Mechanisms of Brain Damage Following Focal Cerebral Ischemia: Changes in Ion Homeostasis and the Importance of Free Radical Formation

In this thesis mechanisms of neuronal damage were investigated in an experimental stroke model i.e focal ischemia. The influence of SD-induced calcium transients on neuronal damage were studied in animals with reduced energy supply. The influence of bioenergetic failure on K+e concentration and Ca2+e homeostasis were also investigated in transient focal ischemia. Moreover, the free radical formation was assessed in focal ischemia/reperfusion, as was the scavenging effect of PBN. Animals with a reduction in energy supply showed prolonged SD-induced calcium transients. These prolonged calcium lead occasionally to mild neuronal damage. Similar SDs are recorded in the ischemic penumbra in focal ischemia, and are probably caused by decreased energy supply. The second bioenrgetic failure in the ischemic focus as recorded in K+e concentration increase occurs at 4-6 h of reperfusion after 2 h MCAO. Treatment with PBN delays or prevent the late increase in K+e level. Recordings of Ca2+e and total calcium content in focal ischemia /reperfusion showed disturbed calcium homeostasis in the cortical core and penumbral zone. Free radical formation were enhanced in the recirculation phase after 2 h MCAO, and treatment with PBN did not reduce these free radical formation. It is concluded that the cellular calcium load in energy compromised brain tissue is tolerated without extensive neuronal damage. This indicates that other mechanisms than only exaggerated calcium transients lead to development of the infarct in focal ischemia. Recordings of Ca2+e and total calcium content in focal ischemia /reperfusion suggest that the tissue is irreversibly damaged after 6 h of recirculation. The lack of an effect of BPN treatment despite its neuroprotective effect may suggest that PBN is effective in small compartments e.g the endothelium

The effect of α-phenyl-tert-butyl nitrone (PBN) on free radical formation in transient focal ischaemia measured by microdialysis and 3,4-dihydroxybenzoate formation

α-phenyl-tert-butyl nitrone (PBN) reduces infarct size, improves recovery of brain energy metabolism and delays the secondary increase in extracellular potassium after focal ischaemia, presumably by trapping OH radicals. We investigated the effect of PBN on the formation of 3,4-dihydroxybenzoic acid (3,4-DHBA) as a measure of OH radical formation, during and following middle cerebral artery occlusion (MCAO). Rats, subjected to 2 h of ischaemia followed by 3 h of recirculation, were injected with either vehicle or PBN (100 mg kg -1 i.p.) prior to MCAO or immediately after recirculation, respectively. The in vivo microdialysis technique was used to collect samples for analysis of 3,4-DHBA by HPLC. The basal levels of 3,4-DHBA were 56-77 nmol L -1 in the four groups. During ischaemia, the formation of 3,4-DHBA decreased by about 50% in all groups. Upon recirculation, a 3-fold rise in 3,4-DHBA formation was seen. At 2 h of recirculation the mean value of 3,4-DHBA in the pretreated, vehicle-injected animals was 125 ± 18 nmol L -1 and in the PBN-injected 145 ± 48 nmol L -1 respectively. When the animals were treated after MCAO either with vehicle or PBN the values at 2 h recirculation were 155 ± 148 and 189 ± 145 nmol L -1, respectively. No statistically significant difference between vehicle- and PBN-injected groups was seen. We conclude that during reperfusion following MCAO, hydroxyl radical formation increases. The increase is not ameliorated by PBN which suggests that PBN does not protect the brain by a general scavenging of OH radicals, although tissue specific actions cannot be excluded

Postischemic exercise attenuates whereas enriched environment has certain enhancing effects on lesion-induced subventricular zone activation in the adult rat

Experimental stroke increases cell proliferation and neurogenesis in the subventricular zone (SVZ) and in the dentate gyrus subgranular zone (SGZ) in the adult mammalian brain. This study examined the effects of postischemic voluntary exercise (running wheel) and environmental enrichment on the SVZ and SGZ 1 week after focal cortical ischemia in adult spontaneously hypertensive rats. Immunohistochemical labeling was performed for incorporation of specific cell markers such as Ki67 and 5-bromodeoxyuridine (proliferating and newborn cells), terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end labeling (apoptotic cells), Sox-2 and glial fibrillary acidic protein (neural stem and progenitor cells), polysialylated neural cell adhesion molecule and doublecortin (neuroblasts). Postischemic exercise and environmental enrichment differentially modulated SVZ cell genesis but lacked effects on the SGZ. Lesion-induced proliferation of neural stem/progenitor cells and neuronal precursors was attenuated in stroke runners without any effects on apoptosis or neuronal migration in the forebrain. Running activity did not affect the SVZ in intact rats. In contrast to postischemic wheel running, postischemic environmental enrichment did not have attenuating effects on the ipsilateral SVZ and increased proliferating putative neural stem cells and neuronal precursors contralaterally. A significant functional improvement, assessed using a rotating pole, was observed only in the postischemically enriched group and was likely due to other types of plasticity than neuronal replacement at this early time point. It may be concluded that in contrast to enriched environment, exercise during the first postischemic week might be detrimental for regenerative processes initiated in the SVZ after stroke

Increased survival of embryonic nigral neurons when grafted to hypothermic rats

Hypothermia can reduce neuronal death caused by ischemia and traumatic brain injury. We therefore investigated whether mild hypothermia in rats receiving a transplant of embryonic mesencephalic rat tissue increases survival of the implanted dopaminergic neurons. Mild hypothermia (32-33°C) during graft implantation and for the following 90 min significantly increased the survival of transplanted dopaminergic neurons to 171% of control values in normothermic (37°C) rats. This demonstrates that treatment of the graft recipient for a relatively short period during and after surgery has a favorable effect on the survival of grafted dopaminergic neurons. These findings may be of importance for clinical neural transplantation trials which are in need of procedures that improve transplant survival. (C) 2000 Lippincott Williams and Wilkins

Cerebral ischemia upregulates vascular endothelin ETB receptors in rat

Background and Purpose-Elevated levels of endothelin 1 (ET 1) have been reported in cerebral ischemia A role for ET may prove more important if the vascular receptors were changed We addressed whether there is any change in ET receptor expression in cerebral ischemia Methods-The right middle cerebral artery (MCA) was occluded in male Wistar rats for 2 hours with the intraluminal filament method The basilar artery and both MCAs were removed after 46 hours of recirculation The contractile responses to ET 1 a combined ETA and ETB receptor agonist and sarafotoxin 6c (S6c) a selective ETB receptor agonist were examined in vitro and ET receptor mRNA was quantified by real time polymerase chain reaction Results-S6c which had no contractile effect per se on fresh or sham operated rat cerebral arteries induced a marked contraction in the occluded MCA (E-m [maximum contraction calculated as percentage of the contractile capacity of 63 5 mmol/L K+] =68+/-68% P<0 0001) while there was no difference in the responses to ET 1 after cerebral ischemia Real time polymerase chain reaction revealed a significant upregulation of both the ETA and ETB receptors (both P<0 05) in the occluded MCA compared with the nonoccluded MCA from the same rats Conclusions-Focal cerebral ischemia in rat induces increased transcription of both ETA and ETB receptors which results in the appearance of a contractile response to the ETB receptor agonist S6c These results suggest a role for ET receptors in the pathogenesis of a vascular component after cerebral ischemia