Aged APP23 mice show a delay in switching to the use of a strategy in the Barnes maze.

Spatial learning and memory deficits in the APP23 transgenic mice have mainly been studied using the Morris water maze (MWM). However learning in the MWM relies on swimming abilities and may be confounded by the stressful nature of this test. We have therefore assessed spatial learning and memory in 12-month-old APP23 using a dry-land maze test developed by Barnes. Mice were given daily learning trials for a total of 41 successive days. After a 12-day interval the mice were re-tested for 4 additional days in order to examine the spatial memory retention. Immediately following this phase, reversal learning was examined for 13 additional days by moving the escape tunnel to the opposite position. During the initial learning phase, APP23 mice showed a significantly longer latency to find the escape tunnel as well as an increased number of errors compared to non-transgenic littermates. These deficits appeared to be due to a delay in switching from a "no strategy" to a spatial strategy. Indeed, this same delay in the use of spatial strategy was observed in the reversal phase of the study. Our results suggest that impairments in APP23 mice in learning and memory maze tests may be due to a specific deficit in the use of spatial strategy.

Characterization of two novel proteins, NgRH1 and NgRH2, structurally and biochemically homologous to the Nogo-66 receptor.

Nogo-66 receptor (NgR) has recently been identified as the neuronal receptor of the myelin-associated proteins Nogo-A, oligodendrocyte protein (OMgp) and myelin-associated glycoprotein (MAG), and mediates inhibition of axonal regeneration both in vitro and in vivo. Through database searches, we have identified two novel proteins (NgRH1 and NgRH2) that turned out to be homologous in their primary structures, biochemical properties and expression patterns to NgR. Like NgR, the homologues contain eight leucine-rich repeats (LRR) flanked by a leucine-rich repeat C-terminus (LRRCT) and a leucine-rich repeat N-terminus (LRRNT), and also have a C-terminal GPI signal sequence. Northern blot analysis showed predominant expression of NgRH1 and NgRH2 mRNA in the brain. In situ hybridization and immunohistochemistry on rat brain slices revealed neuronal expression of the genes. NgRH1 and NgRH2 were detected on the cell surface of recombinant cell lines as N-glycosylated GPI anchored proteins and, consistent with other GPI anchored proteins, were localized within the lipid rafts of cellular membranes. In addition, an N-terminal proteolytic fragment of NgR comprising the majority of the ectodomain was found to be constitutively secreted from cells. Our data indicate that NgR, NgRH1 and NgRH2 constitute a novel receptor protein family, which may play related roles within the CNS.

Increased cerebral infarct volumes in polyglobulic mice overexpressing erythropoietin.

There is increasing evidence that erythropoietin (Epo) has a protective function in cerebral ischemia. When used for treatment, high Epo plasma levels associated with increases in blood viscosity, however, may counteract beneficial effects of Epo in brain ischemia. The authors generated two transgenic mouse lines that overexpress human Epo preferentially, but not exclusively, in neuronal cells. In mouse line tg21, a fourfold increase of Epo protein level was found in brain only, whereas line tg6 showed a dramatic increase of cerebral and systemic transgene expression resulting in hematocrit levels of 80%. Cerebral blood flow (CBF), as determined by bolus tracking magnetic resonance imaging, was not altered in the tg6 line. The time-to-peak interval for the tracer, however, increased approximately threefold in polyglobulic tg6 mice. Immunohistochemical analysis revealed an increase in dilated vessels in tg6 mice, providing an explanation for unaltered CBF in polyglobulic animals. Permanent occlusion of the middle cerebral artery (pMCAO) led to similar perfusion deficits in wild-type, tg6, and tg21 mice. Compared with wild-type controls, infarct volumes were not significantly smaller (22%) in tg21 animals 24 hours after pMCAO, but were 49% enlarged (P < 0.05) in polyglobulic tg6 mice. In the latter animals, elevated numbers of Mac-1 immunoreactive cells in infarcted tissue suggested that leukocyte infiltration contributed to enlarged infarct volume. The current results indicate that moderately increased brain levels of Epo in tg21 transgenic mice were not sufficient to provide significant tissue protection after pMCAO. The results with tg6 mice indicate that systemic chronic treatment with Epo associated with elevated hematocrit might deteriorate outcome after stroke either because of the elevated hematocrit or other chronic effects.

Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat.

Vascular endothelial growth factor (VEGF), a key regulator of vasculogenesis and embryonic angiogenesis, was recently found to be up-regulated in an animal model of stroke. Unlike VEGF, angiopoietin (Ang)-1 and -2, their receptor tie-2, and the associated receptor tie-1 exert their functions at later stages of vascular development, i.e., during vascular remodeling and maturation. To assess the role of the angiopoietin/tie family in ischemia-triggered angiogenesis we analyzed their temporal and spatial expression pattern after middle cerebral artery occlusion (MCAO) using in situ hybridization and immunohistochemistry. Ang-1 mRNA was constitutively expressed in a subset of glial and neuronal cells with no apparent change in expression after MCAO. Ang-2 mRNA was up-regulated 6 hours after MCAO and was mainly observed in endothelial cell (EC) cord tips in the peri-infarct and infarct area. Up-regulation of both Ang-2 and VEGF coincided with EC proliferation. Interestingly, EC proliferation was preceded by a transient period of EC apoptosis, correlating with a change in VEGF/Ang-2 balance. Our observation of specific stages of vascular regression and growth after MCAO are in agreement with recent findings suggesting a dual role of Ang-2 in blood vessel formation, depending on the availability of VEGF.

Plasminogen activation in experimental permanent focal cerebral ischemia.

BACKGROUND: Previous experimental work using in situ zymography has shown very early increased plasminogen activation in ischemic regions after 3 h of ischemia with and without reperfusion. The objective of the present study was to evaluate the time course and extent of plasminogen activation in long-term permanent focal cerebral ischemia. MATERIAL AND METHODS: The middle cerebral artery in male Fisher rats was irreversibly occluded by electrocoagulation. Duration of ischemia was 48, 72, and 168 h. Occlusion was controlled in vivo by MRI at day 2. Plasminogen activation was detected by in situ zymography of 10 microm cryosections with an overlay containing plasminogen and the plasmin substrate caseine. Areas of plasminogen activation were compared to structural lesions (immunohistochemical loss of microtubule-associated protein 2; MAP 2). RESULTS: Compared to controls, increased plasminogen activation was observed in the basal ganglia and the cortex of the ischemic hemisphere after 48, 72, and 168 h (affected area of basal ganglia: 44.5+/-21.9, 70.1+/-2.3 and 66.6+/-2.8%, respectively; affected area of cortex: 63.4+/-9.8, 67.7+/-0.7 and 64.0+/-3.7%, respectively). The duration of ischemia had no significant influence on the extent of plasminogen activation. Areas of increased plasminogen activation significantly overlapped with and exceeded areas of MAP 2 loss (P<0.005). DISCUSSION: Permanent focal cerebral ischemia leads to increased plasminogen activation in ischemic regions. This plasminogen activation remains elevated at persistent levels over days. It may contribute to extracellular matrix (ECM) disruption, secondary hemorrhage, and brain edema in subacute stages of ischemic stroke.

Protective effect of a caspase inhibitor in models for cerebral ischemia in vitro and in vivo.

In primary neuronal-astrocyte cultures from mouse brain, ischemic conditions were simulated by combined oxygen-glucose deprival (OGD) for 2 hrs. This treatment resulted in near complete neuronal damage 24 hrs. later and was accompanied by DNA degradation and apoptotic nuclear morphology. Since caspases are key enzymes in the propagation and execution of programmed cell death, we evaluated the effect of the caspase inhibitor z-VAD-fmk. Damage following 2 hrs. OGD could be reduced by up to 56% with z-VAD-fmk (p<0.05). DNA-fragmentation and caspase activation has been also reported in an in vivo model of cerebral ischemia imitating human stroke. In this model the middle cerebral artery (MCA) is permanently occluded resulting in focal cerebral ischemia and subsequent infarction. Since z-VAD.fmk does not penetrate the blood-brain barrier it was applied intraventricularly as a bolus injection given 30 min. before MCA occlusion which was followed by 24 hrs. of infusion. Infarct volume was determined 48 hrs. after MCA occlusion by means of in vivo magnetic resonance imaging. Z-VAD.fmk dose dependently reduced infarct volume reaching a significant decrease of the cortical infarct by 45% when given as a 120 ng bolus followed by 40 ng/hr. infusion (p<0.05). In summary, our study supports the concept that caspase inhibitors are beneficial in brain ischemia.

Neuron-specific transgene expression of Bcl-XL but not Bcl-2 genes reduced lesion size after permanent middle cerebral artery occlusion in mice.

Protective effects after focal cerebral ischemia were assessed in transgenic mice that overexpress in a neuron-specific fashion mouse Bcl-XL or human Bcl-2. Both Bcl genes were under the control of the same mouse Thy-1 regulatory sequences resulting in very similar expression patterns in cortical neurons. Furthermore, these sequences direct lateonset (i.e. around birth) expression in brain, thus minimizing effects of transgene expression during brain development. Effects on infarct volume were measured using MRI after permanent occlusion of the middle cerebral artery (MCA). When compared to their non-transgenic littermates, Thy1mbcl-XL mice showed a significant 21% reduction in infarct size whereas Thy1hbcl-2 mice did not reveal any reduction. These findings suggest a selective protective advantage of Bcl-XL as compared with Bcl-2 in this mouse model for human stroke.

Biphasic expression of TGF-beta1 mRNA in the rat brain following permanent occlusion of the middle cerebral artery.

Two patterns of transforming growth factor-beta1 (TGF-beta1) expression were identified in brains of normotensive rats following permanent occlusion of the middle cerebral artery (MCAO). First, a relative increase of TGF-beta1 mRNA by 37% was found at 12 h after MCAO in the ipsilateral cingulate cortex as compared to the homotopic contralateral region. The cingulate cortex is located distant from the ischemic territory. Treatment with the glutamate receptor antagonists MK-801 and NBQX did not reduce this expression (34% and 26% increase, respectively). Therefore, peri-infarct depolarization waves were probably not responsible for induction. Secondly, an increase of TGF-beta1 mRNA by 116% was found at 7 days after MCAO within infarcted tissue. This expression was not reduced by the glutamate receptor antagonists MK-801 (increase 140%) and NBQX (increase 137%), either. TGF-beta1 mRNA expression in the cingulate cortex at 12 h after MCAO is possibly mediated by neurons and astroglia and may support cell survival. Expression in the infarcted tissue at 7 days after MCAO is most likely related to the invasion of monocytes and may be involved in the downregulation of inflammatory events, in neoangiogenesis, and in formation of a glial scar around the infarct.

Cell type specific upregulation of vascular endothelial growth factor in an MCA-occlusion model of cerebral infarct.

Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that has been implicated in hypoxia-mediated angiogenesis under physiological and pathological conditions. We used the middle cerebral artery occlusion model (MCAO) in the rat to investigate VEGF mRNA and protein localization, and VEGFR-1 mRNA and VEGFR-2 mRNA expression in cerebral ischemia. By nonradioactive in situ hybridization we observed upregulation of VEGF mRNA and VEGFR-1 mRNA, but not of VEGFR-2 mRNA in the hemisphere ipsilateral to MCA occlusion. VEGF mRNA was upregulated in the periphery of the ischemic area commencing 3 hours (h) after onset of MCAO, reached a peak after 24 h, and remained expressed at lower levels until 7 days (d) after MCAO. Double labelling experiments revealed that the majority of VEGF expressing cells in the penumbra and within the infarct were immunoreactive for Ox-42, Iba-1, and Ed1, but not for GFAP and neurofilament proteins, suggesting that microglial cells/macrophages are the major cell type expressing VEGE Since VEGF was also expressed in Ox-42 immunoreactive cells distant from the infarct (e.g. in the corpus callosum and hippocampus), activated microglial cells expressing VEGF may migrate towards the ischemic stimulus. VEGF protein was also detected on capillaries within the peri-ischemic area, suggesting that VEGF produced and secreted by microglial cells/macrophages binds to its receptors on nearby vascular endothelial cells and initiates an angiogenic response which counterbalances tissue hypoxia. Accordingly, apoptosis of neuroectodermal cells in the penumbra was highly depressed after the onset of angiogenesis. The spatial and temporal correlation between the induction of angiogenesis with VEGF and VEGFR-1 expression suggests that the ischemic upregulation of VEGF represents a physiological response of the brain to counterbalance hypoxia/ischemia in order to protect neuroectodermal tissue.

Attenuated c-fos mRNA induction after middle cerebral artery occlusion in CREB knockout mice does not modulate focal ischemic injury.

To elucidate the mechanism of ischemia-induced signal transduction in vivo, we investigated the effect of the targeted disruption of the alpha and delta isoforms of the cAMP-responsive element-binding protein (CREB) on c-fos and heatshock protein (hsp) 72 gene induction. Permanent focal ischemia was induced by occlusion of the middle cerebral artery of the CREB mutant mice (CREB(-/-), n = 5) and the wild-type mice (n = 6). Three hours after onset of ischemia, the neurologic score was assessed and pictorial measurements of ATP and cerebral protein synthesis (CPS) were carried out to differentiate between the ischemic core (where ATP is depleted), the ischemic penumbra (where ATP is preserved but CPS is inhibited), and the intact tissue (where both ATP and CPS are preserved). There were no significant differences in neurologic score or in ATP, pH, and CPS between the two groups, suggesting that the sensitivity of both strains to ischemia is the same. Targeted disruption of the CREB gene significantly attenuated c-fos gene induction in the periischemic ipsilateral hemisphere but had no effect on either c-fos or hsp72 mRNA expression in the penumbra. The observations demonstrate that CREB expression, despite its differential effect on c-fos, does not modulate acute focal ischemic injury.

Neuronal stress response and neuronal cell damage after cardiocirculatory arrest in rats.

Cardiocirculatory arrest is the most common clinical cause of global cerebral ischemia. We studied neuronal cell damage and neuronal stress response after cardiocirculatory arrest and subsequent cardiopulmonary resuscitation in rats. The temporospatial cellular reactions were assessed by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining of DNA fragments, in situ hybridization (heat shock protein hsp70; immediate early genes c-fos and c-jun), and immunocytochemical (HSP70; and myeloperoxidase, specific marker of polymorphonuclear leukocytes [PMNL]) techniques. Cardiac arrest of 10 minutes' duration was induced in mechanically ventilated male Sprague-Dawley rats anesthetized with nitrous oxide and halothane. After cardiopulmonary resuscitation, animals were allowed to reperfuse spontaneously for 6 hours, 24 hours, 3 days, and 7 days (n = 6 per group). Five sham-operated animals were controls. The TUNEL staining revealed an early onset degeneration in the thalamic reticular nucleus (TRN) at 6 hours that peaked at 3 days. In contrast, degeneration was delayed in the hippocampal CA1 sector, showing an onset at 3 days and a further increase in the number of TUNEL-positive cells at 7 days. A minor portion of TUNEL-positive nuclei in the CA1 sector showed condensed chromatin and apoptotic bodies, whereas all nuclei in the TRN revealed more diffuse staining. After 6 hours of reperfusion, levels of mRNA for hsp70 and c-jun were elevated in circumscribed areas of cortex, in all hippocampal areas, and in most nuclei of thalamus, but not in the TRN. After 24 hours, a strong expression of mRNA for hsp70 and c-jun could be observed in the second layer of the cortex and in hippocampal CA1 sector; hsp70 also was observed in hippocampal CA3 sector. Some animals showed expression of hsp70 and c-jun in the dentate gyrus. After 3 days, hsp70 and c-jun were detected mainly in the CA1 sector of hippocampus. At 7 days, mRNA for both returned to control values. Therefore, delayed cell degeneration in the CA1 sector corresponds to a prolonged expression of hsp70 and c-jun in this area. In situ hybridization studies for c-fos revealed a strong signal in CA3 and dentate gyrus and a less prominent signal in TRN at 6 hours. At 24 hours, CA4 and amygdalae were positive, whereas at 3 and 7 days, the signal reached control levels; no prolonged or secondary expression was observed in the CA1 sector. Immunohistochemical study confirmed translation of HSP70 in various areas corresponding to the detection of mRNA, including the CA1 sector. The number of PMNL increased significantly at 6 hours and 7 days after cardiac arrest; PMNL were distributed disseminately and were not regionally associated with neuronal cell damage. The current data support the view that CA1 neurons might undergo an apoptosis-associated death after cardiac arrest, but PMNL are not directly involved in this process. The marked differences in the time course and the characteristics of TUNEL staining and the neuronal stress response in CA1 sector and TRN point to different mechanisms of neuronal injury in the two selectively vulnerable areas.

Time course of microglia activation and apoptosis in various brain regions after permanent focal cerebral ischemia in mice.

We investigated the temporal course of microglia activation in different brain regions after permanent middle cerebral artery (MCA) occlusion in mice and compared this microglia response with the appearance of apoptotic cells, Microglia activation and morphological changes of microglial cells were visualized using an immunohistochemical method with a polyclonal antibody recognizing the mouse CR3 complement receptor. Cells showing morphological and biochemical features of apoptosis were identified using the terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) method and light microscopy. As early as 30 min after onset of MCA occlusion activated microglia with hypertrophic cell bodies and stout processes were detected in the periphery of the ischemic lesion as identified by diffusion-weighted magnetic resonance imaging. A wider distribution and a progressive increase in the number of activated microglia was found with increasing time. Only few TUNEL-positive cells with apoptotic features were observed within the lesion area at 6 h after onset of cerebral ischemia. From 12 h after MCA occlusion onward a tremendous increase in the number of TUNEL-positive cells was found. Within the thalamus from 24 h onward microglia cells with few processes, irregular morphology and fragmented appearance were detected. Microglia activation in the thalamus progressed up to 4 weeks after MCA occlusion, but had declined after 90 days. Neuronal degeneration in the thalamus as determined by anti-neuronal nuclei immunohistochemistry progressed from 6 days after MCA occlusion onward. Only a few TUNEL-positive cells were found in the thalamus. In summary, microglia activation both in the primary cortical lesion area and in the secondarily affected thalamus preceded the manifestation of tissue injury. These observations encourage further studies on the role of microglia in focal cerebral ischemia.

Differential expression of c-fos and hsp72 mRNA in focal cerebral ischemia of mice.

The heterogeneity of c-fos and hsp72 mRNA expression during focal ischemia was studied in mice by combining in situ hybridization with metabolic imaging. Focal ischemia was produced by middle cerebral artery occlusion for 3 h. The infarct core and the penumbra were differentiated by regional ATP and cerebral protein synthesis (CPS) imaging. hsp72 mRNA expression was restricted to the ischemic penumbra, as defined by the dissociation between preserved ATP and suppressed CPS. c-fos mRNA was expressed not only in the penumbra but also in the peri-ischemic normal brain tissue in which both ATP and CPS were preserved. These data demonstrate a highly selective differential expression of immediate-early and stress-related genes in the peri-infarct surrounding which is explained by different mechanisms of gene induction.

A reproducible model of middle cerebral artery occlusion in mice: hemodynamic, biochemical, and magnetic resonance imaging.

A reproducible model of thread occlusion of the middle cerebral artery (MCA) was established in C57 Black/6J mice by matching the diameter of the thread to the weight of the animals. For this purpose, threads of different diameter (80 to 260 microns) were inserted into the MCA of animals of different weights (18 to 33 g), and the success of vascular occlusion was evaluated by imaging the ischemic territory on serial brain sections with carbon black. Successful occlusion of the MCA resulted in a linear relationship between body weight and thread diameter (r = 0.46, P < 0.01), allowing precise selection of the appropriate thread size. Laser-Doppler measurements of CBF, neurological scoring, and 2,3,5-triphenyltetrazolium chloride staining confirmed that matching of animal weight and suture diameter produced consistent cerebral infarction. Three hours after MCA occlusion, imaging of ATP, tissue pH, and cerebral protein synthesis allowed differentiation between the central infarct core, in which ATP was depleted, and a peripheral penumbra with reduced protein synthesis and tissue acidosis but preserved ATP content. Perfusion deficits and ischemic tissue alterations could also be detected by perfusion- and diffusion-weighted magnetic resonance imaging, demonstrating the feasibility of dynamic evaluations of infarct evolution. The use of multiparametric imaging techniques in this improved MCA occlusion model opens the way for advanced pathophysiological studies of stroke in gene-manipulated animals.

Effect of global system for mobile communication (GSM) microwave exposure on blood-brain barrier permeability in rat.

We investigated the effects of global system for mobile communication (GSM) microwave exposure on the permeability of the blood-brain barrier using a calibrated microwave exposure system in the 900 MHz band. Rats were restrained in a carousel of circularly arranged plastic tubes and sham-exposed or microwave irradiated for a duration of 4 h at specific brain absorption rates (SAR) ranging from 0.3 to 7.5 W/kg. The extravasation of proteins was assessed either at the end of exposure or 7 days later in three to five coronal brain slices by immunohistochemical staining of serum albumin. As a positive control two rats were subjected to cold injury. In the brains of freely moving control rats (n = 20) only one spot of extravasated serum albumin could be detected in one animal. In the sham-exposed control group (n = 20) three animals exhibited a total of 4 extravasations. In animals irradiated for 4 h at SAR of 0.3, 1.5 and 7.5 W/kg (n = 20 in each group) five out of the ten animals of each group killed at the end of the exposure showed 7, 6 and 14 extravasations, respectively. In the ten animals of each group killed 7 days after exposure, the total number of extravasations was 2, 0 and 1, respectively. The increase in serum albumin extravasations after microwave exposure reached significance only in the group exposed to the highest SAR of 7.5 W/kg but not at the lower intensities. Histological injury was not observed in any of the examined brains. Compared to other pathological conditions with increased blood-brain barrier permeability such as cold injury, the here observed serum albumin extravasations are very modest and, moreover, reversible. Microwave exposure in the frequency and intensity range of mobile telephony is unlikely to produce pathologically significant changes of the blood-brain barrier permeability.

Thread occlusion but not electrocoagulation of the middle cerebral artery causes hypothalamic damage with subsequent hyperthermia.

Moderate changes in body temperature can influence the outcome of cerebral ischemic insults and the effect of drugs. Body temperature was measured continuously for 24 hours in rats subjected to permanent occlusion of the middle cerebral artery (MCA) by either coagulation or thread insertion, and the results correlated with the histology of the hypothalamus. The body temperature did not change after MCA occlusion by coagulation and the hypothalamus was intact in all rats. In contrast, the body temperature rapidly increased from about 38 degrees C to more than 39.5 degrees C after MCA occlusion using intraluminal thread, and hyperthermia continued for at least 6 hours in all rats. Histological evaluation revealed neuronal damage in the preoptic area of the hypothalamus in all rats undergoing thread occlusion. Long duration hyperthermia must be prevented after permanent MCA occlusion when the intraluminal thread occlusion model is used in chronic experiments.

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.

Effect of global system for mobile communication microwave exposure on the genomic response of the rat brain.

The acute effect of global system for mobile communication (GSM) microwave exposure on the genomic response of the central nervous system was studied in rats by measuring changes in the messenger RNAs of hsp70, the transcription factor genes c-fos and c-jun and the glial structural gene GFAP using in situ hybridization histochemistry. Protein products of transcription factors, stress proteins and marker proteins of astroglial and microglial activation were assessed by immunocytochemistry. Cell proliferation was evaluated by bromodeoxyuridine incorporation. A special GSM radiofrequency test set, connected to a commercial cellular phone operating in the discontinuous transmission mode, was used to simulate GSM exposure. The study was conducted at time averaged and brain averaged specific absorption rates of 0.3 W/kg (GSM exposure), 1.5 W/kg (GSM exposure) and 7.5 W/kg (continuous wave exposure), respectively. Immediately after exposure, in situ hybridization revealed slight induction of hsp70 messenger RNA in the cerebellum and hippocampus after 7.5 W/kg exposure, but not at lower intensities. A slightly increased expression of c-fos messenger RNA was observed in the cerebellum, neocortex and piriform cortex of all groups subjected to immobilization, but no differences were found amongst different exposure conditions. C-jun and GFAP messenger RNAs did not increase in any of the experimental groups. 24 h after exposure, immunocytochemical analysis of FOS and JUN proteins (c-FOS, FOS B, c-JUN JUN B, JUN D), of HSP70 or of KROX-20 and -24 did not reveal any alterations. Seven days after exposure, neither increased cell proliferation nor altered expression of astroglial and microglial marker proteins were observed. In conclusion, acute high intensity microwave exposure of immobilized rats may induce some minor stress response but does not result in lasting adaptive or reactive changes of the brain.

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.