Hajime Memezawa

Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat.

SummaryIt has become increasingly clear that a stroke lesion usually consists of a densely ischemic focus and of perifocal areas with better upheld flow rates. At least in rats and cats, some of these perifocal (“penumbral”) areas subsequently become recruited in the infarction process. The mechanisms may involve an aberrant cellular calcium metabolism and enhanced production of free radicals. In general, though, the metabolic perturbation in the penumbra requires better characterization. The objective of this article was to define flow distribution in a rat model of reversible middle cerebral artery (MCA) occlusion, so as to allow delineation of the metabolic aberrations responsible for the subsequent infarction. We modified the intraluminal filament occlusion model recently developed by Koizumi et al. (1986), and described in more detail by Nagasawa and Kogure (1989), adopting it for use in both spontaneously breathing and artificially ventilated rats. Successful occlusion of the MCA (achieved in about 9/10 rats) was judged by unilateral EEG depression in ventilated rats, and neurological deficits, such as circling, in spontaneously breathing ones. CBF in the ipsilateral hemisphere was reduced to nearly constant values after 20, 60, and 120 min of occlusion, flow rates in the focus being about 10% and in the perifocal ipsilateral areas about 15–20% of control (contralateral side). When the filament was left in place (permanent occlusion) 2,3,5-triphenyl tetrazolium chloride (TTC) staining and histopathology after 24 h showed a massive infarct on the occluded side, extending from caudoputamen and overlaying cortex to the occipital striate cortex. Animals recirculated after 60 min of MCA occlusion, and allowed to survive 7 days for histopathology, showed infarction of the caudoputamen (lateral part or whole nucleus) in 5/6 animals and selective neuronal necrosis in one animal. The neocortex showed either infarcts, selective neuronal necrosis, or no damage. There was some overlap between neocortical areas which were infarcted and those which were salvaged by reperfusion. In general, though, both the CBF data and the recovery studies with a histopathological endpoint define large parts of the neocortex as perifocal (penumbral) areas which lend themselves to studies of metabolic events leading to infarction.

Focal and Perifocal Changes in Tissue Energy State during Middle Cerebral Artery Occlusion in Normo- and Hyperglycemic Rats

The objective of the present study was to assess changes in cellular energy metabolism in focal and perifocal areas of a stroke lesion and to explore how these changes are modulated by preischemic hyperglycemia. A model for reversible occlusion of the middle cerebral artery (MCA) in rats was used to study changes in energy metabolism. Following MCA occlusion for 5, 15, or 30 min in normoglycemic rats, the tissue was frozen in situ, and samples from the lateral caudoputamen and from two neocortical areas were collected for metabolite analyses, together with a control sample from the contralateral, nonischemic hemisphere. Two other groups, subjected to 30 min of MCA occlusion, were made hyperglycemic by acute glucose infusion or by prior injection of streptozotocin. Enzymatic techniques were used for measurements of phosphocreatine, creatine, ATP, ADP, AMP, glycogen, glucose, pyruvate, and lactate. The neocortex of the contralateral, nonischemic hemisphere had labile metabolites that were similar to those measured in control animals. Ipsilateral neocortex bordering the focus, and thus constituting the “penumbra,” showed mild to moderate ischemic changes. In the “focus” (lateral caudoputamen plus the overlying neocortex), deterioration of energy state was rapid and relatively extensive (ATP content 20–40% of control). After 5 min of occlusion, no further deterioration of metabolic parameters was observed. Substrate levels were markedly reduced, and lactate content rose to ∼10 mM kg−1. In the animals with the most severe energy depletion, no additional accumulation of lactate occurred, suggesting substrate depletion. This was confirmed by the results obtained in the hyperglycemic subjects whose tissue lactate contents rose to ∼20 mM kg−1. However, the energy state of the focus was better preserved in both hyperglycemic groups as compared with the normoglycemic group. It has been shown, in this model, that relatively brief occlusion periods are required to induce infarction. The present results demonstrate that this can occur in spite of the absence of pronounced depletion of energy reserves. After 30 min of MCA occlusion, infarction developed in the lateral caudoputamen, but not in the neocortex. Since a similar perturbation in metabolic state was demonstrated here, other factors must contribute to the degree of tissue damage. The present results suggest that damage is exaggerated by hyperglycemia because it allows additional lactate to accumulate in the partially substrate-depleted tissue.

Hyperthermia nullifies the ameliorating effect of dizocilpine maleate (MK-801) in focal cerebral ischemia.

The present study was inspired by two previous findings from the laboratory. The first was that dizocilpine maleate (MK-801) fails to reduce infarct size when the middle cerebral artery (MCA) is permanently occluded by an intraluminal filament technique in rats. In seeking the reasons for this we measured temperature and found that the body temperature of occluded animals increases to 39.0-39.5 degrees C during the first 2-3 h. In order to explore whether the rise in temperature was responsible for the lack of effect of MK-801, two groups of animals were studied, both containing animals which were subjected to 2 h of transient MCA occlusion and given MK-801 15 min before, as well as 6 and 24 h after ischemia. In one group, temperature was allowed to rise spontaneously during ischemia (39.0-39.5 degrees C). In the other, body temperature was maintained close to normal during ischemia, and for the first 6 h postischemically, by cooling of the ambient air. Infarct volume was assessed by triphenyltetrazolium chloride staining after 48 h of recovery. The results showed that MK-801 failed to reduce infarct size in animals whose body temperature rose during ischemia. In contrast, the drug markedly reduced infarct volume in temperature-controlled animals; in fact, 5/8 animals had no infarcts but selective neuronal damage only. The results suggest that amelioration of focal ischemic damage cannot be expected if body and brain temperature is allowed to rise above normal.

Free radicals and brain damage due to transient middle cerebral artery occlusion: the effect of dimethylthiourea

The objective of this study was to assess whether dimethylthiourea (DMTU), an established free radical scavenger, ameliorates ischaemic damage due to 2–3 h of transient middle cerebral artery (MCA) occlusion, induced by an intraluminal filament. A major point adressed was whether DMTU given before MCA occlusion only delayed the “maturation” of the damage, or if it had a lasting effect on infarct size. The end point was morphological, and either encompassed triphenyltetrazolium chloride (TTC) staining of tissue slices after 24 h or 48 h of recovery, or histopathological assessment of infarct size after 7 days of recovery. In a preliminary series of experiments, rats were subjected to 3 h of MCA occlusion, and infarct volume was assessed by TTC staining after 24 h of recovery. DMTU in a dose of 750 mg/kg reduced infarct volume by more than 50%. However, due to a high mortality rate, that protocol was not subsequently pursued. When the ischaemia duration was reduced to 2 h and the DMTU dose to 400 mg/kg, a similar amelioration of the tissue damage was observed. However, since DMTU reduced a spontaneous rise in body temperature to 39.0–39.5°C, DMTU-treated animals in the main series of experiments with 24 and 48 h of recovery were treated so that they had the same temperature rise as the saline controls. Under such constant temperature conditions, the effect of DMTU at 24 h of recovery was borderline (P= 0.052) and at 48 h it was nil. The lack of a lasting effect of DMTU was supported by the findings on evaluation of infarct area after 7 days of recovery. The results raise the important question whether DMTU, and perhaps other free radical scavengers, delay rather than ameliorate the ischaemic lesion developing after transient MCA occlusion.