The effects of blood pressure and urokinase on brain injuries after experimental.

P u b l i s h e d b y M a n e y P u b l i s h i n g (c ) W . S . M a n e y & S o n L i m i t e dThe effects of blood pressure and urokinase on brain injuries after experimental cerebralinfarction in ratsXunming Ji *{,Ke Li *{,Wenbin Li {,Shuting Li *{,Feng Yan *{,Wei Gong *{andYumin Luo *{*Cerebrovascular Diseases Research Institute,Xuanwu Hospital,Capital Medical University,Beijing,China{Key Laboratory of Neurodegenerative Diseases,Capital Medical University,Ministry of Education,Beijing,China{Beijing Shijitan Hospital,Beijing,ChinaObjective:With the proposal of penumbra theory and development of intra-arterial thrombolysis (such as urokinase),the outcome of ischemic cerebrovascular disease is greatly improved.However,the incidence of hemorrhagic transformation (HT)increased concomitantly,and some studies showed a close relationship between blood pressure and HT.The mechanisms of blood pressure and urokinase effect on the incidence of HT are not clear.In this study,we investigated the effects of the different levels of blood pressure and urokinase on the ischemic lesions,the incidence of HT and the expression of matrix metalloproteinase 9(MMP-9)in the rat ischemia–reperfusion models.Methods:Temporary focal ischemia was induced in male Sprague–Dawley rats using the intraluminal vascular occlusion method.The animals were assigned into four groups (n 511in each group):low blood pressure group (LP),normal blood pressure group (NP),high blood pressure group (HP)and urokinase/high blood pressure group (UKHP).Adnephrin was applied to enhance the mean arterial blood pressure (MABP)at the beginning of reperfusion.MABP was maintained 20mmHg higher than the baseline for 1hour.Sodium nitroprusside was used to decrease MABP by 20mmHg lower than the baseline for 1hour.Both urokinase and adnephrin were used concomitantly in the UKHP group.Neurological deficit scores were evaluated at 2hours (R2h)and 24hours (R24h)after reperfusion.All rats were decapitated,their brains were sliced into 15-m m-thick slices,and the infarct volume and the visible HT were analysed.Three rats in each group were taken for immunohistochemistry and pathological analysis.Results:There was no significant difference in MABP among the groups at the baseline time points (p .0.05),but blood pressure are definitely increased and decreased in the HP,UKHP,and LP groups.Neurological deficit scores showed no significant difference at R2h among the groups (p 50.443).However,neurological deficit scores showed significant differences at R24h among the groups,the neurological deficits scores of rats in the LP group are significantly higher than that in the other pared with that of 2hours after reperfusion,neurological deficit scores deteriorated in the LP group (p 50.047)but was improved in the NP,HP and UKHP groups (p 50.076,0.002,0.017,respectively)at 24hours after reperfusion.The infarct volume in the HP group was apparently smaller than that in the LP group (p 50.006).There was indeed a tendency that HT occurred more frequently in the UKHP group (42.8%)than in the HP (25%)and LP (28.5%)groups.MMP-9expression showed significant increase around the ischemic lesion areas of the UKHP group and significant decrease in the cortical areas of the LP and HP groups but no significant difference in the basal ganglia of rats of all groups.Conclusion:Mild elevation of blood pressure during reperfusion is supposed to improve neurological outcomes in rats following ischemia/reperfusion.The incidence of HT tended to increase with the elevation of blood pressure and the administration of urokinase.Immunohistochemitry analysis indicated that incidence of HT may correlate with excessive expression of MMP-9.[Neurol Res 2009;31:204–208]Keywords:Blood pressure;ischemia/reperfusion;hemorrhagic transformation;urokinaseINTRODUCTIONThe public health is being increasingly threatened by cerebrovascular diseases,among which 80%are ischemic cerebrovascular disease.With the proposal of penumbra theory 1and development of intra-arterialCorrespondence and reprint requests to:Yumin Luo,Cerebrovascular Diseases Research Institute,Xuanwu Hospital,Capital Medical University,45Changchun Street,Beijing 100053,China.[yumin111@]Accepted for publication November 2008.204Neurological Research,2009,Volume 31,March#2009W.S.Maney &Son Ltd10.1179/174313209X393924P u b l i s h e d b y M a n e y P u b l i s h i n g (c ) W . S . M a n e y & S o n L i m i t e dthrombolysis 2,outcome of ischemic cerebrovascular disease is greatly improved.However,the concomitant increasing rate of hemorrhagic transformation (HT)had worsened the neurological status of some patient 3,and some studies had demonstrated the relationship between blood pressure and HT 4.With further devel-opment of endovascular therapy,a new problem rises:how to manipulate the blood pressure of the formerly occluded arteries with recanalization to gain a best risk/benefit ratio to decrease the HT rate and at the same time to improve the regional perfusion efficiently.In this study,we investigate the influence of different blood pressure levels and urokinase on the size of ischemic lesion,the incidence of HT and the expression of matrix metalloproteinase 9(MMP-9)in the rat ischemic–reperfusion model.MATERIALS AND METHODSAnimal modelAll animal experiments were approved by the Institutional Animal Care and Use Committee of Capital Medical University and in accordance with the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals .Temporary focal ischemia was induced in male Sprague–Dawley rats weighing 280–300g using the intraluminal vascular occlusion method as previously described 5.The rats were randomized to four groups (n 511in each group):normal blood pressure group (NP),low blood pressure group (LP),high blood pressure group (HP)and urokinase/high blood pressure group (UKHP).Three rats in each group were used for immunohistochemical and pathological analysis.To ensure the occurrence of ischemia by MCA occlusion,regional cerebral blood flow was monitored using laser Doppler flowmetry (LDF,PeriFlux System 5000;Perimed,Stockholm,Sweden)in the following coordinates:0.5mm anterior and 5.0mm lateral from the bregma.Rectal tempera-ture was controlled at 37.0u C during and after surgery via a temperature-regulated heating pad.Blood pressure was monitored through a measurement (MP100A-CE;BIOPAC Systems,Inc.,Goleta,CA,USA).Blood pressure values were recorded at different time points:5minutes after femoral catheterization (Fc5min),15minutes after occlusion (O15min),beginning of reperfusion (R0min),30minutes after reperfusion (R30min)and 60minutes after reperfusion (R60min).Blood gas,blood glucose and body temperature were also recorded.The animals underwent right MCA occlusion for 120min-utes and then reperfusion for 24hours.After recovering from anesthesia,the animals were maintained in an air-conditioned room at 20u C.Adrenaline (Jiyao Company,TianJin,China)and sodium nitroprusside (Shuanghe Company,Beijing,China)were used to increase or decrease blood pressure in the LP,HP,or UKHP groups.The drugs were administered at the rate of 1ml/h,at 2hours after ischemia and then increase or decrease the blood pressure by 20mmHg for 1hour related to the basic mean arterial blood pressure (MABP).For the NP group,normal saline was applied at the same rate.For the UKHP,urokinase was administered at the same time with adrenaline.Behavioral testsNeurological deficits were scored at 2and 24hours after reperfusion,which were done in blind experi-ments.There were two tests that have been used previously to evaluate various aspects of neurological function,these tests were (1)the postural reflex test,developed by Bederson at al.6to examine upper body posture while the animal is suspended by the tail and (2)the forelimb placing test,developed by De Ryck et al .7to examine sensorimotor integration in forelimb placing responses to visual,tactile and proprioceptive stimuli.Neurological functions were graded on a scale from 0to 12(normal score,0;maximal score,12).Rats with convulsions or sustained disturbances of consciousness were excluded from the study;most of these cases proved to have subarachnoid hemorrhage secondary to suture-induced rupture of the internal carotid artery.Determination of infarct volumeThe animals were killed at 24hours after reperfusion in a carbon dioxide chamber,and the brains were quickly removed and sectioned into eight consecutive coronal slices of 2mm thickness.The slices were stained by immersing into 2%of 5-triphenyl-2H-tetrazolium chloride for 30minutes at 37u C and then fixed with 8%of formalin.The infarct volume of each brain was measured by the Image-ProPlus Analysis Software.Immunohistochemical analysisAnimals were killed in a carbon dioxide chamber at 24hours after reperfusion,and the brains were prepared for freshly frozen coronal sectioning (thickness of 15mm)as described previously 8.For immunohisto-chemical staining of MMP-9,goat anti-MMP-9poly-clonal antibody was used at a dilution of 1:150(Santa Cruz Biotechnology,Inc.,Santa Cruz,CA,USA)Immunohistochemistry was performed using the avi-din–biotin technique (Zhong Shang Jing Qiao Company,Beijing,China).As a negative control,sections were incubated with no primary antibodies.Statistical analysisData were expressed as mean ¡SD.The significant difference between means was assessed by Student’s t -test (single comparisons)or by ANOVA and post hoc Scheffe’s tests (for multiple comparisons).All analyses were performed with SPSS 11.5.A value of p ,0.05was considered significantly.RESULTSMABP valuesThe MABP values at five time points,including 5minutes after femoral catheterization (Fc5min),Effects of blood pressure and urokinase on brain injuries:X.M.Ji et al.Neurological Research,2009,Volume 31,March 205P u b l i s h e d b y M a n e y P u b l i s h i n g (c ) W . 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M a n e y & S o n L i m i t e d15minutes after occlusion (O15min),0minutes after reperfusion (Reperf),30minutes after reperfusion (R30min)and 60minutes after reperfusion (R60min),are shown in Table 1.There was no significant dif-ference among groups in mean artery blood pressure (MABP)at the time points of Fc5min and R0min (p .0.05),but it showed a significant difference at O15min between HP and LP (p ,0.05).MABP at R30min and R60min showed significant differences among groups (p ,0.05).The MABP of the UKHP group showed significant differences comparing with the NP and LP groups (p ,0.05)but showed no significant difference comparing with the HP group (p .0.05).MABP at O15min were significantly different from baseline at Fc5min (t 522.395,p 50.024),but there were no significant difference at R0min and baseline (t 50.922,p 50.365).MABP in LP,HP and UKHP were maintained stably at 20mmHg above or below normal for 1hour,respectively.These results suggest that the blood pressure levels were successfully manipulated in the test groups.There were no significant differences in blood gas/pH,blood glucose and rectal temperature among the four groups (data not shown).Neurological deficit scoresNeurological deficit scores showed no significant difference at R2h among groups (p 50.443).However,the neurological deficit scores of the LP group showed significant increase compared with the NP,HP,and UKHP group at 24hours after reperfusion.When it comes to the comparison within each group between the scores at 2hours and at 24hours after reperfusion,it showed the neurological deficit scores were higher at 24hours after reperfusion than that at 2hours in LP (p 50.047),which indicates neurological deficit scores deteriorated in the LP group.However,the neurological deficit scores were lower at 24hours after reperfusion than that at 2hours in NP,HP and UKHP,especially in HP (p 50.076,0.002and 0.017,respectively),which means the outcomes were improved in the NP,HP and UKHP groups at 24hours after reperfusion (Figure 1).Infarct volumeThe border between infarct and non-infarct tissue was outlined with an image analysis system,and the area of infarction was measured by subtracting the area of the non-lesioned ipsilateral hemisphere from that of the contralateral side.The percentage of the infarct volume was calculated by (contralateral volume 2non-lesioned ipsilateral volume)/contralateral volume.The results showed the percentage of infarct volume in the HP group was lower than LP group significantly (p 50.006),while there was no statistical significance among the other groups (Figure 2).Incidence of HTTable 2showed the rate of HT in LP is 28.5%,0%in NP,25%in HP and 42.8%in UKHP.Since HT cases were not enough in each group,the HT rate could notTable 1:The change of MABP at the different time points in different groups (mean ¡SD,n 58)Group Blood pressure values at different time point,mmHg5minutes after femoralcatheterization15minutes after occlusion Beginning of reperfusion 30minutes after reperfusion 60minutes after reperfusion LP 91.1¡19.596.7¡1486.9¡1561.8¡10.262.1¡7.8NP 79.5¡7.888.1¡8.182.8¡10.782.5¡11.182.1¡8.8HP 82.6¡8.183¡13.376.1¡6.899.6¡5.4101.0¡5.2UKHP79.8¡14.985.5¡6.381.2¡10.894.8¡11.2103.0¡11.5Figure 1:Neurological deficit scores.There was no significantly different of neurological deficit scores at 2hours after reperfusion among the four group.The neurological deficit scores of the LP group versus NP group (*p ,0.05).HP and UKHP groups versus NP (p .0.05).In the LP group,the neurological functional score at 24versus 2hours (a p ,0.05);in the NP group,24hours versus 2hours (b p .0.05);in the HP group,24hours versus 2hours (x p ,0.05);in the UKHP group,24hours versus 2hours (d p ,0.05)Figure 2:The percentage of infarct volume.The percentage of infarct volume in the LP group are larger than HP group (*p ,0.05)Effects of blood pressure and urokinase on brain injuries:X.M.Ji et al.206Neurological Research,2009,Volume 31,MarchP u b l i s h e d b y M a n e y P u b l i s h i n g (c ) W . S . M a n e y & S o n L i m i t e dbe analysed statistically,BUT there was indeed a tendency that HT occurred more often in UKHP than in HP and LP.MMP-9expressionMMP-9expression was significantly increased around the ischemic lesion area in the UKHP group and was significantly decreased in the LP and HP group in the cortical area (Table 3and Figure 3).In the basal ganglia,MMP-9expression was no significantly differ-ent among all groups.DISCUSSIONBlood pressure is one of the most important potentially controllable risk factors in ischemic stroke,and it is also one of the most important prognostic factors.Clinical trials had demonstrated that long-term antihypertensive therapy could reduce incidence of ischemic stroke 9.However,there is still debate on the blood pressure manipulation in cases with acute ischemic stroke,especially those patients undergone intra-arterial throm-bosis.Hence it is important for us to investigate the effects of blood pressure at different levels on the ischemic lesions and HT in rat ischemic–reperfusion model,in order to present some useful information for clinical practice.Many studies had studied on the relationship between blood pressure variation and prognosis in the animal ischemia ischemic stroke models.Saku et al.10intro-duced a cat model:MCA was exposed transorbitally and clipped permanently,then the blood pressure was raised by a balloon catheter previously placed in the descending thoracic artery.Cole et al.4exposed and sutured the rat MCA in two locations through craniot-omy,then investigated the relation among elevated blood pressure,infarct volume and hemorrhagictransformation rate after 180minutes of ischemia and 120minutes of reperfusion 4.However,there are some obvious drawbacks in the animal models mentioned above:(1)both methods are too invasive,thus they will influence the physiological stability of the models;(2)both procedures are very complicated to perform,especially for those without neurosurgical trainings;(3)both methods were performed extra-arterially.In our studies,we chose an intra-arterial thread ischemic–reperfusion model developed by Longa et al.11in 1989,in which the period of the ischemia and reperfusion could be controlled exactly and the ischemic area is stable,thus can well mimic the different statuses of permanent or transient ischemic stroke in human beings.Additionally,the reperfusion procedure is very close to the situation when the occluded artery is recanalized through intra-arterial thrombolysis.The results showed that the physical variations of the rat model,including the blood gas/pH,the rectal temperature and the blood glucose,maintained stable throughout the procedure.Based on the clinical situation,the blood pressure of the rat model in our experiment was raised or lowered by 20mmHg away from the base line.Table 1showed the high blood pressure and low blood pressure in the LP and HP groups are maintained for 1hour stably.There was no significant difference in neurological deficit scores among groups at 2hours after reperfusion,which indicated the stability of our manipulation.LP group showed deterioration in neurological status at 24hours after reperfusion,while other groups showed various degrees of improvement,which suggested thatTable 2:The comparison of HT in different groupsLPNP HP UKHP Number of cases with HT 2023Number of cases without HT5664Table 3:The MMP-9-positive cells in different part of the brain (mean ¡SD,n 53)LPNPHPUKHPCortex 513¡32.8*293¡40.4477¡25.2{{1460¡57.71#Basal ganglia11¡1.710¡110¡4.612¡3*p ,0.05,versus NP group (p 50.003);{p ,0.05,versus NP group (p 50.000);{p .0.05,versus LP group (p 50.952);1p ,0.05,versus NP group (p 50.000);#p ,0.05,versus HP group (p 50.000).Figure 3:MMP-9-positive cells in the cortex and striatum.Standard for the positive cells of (A ,B )LP,(C ,D )NP,(E ,F )HP and (G ,H )UKHP in the cortex and striatum,respectivelyEffects of blood pressure and urokinase on brain injuries:X.M.Ji et al.Neurological Research,2009,Volume 31,March 207P u b l i s h e d b y M a n e y P u b l i s h i n g (c ) W . 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M a n e y & S o n L i m i t e dthe relatively high blood pressure during reperfusion could help to improve neurological status.Powers et al.12thought that normal cerebral vessels possessed the ability to constrict and dilate automatically in response to local perfusion pressure,which may help to stabilize the regional cerebral blood flows regardless of fluctuating blood pressure.When this kind of autoregulation is impaired,local regional cerebral blood flow is mainly dependent on cerebral perfusion pressure.Reduction in MABP will lead to substantial increase in size and severity of the zone of ischemia 13.Based on this theory,elevation of blood pressure during acute ischemic period could improve local cerebral blood flow,thus save the endangered neuronal cells in the penumbral areas.Our experiment also suggested that blood pressure decrease could deteriorate neurolo-gical status.Some studies suggested that blood pressure decrease after experimental cerebral ischemia provides neuro-vascular protection.This may be due to the varied anesthesia methods and the degree that blood pressure was decreased 14.HT is the most dangerous complication with blood pressure manipulation after intra-arterial thrombolysis.Our studies showed that there was a tendency that HT occurred more frequently in UKHP,which could be explained by more severe disruption of local vessel wall and increased infiltration pressure.Immuno-histochemical analysis of MMP-9showed that there were a great number of positive staining cells in the cortical area near the ischemic lesion,while there were only little positive staining cells in the basal ganglia region,which was consistent with the findings of Fujimura 15.Statistical analysis showed that UKHP group had the largest number of positive staining cells in the penumbral areas while the NP group had the smallest.These data suggested that blood pressure changes could affect the expression of MMP-9;how-ever,the mechanism requires further bined analysis of the HT rate and positive staining cell counts in the UKHP group suggested that occurrence of HT was in close relation to expression of MMP-9.Some studies have also reported that the incidence and severity of HT in the rat ischemic/reperfusion model decreased obviously compared with control group,after adminis-tration of the MMP-9inhibitor BB9416,17.The plasma MMP-9concentration in patients with acute cerebral ischemia on admission could predict the occurrence of HT 18.HT tended to occur more frequently in patients with high plasma MMP-9concentration on admission 19.In conclusion,blood pressure changes in rat ischemic/reperfusion model could obviously affect the prognosis.Blood pressure elevation helps to recover from ischemic injury,but at the same time,it increases the incidence of HT;blood pressure lowering worsens the neurological status of the model.The incidence of HT might be related with the expression of MMP-9.ACKNOWLEDGEMENTThis project was supported by the Chinese 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acute ischemic stroke.Stroke 2003;34:40–4619Montaner J,Molina CA,Monasterio J,et al .Matrix metalloprotei-nase-9pretreatment level predicts intracranial hemorrhagic complications after thrombolysis in human stroke.Circulation 2003;107:598–603Effects of blood pressure and urokinase on brain injuries:X.M.Ji et al.208Neurological Research,2009,Volume 31,March。