The temporal evolution of hypoglycemic brain damage

The caudate nucleus and putamen belong to the selectively vulnerable brain regions which incur neuronal damage in clinical and experimental settings of both hypoglycemia and ischemia. We have previously documented the density and distribution of the hypoglycemic damage in rat caudoputamen, but the evolution of the injury, i.e., the sequence of structural changes, has not been assessed. Therefore, in the present study we analyze the light and electron microscopic alterations in the caudoputamen of rats exposed to standardized, pure insults of severe hypoglycemia with isoelectric EEG for 10-60 min, or in rats which, following insults of 30 or 60 min, were allowed to recover for periods from 5 min to 6 months. The hypoglycemic insult produced severe nerve cell injury in the dorsolateral caudoputamen. Immediately after the insult abnormal light neurons with clearing of the peripheral cytoplasm were present. These cells disappeared early in the recovery period, as they do in the cerebral cortex. Dark neurons were also present, but unlike those in the cerebral cortex they did not appear until recovery was instituted. Their number increased for a couple of hours and they became acidophilic within 4-6 h. At this stage, electron microscopy revealed severe clumping of the nuclear chromatin and cytoplasm as well as incipient fragmentation of cell membranes, all these changes indicating an irreversible injury. Within 24 h flocculent densities appeared in the mitochondria and by day 2-3 of recovery the great majority of the medium-sized neurons had undergone karyorrhexis and cytorrhexis, their remnants being subsequently removed by macrophages. After some weeks only large and a few medium-sized neurons remained amidst reactive astrocytes and numerous macrophages. The delay in the appearance of dark, lethally injured medium-sized neurons until the recovery was instituted suggests an effect that does not become apparent until the substrate supply and energy production are restored. Furthermore, it points out again the selectivity of the hypoglycemic nerve cell injury with respect to the type (metabolic characteristics?) and topographic location of the neurons.     

Neurophysiological recovery after hypoglycemic coma in the rat: correlation with cerebral metabolism

Recovery of electroencephalographic activity and somatosensory evoked responses was studied in paralyzed and lightly anesthetized (70% N2O) rats in which profound hypoglycemia had been induced by insulin administration. The duration of severe hypoglycemia was defined as the duration of a flat electroencephalogram (EEG) recording (5, 30, and 60 min, respectively) before restitution with glucose. The restitution period was followed by continuous EEG monitoring and repeated tests for evoked potentials. After 180 min of recovery, the brains were frozen in situ with liquid nitrogen and analyzed for energy metabolism. In accordance with earlier metabolic studies from this laboratory, the recovery after 60 min of severe hypoglycemia was incomplete, with signs of permanent failure of energy metabolism. There was persistent ATP reduction proportional to the duration of the hypoglycemia. The short-term recovery of EEG and sensory evoked responses was proportional to the duration of severe hypoglycemia. The neurophysiological recovery after 5 min of severe hypoglycemia was complete. After 30 min of severe hypoglycemia, the evoked responses recovered but showed a significant prolongation of latency, compared with normal. After 60 min of severe hypoglycemia, no early evoked response and scanty EEG activity were observed. The neurophysiological observations indicate a persistent deficit of synaptic transmission in the somatosensory pathway, including the cortical projection. This can be correlated with neuropathologic changes that are particularly prominent in intermediate cortical layers, as previously shown.     

Cerebral protein synthesis during long-term recovery from severe hypoglycemia

Regional protein synthesis was investigated in the rat brain during long-term recovery from insulin-induced hypoglycemia with 30 min of cerebral electrical silence. At various time intervals up to 14 days after glucose replenishment, animals received a single dose of L-[3,5-3H]tyrosine and were killed 30 min later. Brains were processed for autoradiography using the stripping film technique. Although hypoglycemia sufficiently severe to cause cessation of EEG activity leads to almost complete inhibition of amino acid incorporation in all "vulnerable" forebrain structures (cerebral cortex, hippocampus, caudoputamen), autoradiographs revealed a very specialized sequence with differential posthypoglycemic restoration of biosynthetic activity in certain neuronal cell types. Three major subpopulations could be distinguished: Neurons that fully regained their protein synthetic capacity within 6 h following hypoglycemia (cortical neurons of layer III-VI, large neurons in the caudoputamen, CA3 and CA4 pyramidal neurons, the majority of granule cells of the dentate gyrus) seemed to escape neuronal necrosis. Prolonged impairment of protein synthesis with only partial restoration during the early posthypoglycemic recovery period (CA1 neurons, most small- to medium-sized neurons of the caudoputamen) carried an increased risk of permanent cell damage. The large majority of these neurons, however, showed full recovery of protein synthesis as late as 7 days after hypoglycemia. Neurons with complete lack of amino acid incorporation after 6 h of recovery (granule cells at the crest of the dentate gyrus, small neurons of the dorsolateral caudoputamen) never resumed protein synthesis, regressed, and died. These studies in conjunction with morphological analysis indicate that the sequential recovery of protein synthesis reflects the extent to which neuronal populations are at risk during severe hypoglycemia.     

Heat shock in cultured neurons and astrocytes: correlation of ultrastructure and heat shock protein synthesis

Cultured cerebral cortical neurons and astrocytes were compared after a brief shock. Morphological findings were correlated with the synthesis of the 68 kD heat shock protein (HSP68). Heat shocked neurons demonstrated many severe morphological changes after exposure to temperatures of 43 degrees C for 15 min and 45 degrees C for 10 min. Nuclear membrane 'blebbing' with lysis of the membrane, chromatin clumping, and disappearance of the nucleolus were prominent after both conditions. Lysis of the cell membrane was noted in severely injured neurons; this was more prominent at the higher temperature. In addition, alterations to polyribosomes, Golgi apparatus, rough endoplasmic reticulum and mitochondria were noted in the cytoplasm of neurons after heat shock. In contrast, no significant changes were noted in either the nucleus or cytoplasm of heat shocked astrocytes. The severity of morphological changes in neurons directly correlated with the low level of induction of HSP68 in neurons. Neurons synthesized much less 68 kD heat shock protein than similarly heat shocked astrocytes. We conclude that cultured cerebral cortical neurons are more susceptible to injury after heat shock than heat resistant astrocytes and that one possible mechanism of injury is failure to synthesize adequate amounts of HSP68 after injury.     

Transient focal ischemia in subhuman primates. Neuronal injury as a function of local cerebral blood flow

Unilateral, transient (30, 60, and 120 minutes (min)) middle-cerebral-artery (MCA) occlusion was induced via transorbital craniotomy in 11 waking subhuman primates. Local cerebral blood flow (LCBF) was calculated from hydrogen clearance curves obtained through the use of intracerebral platinum microelectrodes. Unilateral MCA occlusion decreased LCBF in the territory of the ipsilateral MCA. Within minutes of the arterial occlusion all monkeys developed contralateral neurologic deficits that began disappearing three hours (h) after reopening the MCA. Regional ischemia, followed by 24 h of reperfusion, produced varying degrees of tissue vacuolation which correlated (r = 0.60, p less than 0.01, n = 49) with the percent reduction in LCBF multiplied by the occlusion time. Neurons were classified according to the structural features of their perikaryon. A plot of neuron types versus percent vacuolation suggested that normal neurons become increasingly scalloped under increasingly severe ischemic conditions. The number of scalloped neurons decreased precipitously in areas of marked sponginess coincident with the appearance of irreversibly damaged neurons. Local tissue edema values exceeding 30% correlated with irreversible injury to all neurons in the same area. Regional cerebral ischemia of increasing severity was acompanied by increasing numbers of lethally injured neurons.     

Electrolyte shifts between brain and plasma in hypoglycemic coma

Hypoglycemia of sufficient severity to cause cessation of EEG activity (coma) is accompanied by energy failure and by loss of ion homeostasis, the latter encompassing a marked rise in extracellular fluid (ECF) K+ concentration and a fall in ECF Ca2+ concentration. Presumably, ECF Na+ concentration decreases as well. In the present study, the extent that the altered ECF-plasma gradients give rise to net ion fluxes between plasma and tissue is explored. Accordingly, whole tissue contents of Ca2+, Mg2+, K+, and Na+ were measured. The experiments were carried out in anaesthetized and artificially ventilated rats given insulin i.p.; cerebral cortical tissue was sampled at the stage of slow-wave EEG activity, after 10, 30, and 60 min of coma (defined as isoelectric EEG), as well as after 1.5, 6, and 24 h of recovery. In the precomatose animals (with a slow-wave EEG pattern), no changes in electrolyte contents were observed. During coma, tissue Na+ content increased progressively and the K+ content fell (each by 20 mumol g-1 during 60 min). During recovery, these alterations were reversed within the first 6 h. The Mg2+ content remained unchanged. In spite of the appreciable plasma to ECF Ca2+ gradient, no significant calcium accumulation was observed. It is concluded significant calcium accumulation was observed. It is concluded that hypoglycemia leads to irreversible neuronal necrosis in the absence of gross accumulation of calcium in the tissue.     

Hypotension as a complication of hypoglycemia leads to enhanced energy failure but no increase in neuronal necrosis

The hypothesis that arterial hypotension aggravates hypoglycemic brain damage was tested. Thirty minutes of insulin induced hypoglycemia with a flat EEG ("isoelectricity") was compared in seven series of rats. In three series of animals, the energy state of the cerebral cortex was determined at blood pressures of 140, 100 and 80 mm Hg respectively. Hypotension during hypoglycemia exacerbated cortical energy failure. In the fourth to sixth series, blood pressure was adjusted during isoelectricity to 160, 100 and 60 mm Hg, respectively. A seventh series had induced hypotension to 60 mm Hg only in the recovery period. Quantitation of neuronal death was performed in the fourth to seventh series of rats by direct visual counting of acidophilic neurons in sub-serially sectioned brains after one week survival. Although the first three series demonstrated enhanced deterioration of the cerebral energy state with lower blood pressures during hypoglycemia, the fourth to seventh series showed no augmentation of quantitated hypoglycemic neuronal necrosis. The distinct distribution of hypoglycemic brain damage, suggesting a fluid-borne toxin, was present at normal and reduced blood pressures, with no tendency toward an ischemic pattern of pathology. In spite of previously demonstrated reductions of cerebral blood flow to ischemic levels in regions with pronounced loss of autoregulation, no regional exacerbation of neuronal necrosis was seen in these brain areas. It is concluded that hypoglycemic brain damage is distinct from ischemic brain damage, and that the two insults are not additive. Furthermore, moderate hypotension to 60 mm Hg does not aggravate the damage in spite of an enhanced energy failure.     

The role of postischemic recirculation in the development of ischemic neuronal injury following complete cerebral ischemia

Summary The neuronal response to complete cerebral ischemia (CCI) of 5–15 min duration was evaluated at the light and electron microscopic level subsequent to postischemic recirculation periods of up to 60 min. Following postischemic reperfusion, the homogeneous neuronal changes characteristic of permanent CCI were modified into a heterogeneous pattern of selectively vulnerable neuronal responses. Four basic types of neuronal injury were represented within this heterogeneous neuronal population. The Type I neuronal response was most numerous and consisted of chromatin clumping, nucleolar condensation and a breakdown of polysomes. This response may represent a reversal of some of the neuronal changes observed after permanent CCI. In addition to the above changes, Type II neurons contained swollen mitochondria and Golgi saccules which appeared as microvacuoles under the light microscope. Type III neurons displayed varying degrees of neuronal shrinkage and numerous swollen mitochondria. Type IV neurons were markedly shrunken and electron-dense with few identifiable subcellular structures. The distribution of Type I neurons was random but the other neuronal responses occurred in selectively vulnerable brain regions. The number of Type II, III, and IV neurons increased with extended insult durations but were unaffected by the length of recirculation. Ten minutes of CCI represented the threshold for a significant increase in the number of severely altered neurons. These findings suggest that considerable neuronal injury may be present after 10–15 min of CCI, and the lack of a recirculation period following CCI appears to afford the brain parenchyma an extensive degree of structural protection.Supported by PHS Grant NS-12587     

Neuroprotective effects of the sodium channel blocker RS100642 and attenuation of ischemia-induced brain seizures in the rat

Seizurogenic activity develops in many patients following brain injury and may be involved in the pathophysiological effects of brain trauma and stroke. We have evaluated the effects of the use-dependent sodium channel blocker RS100642, an analog of mexiletine, as a neuroprotectant and anti-seizure agent in a rat model of transient middle cerebral artery occlusion (MCAo). Post-injury treatment with RS100642 (0.01-5.0 mg/kg) dose-dependently reduced brain infarction, improved functional recovery of electroencephalographic (EEG) power, and improved neurological outcome following 2 h of MCAo and 24 h recovery. This effect was more potent and offered a larger reduction of brain infarct volume than a maximal neuroprotective dose of mexiletine (10.0 mg/kg). Furthermore, brain seizure activity recorded following 1 h MCAo and 72 h of recovery in injured rats was either completely blocked (30 min pre-MCAo treatment) or significantly reduced (30 min post-MCAo treatment) with RS100642 (1.0 mg/kg) treatment resulting in greater than 60% reduction of core brain infarct. These results indicate that brain seizure activity during MCAo likely contributes to the pathophysiology of brain injury and that RS100642 may be an effective neuroprotective treatment not only to decrease brain injury but also to reduce the pathological EEG associated with focal ischemia.     

Protective effect of lesion to the glutamatergic cortico-striatal projections on the hypoglycemic nerve cell injury in rat striatum

Summary In rat striatum severe hypoglycemia causes an irreversible nerve cell injury, which does not become manifest until during the post-insult recovery period. This injury can be ameliorated by lesions of the glutamatergic cortico-striatal pathway, which suggests that an excitotoxic effect mediated by the glutamatergic input is the likely cause of the posthypoglycemic nerve cell destruction. In this paper we further characterize the protective effect of abolishing the glutamatergic innervation to striatum at the ultrastructural level. Two weeks after a unilateral cortical ablation rats were subjected to 30 min of severe hypoglycemia with isoelectric EEG and killed either immediately after the insult or following 60 min of recovery induced by restoring the blood glucose levels. Immediately after the hypoglycemic insult the structure of striatum was similar on both sides (except for the changes attributable to the ablation); i.e., the neurons and their dendrites had pale cytoplasm with condensed mitochondria, sparse RER and pinpoint ribosomes. After 60 min restitution numerous striatal neurons on the non-protected, non-ablated side had turned variably dark and condensed, whereas under-neath the ablation they remained similar as immediately after hypoglycemia. This sequence indicates that the most likely cause of nerve cell destruction on the non-protected side is the excitotoxic effect mediated by the glutamatergic innervation, which is superimposed on the action of the hypoglycemic insultper se. Furthermore, the primary condensation of neurons and their dendrites indicate existence of another type of acute excitotoxic nerve cell injury which differs from the previously described injury characterized by neuronal swelling.Supported by the Swedish Medical Research Council (projects 12X-07123 and 14X-263), by U.S. Public Health Service (NIH grant no. 2 RO1 NS-07838) and by the Finnish Medical Research Council     

Effects of calcium entry blocker emopamil on postischemic energy metabolism of the isolated perfused rat brain

The effects of emopamil on postischemic energy metabolism and electroencephalographic (EEG) recovery were investigated in the isolated rat brain perfused at either constant pressure or, alternatively, at constant flow rate. Flow rate and perfusion pressure were monitored continuously. The brains were perfused with a fluorocarbon emulsion for 30 min, and after 30 min of ischemia, perfusion was reinstituted for 5, 30, or 60 min. Global cerebral perfusion rate was increased by emopamil throughout the perfusion period and, accordingly, in brains perfused at a constant flow rate, perfusion pressure was reduced by the drug. At constant pressure perfusion, after 5 min after ischemia, cortical levels of creatine-phosphate, adenosine triphosphate (ATR), glucose, glucose-6-phosphate, and fructose-6-phosphate were higher in emopamil-treated brains than in controls, although the levels of adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were reduced. When brains were perfused at constant flow rate, however, emopamil exhibited no effect on brain energy metabolism in the early reperfusion period. Postischemic restoration of high-energy phosphates proved to depend on the flow rate used. After 30 min of postischemic reperfusion, cortical levels of lactate were lower in emopamil-treated brains compared to controls at both constant pressure and constant volume perfusion. Postischemic lactate levels were independent of flow rate and were also reduced when emopamil was only present during reperfusion. The postischemic restoration of cortical EEG activity was improved by the calcium entry blocker.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in pyruvate dehydrogenase complex activity during and following severe insulin-induced hypoglycemia

The effect of severe insulin-induced hypoglycemia on the activity of the pyruvate dehydrogenase enzyme complex (PDHC) was investigated in homogenates of frozen rat cerebral cortex during burst suppression EEG, after 10, 30, and 60 min of isoelectric EEG, and after 30 and 180 min and 24 h of recovery following 30 min of hypoglycemic coma. Changes in PDHC activity were correlated to levels of labile organic phosphates and glycolytic metabolites. In cortex from control animals, the rate of [1-14C]pyruvate decarboxylation was 7.1 +/- 1.3 U/mg of protein, or 35% of the total PDHC activity. The activity was unchanged during burst suppression EEG whereas the active fraction increased to 81-87% during hypoglycemic coma. Thirty minutes after glucose-induced recovery, the PDHC activity had decreased by 33% compared to control levels, and remained significantly depressed after 3 h of recovery. This decrease in activity was not due to a decrease in the total PDHC activity. At 24 h of recovery, PDHC activity had returned to control levels. We conclude that the activation of PDHC during hypoglycemic coma is probably the result of an increased PDH phosphatase activity following depolarization and calcium influx, and allosteric inhibition of PDH kinase due to increased ADP/ATP ratio. The depression of PDHC activity following hypoglycemic coma is probably due to an increased phosphorylation of the enzyme, as a consequence of an imbalance between PDH phosphatase and kinase activities. Since some reduction of the ATP/ADP ratio persisted and since the lactate/pyruvate ratio had normalized by 3 h of recovery, the depression of PDHC most likely reflects a decrease in PDH phosphatase activity, probably due to a decrease in intramitochondrial Ca2+.     

MK801 decreases glutamate release and oxidative metabolism during hypoglycemic coma in piglets.

Hypoglycemic coma increases extracellular excitatory amino acids, which mediate hypoglycemic neuronal degeneration. Cerebral oxygen consumption increases during hypoglycemic coma in piglets. We tested the hypothesis that the NMDA-receptor antagonist dizocilpine (MK801) attenuates the increase in cerebral oxygen consumption during hypoglycemia. We measured EEG, cerebral blood flow (CBF), cerebral oxygen consumption (CMRO(2)) and cortical microdialysate levels of glutamate, aspartate and glycine in pentobarbital-anesthetized piglets during 60 min of insulin-induced hypoglycemic coma. NMDA-receptor distribution was measured by autoradiography. MK801 (0.75 mg/kg i.v.) was given within 5 min after onset of isoelectric EEG. Saline- and MK801-treated normoglycemic control animals were also studied. Brain temperature was maintained at 38.5+/-0.5 degrees C. MK801 prevented the 5--10-fold increase in glutamate and aspartate occurring in saline-treated hypoglycemic animals, and attenuated the increase in CMRO(2). Increases in CBF of 200--400% during hypoglycemic coma were not affected by MK801. MK801 did not alter CBF, CMRO(2) or microdialysate amino acid levels in normoglycemic control animals. Parietal cortex corresponding to microdialysis sites was highly enriched in NMDA receptors, and the density and distribution overall of NMDA receptor binding sites were comparable to that reported in other species. We conclude that NMDA receptor activation plays a central role in hypoglycemia-induced glutamate release, and contributes to increased cerebral oxygen consumption. Neuroprotective effects of MK801 during hypoglycemia in piglets may involve inhibitory effects on glutamate release and oxidative metabolism.     

Effects of insulin-induced hypoglycemia on intracellular pH and impedance in the cerebral cortex of the rat

In order to evaluate changes in extra- and intracellular pH in the brain during hypoglycemia rats were injected with insulin and pH changes evaluated when the EEG showed a slow-wave-polyspike pattern (‘precoma’), or when EEG activity had ceased for 5, 15 or 30 min (‘coma’). Extra- and intracellular acid-base changes were evaluated from pCO₂ and HCO₃-concentrations. In order to allow calculation of intracellular pH (and HCO₃-concentrations) changes in extracellular fluid volume were estimated by measurements of cortical tissue impedance. The main results were as follows.

1. (1) At constant arterial pCO₂ the CSF HCO₃-concentration either rose (15 min of coma) or remained unchanged (all other groups). However, since the cerebrovenous (and tissue) pCO₂ fell, all groups except one (30 min of coma) showed a significant increase in extracellular fluid pH.

2. (2) During severe hypoglycemia, and especially when EEG activity ceased, cortical impedance increased markedly. Calculations with the help of the Rayleigh and Maxwell equations showed that the extracellular fluid volume was reduced to about 50% of control.

3. (3) Intracellular pH increased significantly in precoma and in coma of 15 and 30 min duration. However, pH in the 5 min coma group was significantly lower (but no different from control).

4. (4) In general, the increase in intracellular pH is consistent with previous findings that hypoglycemia is associated with oxidation of endogenous acid metabolites. However, the data suggest that in the initial period of coma acids accumulate by some unidentified mechanism.

Repetitive ischaemia of cat brain: pathophysiological observations

The cumulative effect of repetitive ischaemia on brain injury was studied in halothane-anaesthetized cats submitted to three episodes of global cerebro-circulatory arrest. Ischaemia of 5.0, 7.5 and 10.0 min duration was produced at hourly intervals by intrathoracic clamping of the innominate and subclavian arteries, and the resulting pathophysiological changes were evaluated by recordings on the electroencephalogram (EEG), blood flow and specific gravity. During each episode of ischaemia EEG flattened within 15 s. After ischaemia the latency of EEG recovery increased with the duration and with the number of repetitions of each ischaemic episode, indicating cumulation of electrophysiological impairment. The flow studies revealed a minor degree of hyperaemia after each ischaemic episode, followed by severe hypoperfusion in the caudate nucleus but not in the cerebral cortex. Brain oedema - as assessed by specific gravity measurements - developed in the hippocampus after three episodes of 5 min ischaemia, and in all grey matter structures investigated after three episodes of 10 min ischaemia. To evaluate the resistance of the ischaemically injured brain to respiratory hypoxia, total oxygen was repeatedly reduced to 5% for 5 min. During these episodes EEG activity progressively declined as a function of the length and the repetition of ischaemia. Parallel n.m.r. spectroscopic measurements in the same model have demonstrated that disturbances of brain energy state during the hypoxic episodes are minor even after three episodes of 10 min ischaemia. EEG suppression, in consequence, is an electrical shut-down phenomenon for the maintenance of cerebral energy state under critical conditions of oxygen delivery.     

Changes in local cerebral glucose utilization, DC potential and extracellular potassium in various degree of experimental cerebral contusion

Pathophysiology of the traumatized brain, especially that of cerebral contusion, is very complex and has not been well understood. In recent years, changes in extracellular ion concentration have been known in various pathological conditions such as cerebral concussion, spinal contusion, ischemia, hypoglycemia, epilepsy and spreading depression as one of the triggers to lead to secondary brain damage. To know the metabolic and ionic changes following cerebral contusion, the authors made various degree of cerebral contusion by fluid percussion method, and observed successive changes in EEG, DC potential, extracellular potassium concentration and local cerebral glucose utilization (LCGU). MATERIALS and METHODS: Using 42 male Wistar rats, mild (0.2 kg/cm2), moderate (0.4 kg/cm2) and severe contusion (0.6 kg/cm2) were made in the left lower parietal region of the rats. EEG, DC potential and extracellular potassium concentration (using potassium sensitive glass microelectrode) were monitored for four to five hours after making the contusions. LCGU (by 14C-2-deoxyglucose method) was studied at the time of the negative shift of DC potential. RESULTS: The negative shift of DC potential with EEG suppression was observed at 30 min. to 3 hours after injury. The severer the injury was, the earlier and the more frequent negative shifts appeared. LCGU showed no significant changes in the mild injury group. In the moderate injury group, frequent negative shifts of DC potential associated with EEG suppression were observed. A 20% increase of glucose utilization in the cortex of the lesion side was observed whereas 50% decreases in the subcortical structures were found. In the severe injury group, EEG was suppressed immediately after contusion and had never recovered. DC potential fluctuated and was unstable. The increase of LCGU was noted not only in the cortex of the lesion side but also in some of the subcortical structures (hippocampus, caudate nucleus, dentate nucleus and thalamus). The extracellular potassium concentration rose to 30 mM, being correlated closely with DC potential. DISCUSSION: Increase of LCGU associated with EEG suppression, negative shift of DC potential and elevation in extracellular potassium concentration was thought to be due to spreading depression. It was postulated that spreading depression following cerebral contusion causes energy failure and can lead to secondary brain damage.     

Repetitive ischaemia of cat brain: pathophysiological observations

The cumulative effect of repetitive ischaemia on brain injury was studied in halothane-anaesthetized cats submitted to three episodes of global cerebro-circulatory arrest. Ischaemia of 5.0, 7.5 and 10.0 min duration was produced at hourly intervals by intrathoracic clamping of the innominate and subclavian arteries, and the resulting pathophysiological changes were evaluated by recordings on the electroencephalogram (EEG), blood flow and specific gravity. During each episode of ischaemia EEG flattened within 15 s. After ischaemia the latency of EEG recovery increased with the duration and with the number of repetitions of each ischaemic episode, indicating cumulation of electrophysiological impairment. The flow studies revealed a minor degree of hyperaemia after each ischaemic episode, followed by severe hypoperfusion in the caudate nucleus but not in the cerebral cortex. Brain oedema - as assessed by specific gravity measurements - developed in the hippocampus after three episodes of 5 min ischaemia, and in all grey matter structures investigated after three episodes of 10 min ischaemia. To evaluate the resistance of the ischaemically injured brain to respiratory hypoxia, total oxygen was repeatedly reduced to 5% for 5 min. During these episodes EEG activity progressively declined as a function of the length and the repetition of ischaemia. Parallel n.m.r. spectroscopic measurements in the same model have demonstrated that disturbances of brain energy state during the hypoxic episodes are minor even after three episodes of 10 min ischaemia. EEG suppression, in consequence, is an electrical shut-down phenomenon for the maintenance of cerebral energy state under critical conditions of oxygen delivery.     

Cytosolic free calcium, NAD/NADH redox state and hemodynamic changes in the cat cortex during severe hypoglycemia

Using indo-1, a fluorescent Ca2+ indicator, in vivo fluorometric measurements were made of changes in cytosolic free Ca2+, NAD/NADH redox state, and hemodynamics directly from the cat cortex during and after severe insulin-induced hypoglycemia. Cytosolic free Ca2+ started to increase when the EEG became isoelectric, remained at a significantly high level (p less than 0.05) during the period of isoelectric EEG (IEEG), and recovered to the control level 6 min following an intravenous infusion of glucose. The NAD/NADH redox state oxidized significantly during IEEG and then recovered rapidly to the control level after the glucose infusion. Local cortical blood volume (LCBV) increased gradually during the progression of hypoglycemia, reaching the maximal level (146 +/- 7%) at the end of IEEG, and then started to recover. The mean transit time (MTT) through the cortical microcirculation was shortened during the IEEG (control: 3.84 +/- 0.41 s versus IEEG: 2.73 +/- 0.17 s, p less than 0.05), whereas it was prolonged during the 30-min recovery period (5.68 +/- 0.58 s, p less than 0.05). Local cortical blood flow calculated from the LCBV and MTT showed a twofold increase 5 min into IEEG (201 +/- 27% of control, p less than 0.05), recovered 15 min into the recovery period, and then decreased to 77% of control (p less than 0.05) by 30 min. The data support the hypothesis that hypoglycemic brain damage might be mediated by an elevation of cytosolic free calcium.     

Effects of intra-ischemic blood pressure on outcome from 2-vessel occlusion forebrain ischemia in the rat.

Halothane anesthetized Sprague-Dawley rats underwent 10 min of bilateral carotid artery occlusion with mean arterial pressure (MAP) held at 30, 50 or 60 mmHg. Sham rats did not undergo ischemia. A 7-day recovery interval was allowed. Intra-ischemic electroencephalographic (EEG) changes, behavioral function (Days 5-7), and histologic injury (Day 7) were evaluated. Under similar conditions, cerebral blood flow was determined after 10 min ischemia by the [3H]nicotine indicator fractionation technique. EEG isoelectricity was observed in 11 of 11, 5 of 10, and 2 of 11 rats in the 30 mmHg, 50 mmHg, and 60 mmHg groups respectively. Neither passive avoidance cross-over latencies nor general motor scores were affected by intra-ischemic MAP and no differences from sham performance were observed. The per cent of CA1 neurons counted as dead (left and right hemispheres combined) was significantly affected by intra-ischemic MAP (72, 46 and 28% in the 30 mmHg, 50 mmHg, and 60 mmHg groups, respectively; P less than 0.001). A greater than 50% CA1 neuronal mortality rate was present only in those rats exhibiting EEG isoelectricity. However, the number of rats demonstrating greater than a 25% interhemispheric difference in CA1 neuronal loss was greatest in the 50 mmHg group (P less than 0.02). Hippocampal blood flow decreased in association with severity of hypotension (8 +/- 1, 35 +/- 8, and 48 +/- 2 ml/100 g/min (mean +/- S.E.M.) for 30, 50, and 60 mmHg, respectively; P less than 0.01). Again, however, the greatest variability in blood flow was observed at MAP = 50 mmHg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of S-adenosyl-L-methionine on the cerebral energy metabolism and microcirculation in the rats subjected to transient forebrain ischemia

Effects of S-adenosyl-L-methionine (SAM) on the improvement of cerebral energy metabolism and microcirculation were examined in postischemic rat brain. Male Wistar rats, whose vertebral arteries were electrically cauterized last day, were subjected to forebrain ischemia by temporary clipping of both common carotid arteries. After 60 min of ischemic insult, they were intravenously administered with SAM at doses of 30 or 100 mg/kg; this was followed by recirculation for 60 min. To determine cerebral concentrations of energy metabolites, the brain was frozen in situ. Adenine nucleotides (ATP, ADP, AMP) were assayed by anion-exchange HPLC system, and other metabolites (PCr, glucose, lactate, pyruvate) were analyzed by enzymatic fluorometry. In order to estimate regional cerebral blood flow (rCBF) and glucose utilization, double-tracer autoradiography was undertaken using 14C-iodoantipyrine (14C-IAP) and 18F-fluorodeoxyglucose (18F-FDG). In animals without SAM treatment (60-60 group), energy metabolites did not recover and neither CBF nor glucose uptake restored during 60 min of recirculation. In contrast, in SAM-treated animals (60-60 SAM group), values of the energy metabolites improved significantly and both CBF and glucose uptake recovered, though incompletely. These results indicate that SAM is able to improve postischemic cerebral microcirculation and energy metabolism. For mechanisms of the effects, it is suggested to the enhancement of erythrocyte deformability by phospholipid methylation, the stabilization of mitochondria, and the normalization of injured metabolic reactions. Therefore, we conclude that SAM is able to be effective clinically as a drug treated for the acute phase of cerebrovascular diseases.