Effect of heat shock on neuronal cultures: importance of protein synthesis and HSP72 induction for induced tolerance and survival.

In this study the effects of 30 min heat-shock, ranging from 42 degrees C to 46 degrees C, on survival, protein synthesis and HSP72 expression were investigated in primary rat neuronal cultures. Heat-shock of 44 degrees C resulted in a complete, but transient inhibition of protein synthesis which recovered within 24 h. 46 degrees C heat-shock resulted in an irreversible inhibition of protein synthesis and complete neuronal loss within 24 h. Cycloheximide treatment of neuronal cultures resulted in aggravation of neuronal cell damage after heat-shock of 44 degrees C, indicating that the capacity for recovery of the overall protein synthesis is an important survival factor. In addition, the reduction of neuronal cell damage mediated by heat conditioning was abolished by cycloheximide treatment, indicating that the function of new proteins is important for induced thermotolerance. Induction of the strictly inducible member of the heat-shock protein 70kDa family, HSP72, was found in those few astrocytes which were contaminating the neuronal cell cultures, but not in neurons. These results indicate that newly synthesised proteins other than HSP72 are likely to mediate neuronal protection following heat shock in our experiments. These findings raise the possibility that induced tolerance may not necessarily be mediated by HSP72.

Evidence of apoptosis in primary neuronal cultures after heat shock.

We investigated the effects of 30-min heat shock on survival, DNA degradation, and nuclear morphology of primary rat cortical and hippocampal neurones. In cell cultures which were grown for 8 days in vitro (DIV), only a small portion of neurones showed apoptotic morphology after heat shock of 45 degrees C and typical DNA laddering was not detectable, despite the fact that nearly 50% of the neurones died within 24 h. The majority of the neurones presumably died by necrosis, as indicated by random DNA degradation. In neuronal cultures grown for 15 DIV, heat shock, however, resulted in DNA laddering, occurrence of apoptotic bodies and DNA strand breaks, typical of apoptosis. In these cultures, about 50% of the neurones showed apoptotic morphology following exposure to 45 degrees C in TUNEL and acridine orange staining, whereas glia were not affected in vitality. In addition we were interested whether the highly inducible member of the heat-shock protein family, HSP72, would be induced in apoptotic cells. Double staining for HSP72 and TUNEL revealed concomitant HSP72 induction and occurrence of DNA degradation only in very few neurones in 15-DIV cultures, which were growing adjacent to astrocytes. A clear association of the degenerative process and HSP72 expression, therefore, could not be established. These results demonstrate that environmental stress, such as heat shock, can induce apoptotic death in aged primary cultured neurones. The differentiation state and/or the presence of glial cell elements in the cultures appears to be an important factor for the occurrence of apoptotic features in cultured neurones.

Effect of trophic factors on delayed neuronal death induced by in vitro ischemia in cultivated hippocampal and cortical neurons.

The effect of trophic factors on neuronal survival after 30 min oxygen and glucose deprivation (in vitro ischemia) was studied in primary hippocampal and cortical neuronal cultures of rat. In vitro ischemia was produced at 37 degrees C by placing cultures in glucose-free medium, the oxygen content of which was removed by gassing with pure argon. After in vitro ischemia neurons were allowed to recover either in serum-free minimal essential medium (MEM) or in MEM containing 5% native horse serum, 100 ng/ml basic fibroblast growth factor (bFGF) or 10 ng/ml transforming growth factor-beta 1 (TGF-beta 1), respectively. Cultures that recovered in serum-free medium suffered a progressive type of neuronal injury: survival of either cortical or hippocampal neurons declined from about 60% after 1 h to 50% after 3 h, 40% after 6 h and less than 20% after 24 h. Addition of serum proteins to the incubation medium did not influence early survival (up to 3-6 h) but significantly improved survival after 24 h (more than 40% in both hippocampal and cortical cultures). Addition of TGF-beta 1 and bFGF had only minor effects. These data show that serum reduces delayed ischemic cell death by a mechanism which is different from that of TGF-beta 1 or bFGF protection.

Developmental changes of RNA editing of glutamate receptor subunits GluR5 and GluR6: in vivo versus in vitro.

In the present series of experiments we compared the up-regulation of GluR5 and GluR6 mRNA editing during the transition from the embryonic to the adult state with changes in the extent of editing during neuronal development in vitro. RNA was isolated from rats, from the cerebral cortex, hippocampus and cerebellum of embryonic brains (E19) and adult brains (2 months old), as well as from neurons prepared from the cortex, hippocampus and cerebellum of embryonic brains (E19) and held in tissue culture for 2, 8 or 16 days. Quantification of mRNA editing was achieved by using standards prepared from plasmids with cDNA inserts derived from the edited and unedited state of both GluR5 and GluR6 mRNA. In addition, GluR5 mRNA levels were determined in brain tissue and neuronal cells in culture by quantitative PCR. Developmental changes in the extent of GluR5 and GluR6 mRNA editing were different in vivo compared to in vitro. For GluR5 mRNA editing these differences were most pronounced in cerebellar neurons compared to cerebellar tissue: the extent of GluR5 mRNA editing found in vivo at E19 was significantly down-regulated in cerebellar neurons during the first 8 days in culture, and after 16 days in vitro the extent of editing was still about 50% of that found in the adult state in vivo. For GluR6 mRNA editing these differences were most pronounced in hippocampal neurons compared to the hippocampus in vivo: the extent of GluR6 mRNA editing found in vivo at E19 was significantly down-regulated in vitro during the whole culturing period, most pronounced after 8 days in vivo (to below 40% of that found at E19 and to below 30% of that found in adult hippocampus). GluR5 mRNA levels increased markedly from E19 to the adult brain. However, we could not find any specific pattern of changes in mRNA levels which might account for the development changes in the profile of GluR5 mRNA editing. Comparing developmental changes in the extent of mRNA editing of glutamate receptor subunits may help to elucidate the molecular and regulatory mechanisms of this important editing reaction. Strict control and clear indication of the age of primary neuronal cell cultures used should be required in accounts of electrophysiological or neurotoxicological studies as this would increase comparative usefulness of such experiments, since calcium fluxes through glutamate receptor ion channels are likely to influence the system significantly.

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

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 microM 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.

Susceptibility of hippocampal and cortical neurons to argon-mediated in vitro ischemia.

Neurons from cerebral cortex and hippocampal CA1 sector exhibit a striking difference in vulnerability to transient ischemia. To establish whether this difference is due to the inherent (pathoclitic) properties of these neurons, the ischemic susceptibility was studied in primary cortical and hippocampal cultures by using a new model of argon-induced in vitro ischemia. Neuronal cultures were exposed at 37 degrees C for 10-30 min to argon-equilibrated glucose-free medium. During argon equilibration, Po2 declined to < 2.5 torr within 1 min and stabilized shortly later at approximately 1.3 torr. After 30 min of in vitro ischemia, total adenylate was < 45% and ATP content < 15% of control in both types of culture. Cytosolic calcium activity increased from 15 to 50 nM. Reoxygenation of cultures after in vitro ischemia led to delayed neuronal death, the severity of which depended on the duration of in vitro ischemia but not on the type of neuronal cultures. Energy charge of adenylate transiently returned to approximately 90% of control after 3 h, but ATP content recovered only to 40% and protein synthesis to < 35%. Cytosolic calcium activity continued to rise after ischemia and reached values of approximately 500 nM after 3 h. The new argon-induced in vitro ischemia model offers major advantages over previous methods, but despite this improvement it was not possible to replicate the differences in cortical and hippocampal vulnerability observed in vivo. Our study does not support the hypothesis that selective vulnerability is due to an inherent pathoclitic hypersensitivity.

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

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 micro M 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.

Regional analysis of developmental changes in the extent of GluR6 mRNA editing in rat brain.

The extent of mRNA editing of the kainate receptor subunit GluR6 was evaluated in the cortex, hippocampus and cerebellum of embryonic brains at days 14 and 19 of gestation, in brains of animals aged 4, 25 days, or 3 months, and in hippocampal neurons isolated from embryonic brains at day 19 of gestation and held in tissue culture for 2 or 8 days. Total RNA was isolated and reverse transcribed into cDNA, which was used as template for PCR across the edited base A in TMII of GluR6. The extent of editing was evaluated by restriction digest of PCR products with Bbv 1, gel electrophoresis and image analysis of bands. In all brain structures studied the extent of editing was significantly upregulated during development (P < 0.001). The most pronounced increase in the extent of editing was observed between embryonic days 14 and 19. Highest levels were reached 4 days (94 +/- 1.3%) or 3 months after birth (95 +/- 1.7%) in the cortex and hippocampus, respectively. Notably, in hippocampal neurons held in tissue culture editing was sharply reduced to 67 +/- 3.1% and 29 +/- 3.1% after 2 or 8 days in culture (P < 0.001 vs. the embryonic and adult state). The results illustrate that moderate but significant regional differences exist in the regulation of GluR6 mRNA editing during development (cortex vs. hippocampus and cerebellum). Comparing developmental changes in the extent of editing of AMPA/kainate receptor subunits in vivo and in vitro may help to elucidate the molecular mechanisms of the editing process.

Temporal analysis of the upregulation of GluR5 mRNA editing with age: regional evaluation.

The extent of mRNA editing of the kainate receptor subunit GluR5 was evaluated in tissue samples taken from the cerebral cortex, hippocampus and cerebellum of rat brain and in cortical neurons held in tissue culture, by PCR amplification of GluR5 cDNA across the edited base and restriction analysis of the amplification product with Bbv 1. Samples were taken from embryonic brains of rats at day 21 of gestation and from brains 4 days, 25 days and 3 month after birth. Cortical neurons were isolated from the tissue at day 19 of gestation and kept for 2 or 8 days in culture. The extent of editing was sharply upregulated during development in all brain structures studied. In the cortex and hippocampus the extent of editing exhibited already the adult state 4 days after birth. In the cerebellum, in contrast, the extent of editing was still 42 +/- 11.4% 25 days after birth but 82 +/- 6.2% in the adult state. In neurons held in tissue culture for up to 8 days, upregulation of editing did not take place. It is concluded that GluR5 editing is differently regulated in different brain structures and that the developmental changes observed in vivo are blocked when cells are kept in vitro.

Delayed neuronal death after brief histotoxic hypoxia in vitro.

The effect of three metabolic inhibitors--iodoacetate, potassium cyanide, and potassium arsenate--on neuronal viability was studied in primary rat cortical and hippocampal CA1 neuronal cultures. Iodoacetate (0.1 mM) applied for 5 min to 8-day-old cultures resulted in delayed neuronal death within 3-24 h in cortical and hippocampal CA1 neurons. Neuronal degeneration was preceded by transient inhibition of energy metabolism to approximately 40% and a permanent inhibition of protein synthesis to approximately 50%. The inhibition of protein synthesis and the neuronal death were prevented by the free radical scavenger vitamin E but not by the glutamate antagonist MK-801. Removal of calcium during iodoacetate exposure could not protect against toxicity, and there was no increase of intracellular calcium concentration during and shortly after iodoacetate treatment. Cyanide and arsenate produced only partial neuronal degeneration, even at a dose of 10 mM. These observations demonstrate that brief exposure of neurons to low concentrations of iodoacetate produces a delayed type of neuronal death that is not mediated by either calcium or glutamate. The therapeutic effect of vitamin E points to a free-radical mediated injury and suggests that this type of pathology may also be involved in delayed neuronal death after transient energy depletion in vivo.

Effect of serum on intracellular calcium homeostasis and survival of primary cortical and hippocampal CA1 neurons following brief glutamate treatment.

Glutamate neurotoxicity was studied in primary neuronal cultures prepared from rat cerebral cortex and hippocampal CA1 sector. Neurons were cultivated with 5% native horse serum and then exposed to 0.1 or 1.0 mM glutamate for 5 min. Subsequently, neurons were allowed to recover for 24 hours either in the presence or in the absence of 5% native horse serum. In the absence of serum, neurons showed morphological signs of degeneration and exhibited marked loss of vitality as tested by vital staining and release of lactate dehydrogenase (LDH). In contrast, when neurons were cultivated in the presence of serum, no degenerative changes were seen and the neurons survived. Heat inactivated serum did not prevent neuronal death but addition of basic fibroblast growth factor (bFGF) or transforming growth factor-beta 1 (TGF-beta 1) had the same protective effect as native serum. Measurements of intracellular calcium activity ([Ca2+]i) with the indicator dye fura-2 revealed a sharp increase during glutamate exposure. In the absence of serum, [Ca2+]i returned to near control within 5 min but it secondarily increased after 1 hour to almost the same level as during glutamate exposure. This delayed increase was more pronounced in CA1 than in cortical neurons, it correlated linearly with the initial rise during glutamate exposure, and it was greatly reduced in the presence of serum. These observations suggest that glutamate neurotoxicity in vitro is a function of the delayed and not of the primary rise of intracellular calcium activity, and that trophic factors prevent neurotoxicity by attenuating this delayed response.

Developmental changes in the extent of RNA editing of glutamate receptor subunit GluR5 in rat brain.

The extent of RNA editing of the glutamate receptor subunit GluR5 mRNA was evaluated in the cortex, hippocampus and cerebellum of embryonic rat brains, aged 19 days, and adult brains, aged 2-3 months. RNA was isolated and transcribed into cDNA, which was used as template for amplifying a PCR product across the edited region of the GluR5 subunit. Quantification of editing was performed by restriction digest of PCR products with BbvI and image analysis of bands obtained after electrophoresis. The extent of editing was significantly lower in the embryonic state as compared to the adult state. These differences were most pronounced in the cerebellum where the extent of editing amounted to 27 +/- 8% and 78 +/- 2% in the embryonic and adult state, respectively (P < 0.001). Since expression of GluR5 is relatively high in brain areas of neuronal differentiation and synapse formation it is concluded that un-edited GluR5 may play a role in the development of the nervous system.

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.

Ultrastructural localization of calcium in ischemic hippocampal slices: the influence of adenosine and theophylline.

Calcium was localized ultrastructurally with the use of the modified oxalate-pyroantimonate reaction in the CA1 region of rat hippocampal slices. Ten-minute ischemia (incubation with anoxic and glucose-free medium) followed by 30 min reoxygenation resulted in mitochondrial calcium sequestration and ultrastructural damage. The addition of the adenosine receptor antagonist, theophylline, worsened the ischemia-induced morphological changes and particularly exaggerated the Ca2+ loading in the postsynaptic dendrites. In contrast, adenosine protected against ischemia-induced changes. The results suggest that adenosine exerts its neuroprotective action largely by maintaining intracellular calcium-homeostasis.

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

Transient 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamine stimulates growth in rat cerebral endothelial cell culture.

Endothelial cells were isolated from rat cerebral cortices using combined enzymatic digestions and Percoll gradient centrifugation. Primary cultures were subsequently grown on collagen-covered dishes in a medium containing 20% fetal calf serum and 0.6 mmol glutamine. The majority of cultures became confluent by day 7 or 8, but some could not reach confluence. The cells were fusiform in shape and exhibited immunoreactivity to factor VIII-related antigen and binding to the lectin Griffonia simplicifolia. Exposure of cultures to media containing 2.6 mmol glutamine resulted in accelerated growth (in cultures were confluent at days 3-4) and change in culture morphology, namely the formation of circular, cell-free areas. However, this treatment did not restore gamma-glutamyl transpeptidase activity that was lost during cultivation. As for other amino acids, asparagine was less potent, glycine and phenylalanine failed to mimic the glutamine effect. In summary, glutamine stimulates growth of cerebral endothelial cells in vitro and so it may supplement for other growth factors in the culture media.

Protective effect of adenosine and a novel xanthine derivative propentofylline on the cell damage after bilateral carotid occlusion in the gerbil hippocampus.

The role of adenosine in the development of ischemia induced pathological changes has been examined in Mongolian gerbils. A dramatic increase in the concentrations of adenosine, inosine and hypoxanthine was detected by microdialysis in the dorsal part of hippocampus and in the striatum immediately after 5 min bilateral occlusion of the carotid arteries. From a resting value of about 0.5 microM the concentration of adenosine increased to more than 10 microM. The adenosine levels became normalized within 30 min after ischemia. Inosine and hypoxanthine levels were higher and they increased and also returned towards control somewhat later than adenosine. A second occlusion resulted in a similar but somewhat smaller increase in purine levels. Carotid occlusion for up to 12 min had no major, lasting effect on the binding to adenosine A1-receptors in the CA-regions of the hippocampus, as determined by autoradiography. Neuronal and vascular changes (degeneration of neurons, mitochondrial destruction and ribosomal disaggregation, astroglial oedema) due to ischemia (3-12 min, followed by 48 h recirculation) was studied with light and electron microscopy in the selectively vulnerable CA1 area of hippocampus. In one series of experiments the adenosine antagonist theophylline (20 mg/kg i.p.), given 15 min prior to a 5 min occlusion, significantly enhanced the ischemia induced changes. In another experiment the adenosine uptake inhibitor propentofylline (HWA 285, 10 mg/kg), injected 15 min before a 12 min carotid occlusion, reduced the neuronal (90%) and astroglial changes (84%) due to ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Dexamethasone treatment attenuates the development of ischaemic brain oedema in gerbils.

Transient global forebrain ischaemia was produced in Mongolian gerbils by occluding both common carotid arteries for 10 min followed by 48 h recirculation. Dexamethasone, 5 mg/kg i.p., was given 5 h before the occlusion and every 12 h thereafter. After occlusion an increase in water, sodium and calcium content was found in the parietal cortex and hippocampus, while the concentration of potassium decreased. Exudation of plasma albumin was not found in the brain. The activity of Na+, K(+)-ATPase decreased in the hippocampus. Morphological signs of cerebral oedema were also observed, both in the CA1 region of the hippocampus and in the cortex. Dexamethasone treatment prevented the accumulation of water, sodium and calcium in the ischaemic brain. It also attenuated the oedematous morphological changes of the blood-brain barrier. Thus dexamethasone treatment may also have therapeutic relevance in the acute, high-risk phase of patients suffering from repetitive, transitoric cerebral ischaemia.

Kinetics of protein phosphorylation in microvessels isolated from rat brain: modulation by second messengers.

The role of second messengers in the regulation of protein phosphorylation was studied in microvessels isolated from rat cerebral cortex. The phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the kinetics of 32P incorporation into specific protein substrates were evaluated by computer-aided x-ray film densitometry. With the use of this method, Ca2+-calmodulin (CAM)-, Ca2+/phospholipid (PK C)-, cyclic GMP (cGMP)-, and cyclic AMP (cAMP)-dependent protein kinases were detected. CAM-dependent protein kinase proved to be the major phosphorylating enzyme in the microvascular fraction of the rat cerebral cortex; the activity of cGMP-dependent protein kinase was much higher than that of the cAMP-dependent one. Autophosphorylation of both the alpha- and beta-subunits of CAM-dependent protein kinase and the proteolytic fragment of the PK C enzyme was also detected. The kinetics of phosphorylation of the individual polypeptides indicate the presence in the cerebral endothelium of phosphoprotein phosphatases. The phosphorylation of proteins in the cerebral capillaries was more or less reversible; the addition of second messengers initiated a very rapid increase in 32P incorporation, followed by a slow decrease. Because the intracellular signal transducers like Ca2+ and cyclic nucleotides are frequently regulated by different vasoactive substances in the endothelial cells, the modified phosphorylation evoked by these second messengers may be related in vivo to certain changes in the transport processes of the blood-brain barrier.

Reverse pinocytosis induced in cerebral endothelial cells by injection of histamine into the cerebral ventricle.

Histamine dihydrochloride (10 micrograms of 500 micrograms/ml) was infused during 1 min into the lateral cerebral ventricle of rats, which resulted in a significant stimulation of pinocytosis in the endothelial cells. Systemic injections of mepyramine or metiamide could not prevent this activation. In contrast, ranitidine, injected with histamine was able to inhibit the stimulation of pinocytosis. Albumin exudation from the blood was not found. There was also no change in water and electrolyte contents of the brain tissue. The results suggest that histamine reaching the abluminal membrane can activate the pinocytosis in the cerebral endothelial cells in the reverse direction, i.e., from brain to blood, without opening the blood-brain barrier.