Ping-An Li

Amelioration by cyclosporin A of brain damage in transient forebrain ischemia in the rat

The immunosuppressant drug cyclosporin A (CsA) is considered to be inherently protective in conditions of ischemia, e.g. in hepatic and cardiac tissue. However, investigations of effects of CsA on neuronal tissue have been contradictory, probably because the blood-brain barrier (BBB) is virtually impermeable to CsA. In the present study, we exploited the finding that the insertion of a syringe needle into brain parenchyma obviously disrupts the BBB and allows influx of CsA, and explored whether CsA, given as intraperitoneal injections daily for 1 week before and 1 week after forebrain ischemia of 7 or 10 min duration, ameliorates the damage incurred to the hippocampal CA 1 sector. In other experiments, the needle insertion and the first i.p. injection of CsA were made 30 min after the start of recirculation, with continued daily administration of CsA during the postinsult week. In animals which were injected with CsA in daily doses of 10 mg kg-1, but in which no needle was inserted, the drug failed to ameliorate CA1 damage, whether the ischemia had a duration of 7 or 10 min. Likewise, needle insertion had no effect on CA1 damage if CsA was not administered. In contrast, when CsA was given to animals with a needle insertion, CA1 damage was dramatically ameliorated, whether treatment was initiated 1 week before ischemia, or 30 min after the start of recirculation. The effect of CsA seemed larger than that of any other drug proposed to have an anti-ischemic effect in forebrain/global ischemia. Injection of tritiated CsA in one animal with BBB disruption lead to detectable radioactivity throughout the ventricular system, suggesting a generalised increase of the entry of CsA across the BBB. The results demonstrate that immunosuppressants of the type represented by CsA markedly ameliorate delayed neuronal damage after transient forebrain ischemia, provided that they can pass the BBB. It is discussed whether the effect of the drug is one involving calcineurin, a protein phosphatase, or if CsA counteracts a permeability transition of the inner mitochondrial membrane, assumed to occur in response to adverse conditions, e.g. gradual accumulation of Ca2+ in the mitochondria in the postischemic period.

Production of hydroxyl free radical by brain tissues in hyperglycemic rats subjected to transient forebrain ischemia.

Preischemic hyperglycemia is known to aggravate brain damage resulting from transient ischemia. In the present study, we explored whether this aggravation is preceded by an enhanced formation of reactive oxygen species (ROS) during the early reperfusion period. To that end, normo- and hyperglycemic rats were subjected to 15 min of forebrain ischemia and allowed recovery periods of 5, 15, and 60 min. Sodium salicylate was injected intraperitoneally in a dose of 100 mg/kg, and tissues were sampled during recirculation to allow analyses of salicylic acid (SA) and its hydroxylation products, 2,3- and 2,5-dihydroxybenzoate (DHBA). Tissue sampled from thalamus and caudoputamen in normoglycemic animals failed to show an increase in 2,3- or 2,5-DHBA after 5 and 15 min of recirculation. However, such an increase was observed in the neocortex after 60 min of recirculation, with a suggested increase in the hippocampus as well. Hyperglycemia had three effects. First, it increased 2,5-DHBA in the thalamus and caudoputamen to values exceeding normoglycemic ones after 15 min of recirculation. Second, it increased basal values of 2,5- and total DHBA in the neocortex. Third, it increased the 60-min values for 2,5- and total DHBA in the hippocampus. These results hint that, at least in part, hyperglycemia may aggravate damage by enhancing basal- and ischemia-triggered production of ROS.

Hyperglycemia-exaggerated ischemic brain damage following 30 min of middle cerebral artery occlusion is not due to capillary obstruction.

Transient focal ischemia of brief duration (15-30 min) gives rise to brain damage. In normoglycemic animals this damage usually consists of selective neuronal necrosis (SNN), and is largely confined to the lateral caudoputamen. In hyperglycemic subjects damage occurs more rapidly, involves also neocortical areas, and is often of the pan-necrotic type (infarction). Since experiments on forebrain ischemia of 30 min duration suggest that microcirculatory compromise develops during recirculation, we studied whether focal ischemia of the same duration, followed by reperfusion for 1, 2 or 4 h, leads to microcirculatory dysfunction. To test this possibility, we fixed the tissue by perfusion and counted the number of formed elements (leukocytes, macrophages and erythrocytes) in capillaries and postcapillary venules. Furthermore, capillary patency was evaluated following in vivo injection of Evans blue. Histopathological examination of tissue fixed by perfusion after 1, 2 and 4 h of recirculation showed an increasing density of SNN in the caudoputamen of normoglycemic animals. Hyperglycemic, but not normoglycemic, animals showed pan-necrotic lesions (infarction) after 4 h of recirculation. As a result, the total volume of tissue damage (SNN plus infarction) was larger in hyper- than in normoglycemic animals at 2 and 4 h of recirculation. In addition, hyperglycemic animals showed involvement of neocortex which increased with the time of reperfusion. In the ischemic hemisphere, between 5 and 10% of counted capillaries contained formed elements. However, since hyperglycemic animals contained an equal (or smaller) amount of cells the results did not suggest that capillary plugging could explain the aggravated damage. Moreover, both normo- and hyperglycemic animals showed close to 100% capillary patency. The results thus fail to support the notion that the aggravation of focal ischemic damage by hyperglycemia is due to obstruction of microvessel by swelling or leukocyte adherence.

Early release of cytochrome C and activation of caspase-3 in hyperglycemic rats subjected to transient forebrain ischemia

The mechanisms underlying the aggravating effect of hyperglycemia on brain damage are still elusive. The present study was designed to test our hypothesis that hyperglycemia-mediated damage is caused by mitochondrial dysfunction with mitochondrial release of cytochrome c (cyt c) to the cytoplasm, which leads to activation of caspase-3, the executioner of cell death. We induced 15 min of forebrain ischemia, followed by 0.5, 1, and 3 h of recirculation in sham, normoglycemic and hyperglycemic rats. Release of cyt c was observed in the neocortex and CA3 in hyperglycemic rats after only 0.5 h of reperfusion, when no obvious neuronal damage was observed. The release of cyt c persisted after 1 and 3 h of reperfusion. Activation of caspase-3 was observed after 1 and 3 h of recovery in hyperglycemic animals. No cyt c release or caspase-3 activation was observed in sham-operated controls while a mild increase of cyt c was observed in normoglycemic ischemic animals after 1 and 3 h of reperfusion. The findings that there is caspase activation and cyt c relocation support a notion that the biochemical changes that constitute programmed cell death occur after ischemia and contribute, at least in part, to hyperglycemia-aggravated ischemic neuronal death.

Effects of Streptozotocin-Induced Hyperglycemia on Brain Damage Following Transient Ischemia ☆

Hyperglycemia is known to aggravate ischemic brain damage. The present experiments were undertaken to explore whether hyperglycemia caused by streptozotocin-induced diabetes exacerbates brain damage following transient brain ischemia as it does in animals acutely infused with glucose. Experimental diabetes was induced by injection of streptozotocin in rats which were subjected to 10 min of forebrain ischemia either 1 week (1-wk) or 4 weeks (4-wk) after the induction of diabetes. Normoglycemic rats exposed to the same duration of ischemia and sham-operated diabetic rats served as controls. The animals underwent evaluation of clinical outcome and histopathological analysis of brain damage. Postischemic seizures developed in 35.3 and 42.1% of 1-wk and 4-wk diabetic hyperglycemic animals, respectively. The incidence of seizure was not different between the two groups. None of the diabetic animals with plasma glucose concentrations below 12 mM exhibited seizure activity. The extent and distribution of brain damage were similar between 1-and 4-wk diabetic animals. In the CA1 and in the subicular regions of hippocampus, both diabetic hyperglycemic and normoglycemic animals showed 70-80% cell death. Diabetic hyperglycemic animals had more severe neuronal necrosis in the parietal cortex than normoglycemic animals. In diabetic hyperglycemic animals, neuronal damage involved additional brain structures, e.g., cingulate cortex, thalamus nuclei, substantia nigra, pars reticulata, and the hippocampal CA3 sector, i.e., structures in which neurons were not affected in normoglycemic ischemic subjects at this duration of ischemia. These findings demonstrate that diabetic hyperglycemic animals frequently develop postischemic seizures and that streptozotocin-induced hyperglycemia results exacerbated postischemic brain damage of the same density and distribution as in acutely glucose-infused animals.

Is Neuronal Injury Caused by Hypoglycemic Coma of the Necrotic or Apoptotic Type

In this study, we explored if a 30 minute period of hypoglycemic coma yields damage which shows some features associated with apoptosis. To that end, we induced insulin-hypoglycemic coma of 30 min duration, and studied brain tissues after the coma period, and after recovery period of 30 min, 3 h, and 6 h. Histopathological data confirmed neuronal damage in all of the vulnerable neuronal populations. Release of cytochrome c (cyt c), assessed by Western Blot, was observed in the neocortex and caudoputamen after 3 and 6 h of recovery. In these regions, the caspase-like activity increased above control after 6 h of recovery. By laser-scanning confocal microscopy, a clear expression of Bax was observed after 30 min of coma in the superficial layers of the neocortex, reaching a peak after 30 min of recovery. Punctuate immunolabeling surrounding nuclei in soma and dendrites in cortical pyramidal neurons likely represents mitochondria, which suggests that Bax protein assembled at the surface of mitochondria in vulnerable neocortical neurons. It is concluded that although previous morphological data have suggested that cells die by necrosis, neuronal damage after hypoglycemic coma shows some features of apoptosis.

Phosphorylation of extracellular signal-regulated kinase after transient cerebral ischemia in hyperglycemic rats.

The present study was undertaken to investigate whether extracellular signal-regulated kinase (ERK) was involved in mediating hyperglycemia-exaggerated cerebral ischemic damage. Phosphorylation of ERK 1/2 was studied by immunocytochemistry and by Western blot analyses. Rats were subjected to 15 min of forebrain ischemia, followed by 0.5, 1, and 3 h of reperfusion under normoglycemic and hyperglycemic conditions. The results showed that in normoglycemic animals, moderate phosphorylation of ERK 1/2 was transiently induced after 0.5 h of recovery in cingulate cortex and in dentate gyrus, returning to control values thereafter. In hyperglycemic animals, phosphorylation of ERK 1/2 was markedly increased in the cingulate cortex and dentate gyrus after 0.5 h of recovery, the increases being sustained for at least 3 h after reperfusion. Hyperglycemia also induced phosphorylation of ERK 1/2 in the hippocampal CA3 sector but not in the CA1 area. Thus, the distribution of phospho-ERK 1/2 coincides with hyperglycemia-recruited damage structures. The results suggest that hyperglycemia may influence the outcome of an ischemic insult by modulating signal transduction pathways involving ERK 1/2.

The effect of α-phenyl-N-tert-butyl nitrone on bioenergetic state in substantia nigra following flurothyl-induced status epilepticus in rats

Status epilepticus (SE), i.e. ongoing seizures of more than 30 min duration, gives rise to bilateral pan-necrotic lesions of the substantia nigra, pars reticulata (SNPR). These are known to be preceded by an initial increase, followed by a depression of metabolic rate, and by failure of the bioenergetic state, suggesting mitochondrial dysfunction. We have previously shown that the spin trap alpha-phenyl-N-tert-butyl nitrone (PBN) prevents the lesions caused by 45 min of SE from occurring, in spite of ongoing seizure activity. In this article, we demonstrate that PBN, given 30 min before seizure induction, reduces or prevents the decrease in ATP concentration and adenylate energy charge, without significantly reducing the amount of lactate accumulated, or the decrease in intracellular pH (pHi). The results suggest that the spin trap nitrone preserves the structural and functional integrity of SNPR neurons by protecting the mitochondria against oxidative damage.

Capillary patency after transient middle cerebral artery occlusion of 2 h duration

Reperfusion after transient focal ischemia of 2 h duration is followed by secondary bioenergetic failure after 4 h of reperfusion. The objective of the present study was to explore whether or not this secondary deterioration is due to secondary microcirculatory compromise. Normal fasted rats were subjected to 2 h of MCA occlusion and allowed reperfusion for 2, 4, 6 and 8 h. At predetermined reperfusion times, rats were injected with Evans blue and decapitated. Capillary patency was determined using a fluorescent double-staining technique. No capillary perfusion deficits were detected in the ischemic neocortical penumbra, neocortical focus or striatal focus. We concluded that the secondary deterioration of bioenergetic state is not due to microcirculatory compromise. Since hyperglycemic animals show pan-necrotic lesions, a hyperglycemic group was added at 8 h of reperfusion to test if the adverse effect of hyperglycemia on ischemic damage is related to capillary compromise. The results showed that, in hyperglycemic rats, capillary perfusion in the striatal focus was compromised after 8 h of recirculation following 2 h of MCA occlusion. It is concluded that when normoglycemic rats are subjected to 2 h of MCA occlusion, capillary patency is not affected during the first 4-6 h of reflow. At 8 h of reflow, though, particularly in hyperglycemic rats, microcirculation is compromised in the caudoputamenal focus, probably reflecting infarction.

Effects of intracarotid arterial injection of cyclosporin A and spontaneous hypothermia on brain damage incurred after a long period of global ischemia

A recent study showed that a single intracarotid arterial injection of cyclosporin A (CsA) can dramatically reduce infarct volume in rats subjected to transient focal ischemia. The present experiments were undertaken to investigate whether intracarotid arterial injection of CsA reduces brain damage after global ischemia. Since hypothermia is also an efficacious factor in preventing ischemic brain damage, in the second part of the experiments we tested whether a combination of hypothermia and CsA would provide additional brain protection. Global ischemia of a 30-min duration was induced in the rat. CsA (10 mg/kg) was injected into the carotid artery immediately after reperfusion. Hypothermia was instituted after ischemia by allowing spontaneous head temperature to fall to 30-32 degrees C, while body temperature was upheld at 37 degrees C. The results demonstrated that vehicle-treated animals could not survive beyond 1-2 days after reperfusion, and the histopathological outcome in a separate group of rats perfusion-fixed after 1 day reperfusion showed 80-100% brain damage in the caudoputamen, and in the hippocampal CA1, CA3, CA4 and dentate gyrus subregions. Microinfarction and grade 3 damage were frequently observed in the cingulate and parietal cortex and in the thalamus. CsA moderately prolonged animal survival to 3 days after reperfusion and reduced brain damage to grade 2 in the cortical areas and the thalamus. Hypothermia further increased animal survival to at least 6 days after reperfusion and reduced brain damage to 30% in the caudoputamen, to close to zero in the CA3, CA4, and dentate gyrus, and to grade 1-2 in the cortical areas and the thalamus. The combination of hypothermia and CsA did not give additional protection.