened horizontally beyond a cage filled with dry wooden chips. Both forepaws were placed on the rope and scoring was as follows: 1 (both paws could grip the rope), 2 (only one paw could grip the rope) or 3 (no paws could grip).
Rats were sacrificed 24 h after the modeling and the infarct areas were assessed with TTC staining. The result showed that the infarct volume in the vehicle-treated group was 38.37 ± 2.88% which was close to the traditional embolic stroke model. t-PA treatment induced detectable recovery from the MCA embolism determined either by TTC or HE stained (Fig. 2A–C).
2.7. Inclined plane test
3.4. Evaluation of the thrombolytic time window
Motor performance was measured using a sliding apparatus [13]. The sliding apparatus had a 60 × 40 cm wood plane that could be inclined at an angle of 0° (horizontal) to 90°. There is a 25 × 15-cm rough plastic zone in the middle of the plane for the rat to grip. Each rat was initially placed on the 80°-angled incline plane 3 times to preaccommodate. Each trial was performed by calculating the time which the rat held its position in the plastic zone. Holding 120 s or longer was counted as 120 s. Each trial was performed after a 1-min interval. During this time the rat was returned to a cage filled with dry wooden chips, with which the limbs and body were wiped and dried so as not to influence the friction coefficient by urine and feces. Trials in which the rat held its position with one or more limbs slipping out of the plastic zone were not included in the result.
Delayed t-PA administration at 2, 4 and 6 h postischemia shows variety outcomes. t-PA treatment at 2–4 h restored perfusion reduced 24-h infarct volumes significantly, but no measurable improvement with delayed t-PA treatment at 6 h postischemia was observed (Fig. 3A–B). t-PA administration improved all four behavior tests compared to the vehicle group at 2 h and 4 h postischemia. However, no measureable change in behavior tests between the groups at 6 h after the thrombosis was observed.
2.8. Quantification of infarction volume After the neurological deficit test, rats were euthanized and decapitated and the brains were rapidly removed for the determination of infarct size. Brains were perfused with saline, and 8 coronal sections (2 mm thick) were stained with 0.5% 2,3,5-triphenyltetrazolium chloride (TTC) and fixed in 4% paraformaldehyde solution to quantify the percentage of infarct volumes. Edema correction was performed as previously described [7]. 2.9. Data and statistical analysis All results are presented as mean ± SEM. In most cases, statistical analysis was obtained by one-way analysis of variance (ANOVA) post-hoc Newman–Keuls analysis for multiple comparisons with SPSS. For comparison of the mortality between t-PA and vehicle, a χ2 test was performed with SPSS. P b 0.05 was set as statistically significant.
4. Discussion In this study, we demonstrated a standard method for performing an improved embolic stroke model in rats, in which the origin of the MCA territory is occluded by fresh thrombus. The major advantage of this model is: the occlusion of the MCA region with thrombus formed in CCA is similar to thromboembolic stroke in humans. This model does not require craniectomy and uses newly formed autologous thrombus in vivo, which is suitable for performing preclinical investigation of thrombolytic therapy. Compared with the traditional embolic stroke model, this model could also develop a reproducible and predictable infarct volume within the territory supplied by the MCA. To perform this model, the well-shattered thrombus and its lodgment is the key to success. Previous studies demonstrated that the dried blood clot introduction is difficult to manage. It could be easily lead to variations in infarct size and affected brain regions [3,14]. In this study, we demonstrated how to prepare thrombosis in CCA by constant current for 1.00 mA, lasting 225 s. This condition only applied on rats weighted 250-290 g according to our preliminary study (data not shown). To precisely lodge the thrombus and produce a reproducible embolic stroke model, CCA should be closed for 10–15 min right after the black shattered clots channeled into ICA. This is because the negative pressure caused by Willis circulation would draw the clots into the MCA zone. The shattered
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Fig. 2. The brain lesion was measured by TTC-staining (A) and HE-staining (C). t-PA was infused into the EJV right after the infarction and the brain tissue was fetched and subjected to each process 24 h after the ischemia onset. Data represent infarct volume as percent of ipsilateral hemisphere volume and values are means ± SEM of 5 experiments.
thrombus was also readily visualized at the origin of the MCA in all salinetreated rats but was largely dissolved in all t-PA-treated rats at 24 h after embolization. To evaluate the success of our model, the CBF, four animal behavior tests and the distribution of cerebral infarction were assessed. After the infarction, CBF was decreased to 30% of baseline and this value continued to drop to 20% at 6 h postischemia, consistent with previous reports [4, 15]. Without thrombolysis, 40% of the animals died within the first 3 d because of brain hemorrhage or edema. t-PA treatment significantly restored focal CBF, enhanced the survival rate and improved behavior recovery. Moreover, we demonstrated that the tissue lesion was produced mostly within the MCA territory as seen in the neocortex and striatum regions, and t-PA treatment significantly reduced the infarct size at 24 h after stroke. Together, our data demonstrated that the thrombus could be lysed by effective thrombolytic agents in at least 4 h postischemia. Finally, we note that there are several technical concerns that may hinder the success of the embolic MCAO model. A common problem encountered in performing the embolic MCAO model is early spontaneous reperfusion after embolization. The occurrence of spontaneous reperfusion is likely to be associated with the fragile extravascular coagulated clot used to occlude the MCA [3,11,14]. We believe that this method could meet the need of delayed thrombolysis study for the focal CBF
remains steady over at least 6 h. Animal behavior tests and brain lesion can be observed 24 h after the embolism. Thrombolysis with t-PA at 2– 4 h restored focal CBF, promoted behavior tests and reduced 24-h infarct volumes, but the lesion was obtained with delayed 6-h t-PA administration. This was probably due to the ischemia-reperfusion damage caused by delayed thrombolysis. 5. Conclusion The techniques presented in this article should help investigators to overcome technical problems in embolic stroke research. The surgery produced highly reproducible infarct size by fresh thrombus in a short time period (about 20–25 min) according to the protocol. Disclosure/conflict of interest There is no conflict of interest. Acknowledgments This work was funded by the Major Scientific and Technological Special Project for “Significant New Drugs Creation” (no. 2013ZX09508104), the National Natural Science Foundation of China (no. 81102444) and the
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Fig. 3. Delay t-PA treatment in thromboembolic stroke model. t-PA was infused into the EJV 2-6 h after the infarction and the focal CBF (A), infarct volume (B), neurologic deficit (C), rotating rod test (D), forelimb function test (E) and inclined plane test (F) were carried out. All data are presented as means ± SEM of 10 experiments.
Central Public Scientific Research Institution Fundamental Project (no. 2014CX05). References [1] R. Bhatia, M.D. Hill, N. Shobha, B. Menon, S. Bal, P. Kochar, T. Watson, M. Goyal, A.M. Demchuk, Low rates of acute recanalization with intravenous recombinant tissue plasminogen activator in ischemic stroke: real-world experience and a call for action, Stroke 41 (2010) 2254–2258. [2] G.J. del Zoppo, R.T. Higashida, A.J. Furlan, M.S. Pessin, H.A. Rowley, M. Gent, PROACT: a phase II randomized trial of recombinant pro-urokinase by direct arterial delivery in acute middle cerebral artery stroke. PROACT investigators. Prolyse in acute cerebral thromboembolism, Stroke 29 (1998) 4–11. [3] V.A. Dinapoli, C.L. Rosen, T. Nagamine, T. Crocco, Selective MCA occlusion: a precise embolic stroke model, J. Neurosci. Methods 154 (2006) 233–238. [4] U. Dirnagl, B. Kaplan, M. Jacewicz, W. Pulsinelli, Continuous measurement of cerebral cortical blood flow by laser-Doppler flowmetry in a rat stroke model, J. Cereb. Blood Flow Metab. 9 (1989) 589–596. [5] W. Hacke, M. Kaste, E. Bluhmki, M. Brozman, A. Davalos, D. Guidetti, V. Larrue, K.R. Lees, Z. Medeghri, T. Machnig, D. Schneider, R. von Kummer, N. Wahlgren, D. Toni, E. Investigators, Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke, N. Engl. J. Med. 359 (2008) 1317–1329. [6] M.N. Hoda, W. Li, A. Ahmad, S. Ogbi, M.A. Zemskova, M.H. Johnson, A. Ergul, W.D. Hill, D.C. Hess, I.Y. Sazonova, Sex-independent neuroprotection with minocycline after experimental thromboembolic stroke, Exp. Trans. Stroke Med. 3 (2011) 16. [7] K.Y. Lee, O.N. Bae, K. Serfozo, S. Hejabian, A. Moussa, M. Reeves, W. Rumbeiha, S.D. Fitzgerald, G. Stein, S.H. Baek, J. Goudreau, M. Kassab, A. Majid, Asiatic acid attenuates infarct volume, mitochondrial dysfunction, and matrix metalloproteinase-9 induction after focal cerebral ischemia, Stroke 43 (2012) 1632–1638.
[8] W. Li, Z. Qu, R. Prakash, C. Chung, H. Ma, M.N. Hoda, S.C. Fagan, A. Ergul, Comparative analysis of the neurovascular injury and functional outcomes in experimental stroke models in diabetic Goto-Kakizaki rats, Brain Res. 1541 (2013) 106–114. [9] S.R. Martini, T.A. Kent, Hyperglycemia in acute ischemic stroke: a vascular perspective, J. Cereb. Blood Flow Metab.: Off. J. Int. Soc. Cereb. Blood Flow Metab. 27 (2007) 435–451. [10] V.L. Roger, A.S. Go, D.M. Lloyd-Jones, E.J. Benjamin, J.D. Berry, W.B. Borden, D.M. Bravata, S. Dai, E.S. Ford, C.S. Fox, H.J. Fullerton, C. Gillespie, S.M. Hailpern, J.A. Heit, V.J. Howard, B.M. Kissela, S.J. Kittner, D.T. Lackland, J.H. Lichtman, L.D. Lisabeth, D.M. Makuc, G.M. Marcus, A. Marelli, D.B. Matchar, C.S. Moy, D. Mozaffarian, M.E. Mussolino, G. Nichol, N.P. Paynter, E.Z. Soliman, P.D. Sorlie, N. Sotoodehnia, T.N. Turan, S.S. Virani, N.D. Wong, D. Woo, M.B. Turner, American Heart Association Statistics C, Stroke Statistics S, Heart disease and stroke statistics — 2012 update: a report from the American Heart Association, Circulation 125 (2012) e2–e220. [11] K.M. Sicard, M. Fisher, Animal models of focal brain ischemia, Exp. Trans. Stroke Med. 1 (2009) 7. [12] S. van Gorp, M. Leerink, S. Nguyen, O. Platoshyn, M. Marsala, E.A. Joosten, Translation of the rat thoracic contusion model; part 2 — forward versus backward locomotion testing, Spinal Cord 52 (2014) 529–535. [13] F. Yonemori, T. Yamaguchi, H. Yamada, A. Tamura, Evaluation of a motor deficit after chronic focal cerebral ischemia in rats, J. Cereb. Blood Flow Metab. 18 (1998) 1099–1106. [14] R.L. Zhang, M. Chopp, Z.G. Zhang, Q. Jiang, J.R. Ewing, A rat model of focal embolic cerebral ischemia, Brain Res. 766 (1997) 83–92. [15] H. Zhu, X. Fan, Z. Yu, J. Liu, Y. Murata, J. Lu, S. Zhao, K.A. Hajjar, E.H. Lo, X. Wang, Annexin A2 combined with low-dose tPA improves thrombolytic therapy in a rat model of focal embolic stroke, J. Cereb. Blood Flow Metab.: Off. J. Int. Soc. Cereb. Blood Flow Metab. 30 (2010) 1137–1146.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
A novel embolic middle cerebral artery occlusion model induced by thrombus formed in common carotid artery in rat Yin-Zhong Ma a, Li Li a, Jun-Ke Song a, Zi-Ran Niu a, Hai-Feng Liu b,c, Xiang-Shan Zhou c, Fu-Sheng Xie b, Guan-Hua Du a,⁎ a b c
Beijing Key Laboratory of Drug Target and Screening Research, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China Shandong Ehua Biopharmaceutical Co., Ltd., Shandong 252201, China Shandong Dong-e E-Jiao Co., Ltd., Shandong 252201, China
a r t i c l e
i n f o
Article history: Received 23 June 2015 Received in revised form 31 August 2015 Accepted 21 September 2015 Available online 5 October 2015 Keywords: Stroke t-PA Thrombolytic Cerebral embolism Therapeutic time window Disease model
a b s t r a c t Stroke is a major cause of death and disability worldwide. However, treatment options to date are very limited. To meet the need for validating the novel therapeutic approaches and understanding the physiopathology of the ischemic brain injury, experimental stroke models were critical for preclinical research. However, commonly used embolic stroke models are reluctant to mimic the clinical situation and not suitable for thrombolytic timing studies. In this paper, we established a standard method for producing a rat embolic stroke model with autologous thrombus formed within the common carotid artery (CCA) by constant galvanic stimulation. Then the thrombus was shattered and channeled into the origin of the MCA and small (lacunar) artery. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured at 2, 4 and 6 h postischemia. This model developed a predictable infarct volume (38.37 ± 2.88%) and gradually reduced blood flow (20% of preischemic baselines) within the middle cerebral artery (MCA) territory. The thrombus occluded in the MCA was able to be lysed by a tissue-type plasminogen activator (t-PA) within 4 h postischemia. The techniques presented in this paper would help investigators to overcome technical problems for stroke research. © 2015 Published by Elsevier B.V.
1. Introduction Stroke is one of the leading causes of death and disability worldwide, but until now the treatment options for acute stroke remain limited [10]. To develop the novel therapeutic approaches and understanding the physiopathology of the ischemic brain injury, experimental strokes were valuable tools. An ideal experimental stroke model needs to have two features: occluded by autologous blood clots which can be lysed by thrombolytics, such as t-PA [1]; the thrombosis and infarction closely simulate human ischemic stroke. At present, two major types of ischemic stroke models have been developed: intraluminal suture middle cerebral artery occlusion (MCAO) and embolic MCAO. The intraluminal MCAO was developed by Koizumi in 1986 to simulate this impactful human pathology in rats. A modification of the MCAO method was later presented by Longa (Longa et al., 1989). Both techniques have been widely used to identify molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities. However, it does not reliably reproduce the
inhomogeneous vascular findings at the microvascular and macrovascular levels in acute territorial stroke [2]. Furthermore, the therapeutic window for therapeutic options may be substantially shorter in this model than in humans [5,9]. Embolic MCAO used blood drawn from the animal to form autologous clots of specific size and composition in vitro and are subsequently (e.g. 24 h later) introduced into the cerebral circulation via a cannula inserted and advanced along the internal carotid artery. Embolic MCAO causes most human strokes and therefore models that simulate this type of occlusion are useful for testing new thrombolytic agents. However, the current embolic model failed to mimic the thrombosis and thrombusaging since the thrombus was formed from dried blood clots in vitro or other artificial emboli. In this article, we demonstrated a standard method for producing embolic stroke with thrombus formed by galvanic stimulation in CCA in rats. This model could develop a predictable infarct volume within the MCA territory. The technique presented in this article was suitable for the study of thrombolytic time window. 2. Materials and methods
Abbreviations: t-PA, tissue-type plasminogen activator; CCA, common carotid artery; MCAO, middle cerebral artery occlusion; ECA, external carotid artery; ICA, internal carotid artery; PPA, pterygopalatine artery; PU, perfusion unit; CBF, cerebral focal perfusion. ⁎ Corresponding author. E-mail address: [email protected] (G.-H. Du).
http://dx.doi.org/10.1016/j.jns.2015.09.362 0022-510X/© 2015 Published by Elsevier B.V.
2.1. Experimental animals Male Sprague–Dawley rats (250–290 g) were purchased from Vital River Laboratory Animal Technology Co., Beijing. All animal experiments
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Y.-Z. Ma et al. / Journal of the Neurological Sciences 359 (2015) 275–279
were approved by the Institutional Animal Care and Use Committee of the Peking Union Medical College and in accordance with the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals. All animals were acclimatized for 2 d or more prior to surgery.
2.2. Experimental embolic stroke models and treatments Sterilize all surgical tools by autoclaving (minimum 121°°C, 15 psi, for 20 min). Sanitize the surgery table and associated surgical equipment using 75% ethanol. Anesthetize the rat with isoflurane (5% for induction, 2–3% for maintenance) in 70% N2O and 30% O2 by a face mask. Confirm anesthesia by a toe pinch. Shave the fur on the ventral neck and head regions with electric clippers to expose the skin areas. Temperature of the rat was maintained at 36.5–37.5°°C using a homeothermic blanket control unit. Disinfect the shaved skin and surrounding fur with 10% povidone– iodine followed by 75% ethanol. Make a 2-cm-long midline incision on the neck. Use retractors to expose the surgical field and dissect the right CCA, external carotid artery (ECA), internal carotid artery (ICA), and pterygopalatine artery (PPA). Dissect the CCA free from the surrounding nerves and fascia (without harming the vagal nerve). Wipe dry the CCA with sterile cotton-wool mops. As was shown in Fig. 1A, three artery clamps were used to close PPA, ECA and the right CCA (the distal end). Put the CCA into the electric clamp from the YLS-14B thrombus formation tester (Jinan Yiyan Science & Technology Development Co., Ltd) and initiated the galvanic stimulation (1.00 mA) for 225 s. The thrombus could be observed (about 5 mm-long, dark black) after the clamp was removed. The thrombus was smashed 5–10 times by an ophthalmic forceps with serrated soft tip. Arteriopalmus was re-emerging when the thrombus pieces were
smashed into homogeneous pieces. The artery clamp on the right CCA was relieved for 1–3 s to flush the comminuted thrombus into the ICA and the CCA was reclipped for another 10–15 min to channel the thrombus into the MCA/lacunar artery by Willis circulation. Recheck the recanalization of CCA after relief of all the artery clamps. Closed the midline incision by suture. Laser-Doppler flowmetry (2 mm posterior, 5 mm lateral to bregma) was used to monitor cerebral perfusion throughout the surgery. Only rats that showed sustained ischemia to less than 20% of pre-embolic baselines were included. Animals with spontaneous revascularization before thrombolysis were excluded (6 of a total of 67 rats). Exclusion took place before assignment into the various treatment groups: vehicle saline injected at 2–6 h after ischemia (n = 10); t-PA infused as a 10% bolus, and the remainder was infused continuously over a 30 min interval at 2–6 h after ischemia (n = 10).
2.3. Monitoring on cerebral focal perfusion (CBF) Focal CBF was monitored by means of laser-Doppler flowmetry (Perimed, Sweden) throughout the surgery. Prior to thrombus occlusion, a 1.2 cm long midline incision was made in the scalp to expose the skull bone. The tissues on the skull bone were removed with a dental scraper and sterile cotton-wool mops. The laser-Doppler probe was sticked at 2 mm posterior and 5 mm lateral to the bregma above the intact dura. The perfusion unit (PU) value was recorded for 5 min as the baselines. The results were calculated as the percentage change of PU values at the endpoint compared with each baseline. The scalp incision was closed by suture after the measurement. Special attention should be paid to avoid the confounding effects of superfluous light, heterogeneous distribution of superficial blood vessels, and movement artifacts on laser-Doppler flowmetry.
Fig. 1. The CBF and mortality rate was measured in thromboembolic stroke model after the thrombolytic therapy. (A) Schematic diagram of the surgery. (B) Focal CBF was measured 0.25–6 h after the infarction continuously. t-PA treatment significantly restores the (C) focal CBF and lowers the (D) mortality rate. Data represent CBF as percentage change from postischemia baseline and values are means ± SEM of 10 experiments.
Y.-Z. Ma et al. / Journal of the Neurological Sciences 359 (2015) 275–279
2.4. Neurological deficit tests
3. Result
Neurological deficits in the animals were assessed 24 h after the surgery using a modified 5-point Bederson scale, as described in detail previously [6]. The scoring was as follows: 0, no deficit; 1, forelimb flexion deficit on contralateral side; 2, decreased resistance to lateral push and torso turning to the ipsilateral side when held by the tail; 3, very significant circling to the affected side and reduced capability to bear weight on the affected side; 4, animal rarely moves spontaneously and prefers to lay down or stay at rest.
3.1. General features of the embolic stroke model
2.5. Rotating rod test A rotating rod apparatus was modified and used to assess motor performance (diameter increased from 10 cm to 13 cm and lined with 1.5 mm thick rubber) [12]. Rats were placed on the elevated rotating rod, which was started at 22 rpm over 60 s. Timing started after hindpaw movement was observed. The scoring was as follows: 0 (walked on rod for more than 60 s), 1 (walked on rod for less than 60 s), 2 (fell off right after the rotation started), or 3 (fell off before the rotation started).
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The physiological parameters remained within the normal range in all groups. In the vehicle-treated group, there were 3 rats that died 1 d later of modeling and 4 in 10 rats died in 5 d. The overall mortality was 40% in vehicle-treated stroke-only rats. By contrast, the t-PA-treated animals had a significantly lower mortality (2 of 10 died in 5 d) as compared with the vehicle group. As was shown in Fig. 1B, once after the occlusion, cerebral perfusion in the middle cerebral artery territory dropped below 80% of preischemic baselines gradually. t-PA treatment significantly elevated the focal CBF and decreased the mortality to 20% (Fig. 1C–D). 3.2. Success rate of model In all stroke-only rats (67 rats), 11 rats died of intracranial hemorrhage and edema in 24 h later of modeling, and 6 rats were excluded from the success rate account as these rats were found to have spontaneous revascularization before thrombolysis. The success rate of our experimental embolic stroke model is 74.6%.
2.6. Forelimb function test 3.3. Analysis of brain lesion A grip strength meter was modified and used to assess the hemiparesis and strength of the forelimb [8]. A rope of 0.5 mm diameter and 20 cm long was fastened horizontally beyond a cage filled with dry wooden chips. Both forepaws were placed on the rope and scoring was as follows: 1 (both paws could grip the rope), 2 (only one paw could grip the rope) or 3 (no paws could grip).
Rats were sacrificed 24 h after the modeling and the infarct areas were assessed with TTC staining. The result showed that the infarct volume in the vehicle-treated group was 38.37 ± 2.88% which was close to the traditional embolic stroke model. t-PA treatment induced detectable recovery from the MCA embolism determined either by TTC or HE stained (Fig. 2A–C).
2.7. Inclined plane test
3.4. Evaluation of the thrombolytic time window
Motor performance was measured using a sliding apparatus [13]. The sliding apparatus had a 60 × 40 cm wood plane that could be inclined at an angle of 0° (horizontal) to 90°. There is a 25 × 15-cm rough plastic zone in the middle of the plane for the rat to grip. Each rat was initially placed on the 80°-angled incline plane 3 times to preaccommodate. Each trial was performed by calculating the time which the rat held its position in the plastic zone. Holding 120 s or longer was counted as 120 s. Each trial was performed after a 1-min interval. During this time the rat was returned to a cage filled with dry wooden chips, with which the limbs and body were wiped and dried so as not to influence the friction coefficient by urine and feces. Trials in which the rat held its position with one or more limbs slipping out of the plastic zone were not included in the result.
Delayed t-PA administration at 2, 4 and 6 h postischemia shows variety outcomes. t-PA treatment at 2–4 h restored perfusion reduced 24-h infarct volumes significantly, but no measurable improvement with delayed t-PA treatment at 6 h postischemia was observed (Fig. 3A–B). t-PA administration improved all four behavior tests compared to the vehicle group at 2 h and 4 h postischemia. However, no measureable change in behavior tests between the groups at 6 h after the thrombosis was observed.
2.8. Quantification of infarction volume After the neurological deficit test, rats were euthanized and decapitated and the brains were rapidly removed for the determination of infarct size. Brains were perfused with saline, and 8 coronal sections (2 mm thick) were stained with 0.5% 2,3,5-triphenyltetrazolium chloride (TTC) and fixed in 4% paraformaldehyde solution to quantify the percentage of infarct volumes. Edema correction was performed as previously described [7]. 2.9. Data and statistical analysis All results are presented as mean ± SEM. In most cases, statistical analysis was obtained by one-way analysis of variance (ANOVA) post-hoc Newman–Keuls analysis for multiple comparisons with SPSS. For comparison of the mortality between t-PA and vehicle, a χ2 test was performed with SPSS. P b 0.05 was set as statistically significant.
4. Discussion In this study, we demonstrated a standard method for performing an improved embolic stroke model in rats, in which the origin of the MCA territory is occluded by fresh thrombus. The major advantage of this model is: the occlusion of the MCA region with thrombus formed in CCA is similar to thromboembolic stroke in humans. This model does not require craniectomy and uses newly formed autologous thrombus in vivo, which is suitable for performing preclinical investigation of thrombolytic therapy. Compared with the traditional embolic stroke model, this model could also develop a reproducible and predictable infarct volume within the territory supplied by the MCA. To perform this model, the well-shattered thrombus and its lodgment is the key to success. Previous studies demonstrated that the dried blood clot introduction is difficult to manage. It could be easily lead to variations in infarct size and affected brain regions [3,14]. In this study, we demonstrated how to prepare thrombosis in CCA by constant current for 1.00 mA, lasting 225 s. This condition only applied on rats weighted 250-290 g according to our preliminary study (data not shown). To precisely lodge the thrombus and produce a reproducible embolic stroke model, CCA should be closed for 10–15 min right after the black shattered clots channeled into ICA. This is because the negative pressure caused by Willis circulation would draw the clots into the MCA zone. The shattered
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Fig. 2. The brain lesion was measured by TTC-staining (A) and HE-staining (C). t-PA was infused into the EJV right after the infarction and the brain tissue was fetched and subjected to each process 24 h after the ischemia onset. Data represent infarct volume as percent of ipsilateral hemisphere volume and values are means ± SEM of 5 experiments.
thrombus was also readily visualized at the origin of the MCA in all salinetreated rats but was largely dissolved in all t-PA-treated rats at 24 h after embolization. To evaluate the success of our model, the CBF, four animal behavior tests and the distribution of cerebral infarction were assessed. After the infarction, CBF was decreased to 30% of baseline and this value continued to drop to 20% at 6 h postischemia, consistent with previous reports [4, 15]. Without thrombolysis, 40% of the animals died within the first 3 d because of brain hemorrhage or edema. t-PA treatment significantly restored focal CBF, enhanced the survival rate and improved behavior recovery. Moreover, we demonstrated that the tissue lesion was produced mostly within the MCA territory as seen in the neocortex and striatum regions, and t-PA treatment significantly reduced the infarct size at 24 h after stroke. Together, our data demonstrated that the thrombus could be lysed by effective thrombolytic agents in at least 4 h postischemia. Finally, we note that there are several technical concerns that may hinder the success of the embolic MCAO model. A common problem encountered in performing the embolic MCAO model is early spontaneous reperfusion after embolization. The occurrence of spontaneous reperfusion is likely to be associated with the fragile extravascular coagulated clot used to occlude the MCA [3,11,14]. We believe that this method could meet the need of delayed thrombolysis study for the focal CBF
remains steady over at least 6 h. Animal behavior tests and brain lesion can be observed 24 h after the embolism. Thrombolysis with t-PA at 2– 4 h restored focal CBF, promoted behavior tests and reduced 24-h infarct volumes, but the lesion was obtained with delayed 6-h t-PA administration. This was probably due to the ischemia-reperfusion damage caused by delayed thrombolysis. 5. Conclusion The techniques presented in this article should help investigators to overcome technical problems in embolic stroke research. The surgery produced highly reproducible infarct size by fresh thrombus in a short time period (about 20–25 min) according to the protocol. Disclosure/conflict of interest There is no conflict of interest. Acknowledgments This work was funded by the Major Scientific and Technological Special Project for “Significant New Drugs Creation” (no. 2013ZX09508104), the National Natural Science Foundation of China (no. 81102444) and the
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Fig. 3. Delay t-PA treatment in thromboembolic stroke model. t-PA was infused into the EJV 2-6 h after the infarction and the focal CBF (A), infarct volume (B), neurologic deficit (C), rotating rod test (D), forelimb function test (E) and inclined plane test (F) were carried out. All data are presented as means ± SEM of 10 experiments.
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