Therapeutic Effect Of Partial Reperfusion On Acute Cerebral Ischemia In Rats

IntroductionIschemic stroke is a dynamic process triggered by the sudden loss of blood circulation to an area of the brain, resulting in a corresponding loss of neurologic function. It has been estimated that approximately 70% of ischemic strokes are caused by acute occlusion of a large intracranial or extracranial cerebral artery. Thus urgent reperfusion therapy, aimed at restoration or improvement of perfusion to the ischemic area, is a principle therapeutic target in the acute phase of ischemic stroke. Thrombolytic stroke therapy is based on the "recanalization hypothesis," i.e., that reopening of occluded vessels improves clinical outcome in acute ischemic stroke through regional reperfusion and salvage of threatened tissues. Indeed, intravenous administration of recombinant tissue plasminogen activator (rtPA) as a procedure for thrombolysis is currently the only FDA-approved medical therapy for treatment of patients with acute ischemic stroke. Though intravenous thrombolytic therapy for acute stroke is now generally accepted, it has also been challenged. Recanalization may exacerbate tissue injury by promoting reperfusion (hyperperfusion), excessive cerebral edema, and symptomatic intracerebral hemorrhage (SIH), producing a so-called "cerebral reperfusion injury". The SIH was seen in 6% of patients treated in the original National Institute of Neurological Disorders and Stroke trial that led to rtPA's approval and was subsequently confirmed in multiple postmarketing studies. Ischemic/reperfusion injury is the major obstacle toward the goal of extending this treatment modality to a higher proportion of patients with acute ischemic stroke.The cerebral circulation is a unique vascular bed in that the extracranial and large intracranial pial vessels contribute significantly to cerebrovascular resistance and respond myogenically to changes in perfusion pressure. The contractile response of the cerebral arteries to pressure contributes to autoregulation of cerebral blood flow (CBF) and is facilitated by vascular smooth muscle that contracts to increased pressure and relaxes in response to decreased pressure. It has been demonstrated that reperfusion following transient cerebral ischemia causes autoregulatory loss and hyperperfusion due to a diminished CVR that exposes the microcirculation to excessive perfusion pressure. In addition, treatment with tissue plasminogen activator significantly diminished myogenic reactivity in isolated cerebral arteries, a result that was additive if arteries were exposed to ischemia. Several animal studies suggest that the restoration of cerebral circulation consistently results in a hyperperfusion phase. Hyperperfusion may contribute to the development of reperfusion injury by causing brain edema or hemorrhage.Controlled reperfusion as an efficiency strategy has been used successfully in many organs and tissues for preventing reperfusion jury, such as pulmonary injury, myocardium, limb and kidney. However, since it is difficult to control CBF of a certain region without affecting other areas in cerebral circulation than in others, due to the Willis Cycle, this method was not studied in treatment of experimental cerebral ischemia. Based on this, we developed a partial reperfusion technique which can limit the focal cerebral blood flow to test whether controlled reperfusion reduces the cerebral ischemic/reperfusion injury and improves neurological deficit, and the potential mechanisms involved will also be discussed.This paper includes two parts. Effects of ischemia and rtPA on cerebrovascular autoregulation were studied in part one, which including 2 experients:In first experiment, changes of affected artery autoregulation after focal cerebral ischemia were studied. In second experiment, combined effects of cerebral ischemia and tissue plaminogen activator on cerebrovascular autoregulation were investigated. Therapeutic effects of partial reperfusion technique combined with rtPA on experimental cerebral ischemia were studied in part two, which including 3 experiments:In first experiment, we investigated wherther partial reperfusion is effective on preventing reperfusion injury following experimental cerebral ischemia. In second experiment, combined effects of partial reperfusion and tissue plasminogen activator on cerebral blood flow and reperfusion injury after transient cerebral ischemia were studied. In third experiment, we confirmed that partial reperfusion reduces ischemia/reperfusion injury following delayed thrombolysis in rat embolic stroke model.Part-ⅠEffects of ischemia and rtPA on cerebrovascular autoregulationExperiment-1 Changes of affected artery autoregulation after focal cerebral ischemiaObjective:To investigate the effect of cerebral ischemia on affected artery autoregulation, and evaluate the probably mechanisms of ischaemia-reperfusion injury.Methods:Intraluminal suture ischemic model was used by occlusion of left middle cerebral artery in rats. Two hours later, the middle cerebral artery segments were isolated from both ischemia and control groups for measurement of changes in vessel diameter induced by increasing pressure and vasoactive compounds. And then, distensibility, myogenic tone, reactivity to 5-HT and ACh were calculated and compared between groups.Results:In lower pressure range (20mmgHg,40mmHg and 60mmHg) ischemic vessels showed an increased myogenic tone (P<0.01 vs control group, for all) and decreased diameter (P<0.05 vs control group, for all); In higher pressure range (100mmgHg and 120mmHg), ischemic vessels showed an increased diameter(P<0.05 vs control group, for both), distensibility (P<0.01 vs control group, for both) and decreased myogenic tone(P<0.01 vs control group, for both); In normal pressure range (80mmHg), myogenic tone and diameter were not altered after ischemia (P>0.05 vs control group, for both). Both groups constricted to 5-HT and dilated to ACh, however, the response was significantly diminished after ischemia(5-HT: F=97.596, P=0.000; ACh:F=35.793, P=0.000).Conclusion:These findings demonstrate that contractile and diastolic function of affected artery was impaired after ischemia, a result that may contribute to ischemia-reperfusion injury by losing upstream cerebrovascular resistance and increasing perfusion on the microcirculation.Experimental-2 Combined effects of ischemia with tissue plaminogen activator on cerebrovascular autoregulationObjective:To investigate the combined effects of ischemia and tissue plasminogen activator on vasoactive function of affected arteries and evaluate the probably mechanisms of the secondary hemorrhage after thrombolysis.Methods:Intraluminal suture ischemic model was used by occlusion of right middle cerebral artery in rats. Ninety minutes later, the middle cerebral artery segments were isolated for measurement of changes in vessel diameter induced by increasing pressure and vasoactive compounds. Arteries were either nonischemic (control; n=6), nonischemic and perfused with rtPA for 50-60mins (rtPA; n=6), ischemic (ISC; n=8), or ischemic and perfused with rtPA for 50-60mins (rtPA-ISC; n=8). And then, myogenic tone, reactivity to 5-HT and ACh were calculated.Results:Control arteries increased myogenic tone from 60mmHg to 120mmHg (P=0.000) and responded myogenic reactivity, all other groups decreased tone at 120mmHg (P<0.05 vs.control for all) and lost myogenic reactivity; Diameters of control arteries showed the least (P<0.05 vs.all three others), rtPA-ISC group showed the largest (P<0.05vs.all three others); All arteries constricted to 5-HT and dilated to ACh, however, the response was diminished in all groups compared with control (P<0.05 for all other groups); rtPA-ISC responded the least (P<0.05 vs. all other groups).Conclusion:These findings demonstrate that vasoactive function of affected artery was impaired after ischemia, and the impaired function was increased in the presence of tissue plasminogen activator. A result that may contribute to intracerebral heamorrhage by losing upstream cerebrovascular resistance and increasing perfusion on the microcirculation after thrombolysis.Part-ⅡTherapeutic effects of partial reperfusion technique combined with rtPA on experimental cerebral ischemiaExperiment-1 Effects of partial reperfusion on cerebral blood flow and the secondary heamorrhage after focal cerebral ischemia in rats Background and objective:Controlled reperfusion as an efficiency strategy has been used successfully in other organs and tissues for preventing reperfusion jury. In the present study, we investigated whether partial reperfusion is effective on preventing reperfusion injury following experimental cerebral ischemia.Methods:A total of 19 Male Wistar rats who suffered 3 hours cerebral ischemia were treated with partial reperfusion, complete reperfusion or no reperfusion (control).Results:A transient postischemic hyperperfusion (127±16%) followed by sustained reduction in CBF was recorded by laser Doppler flowmetry in complete reperfusion group, and partial reperfusion inhibited this postischemic hyperperfusion (85±17%). After 1 h of reperfusion, complete reperfusion produced a significant intracerebral heamorrhage (689±196μg vs.323±17μg; P<0.05) and brain edema (1.44±0.10 vs. 1.20±0.08; P<0.05) compared with control, and partial reperfusion didn't exacerbate intracerebral haemorrhage (384±74μg) and brain edema (1.30±0.12) compared with control group (P>0.05, for both), it furthermore reduced the complete reperfusion related haemorrhage and brain edema (P<0.05 vs. complete reperfusion, for both).Conclusion:Reperfusion injury is the major obstacle of recanalization therapy for patients with acute ischemic stroke. This study demonstrats that partial reperfusion is an effective strategy for prevention of postischemic hyperperfusion and the secondary hemorrhage and brain edema.Experiment-2 Combined effects of partial reperfusion and tissue plasminogen activator on cerebral blood flow and reperfusion injury after transient cerebral ischemia in rats Background and objective:Reperfusion injury is the major obstacle of thrombolysis therapy for patients with acute ischemic stroke. Postischemic hyperperfusion as a risk factor of reperfusion injury is recognized as a frequent occurrence in stroke and a severe complication of endarterectomy. Stepwise revascularization which prevents abrupt improvement in reperfusion is an efficiency strategy for preventing hyperperfusion and the secondary reperfusion injury in endarterectomy procedures. Based on the same principle, blood flow limitation technique was introduced and used to determine the combined effects of partial reperfusion and tissue plasminogen activator on cerebral blood flow and the development of the secondary haemorrhage and brain edema after transient cerebral ischemia.Methods:Rats who suffered 3 h middle cerebral artery occlusion were treated with partial reperfusion (part), partial reperfusion with tissue plasminogen activator (part-rtPA), complete reperfusion (comp) and complete reperfusion with tissue plasminogen activator (comp-rtPA). Cerebral blood flow, secondary haemorrhage and brain edema were compared among the groups.Results:Partial reperfusion significantly inhibited the early postischemic hyperperfusin (partial groups vs. complete groups, P<0.05 for all) and reduced brain edema (partial groups vs. complete groups, P<0.05 for all). Compared with comp group, part group reduced the secondary hemorrhage (P=0.045). Furthermore, part-rtPA also improved the subsequent hypoperfusion compared with comp-rtPA group (P=0.032).Conclusion:Partial reperfusion combined with tissue plasminogen administration is an efficiency therapeutic strategy for 3 hour's cerebral ischemia in rats. Experiment-3 Therapeutic effect of controlled reperfusion combined with tissue plasminogen activator on delayed embolic stroke in ratsObjective:To study the effects of controlled reperfusion combined with tissue plasminogen activator on delayed embolic stroke in rats.Methods:Controlledl reperfusion technique was used combined with tissue plasminogen activator to treat rats who suffered 4 h embolic stroke. Neurological function and infarct volume were evaluated and compared with rtPA (just using tissue plasminogen activator) and control group (saline alone) at 6 h after treatment.Results:Partial reperfusion combined tissue plasminogen activator group showed a improved (CBF t=-2.668, P=0.037) than before suture insertion, showed a better neurological function (9±5 vs.14±4, P<0.05) and a lower infarct volume% (5.4±2.6% vs.19.4±4.3%, P<0.05) than rtPA group, and also showed a lower infarct volume%(5.4±2.6% vs.13.3±3.1%, P<0.05) compared control group.Conclusion:Partial reperfusion combined with rtPA administration is an efficiency therapeutic strategy for delayed embolic stroke in rats.