Establishment Of Ischemic Stroke Model With Interventional Techniques And Imaging Evaluation In Canine

Background and purpose:Ischemic stroke is one of the leading causes of death and the main reason for long-term disability. An appropriate animal model of ischemic stroke is urgently required for understanding the exact pathophysiological mechanism of stroke and testing any new therapeutic regimen. The purposes of our serial experiments were:(1) to establish a canine stroke model occluding middle cerebral artery (MCA) and blocking ipsilateral internal carotid artery (ICA), and assess the infarct lesions by magnetic resonance imaging (MRI).(2) to establish a canine lacunar infarction model by means of occluding the proximal middle cerebral artery with single autologous clot, and analyze its feasibility and reproducibility.(3) to evaluate the potential efficacy of FLAIR-related techniques in identifying the onset time of cerebral ischemia in a canine embolic stroke model.(4) to evaluated the temporal change of the relative DWI signal intensity (rDWI), relative ADC value (rADC), relative FLAIR signal intensity (rFLAIR), and relative T2signal intensity (rT2), and further compare their diagnostic value in identifying the hyperacute lesions based on the embolic canine model with clear onset time.Materials and methods:(1) Fifteen healthy adult beagle dogs were used in this study. Under general anesthesia, bilateral internal carotid angiography was performed with5F catheter to evaluate the brain vascular anatomy. Stroke model was generated by injecting two autologous clots into MCA, followed by ipsilateral ICA blockade (ilICAB) for two hours using a catheter in adult beagle dogs. Outcome measurements included24-hour and7-day post-occlusion T2-weighted imaging (T2WI)-based infarct volume calculation. In addition, pial collateral score, canine neurobehavioral score and histopathologic results were documented.(2) Eight healthy adult beagle dogs were used. Then, the lacunar infarction model was generated by injecting single autologous clot to occluding the proximal middle cerebral artery after anatomic cerebral angiography. Outcome measurements included the location and size of infarction lesions on T2-weighted imaging (T2WI). In addition, canine neurobehavioral scores at24hours after occlusion were documented. On the seventh day, the histopathologic examination was done following repeated cerebral angiography.(3) An embolic ischemic model was generated with an autologous clot in24beagle dogs. Both FLAIR and DWI were performed at3hours,4hours,5hours,6hours, and24hours after embolization, respectively. Visual "DWI-FLAIR mismatch" was defined as hyperintense signal detected on DWI but not on FLAIR. The relative signal intensity of FLAIR-positive lesions and the degree of DWI-FLAIR mismatch was calculated as relative FLAIR=relative signal intensity of FLAIR positive lesions, mismatch degree=(100-VFLAIR/VDWI) x100%.(4) Cerebral ischemic models were established in twenty-four adult healthy beagle dogs. Imaging acquisitions including DWI, FLAIR, T2WI, ADC were performed serially at3,4,5,6and24h after the left MCA embolization, respectively. DWI+, FLAIR+or T2+were defined as new hyperintense signals detected on DWI, FLAIR or T2images. Interobserver and intraobserver agreement for quantitative judgement of rDWI, rADC, rFLAIR, and rT2was assessed with Pearson correlation coefficient. ROC curves for rDWI, rADC, rFLAIR and rT2value in identifying hyperacute lesions within4h were paired compared with Wilcoxon analyze. P<0.05was considered statistically significant. Statistical analysis was carried out with the SPSS16.0.Results: (1) Twelve dogs (12/15) with left middle cerebral artery occlusion (MCAO) and ilICAB survived7days without complications or casulties, and MCA got reperfused at7days after occlusion. High signal intensity in the basal ganglia and cerebral cortex on T2WI was initially observed in each dog at6hours after procedure. The mean percentage hemispherical infarct volume corrected for edema in all dogs on T2WI at24hours after occlusion was12.99±1.57%, and the degree of variability was12.08%. The infarct volumes at24hour correlated with pial collateral scores and canine neurobehavioral scores well.(2) Six dogs were successfully created the lacunar infarction models with the proximal middle cerebral artery occlusion. The technical success rate was75%. All these dogs survived7days without complications or casulties, and MCA got reperfused at7days after occlusion. High and small size signal intensity in the basal ganglia and cerebral cortex on T2WI was observed in all dogs at6hours post procedure. The mean size of these infarction lesions was5.8±0.57mm (range4.7mm to9.6mm) at24hours. The mean canine neurobehavioral score was3.8±0.40at24hours after procedure. The size of lacunar infarction lesions at24hours was not significant correlated with the canine neurobehavioral scores (r2=0.0635, P=0.630). The lacunar infarction lesions were confirmed by pathology eventually.(3) The ischemic model was successfully established in20animals. FLAIR-positive lesions were seen in3,11,16,19, and20beagle dogs at5time points after embolization, respectively. There was significant correlation between the relative FLAIR and the onset time, degree of DWI-FLAIR mismatch and the onset time (relative FLAIR:r=+0.42;95%CI,0.20-0.60; mismatch degree:r=-0.85;95%CI,0.89-0.78). Receiver operating characteristic curves showed that the degree of DWI-FLAIR mismatch could identify the hyperacute ischemic lesions with a sensitivity range from1.00to0.76; visual DWI-FLAIR mismatch sensitivity ranged from0.85to0.39, whereas specificity was0.83-0.95versus0.85-1.00.(4) Four dogs with incomplete proximal MCA embolization were excluded for further analysis. Twenty ischemic models were successfully established. All rSI values were linearly correlated to time with significance until24h after model establishment (P<0.05). Paired comparison of ROC curves showed that significant difference was found between rADC and other three rSIs (P<0.0001). However, no significant difference was found among rDWI, rT2and rFLAIR.Conclusions:(1) This canine ischemic stroke model with combined MCAO and ilICAB reported here proven to be highly feasible and reproducible in large animal settings. Preliminary findings indicated that the infarct volumes at24-hour postocclusion were correlated with leptomeningeal collateral scores and canine neurobehavioral scores. This model may serve as a powerful preclinical ischemic stroke model to overcome the translational gap that exists between basic science and clinical research.(2) This canine lacunar infarction model with proximal middle cerebral artery occlusion with single autologous clot reported here proven to be highly feasible and reproducible. This model may serve as a bridge to understand the exact pathophysiological mechanism of human lacunar infarction stroke, which could provide the basic science for further clinical research.(3) The relative FLAIR and DWI-FLAIR mismatch values were useful in predicting the onset time in this canine embolic stroke model. The degree of DWI-FLAIR mismatch proposed in this study could be a good indicator with high sensitivity for identifying the hyperacute ischemic stroke.(4) Based on this embolic ischemic model with clear onset time of stroke, we found that the rDWI, rFLAIR and rT2may be helpful to age the ischemic events with similar diagnostic value. However, rADC does not seem comparable based on our embolic canine model.