U. Oschlies

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.

Relation of neuronal endoplasmic reticulum calcium homeostasis to ribosomal aggregation and protein synthesis: implications for stress-induced suppression of protein synthesis

Results from experiments performed with permanent non-neuronal cell lines suggest that endoplasmic reticulum (ER) calcium homeostasis plays a key role in the control of protein synthesis (PS). It has been concluded that disturbances in ER calcium homeostasis may contribute to the suppression of PS triggered by a severe metabolic stress (W. Paschen, Med. Hypoth., 47 (1996) 283-288). To elucidate how an emptying of ER calcium stores of these cells would effect PS and ribosomal aggregation of non-transformed fully differentiated cells, experiments were run on primary neuronal cell cultures. ER calcium stores were depleted by treating cells with thapsigargin (TG, a selective, irreversible inhibitor of ER Ca(2+)-ATPase), cyclopiazonic acid (CPA, a reversible inhibitor of ER Ca(2+)-ATPase), or caffeine (an agonist of ER ryanodine receptor). Changes in intracellular calcium activity were evaluated by fluorescence microscopy using fura-2-loaded cells. Protein synthesis was determined by measuring the incorporation of [3H]leucine into proteins. The degree of aggregation of ribosomes was evaluated by electron microscopy. TG induced a permanent inhibition of PS to about 10% of control which was only partially reversed within 2 h of recovery. CPA caused about 70% inhibition of PS, and PS recovered completely 60 min after treatment. Caffeine produced an inhibition of PS to about 50% of control. Loading cells with the calcium chelator BAPTA-AM (33.3 microM) alone suppressed PS without reversing TG- or caffeine-induced inhibition of PS, indicating that the suppression of PS was caused by a depletion of ER calcium stores and not by an increase in cytosolic calcium activity. TG-treatment of cells induced a complete disaggregation of polysomes which was not reversed within the 4 h recovery period following TG-treatment. After caffeine treatment of cells, we observed a heterogenous pattern of ribosomal aggregation: in some neurons ribosomes were almost completely aggregated while in other cells a significant portion of polyribosomes were disaggregated. The results indicate that a depletion of neuronal ER calcium stores disturbs protein synthesis in a similar way to the effects of transient forms of metabolic stress (ischemia, hypoglycemia or status epilepticus), thus implying that a disturbance in ER calcium homeostasis may contribute to the pathological process of stress-induced cell injury.

The effect of dexamethasone on serum protein extravasation and edema development in experimental brain tumors of cat.

SummaryExperimental brain tumors were produced in 20 cats by stereotaxic xenotransplantation of a blastomatous glial cell clone into the internal capsule of the left hemisphere. Ten of these animals were treated after 2 weeks with a single injection of 10 mg dexamethasone in crystalline suspension. Three weeks after xenotransplantation vascular permeability was studied by electron microscopy with horseradish peroxidase as the barrier tracer (four animals), and extravasation of serum proteins was visualized by immunohistochemistry, using an image processing system (16 animals). In animals used for immunohistochemistry, the water content of peritumoral brain tissue was also determined.In both treated and untreated animals, spherical tumors with a diameter of about 10 mm were present at the implantation site. Extravasation of horseradish peroxidase was detected only in the tumor, but there was accumulation of serum proteins both in the tumor and the peritumoral white matter. Edema, in consequence, originated mainly in the tumor from where it spread into the surrounding brain tissue. Corticosteroid therapy reduced the water content of peritumoral brain tissue but did not affect increased barrier permeability of tumor vessels, and only slightly improved peritumoral accumulation of serum proteins. It is concluded that amelioration of tumor edema by corticosteroids cannot result solely from tightening of the blood-brain barrier to circulating macromolecules but must be due to an active restoration of cerebral water homeostasis despite persisting serum protein extravasation.

Mitochondrial calcium sequestration in cortical and hippocampal neurons after prolonged ischemia of the cat brain.

SummaryAdult normothermic cats were submitted to 1- h complete cerebrocirculatory arrest, followed by blood recirculation for 6–8 h. Two groups of animals could be distinguished: In one group electrocorticogram and somatically evoked primary cortical potentials steadily recovered after ischemia, and in another electrophysiologic recovery was absent. At the end of the recirculation period, calcium content was measured in tissue samples taken from cerebral cortex and hippocampus, and compared with mitochondrial calcium sequestration as assessed by electron-microscopic cytochemistry. Protein content of cortex and hippocampus was also determined for evaluation of tissue swelling. The two regions were selected because previous experiments had revealed that in animals with electrophysiologic recovery cerebral cortex remains intact although hippocampus is selectively injured, whereas in animals without electrophysiologic recovery both cerebral cortex and hippocampus are damaged.In animals with functional recovery, neither calcium content nor mitochondrial calcium sequestration were significantly increased in either cerebral cortex or hippocampal subfield CA1. Only in dentate gyrus a minor degree of mitochondrial calcium sequestration was present. Calculation of tissue swelling revealed no change in cerebral cortex, but a volume increase by 18% in hippocampus, indicating development of brain edema in this region. In animals without functional recovery tissue calcium significantly increased both in cortex and hippocampus (by 49% and 73% of control, respectively), and there was significant mitochondrial calcium accumulation in both regions. Calculated brain swelling in these animals amounted to 16% and 26% in cortex and hippocampus, respectively.The results obtained do not support the hypothesis that selective vulnerability of hippocampus is the consequence of neuronal calcium overload but rather indicate that calcium accumulation is an unspecific epiphenomenon of irreversible cell injury.

Glutamate-induced ribosomal disaggregation and ultrastructural changes in rat cortical neuronal culture : protective effect of horse serum

Differentiated primary cortical neuronal cultures of rat were exposed for 5 min to 0.1 and 1.0 mM glutamate. In cultures maintained in serum-free medium after glutamate exposure, ribosomes completely disaggregated and neurons died within 24 h already after 0.1 mM glutamate. Addition of 5% horse serum to the culture medium prevented both ribosomal disaggregation and neuronal death even after exposure to 1.0 mM glutamate. Glutamate toxicity in vitro requires removal of serum-associated growth factors from the incubation medium and, therefore, may not be representative for neuronal vulnerability in vivo.

Barbiturate promotes post-ischemic reaggregation of polyribosomes in gerbil hippocampus

A brief period of cerebral ischemia is followed by severe inhibition of protein synthesis which is slowly reversed in the resistant but not in the selectively vulnerable regions of the brain. Inhibition occurs at the translational level, as evidenced by the disaggregation of ribosomes into monosomes. In order to evaluate the importance of this disturbance for the evolution of ischemic injury, the effect of the neuroprotective drug, pentobarbital, on ribosomal aggregation was studied in gerbils subjected to 5 min bilateral carotid artery occlusion. Pentobarbital (50 mg/kg, i.p.) was applied shortly after the ischemia, and the aggregational state of ribosomes was investigated by electron microscopy after recirculation times ranging from 15 min to 1 day. Pentobarbital treatment did not prevent the initial post-ischemic disaggregation but promoted the subsequent reaggregation in the selectively vulnerable neurons. This suggests that post-ischemic application of barbiturates exerts its beneficial effect by reversing the post-ischemic block of ribosomal reaggregation in vulnerable regions.

Putrescine content and structural defects in isolated fractions of rat brain after reversible cerebral ischemia

Reversible cerebral ischemia was produced in rats by occluding both vertebral and both carotid arteries. Following 30 min of ischemia, brains were recirculated for 24 h. The hippocampus, the striatum, and the cortex were sampled, homogenized, and fractionated on a discontinuous sucrose gradient. The fractions were evaluated morphologically by electron microscopy and biochemically by measuring the activity of marker enzymes. Putrescine was extracted from the isolated fractions and measured quantitatively using HPLC and a fluorescence detector. In the total tissue homogenate of control animals putrescine content amounted to 72.0 +/- 3.1, 70.2 +/- 7.6, and 72.7 +/- 2.1 pmol/mg protein in samples prepared from the cortex, the hippocampus, and the striatum, respectively. In the mitochondrial fraction the content was lower, while in the synaptosomal fraction and in myelin it was higher than that in total tissue homogenate. Following cerebral ischemia there was a 6- to 10-fold increase in putrescine in tissue homogenate: In the cortex it increased to 429 +/- 24 pmol/mg protein, in the hippocampus to 585 +/- 70 pmol/mg protein, and in the striatum to 718 +/- 98 pmol/mg protein. Among the isolated fractions the highest levels of putrescine were found in synaptosomes from the striatum (663 +/- 196 pmol/mg protein), followed by the hippocampus (500 +/- 125 pmol/mg protein) and the cerebral cortex (349 +/- 45 pmol/mg protein). This order correlated to the degree of morphological injury which was most pronounced in the striatum and the hippocampus and less in the cerebral cortex. The results of the present study provide further evidence of a relationship between postischemic putrescine levels and the extent of ischemia-induced neuronal injury.

Serum prevents glutamate-induced mitochondrial calcium accumulation in primary neuronal cultures

Abstract The effect of serum proteins on glutamate-induced mitochondrial calcium accumulation was studied in primary cortical and hippocampal cultures using oxalate-pyroantimonate staining with electron microscopy. Cultures were prepared from rat embryos on gestational day 17–19 and cultivated for 8 days in minimal essential medium (MEM) containing 5% native horse serum. At this time cultures were exposed for 5 min to 100 μM or 1.0 mM glutamate, followed by recovery in either serum-free or serum-containing culture medium. Mitochondrial calcium accumulation was assessed before glutamate treatment, at the end of glutamate exposure, and after 5 min, 30 min, 6 h and 24 h of recovery. Under control conditions and at the end of glutamate exposure, mitochondria contained only a few calcium deposits. If cultures were placed in serum-free medium after glutamate treatment, mitochondria were progressively loaded with calcium. At 5 min after glutamate exposure mitochondrial calcium deposits were prominent in both cortical and hippocampal cultures, followed by a further steady increase and neuronal death within 24 h. When cultures were allowed to recover after glutamate treatment in serum-containing MEM, calcium sequestration and ultrastructural changes of mitochondria were essentially absent, and neurons survived. No differences between cortical and hippocampal cultures were observed. The data demonstrate that prevention of glutamate neurotoxicity by serum proteins is associated with prevention of post-glutamate mitochondrial calcium accumulation.

Early ultrastructural changes after brief histotoxic hypoxia in cultured cortical and hippocampal CA1 neurons.

Abstract Primary cortical and hippocampal neuronal cultures submitted to brief histotoxic hypoxia suffer delayed neuronal death after 24 h [Uto et al. (1995) J Neurochem 64: 2185–2192]. In this study the ultrastructural changes were monitored during the first 6 h following 5-min histotoxic hypoxia induced by exposure to 100 μM iodoacetate. In both cortical and hippocampal CA1 neurons, disaggregation of ribosomes was the earliest sign of histotoxic pathology. Vacuolizations of mitochondria, endoplasmic reticulum and Golgi apparatus, as well as fragmentation and disintegration of neurofilaments followed later. Signs of apoptotic nuclear degeneration were absent. Our observations demonstrate that, similar to that seen in ischemia, disaggregation of ribosomes after brief histotoxic hypoxia is one of the first pathological alterations heralding delayed neuronal death.