Petra Bonnekoh

Immunocytochemical Study of an Early Microglial Activation in Ischemia

Transient arrest of the cerebral blood circulation results in neuronal cell death in selectively vulnerable regions of the rat brain. To elucidate further the involvement of glial cells in this pathology, we have studied the temporal and spatial distribution pattern of activated microglial cells in several regions of the ischemic rat brain. Transient global ischemia was produced in rats by 30 min of a four-vessel occlusion. Survival times were 1, 3, and 7 days after the ischemic injury. The microglial reaction was studied immunocytochemically using several monoclonal antibodies, e.g., against CR3 complement receptor and major histocompatibility complex (MHC) antigens. Two recently produced monoclonal antibodies against rat microglial cells, designated MUC 101 and 102, were also used to identify microglial cells. Following ischemia, the microglial reaction was correlated with the development of neuronal damage. The earliest presence of activated microglial cells was observed in the dorsolateral striatum, the CA1 area, and the dentate hilus of the dorsal hippocampus. However, the microglial reaction was not confined to areas showing selective neuronal damage, but also occurred in regions that are rather resistant to ischemia, such as the CA3 area. Particularly in the frontoparietal cortex, the appearance of MHC class II–positive microglial cells provided an early indication of the subsequent distribution pattern of neuronal damage. The microglial reaction would thus seem to be an early, sensitive, and reliable marker for the occurrence of neuronal damage in ischemia.

Prevention of Postischemic Hyperthermia Prevents Ischemic Injury of CA1 Neurons in Gerbils

Halothane-anesthetized Mongolian gerbils were submitted to 5-min bilateral carotid artery occlusion. After ischemia, halothane anesthesia was continued for various periods of up to 85 min, and the degree of CA1 neuronal injury was estimated 7 days later by counting the number of surviving pyramidal cells. During ischemia and postischemic halothane anesthesia, rectal and cranial temperature was kept at control level (37.7 and 37.0°C, respectively) using a feedback-controlled heating system. When anesthesia was discontinued after ischemia, transient hyperthermia occurred. In animals with 0- and 15-min postischemic halothane anesthesia, both cranial and rectal temperature rose by ∼1.5°C, and the number of surviving CA1 neurons amounted to <25% of control. After 45- or 85-min postischemic anesthesia, hyperthermia was significantly reduced and the number of surviving neurons increased to 65 and 89%, respectively. The protective effect of postischemic anesthesia was lost when anesthetized animals were submitted to the same hyperthermic profile as nonanesthetized ones, using a feedback-controlled heating system (16% surviving neurons in hyperthermia vs. 89% in normothermia, respectively). These observations demonstrate that postischemic anesthesia with 1% halothane protects against delayed neuronal death by preventing postischemic hyperthermia and not by its anesthetic effects.

Microglial Reaction in the Rat Cerebral Cortex Induced by Cortical Spreading Depression

The response of microglial cells to cortical spreading depression (CSD) was studied in rat brain by immunocytochemistry. CSD was elicited for one hour by the topical application of 4M potassium chloride solution and the microglial reaction examined immunocytochemically after 4, 16, 24 and 72 hours. CSD was sufficient to induce a microglial reaction throughout the cortex at 24 hours. Activated microglial cells furthermore showed a striking de‐novo expression of major histocompatibility complex class II antigens. In contrast, no microglial reaction was observed in the cortex of sham‐operated animals. This microglial reaction in response to CSD was not associated with histologically detectable neuronal damage. These results support the view that microglial cells are extremely sensitive to changes of the brain microenvironment. Their activation may be related to changes of ion homeostasis in the brain which are not sufficient to trigger neuronal injury.

[14C]leucine incorporation into brain proteins in gerbils after transient ischemia: relationship to selective vulnerability of hippocampus.

Abstract: Regional [14C]leucine incorporation into brain proteins was studied in gerbils after global ischemia for 5 min and recirculation times of 45 min to 7 days, using a combination of quantitative autoradiography and biochemical analysis. After recirculation for 45 min, incorporated radioactivity was reduced to ∼20–40% of control values in all ischemic brain regions. Specific activity of the tracer, in contrast, was increased, a finding indicating that the reduced incorporation of radioactivity was not due to reduced tracer influx from plasma or a dilution of the tracer by increased proteolysis. After recirculation for 6 h, [14C]leucine incorporation returned to control levels in all regions except the CA1 sector of the hippocampus, where it amounted to <50%. After 1 day, protein synthesis in the CA1 sector returned to ∼70% of control values, followed by a secondary decline to <50% after 3 days and returned to near control values after 7 days. Histological evaluations revealed selective neuronal death in the CA1 sector of the hippocampus after 3 days of recirculation. The complex time course of protein synthesis in the CA1 sector suggests a biphasic mode of injury, which may be related to similar changes of calcium homeostasis. The final return to near normal after CA1 neurons have disappeared is explained by astroglial proliferation and demonstrates that at this time protein synthesis is not a marker of neuronal viability.

LOCOMOTOR HYPERACTIVITY AND HIPPOCAMPAL CA1 INJURY AFTER TRANSIENT FOREBRAIN ISCHEMIA OF GERBILS

The influence of a repeated transient forebrain ischemia on the development of post-ischemic locomotor hyperactivity was determined in the gerbil. Animals were subjected to two episodes of 5 min bilateral carotid artery occlusion in halothane anesthesia separated by one week. By using an animal activity monitor for counting spontaneous movements, the locomotor activity was assessed before and after each ischemic period. Seven days after ischemia the number of intact hippocampal CA1 neurons was counted from histological sections. Following the first 5 min ischemia a phase of locomotor hyperactivity of more than 20-fold of control was observed. One week after ischemia less than 20% of CA1 neurons had survived. Exposure of gerbils at this time to a second 5 min ischemic episode did not cause any increased locomotor activity. These findings support the assumption that post-ischemic locomotor hyperactivity is a symptom of the acutely injured but still functionally active CA1 sector of hippocampus.

Selective vulnerability in the gerbil hippocampus: Morphological changes after 5-min ischemia and long survival times

SummaryThe morphology of the hippocampus of Mongolian gerbils was investigated by light and electron microscopy after 5-min forebrain ischemia and survival times of up to 10 months. After 3 weeks recirculation only 5.8% of pyramidal neurons of the CA1 (cornu ammonis 1) sector had survived but the thickness of the inner and outer hippocampal layers did not change. After recirculation times of 6 and 10 months the number of surviving neurons declined no further but all layers of the CA1 subfield shrank markedly. Ultrastructurally, many but not all surviving CA1 neurons were altered. After 3 weeks both “dark” and “pale” type neurons were present, while after 6 and 10 months only the “pale” type of injury persisted. Axonal enlargements and myelin breakdown were observed at all survival times up to 10 months of recirculation. The astrocytes of CA1 sector contained numerous glial fibrils which were most pronounced after the longer recirculation times. The stratum radiatum presented intact presynaptic terminals densely packed with an abundance of clear vesicles even after survival of 10 months. Initially, morphologically damaged postsynaptic structures were still attached to these terminals but they disappeared after longer recirculation times. However, even after 10 months some intact synapses were observed involving dendrites which probably originated from surviving CA1 neurons. In CA3 sector and dentate gyrus no ultrastructural changes occurred at any survival time. The close association of surviving CA1 neurons with intact presynaptic terminals and reactive glial cells may be of importance for the development of epileptogenic foci which are characteristic of this particular brain region.

The microglial reaction in the rat hippocampus following global ischemia: immuno-electron microscopy

SummaryTransient arrest of the cerebral circulation leads to neuronal cell death in selectively vulnerable regions of the central nervous system. It has recently been shown at the light microscopical level that neuronal necrosis is accompanied by a rapid microglial reaction in ischemia (Gehrmann et al. (1992) J. Cereb. Blood Flow Metab. 12:257–269). In the present study we have examined the postischemic microglial reaction in the dorsal rat hippocampus at the ultrastructural level using immuno-electron microscopy. Global ischemia was produced by 30 min of four-vessel occlusion and the microglial reaction then studied after 8, 24 and 72 h. In sham-operated controls microglial cells were not phagocytic; they were randomly distributed throughout the neuropil and occasionally made contacts with other structures such as dendrites in CA1. Ultrastructural signs of activation were observed from 1 day postlesion onward. Reactive microglial cells were consistently seen to phagocytose degenerating neurons particularly in the CA1 stratum pyramidale and in the CA4 sector. They were sometimes interposed between two morphologically distinct types of CA1 neurons, i.e., “dark” (degenerating) and “pale” (surviving) types of neurons. Phagocytic microglial cells also became positive for major histocompatibility complex (MHC) class II antigens at these locations from 1 day after ischemia onward. Furthermore, activated microglial cells were frequent along degenerating dendrites in the stratum radiatum of CA1. After survival times of up to 72 h microglial cells, but not astrocytes, were occasionally observed to undergo mitosis. In addition to their random distribution across the neuropil, microglial cells were frequently observed in a perivascular position under normal conditions. These perivascular microglial cells rapidly expressed MHC class II antigens, extended broad cellular processes and showed signs of phagocytic activity from 1 day onward. These results demonstrate that upon ischemic injury microglial cells proliferate and are rapidly recruited to the site of injury. By virtue of their pronounced cytotoxic potential, microglial cells could be further involved in mediating tissue destruction in ischemia, thus constituting the main immuneffector cell population in this pathological state.

Temperature effect on immunostaining of microtubule-associated protein 2 and synaptophysin after 30 minutes of forebrain ischemia in rat.

SummaryThe regional distribution of the postsynaptic microtubule-associated protein 2 (MAP2) and the presynaptic marker protein synaptophysin was investigated by immunohistochemistry in brains of rats submitted to 30-min forebrain ischemia by four-vessel occlusion. The following brain temperature profiles during ischemia were compared: (1) constant brain temperature of 36°C (normothermia; n=5); (2) spontaneous temperature decline from 36° to 31°C (spontaneous hypothermia; n=5) and (3) constant temperature of 30°C (induced hypothermia; n=5). Normothermia was produced by exposing the ischemic head to an external heat source, and induced hypothermia by cooling the head with liquid nitrogen vapours. Sham-operated animals were either kept at ambient temperature or exposed to the same heat source, as required for maintaining normothermia during ischemia. Seven days after sham operation or ischemia, brains were fixed by perfusion and processed for immunohistochemistry using monoclonal antibodies against MAP2 and synaptic vesicle-specific protein (synaptophysin). Normothermic ischemia resulted in complete loss of MAP2 immunostaining in the whole hippocampus, spontaneous hypothermic ischemia in complete loss of MAP2 in CA1 sector, and induced hypothermic ischemia only in variable loss of MAP2 in CA1 sector. Post-ischemic immunostaining of synaptophysin revealed a temperature-dependent increase in stratum lacunosum-moleculare of CA1 sector, the density of which correlated inversely with MAP2 staining. Comparison with morphological alterations showed a close relationship between loss of MAP2 staining and histological injury. The post-ischemic activation of synaptophysin may reflect regenerative processes associated with synaptic remodelling and, therefore, is an indirect marker of the severity of ischemic injury.

Neuronal Damage after Repeated 5 Minutes of Ischemia in the Gerbil is Preceded by Prolonged Impairment of Protein Metabolism

The effect of single or repeated episodes of cerebral ischemia on protein biosynthesis and neuronal injury was studied in halothane-anesthetized gerbils by autoradiography of [14C]leucine incorporation into brain proteins and light microscopy. For quantification of the protein synthesis rate, the steady-state precursor pool distribution space for labeled and unlabeled free leucine was determined by clamping the specific activity of [14C]leucine in plasma, and by measuring free tissue leucine in samples taken from various parts of the brain. Control values of protein synthesis were 14.6 ± 2.2, 5.8 ± 2.3, 14.2 ± 3.1, and 10.0 ± 3.8 nmol g−1 min−1 (means ± SD) in the frontal cortex, striatum, CA1 sector, and thalamus, respectively. Following a single episode of 5 or 15 min of ischemia, protein synthesis recovered to normal in all brain regions except the CA1 sector, where it returned to only 50% of control after 6 h and to less than 20% after 3 days of recirculation. After three episodes of 5 min of ischemia spaced at 1 h intervals, protein synthesis remained severely suppressed in all brain regions after both 6 h and 3 days of recirculation. Inhibition of protein synthesis after 6 h predicted histological injury after 3 days of recirculation. In animals submitted to a single episode of 5 or 15 min of ischemia, histological damage was restricted to the CA1 sector but injury occurred throughout the brain after three episodes of 5 min of ischemia. These observations demonstrate that persisting inhibition of protein synthesis following cerebral ischemia is an early manifestation of neuronal injury. Prevention of neuronal injury requires restoration of a normal protein synthesis rate.

Ornithine decarboxylase in reversible cerebral ischemia: an immunohistochemical study

SummaryAnesthetized Mongolian gerbils were subjected to 5-min ischemia and 8 h of recirculation. Vibratiom sections were taken for studying changes in ornithine decarboxylase (ODC) immunoreactivity using an antiserum to ODC, and tissue samples were taken for measuring ODC activity. After 5-min ischemia and 8-h recirculation ODC activity increased 11.5-, 5.9-, and 7.9-fold in the cerebral cortex, striatum and hippocampus, respectively (P≤0.05 to 0.01). In the cortex, striatum and hippocampus of control animals immunoreactivity was low but clearly above the detection limit. The reaction was confined to neurons. After 5-min ischemia and 8-h recirculation a sharp increase in immunoreactivity was observed confined to neurons, indicating that the postischemic activation of polyamine metabolism is a neuronal response to ischemia. The immunoreactivity was markedly increased in the perinuclear cytoplasm and the dendrites. In the striatum the density of neurons exhibiting a sharp increase in immunoreactivity was more pronounced in the lateral than in the ventral part. In the hippocampus a strong reaction was present in all subfields but the CA1 subfield was particularly affected. The present study demonstrates for the first time that biosynthesis of a protein is markedly activated during the first 24 h of recirculation after 5-min cerebral ischemia of gerbils even in the vulnerable CA1 subfield, in which the overall protein synthesis is sharply reduced at the same time. Studying polyamine metabolism after ischemia may, thus, provide new information about the basic molecular mechanisms responsible for the altered gene expression after metabolic stress.