Marika Kiessling

Intravenous Brain-Derived Neurotrophic Factor Reduces Infarct Size and Counterregulates Bax and Bcl-2 Expression After Temporary Focal Cerebral Ischemia

Background and Purpose Pretreatment with intraventricular brain-derived neurotrophic factor (BDNF) reduces ischemic damage after focal cerebral ischemia. In this experiment we studied the effect of intravenous BDNF delivered after focal cerebral ischemia on neurological outcome, infarct size, and expression of proapoptotic and antiapoptotic proteins Bax and Bcl-2, respectively. Methods With the use of the suture occlusion technique, the right middle cerebral artery in rats was temporarily occluded for 2 hours. Thirty minutes after vessel occlusion, BDNF (300 &mgr;g/kg per hour in vehicle; n=12) or vehicle alone (n=13) was continuously infused intravenously for 3 hours. After 24 hours the animals were weighed and neurologically assessed on a 5-point scale. The animals were then killed, and brains underwent either 2,3,5-triphenyltetrazolium chloride staining for assessment of infarct volume or paraffin embedding for morphology and immunohistochemistry (Bax, Bcl-2). Results Physiological parameters (mean arterial blood pressure, Po2, Pco2, pH, body temperature, glucose) and weight revealed no difference between groups. Neurological deficit was improved in BDNF-treated animals versus controls (P <0.05, unpaired, 2-tailed t test). Mean±SD infarct volume was 229.7±97.7 mm3 in controls and 121.3±80.2 mm3 in BDNF-treated animals (P <0.05, unpaired, 2-tailed t test). Cortical infarct volume was 155.5±78.5 mm3 in the placebo group and 69.9±50.2 mm3 in the BDNF-treated group (P <0.05, unpaired, 2-tailed t test). Subcortical infarct volume was 74.1±30.6 mm3 in the placebo group and 51.1±26.8 mm3 in the BDNF-treated group (P =NS). Bax-positive neurons were significantly reduced in the ischemic penumbra in BDNF-treated animals (P <0.05, unpaired, 2-tailed t test), whereas Bcl-2–positive neurons were significantly increased in this area (P <0.001, unpaired, 2-tailed t test). Conclusions This study demonstrates a neuroprotective effect of BDNF when delivered intravenously after onset of focal cerebral ischemia. As shown here, one possible mechanism of action of neuroprotection of BDNF after focal ischemia appears to be counterregulation of Bax/Bcl-2 proteins within the ischemic penumbra.

Effect of Brain-Derived Neurotrophic Factor Treatment and Forced Arm Use on Functional Motor Recovery After Small Cortical Ischemia

Background and Purpose— Both the administration of growth factors and physical therapy such as forced arm use (FAU) are promising approaches to enhance recovery after stroke. We explored the effects of these therapies on behavioral recovery and molecular markers of regeneration after experimental ischemia. Methods— Rats were subjected to photothrombotic ischemia: sham (no ischemia), control (ischemia), brain-derived neurotrophic factor (BDNF; ischemia plus BDNF, 20 &mgr;g), and FAU (ischemia plus FAU, 1-sleeve plaster cast ipsilateral limb). Animals survived 1 or 6 weeks and underwent behavioral testing (Rotarod, beam balance, adhesive removal, plantar test, neuroscore). After the rats were killed, brain sections were immunostained for semiquantitative analysis of MAP1B, MAP2, synaptophysin, GFAP expression, and quantification of infarct volumes. Results— Infarct volumes were not different between the groups 1 or 6 weeks after ischemia. BDNF-treated animals had better functional motor recovery (Rotarod, beam balance, neuroscore) compared with all other groups (P <0.05). There was no significant adverse effect of early FAU treatment on motor recovery, although sensorimotor function (adhesive removal test) was impaired (P <0.05). There were no differences between groups as measured by nociception of the left and right forepaw (plantar test). BDNF treatment transiently induced MAP1B expression in the ischemic border zone and synaptophysin expression within the contralateral cortex 6 weeks after ischemia (P <0.05). Both BDNF and FAU reduced astrogliosis compared with controls (P <0.05). Conclusions— Postischemic intravenous BDNF treatment improves functional motor recovery after photothrombotic stroke and induces widespread neuronal remodeling. Early FAU treatment after stroke does not increase infarct size, impairs sensorimotor function, but leaves motor function unchanged. Postischemic astrogliosis was reduced by both treatments.

Antitumor Vaccination of Patients With Glioblastoma Multiforme: A Pilot Study to Assess Feasibility, Safety, and Clinical Benefit

PURPOSE Prognosis of patients with glioblastoma is poor. Therefore, in glioblastoma patients, we analyzed whether antitumor vaccination with a virus-modified autologous tumor cell vaccine is feasible and safe. Also, we determined the influence on progression-free survival and overall survival and on vaccination-induced antitumor reactivity. PATIENTS AND METHODS In a nonrandomized study, 23 patients were vaccinated and compared with nonvaccinated controls (n = 87). Vaccine was prepared from patients tumor cell cultures by infection of the cells with Newcastle Disease Virus, followed by gamma-irradiation, and applied up to eight times. Antitumor immune reactivity was determined in skin, blood, and relapsed tumor by delayed-type hypersensitivity skin reaction, ELISPOT assay, and immunohistochemistry, respectively. RESULTS Establishment of tumor cell cultures was successful in approximately 90% of patients. After vaccination, we observed no severe side effects. The median progression-free survival of vaccinated patients was 40 weeks (v 26 weeks in controls; log-rank test, P = .024), and the median overall survival of vaccinated patients was 100 weeks (v 49 weeks in controls; log-rank test, P < .001). Forty-five percent of the controls survived 1 year, 11% survived 2 years, and there were no long-term survivors (> or = 3 years). Ninety-one percent of vaccinated patients survived 1 year, 39% survived 2 years, and 4% were long-term survivors. In the vaccinated group, immune monitoring revealed significant increases of delayed-type hypersensitivity reactivity, numbers of tumor-reactive memory T cells, and numbers of CD8(+) tumor-infiltrating T-lymphocytes in secondary tumors. CONCLUSION Postoperative vaccination with virus-modified autologous tumor cells seems to be feasible and safe and to improve the prognosis of patients with glioblastomas. This could be substantiated by the observed antitumor immune response.

Induction of immediate early gene encoded proteins in the rat hippocampus after bicuculline-induced seizures: differential expression of KROX-24, FOS and JUN proteins.

Immunocytochemistry with specific antisera was used to assess regional levels of six immediate early gene encoded proteins (KROX-24, c-FOS, FOS B, c-JUN, JUN B and JUN D) in the rat hippocampus after 15 min of bicuculline-induced seizures. Serial sections of the dorsal hippocampus were examined at various postictal recovery periods up to 24 h. The results demonstrate a complex temporal and spatial pattern of immediate early gene synthesis and accumulation. Three major categories of immediate early gene products could best be distinguished in the dentate gyrus: KROX-24 and c-FOS showed a concurrent rapid rise with peak levels at 2 h and a return to baseline levels within 8 h after seizure termination. FOS B, c-JUN and JUN B levels increased more gradually with peak intensities in the dentate gyrus reached at 4 h. These immediate early gene products showed above normal levels in various hippocampal subpopulations up to 24 h. JUN D exhibited the most delayed onset combined with a prolonged increase of seizure-induced immunoreactivity. Irrespective of this differential temporal expression profile of individual transcription factors, the sequence of induction in the hippocampal subpopulations was identical for all immediate early gene-encoded proteins examined: first in the dentate gyrus granule cells followed by CA1 and CA3 neurons, respectively. Our data indicate an asynchronous synthesis of several immediate early gene-encoded proteins in the brain after status epilepticus. FOS and JUN proteins act via homo- or heterodimer complexes at the AP-1 and other DNA binding sites. The different time-courses for individual immediate early gene products strongly suggest, that at different time-points after status epilepticus, different AP-1 complexes are effective. In vitro studies have shown that different AP-1 complexes possess different DNA binding affinities as well as different transcriptional regulatory effects. Our results suggest that these molecular mechanisms are also effective in vivo.

Induction and suppression of immediate early genes in specific rat brain regions by the non-competitive N-methyl-D-aspartate receptor antagonist MK-801

The expression pattern of six different immediate early gene-encoded proteins was examined in the rat forebrain after intraperitoneal administration of MK-801, a non-competitive N-methyl-D-aspartate receptor antagonist, at doses of 3 mg/kg and 0.3 mg/kg, respectively. Following MK-801 treatment, the presence of c-FOS, FOS B, KROX-24, c-JUN, JUN B, and JUN D were investigated by immunocytochemistry with specific antisera at different time intervals up to 48 h. Selective induction of all six immediate early genes was found in layer III neurons of the posterior cingulate and retrosplenial cortex. More complex effects were observed in the neocortex: MK-801 did not influence constitutive expression of different FOS and JUN proteins, but caused marked induction of c-FOS, FOS B, JUN B and JUN D, mainly in layer IV, but also in layers V and VI. In contrast, strong neocortical constitutive expression of KROX-24 was almost abolished by MK-801 administration, and replaced by an expression pattern similar to that of FOS and JUN proteins. Subcortical areas such as the hypothalamus and thalamus demonstrated an induction of a subset of immediate early genes (c-fos, fos B, Krox-24, jun B). Injection of MK-801 caused the same distributional pattern of immediate early gene expression irrespective of the dose given, but the extent of changes was stronger after 3 mg/kg, and altered levels of immunoreactivity persisted longer. In many experimental paradigms, immediate early genes are induced by N-methyl-D-aspartate receptor-mediated mechanisms. This induction can readily be blocked by N-methyl-D-aspartate receptor antagonists like MK-801. Our data, however, indicate that MK-801 itself causes immediate early gene expression in specific neuronal populations. In the present study MK-801-elicited expression of immediate early gene-encoded proteins seems to identify reversibly injured neurons, mainly in layer III of the posterior cingulate and retrosplenial cortex. These neurons have previously been shown to be the principal target of N-methyl-D-aspartate receptor antagonist toxicity. Since immediate early gene induction precedes heat-shock protein expression as well as pathomorphological changes, and is induced in additional cortical cell populations, it seems to be a more rapid and more sensitive indicator of non-lethal neuronal injury.

Spatiotemporal Induction of Immediate Early Genes in the Rat Brain after Limbic Seizures: Effects of NMDA Receptor Antagonist MK‐801

Fos, jun and krox belong to multigene families coding for transcription factors. These cellular immediate early genes (IEGs) are thought to be involved in coupling neuronal excitation to changes of target gene expression. Immunocytochemistry with specific antisera was used to assess regional levels of six IEG‐encoded proteins (c‐Fos, Fos B, Krox‐24, c‐Jun, Jun B, Jun D) in the rat forebrain after kainic acid‐induced limbic seizures. The results demonstrate a complex spatial pattern of IEG induction and/or suppression in limbic and non‐limbic structures. The sequence of induction within hippocampal subpopulations was identical for all IEGs investigated, following the order dentate gyrus, CA1 and CA3, and irrespective of different temporal profiles for individual transcription factors. Since Fos and Jun proteins act via homo‐ and heterodimer complexes at specific DNA sites, our data imply that the postictal combinatorial changes of these dimers allow a sequential and differential regulation of target gene expression in specific forebrain regions. Pretreatment with the non‐competitive NMDA receptor antagonist MK‐801 did not affect kainate‐induced expression of IEGs in the limbic system, indicating that IEG induction in these regions is mediated by high‐affinity kainate and AMPA receptors rather than NMDA receptors. In contrast, MK‐801 abolished IEG induction in the somatosensory cortex and striatum, suggesting that IEG expression in non‐limbic neurons occurs transsynaptically and is mediated by NMDA receptors.

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

Abstract 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.

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.

Selective c‐JUN Expression in CA1 Neurons of the Gerbil Hippocampus during and after Acquisition of an Ischemia‐Tolerant State

The selective delayed neuronal death of CA1 pyramidal cells after transient global ischemia in the gerbil brain can be prevented by preconditioning with a short sublethal period of ischemia 1–7 days prior to a subsequent, usually lethal ischemia of 5 min duration. Since changes of neuronal gene expression may play a crucial role in this tolerance induction, we investigated the postischemic expression profile of the fos, jun and Krox transcription factor families. We have previously reported that a single 5 min period of cerebral ischemia does not cause a de novo synthesis of immediate early gene (IEG) encoded proteins in CA1 neurons. In the present study, two experimental groups of Mongolian gerbils were investigated: one group was subjected to a single tolerance‐inducing 2.5 min period of ischemia by bilateral occlusion of the common carotid artery. The second (combined ischemia) group was subjected to 2.5 min of ischemia, followed by 5 min of ischemia 4 days later. Postischemic expression of c‐FOS, FOS B, c‐JUN, JUN B, JUN D and KROX‐24 was investigated by in situ hybridization and immunocytochemistry up to 48 h of recirculation. In contrast to a single 5 min period of ischemia, 2.5 min caused a postischemic expression of c‐JUN protein, but no other IEGs, in CA1 neurons (peak at 6 h). Similarly, a selective but delayed c‐JUN expression (peak at 18 h) was observed in animals subjected to combined ischemia. These results indicate that the induction of an endogenous neuroprotective state in CA1 neurons is associated with the activation of a genetic program which involves the expression of specific transcription factors.

Amplification and differential expression of members of the erbB-gene family in human glioblastoma.

SummaryThe objective of the present study was to determine the frequency of amplifications of three different members of theerbB gene family in human glioblastoma multiforme (GBM). We investigated 47 glial tumors (37 GBM WHO grade IV, 5 anaplastic astrocytomas WHO III and 5 astrocytomas WHO II) by Southern and Western analysis, and immunocytochemistry. Gene amplification oferbB genes in human malignant gliomas was restricted to the EGF receptor (EGFR) gene,erbB-1. We found amplification of the EGFR gene in 49% (18/37) of GBM but not in the astrocytomas WHO II/III. TheerbB-2 anderbB-3 genes showed no amplification in the tumor specimens investigated in this study. At the protein level we found overexpression of the EGF receptor in 86% (32/37) by Western analysis and in 92% (34/37) by immunocytochemistry. Expression of the ERBB2 protein was present in 54% (20/37) but immunoreactivity was much weaker than for EGF receptor and in most cases barely detectable by Western analysis and immunocytochemistry. The ERBB3 protein was not expressed in the glial tumors investigated in this study. Of the threeerbB genes only gene amplification and overexpression of the EGF receptor seems to have an impact on tumor progression of human gliomas. Our data from immunohistochemistry indicate that ERBB2 expression in GBM is closely correlated with EGF receptor levels and is therefore not useful as an independent prognostic parameter.