| Object1.To establish a stable, reproducible, standardized middle cerebral artery occlusion (MCAO) model with suture method in rabbit, and approach a method for quantitative detection of the scope and volume of acute focal cerebral ischemia by using trends of EEG [amplitude integrated EEG (aEEG) and band power] and modified neurological severity scores (mNSS).2.To establish a carotid artery selective brain hypothermia model of cold saline infusion in rabbit, explore appropriate perfusion temperature, perfusion speed and perfusion time, and approach the impact of cold saline infusion on vital signs, blood gas, serum sodium and blood.3.Suitable animals with aEEG screening after MCAO were treated with selective brain hypothermia through carotid artery infusion of cold saline. To approach the impact of selective brain hypothermia on intracranial pressure, cerebral perfusion pressure, infarct volume, brain edema, mNSS, specific biochemical indicators of brain damage and so on, and evaluate the protection of selective brain hypothermia on acute focal cerebral ischemia. Materials and Methods1.Random design,36 male New Zealand white rabbits were randomly assigned to be vascular cast, trypan blue staining, MCAO model (n=29) and sham operation (n=5) treatment. We made suture MCAO model with monitoring of brain function. According to TTC staining MCAO group was divided into the cortex+basal ganglia infarction (n=11),basal ganglia infarction (n=7), subarachnoid hemorrhage (n=5) and non-lesion subgroups (n=6). Physiological differences of subgroups were compared before or after MCAO. Infarct volume was measured with image analysis system of ImageTool. Differences of weight or suture indicators were compared in subgroups. The relationship between the indicators and the relative infarct volume was analyzed. Continuous monitoring of brain function, differences of aEEG or band power in subgroups were compared before or after MCAO respectively, and their differences before and after MCAO in each subgroup were analysed respectively. The relationship between aEEG decline and the relative infarct volume was analysed. Neurological injurys were detected with mNSS by two observers after 24h and 48h respectively. The consistency of the two observers' scores and the relationship between mNSS and relative infarct size were analysed. Preoperative comparisons in subgroups using one-way analysis of variance (One-way ANOVA), postoperative comparisons using analysis of covariance, two groups' comparisons using LSD method; comparison before and after operation in the same group using paired t test; measurement data correlation analysis using Pearson correlation and linear regression, rank correlation using Spearman correlation analysis; aEEG on diagnosing cortical cerebral infarction involving the use of ROC analysis, and diagnosed according to profession Youden index point, comparison of the diagnostic value between two methods using Z test; evaluation of two observers' mNSS results using Kappa consistency test. 2. Random design,20 male New Zealand white rabbits were randomly divided into 50ml/h reperfusion group (I1 group, n=5),99ml/h reperfusion group (I2 group, n=5) and integrated perfusion group (I3 group, n=10). Carotid artery catheter, selective infusion of cold saline (6.0±0.5℃),compared differences of cooling efficiency between 50ml/h and 99ml/h reperfusion groups, according to the cooling efficiencies of I1 and I2 groups to develop integrated cooling program. Explore the impact of brain temperature changes on rectal temperature during hypothermia treatment in integrated cooling group and relationship between the two.Compare changes of heart rate, mean arterial pressure, haematocrit, sodium concentration, blood gas and other indicators during hypothermia in integrated perfusion group, and analysis the relationship between brain temperature and various indicators. Morphological changes were observed after perfusion situation. Repeated measure analysis of brain temperature was used in different time points with different speeds; and also used in different time points with different areas; repeated measure analysis of various physiological indicators in different time points were used respectively; with continuous infusion of cold saline, the relationship between brain temperature and rectal temperature, heart rate, mean arterial pressure, haematocrit, sodium ion concentration or blood gas, using Pearson correlation analysis.3.Random design,32 male New Zealand white rabbits were randomly divided into MCAO group (M group, n=8), MCAO + cold saline infusion group (30min and 60min perfusion subgroups, I1 and I2 groups, each 8 rabbits), MCAO+38℃saline infusion group (I3 group, n=8). After establishing MCAO model, suitable animals (aEEG upper bound decrease>0.261)were selected to be different perfusion groups, comparing differences of physiological indicators in groups at each time point. With intraoperative invasive monitoring of intracranial pressure, ICP changes were analyzed in different time points and different groups, so did cerebral perfusion pressure. Infarct volume and edema extent were measured with ImageTool by TTC staining 48h after MCAO. According to the formula, the relative infarct volume was calculated out, and its difference in groups was analyzed, so die the cerebral edema index.24h and 48h after MCAO, mNSS were applied for evaluating neurological damage in each group to compare the differences in groups and the difference between the two time points. Concentrations of NSE, S-100βand MMP-9 in peripheral blood were measured with the method of ELISA respectively, comparing the differences in groups of three indicators. Repeated measure analysis of ICP was used in different time points with different infusion option, so did cerebral perfusion pressure; after selective cold saline infusing, comparisons in groups of various indicators using one-way analysis of variance (One-way ANOVA), two groups' comparisons using LSD method; the comparison of estimated and actual volume of infarction in each group using two independent sample t test; the comparison of 24h and 48h mNSS score in each group using paired samples t test.Results1.Rabbit Willis circle integrity, similar to human. Achievement ratio of MCAO model with suture method was 62.07%(18/29), which involved the cortex and basal ganglia in 11 cases accounted for 37.93%(11/29), only involving the basal ganglia accounted for 24.38% (7/29). The baseline data were balanced in 4 subgroups (P>0.05). Blood glucose of cortex + basal ganglia infarction after MCAO was significantly higher than basal ganglia infarction and non-lesion group (both P<0.05). The difference of body weight in groups was significant (F=3.608, P=0.017), no lesion group were significantly lower than the weight of the cortex + basal ganglia infarction and basal ganglia infarction group (both P<0.05).The depth difference of suture insertion was significant (F=4.985,P=0.008), cortex + basal ganglia infarction was significantly lighter than subarachnoid hemorrhage group and was significantly deeper than non-lesion group (both P<0.05). In cortex + basal ganglia infarction group, body weight, head diameter of suture, depth of suture insertion and relative infarct volume of the linear regression equation was significant (F=4.488, P=0.047), coefficient of determination was 0.658, relative infarction volume effect in decreasing order, depth of suture insertion, body weight and head diameter of suture.AEEG differences of upper bound, lower bound or bandwidth were significant in groups (F values were 14.059,12.698 and 12.827, respectively, all P<0.001).In cortex + basal ganglia infarction, aEEG upper bound, lower bound and bandwidth were significantly smaller than the other groups (all P<0.05);after MCAO, aEEG upper bound, lower bound and bandwidth decreased by 50.02%,52.02% and 46.27% respectively (all P<0.001).AEEG decreases of upper bound, lower bound and bandwidth showed a positive correlation with the relative infarct volume, and linear regression equations were significant (coefficients of determination were 0.518, 0.473 and 0.423,P values were 0.012,0.019 and 0.030 respectively).There was great potential of aEEG for diagnosing ischemia.After MCAO, ABP difference of a wave in groups was significant (F=2.870, P=0.041),ABP values of a wave in cortex + basal ganglia infarction were significantly less than subarachnoid hemorrhage group, no lesion group and control group respectively (all P<0.05). It was significant that ABP differences of a wave between before and after MCAO were compared in groups (F=5.879, P=0.001). After MCAO, ABP of a wave decreased significantly by 69.63% in cortex + basal ganglia infarction (P<0.001),which was significantly larger than the other four groups (all P<0.05). After MCAO, ABP difference ofβwave in groups was significant (F=2.724, P=0.049), ABP values ofβwave in cortex + basal ganglia infarction were significantly less than basal ganglia infarction and no lesion group (both P<0.05). After MCAO, ABP ofβwave decreased significantly by 71.29% in cortex + basal ganglia infarction (P=0.003).After MCAO, ABP difference of 0 wave in groups was significant (F=4.018, P=0.011),ABP values of 0 wave in cortex + basal ganglia infarction were significantly less than the other groups (all P<0.05). It was significant that ABP differences of 0 wave between before and after MCAO were compared in groups (F=6.911,P<0.001).After MCAO, ABP ofθwave decreased significantly by 66.65% in cortex + basal ganglia infarction (P=0.001), which was significantly larger than the other four groups (all P<0.05). Differences of relative band power before and after MCAO in groups were not statistically significant.Assessment of neurological injury with mNSS had a good Kappa consistency, Kappa values of 0.746-1.000, all P<0.001.Score and relative infarct volume was positively correlated, Spearman rank correlation coefficient of 0.731,significant correlation (P-0.011).2. Brain temperature difference was significant in different time points (F=175.730, P<0.001).99ml/h and 50ml/h two-speed selective infusion of cold saline can significantly reduce the brain temperature (F values were 108.926 and 80.459, both P<0.001).Speed of decline in brain temperature 99ml/h group was significantly higher than 50ml/h group (F=27.820, P=0.001),except before perfusion, at each time point brain temperature 99ml/h group was significantly lower than 50ml/h group (all P<0.01).In integrated perfusion group,brain temperature difference at different time points was significant (F=206.462, P<0.001),with perfusion time going, brain temperature decreased significantly (F=199.366, P<0.001).Brain temperature at each time point was significantly lower than rectal temperature (F=546.385,P<0.001).Rectal temperature was significantly decreased with the perfusion time going (F=19.608, P<0.001),but always maintained at normal levels (38-40℃).In the course of selective infusion of cold saline brain temperature and rectal temperature was no correlation (r=0.607, P=0.111).In all time points, heart rate, mean arterial pressure, haematocrit, sodium concentration and blood gas analysis, no significant differences (all P>0.05), there was no correlation between brain temperature and indicators (all P>0.05).48h after perfusion in integrated perfusion group, all scores with mNSS were 0, no neurological injury performance. TTC and HE staining were both normal.3.Baseline data between groups balanced (all P>0.05). Intracranial pressures in different time points were significantly different (F=85.003, P<0.001);intracranial pressure between the groups was statistically significant difference (F=6.891, P=0.001).After the selective infusion of cold saline (3h after MCAO) in each time point, intracranial pressure between the groups were statistically significant differences (all P<0.01).Cerebral perfusion pressure in different time points were significantly different (F=36.048, P<0.001).There was significant difference of relative infarct volume between the groups (F=8.241,P<0.001),12 group relative infarct volume was significantly lower than other groups (all P<0.05), while significantly lower than the estimated infarct volume (t=-3.162, P=0.007). There was significant difference of edema index between the groups (F=4.181,P=0.015),12 group edema index was significantly lower than the M and 13 groups (both P<0.05). 24h and 48h mNSS results were significantly different between the groups (F values were 4.087 and 6.701,P values were 0.016 and 0.002 respectively), of which 12 group mNSS results at two time points were the lowest,48h score were significantly lower than the other groups (all P<0.05). NSE, S-100β, MMP-9 were significant differences between the groups (F values were 5.806,4.749 and 3.789, P values were 0.003,0.008 and 0.021 respectively), three indicator concentrations of I1 and 12 groups were statistically significant lower than M group respectively (both P<0.05), NSE concentrations of I1 and 12 groups were statistically significant lower than 13 group (both P<0.05).Conclusions1.Establish a rabbit standard MCAO model with suture method, which is a stable, reproducible model of focal cerebral ischemia.2. Under the premise of the fixed suture tip diameter (0.5-0.6mm), the body weight and the depth of suture placement could affect the success rate of MCAO modeling, and the volume of cerebral infarction. Body weight> 2.5kg, depth of suture placement between 5.5-6.0cm, model success rate was highest.3.AEEG upper bound, lower bound and bandwidth significantly decreased indicating success of MCAO model and lesions involving the cortex.4. When lesion involving the cortex,α,β,θwave ABP after MCAO was significantly reduced compared with that before.5.AEEG decline of upper bound, lower bound or bandwidth was respectively positive correlation with the relative infarct volume, all regression equations were significant. AEEG decline can estimate the relative infarct volume.6. AEEG had high sensitivity and specificity for diagnosing cortical infarction, which could be used to select successful MCAO model.7.MNSS is a consistent, reliable and positively correlated with relative infarct volume scoring system for evaluating neurological function.8.Intra-arterial selective infusion of cold saline can rapidly reduce brain temperature, while maintaining rectal temperature, heart rate, mean arterial pressure, haematocrit, Na+ concentration and blood gas balance. This is an efficient, safe, reproducible method of selective hypothermia.9. Intra-arterial selective infusion of cold saline for 60min can significantly reduce the intracranial pressure, increased cerebral perfusion pressure, during the acute phase play a role in brain protection. 10. The estimated volume with aEEG decline was similar with the relative infarct volume with TTC staining, Intra-arterial selective infusion of cold saline for 60min significantly reduced infarct volume and edema.11.Intra-arterial selective infusion of cold saline for 60min can significantly decrease the 24h and 48h mNSS score, which is conducive to the recovery of neurological function after MCAO.12. Intra-arterial selective infusion of cold saline for 60min can significantly reduce the NSE, S-100β,MMP-9 concentration, reduce neurons and glial cells damage, protect the blood-brain barrier, and inhibit vasogenic edema.The originalities of this current project lie in:reveal1.Find that aEEG upper bound, lower bound and bandwidth significantly decreased when lesions involved the cortex in MCAO model; aEEG decline was positive correlation with the relative infarct volume and could be used to estimate infarct volume according to the regression equation; aEEG had high sensitivity and specificity for diagnosing cortical infarction, which could be used to select successful MCAO model.2. Reveal that rabbit mNSS had high consistency and credibility and was positive correlation with relative infarct volume.3.Establish an efficient, safe, reproducible rabbit selective cerebral hypothermia model using infusing cold saline through carotid artery.4. Find in acute focal cerebral ischemia, selective cerebral hypothermia 60min through internal carotid artery infusing cold saline could significantly degrade intracranial pressure, increase cerebral perfusion pressure, reduce infarction volume, inhibit edema and improve neurological outcome.5.Find in acute focal cerebral ischemia, selective cerebral hypothermia 60min through internal carotid artery infusing cold saline could significantly reduce blood NSE, S-100β, MMP-9 concentration, protect neurons, glial cell and blood-brain barrier and inhibit vasogenic edema. |
PDF Full Text Request