Chronic hypoxia induced ultrastructural changes in the rat adrenal zona glomerulosa.

The adrenal cortex plays an important role in adaptation to various forms of stress, including hypoxia. While physiological changes in the aldosterone metabolism during hypoxia have been extensively described, few studies have focused on the morphological changes in the adrenal glands under chronic hypoxia. We studied the ultrastructure of the zona glomerulosa of 6-month-old Wistar rats exposed to chronic normobaric hypoxia. Animals were divided into two groups: control (n=12) and hypoxic (n=12). In this latter group, the animals were kept at 7% O2 concentration after a gradual adaptation (21, 15, 12, 10, 8, 7 vol% O2). The duration of the study was 112 days. In comparison with normoxic rats, body weight and adrenal gland weight of hypoxic animals was significantly reduced by 18.5% (p=0.006) and 14.7% (p=0.001) respectively. The thickness of the zona glomerulosa decreased due to atrophy of cells. The main ultrastructural changes observed were: 1) a decrease in, or complete elimination of, lipid droplet content; 2) a marked increase in lysosome number; and 3) the presence of giant mitochondria. Our findings show that rats fail to adapt to severe chronic hypoxia. The ultrastructural changes in the zona glomerulosa found in the present study could reflect changes in the aldosterone pathway.

Short-term neuropathological aspects of in vivo suicide gene transfer to the F98 rat glioblastoma using liposomal and viral vectors.

To date, only few preclinical protocols on liposomal suicide gene transfer in tumors have been published, none of which directly compared viral to liposomal vectors in terms of immunoreactivity and efficacy. We thus studied the neuropathological alterations in 80 rats being treated for glioblastoma using liposomal and, for comparison, adenoviral and retroviral suicide gene transfer approaches to identify vector-associated efficacy and toxicity for further clinical studies. 62 rats served as controls. F98 tumors were established in Fisher rats and transfected in vivo with the thymidine kinase gene of herpes simplex virus (HSVtk) by a single intratumoral application and an implanted intratumoral continuous delivery system. Three days later ganciclovir was given intraperitoneally for 14 days. The animals were sacrificed 17 days post completed gene transfer. Brains were examined histologically and immunohistochemically using markers for immunocompetent cells. Ten animals showed complete tumor regression; they all belonged to the liposomal and adenoviral groups. In 6 of 10 experimental groups considerable numbers of lymphocytes along the margins of the regression cavities could be observed. Control animals of the liposomal and adenoviral groups showed only little lymphocytic infiltration, underlining the minimal immunogenicity of these carriers. In contrast, the retroviral control group featured a high lymphocyte infiltration. In summary, this study indicates that, in terms of both efficacy and immunoreaction, liposomes are as appropriate as adenoviruses in the treatment of rat glial tumors using suicide gene transfer strategies.

Voltage- and gamma-aminobutyric acid-activated membrane currents in the human medulloblastoma cell line MHH-MED-3.

The whole-cell patch clamp technique was used to characterize voltage- and neurotransmitter-activated currents in the medulloblastoma cell line MHH-MED-3 and cells from tissue slices and primary cultures of two medulloblastoma biopsies. These preparations revealed similar electrophysiological properties. All tested cells displayed 4-aminopyridine-sensitive delayed rectifying K(+) currents, gamma-aminobutyric acid(A) receptor-mediated Cl(-) currents and most of them inward rectifier K(+) currents. Transient inward currents were mainly carried by low-voltage activated T-type Ca(2+) channels in MHH-MED-3 cells, and tetrodotoxin-sensitive Na(+) channels in cells from the primary culture. From these characteristics we conclude that medulloblastoma cells share physiological features with developing cerebellar granule cells at an immature stage.

Regional differences of cerebrovascular reactivity effected by calcium channel blocker - dotarizine.

Dotarizine, a novel antimigraine prophylactic drug, is chemically related to Diphenylbutylpiperazines, which are known to have Ca(2+)-antagonistic, alpha-adrenolytic and antihistaminic properties. Additionally, Dotarizine exhibits strong 5-HT2 receptor-specific antiserotoninergic properties. The vasostabilizing effect of Dotarizine on cerebrovascular reactivity during different ventilation conditions was demonstrated in various in vitro and in vivo studies. In the presented study, the effect of chronic oral administration of the drug on vascular reactions of different areas of cerebral vessels following hyperventilation was investigated. The experiments were carried out on two groups of experimental animals (rabbits). In the first group (6) 25 mg/kg of Dotarizine dissolved in 0.25% agar was administered orally for 5 days twice daily. The control group of animals (6) was fed with agar of the same concentration according to the same time schedule. During the experiment, 15 min hyperventilation was performed and blood flow velocity (BFV) in the middle cerebral artery (MCA) and the basilar artery (BA) was recorded using Transcranial Doppler apparatus (TCD) before and after hyperventilation state. The obtained results revealed a strong antivasoconstrictive effect of Dotarizine on cerebral vessels reactivity during hyperventilation. In the control experimental group, the 15 min hyperventilation caused a decrease in the mean BFV in MCA and BA by 36 and 14%, respectively, and in the drug-treated group under the same ventilation conditions the decrease of the mean BFV in BA was only 6% and even a slight increase (8% as compared with control values) of BFV in MCA was observed. Comparison of the pulsatility index (PI) values demonstrated a significant decrease of vascular resistance in MCA in the Dotarizine-treated group of animals (P<0.1). From the obtained results it can be concluded that chronic oral administration of a novel compound (Dotarizine) diminishes the vasoconstrictive effect of hyperventilation on cerebral vessels in rabbits. The influence of this drug demonstrates regional differences in the cerebrovascular reactivity and it appears to change the vascular resistance in the small arteries of the cerebrovascular system. Thus, it can be recommended as a good prophylactic antimigraine compound due its vasostabilizing properties.

Vasostabilizing effect of Dotarizine (Ca(2+)-channel blocker) on cerebrovascular reactivity in rabbits.

Disturbances of the cerebrovascular reactivity in cases of migraine with aura are well-known. It has been suggested that the vasostabilizing effects of novel prophylactic pharmaceuticals are determined by their antiserotoninergic and/or nitric oxide releasing properties. Dotarizine, a representative of Ca2+ channel blockers from diphenilbutilpiperazines group also reveals antiserotoninergic 5-HT2A and 5-HT2C receptor-specific properties. The vasodilatatory and antivasoconstrictive properties of this compound were reported previously. In this study the efficacy of Dotarizine chronic oral administration on cerebrovascular reactivity during hyperventilation was examined with respect to its duration of action. Experiments were carried out on 13 rabbits. There was an interval of two days between a five days compound administration and performed hyperventilation. Blood flow velocities (BFV) in the middle cerebral artery (MCA) and basilar artery (BA) were measured in control conditions, after 10 min hyperventilation and in the tenth minute of recovery of normoventilation. Our data reveal a decrease of antivasoconstrictive properties of Dotarizine between its administration and vasoconstrictive test. Subsequent normoventilation showed a distinct vasostabilising effect of this compound with evident regional differences in its influence on cerebral vessels. Thus Dotarizine might be useful as prophylactic medication in migraine therapy, due to its Ca2+ channel blocking and antiserotoninergic properties, but the time-frame of its efficacy has to be defined.

Brain-derived neurotrophic factor and neurotrophin-3 levels in Alzheimer's disease brains.

In the present study, we investigated the levels of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in post-mortem brain tissue of Alzheimer's disease (AD) patients, and we observed a significant increase of BDNF concentration in hippocampus and parietal cortex of AD patients, as well as a negative correlation between NT-3 levels and age in hippocampus and putamen of control subjects, and for BDNF in frontal cortex. A defining feature of AD is the post-mortem identification of neuritic plaques and neurofibrillary tangles in the brain, however, a more significant neuropathological finding is the degeneration of cholinergic neurones of the basal forebrain, critically involved in memory and cognition. Neurotrophic factors are partly responsible for the maintenance of neuronal function and structural integrity in the adult brain. Our results provide, therefore, evidence that, under conditions of progressive neurodegeneration the brain stimulates the over expression of certain neurotrophic factors as a possible mechanisms of compensation, and that during senescence the expression of these molecules is regulated.

Effects of chronic deep hypoxia on the expression of nitric oxide synthase in the rat brain.

Experimental studies in extreme hypoxic conditions affecting the brain have been performed mainly in acute but not chronic models. Twenty rats were housed and exposed to decreasing concentrations of oxygen (from 21% to 7% over 130 days) and ten normal rats were used as control. Paraffin slices from representative sections containing cerebral cortex, cerebellum, striatum, hippocampus, thalamus and hypothalamus were incubated with antisera against nitric oxide synthase. Cortex and striatum showed small randomly distributed positive neurons with bipolar features, in greater numbers in the hypoxic group (p < 0.02). The granular layer of the cerebellum showed a strongly positive rim around some cell nuclei. Purkinje cells were immunopositive in hypoxic rats. Hipoccampal, thalamic and hypothalamic nuclei showed no quantitative differences in the number of positive neurons. The increased number of blood vessels and their dilation observed in some brain regions in hypoxic rats, mainly in ventral striatum, lead us to hypothesise that NOS may be overexpressed and act at these sites as vasomodulator and/or mediator of secondary cell injury affecting selective neuronal populations. We conclude that prolonged periods of adaptation to deep hypoxia reduces the effect of hypoxia on the upregulation of NOS in the brain tissue.

Effect of oral administration of dotarizine on cerebrovasculature subjected to constriction followed by dilatation in rabbits.

In the study presented the effect of Dotarizine on blood flow velocity in cerebral arteries - in middle cerebral artery (MCA), and basilar artery (BA)- was investigated and compared utilising transcranial Doppler sonography during normoventilation, 15 min hyperventilation with subsequent 3 min anoxia in anaesthetized rabbits. In the Dotarizine treated group (12 rabbits) 25 mg/kg of Dotarizine dissolved in 0,25% agar was administered orally for five days twice daily. In the control group (9 rabbits) animals were fed with agar of the same concentration. The results revealed that decrease of flow velocity caused by hyperventilation and increase during anoxia were less pronounced in the Dotarizine treated group than in control group of animals. A difference between changes of flow velocity in MCA and BA during anoxia was found and the different reactivity of both vessels was established.

Novel twelve-generation kindred of fatal familial insomnia from germany representing the entire spectrum of disease expression.

We present a novel large German kindred of fatal familial insomnia (FFI) consisting of three branches and comprising more than 800 individuals of 12 generations, the largest pedigree of any familial prion disease known today. There is a wide spectrum of clinical presentations leading to misdiagnoses of Olivo-Ponto-Cerebellar Atrophy (OPCA), Parkinson's or Alzheimer's disease in addition to Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker (GSS) syndrome. Molecular genetic analysis of the prion protein gene (PRNP) confirmed the mutation D178N segregating with methionine at the polymorphic codon 129 of PRNP in all 7 patients examined. This polymorphism at codon 129 is supposed to discriminate between familial CJD (fCJD) and FFI; the 129M allele determines FFI and 129V fCJD. Furthermore, heterozygosity at this site appears to induce prolonged disease duration as compared to the homozygous condition. The variability of the clinical and pathological findings documented for our patients indicates the difficulty in establishing the diagnosis of FFI on clinical and on pathological grounds alone. In three cases (IX-97, XI-21, V-2) followed up by us prospectively insomnia was an early and severe symptom; however, in case notes analyzed retrospectively this symptom was frequently missed. In contrast to previous reports and in agreement with recent studies we cannot confirm a clear relationship between the status of the M/V polymorphism at codon 129 and the age-of-onset of this disease.

Heart mitochondria in rats submitted to chronic hypoxia.

The effect of prolonged exposure to normobaric hypoxia on the mitochondria of myocard of rats exposed for several weeks to 8 and 7% O2 has been morphometrically evaluated. Twelve male Wistar rats housed in Nalgene cages (2 per cage) with a batch of six cages placed in plexiglass chambers were maintained in air/N2 mixtures containing different concentrations of O2. Six animals kept in similar cages under normoxia served as controls. When at day 60 the FIO2 was reduced to 8%, the weight increase stagnated and after the 81st test day, on which the hypoxic animals were subdivided into 8% and 7% groups the weight curve showed a decrease in the mean body weight for both groups. The arrest and the following loss of weight beyond the 85th day may be interpreted as the expression of a limit reached in the compensation capacity. In the 8%-group the shape of the mitochondria varied more markedly often with budding and furrowing of the surface. In the 7%-group bizarre shapes and wide variations in size with a decided shift towards larger mitochondria were noteworthy. While rats kept under 8% oxygen exhibited a numerical increase in myocardial mitochondria compared to controls, the mitochondria of the 7%-group were numerically reduced. The results suggest that hypoxia of 8% oxygen is compensatable, if only to some extent, by an increasing surface of mitochondrial membranes, and that further reduction of oxygen causes compensation mechanisms to fail as seen by the severe alterations of the mitochondrial population of the cardiomyocyte in the 7%-group.

Craniocerebral trauma induces hemorheological disturbances.

Several mechanisms are involved in the development of secondary ischemic brain damage, including microthrombi formation, which is thought to play a prominent role. Ninety-four autopsy cases were macro- and microscopically examined by specific staining for fibrin, 74 of which showed cortical contusion after a craniocerebral trauma. Twenty cases with no neurological pathology were used as controls. Traumatic cases comprised 52 males and 22 females, with a mean age of 48 years; most cases died in the first 48 h. The total number of fibrinous microthrombi in a slice of each hemisphere was determined. The mean number of microthrombi found in contused hemisphere was 152 (37-283), with 88 in the contralateral hemisphere (21-139) as compared to 13 (0-27) in control cases. Differences were statistically significant. Globular microthrombi or "shock bodies" (2-60 micro diameter) were present in five cases. Enhanced presence of microthrombi in contused brain areas, higher incidence in young people, an increase in the amount of microthrombi up to the 9th day after injury and involvement of the contralateral hemisphere free of contusion foci were all demonstrated. Microthrombi would therefore seem to be one of the central secondary events after brain trauma to bear in mind when designing treatment strategies.

Changes in glucidic radicals in contused human brains.

Changes in carbohydrate metabolism in brain injury and the involvement of numerous glycoproteins in the subsequent restoration phenomena orientated this paper towards reporting on glucidic radical distribution in postinjured brain tissue. Samples from nine patients suffering severe head injuries and three matched controls were studied. Autopsies were performed up to 24 h postmortem; brains were fixed in formalin and samples were taken from contusional, pericontusional and different brain regions including the anterior cingulum and corpus callosum, parasagittal gyrus, putamen, hippocampus and opercular areas embedded in paraffin. Hematoxylin-eosin staining, immunoreaction with glial fibrillary acid protein (GFAP) and biotin-conjugated lectins (RCA, UEA, PNA, concanavalin A (Con A) and WGA) were used. Contusion and related phenomena such as ischemia induced changes in lectin expression in several elements of the nervous tissue. Endothelial cells of contused areas were positive for RCA, UEA and progressively for WGA and Con A, which could be related to hemorheological disturbances inducing secondary brain damage. Neurons in affected areas were also stained for Con A and UEA, with some processes being delineated. Axonal swellings showed particular affinity to any lectin. Reactive astrocytes displaying only mild staining to WGA in cell bodies were strongly positive for GFAP, showing different patterns of reactivity in the cortex and in white matter.

The effect of Dotarizine--(Ca2+ channel blocker)--on vascular reactivity and ultrastructure of cerebral capillaries in animals subjected to anoxia.

Dotarizine--the novel piperazine derivative--belongs to wide spectrum Ca2+ channel antagonists. It was reported to have strong vasodilatatory and antiserotoninergic activities. Comparing with other Ca2+ channel blockers Dotarizine was found to have lower oral toxicity. In the present study the influence of the oral administration of the novel compound on the blood flow velocity changes in different cerebral arteries--in basilar artery (BA) and middle cerebral artery (MCA)--was investigated under hypoxic conditions. The ultrastructural morphological changes of intracerebral vessels endothelium in treated and untreated anoxic animal groups were also demonstrated. The experiments were carried out on rabbits. In the experimental group 25 mg/kg of Dotarizine dissolved in 0.25% agar was administered orally three times at the 10 hours' intervals. The sham group of animals was fed with agar of the same concentration. During anoxic conditions strong vasodilatory effects were observed in both investigated vessels of drug-treated animals. In the experimental group marked ultrastructural differences in parenchymal vessel endotheliumin comparison to sham group were revealed. Thus, the oral administration of Dotarizine might have effect on the various parts of the cerebrovascular system and can play significant role in improvement of various cerebrovascular disorders.

Upregulation of vascular endothelial growth factor in severe chronic brain hypoxia of the rat.

The vascular endothelial growth factor (VEGF) has been shown to be upregulated in acute hypoxia. Although an increase in blood vessel number has been described in severe chronic brain hypoxia, it is unclear whether VEGF is upregulated in this condition. We therefore investigated male inbred Wistar rats, which were exposed for 9 to 13 weeks to decreasing amounts of oxygen, down to 7% O2 (15%: 15 days; 12%, 10%, respectively; 8%: 1 day, 3 weeks, respectively; 7%: 4 weeks). The expression of VEGF was studied by Northern analysis and in situ hybridization in frozen sections of cerebral cortex, hippocampus and cerebellum in six chronic hypoxic and two control rats. We found a marked upregulation of VEGF mRNA in all brain regions investigated, being strongest in cerebral cortex and cerebellum. Our results suggest a potential role of VEGF for vascular growth and vascular permeability observed in chronic cerebral hypoxia.

The histopathological and clinical relevance of apoptotic cell death in medulloblastomas.

We studied apoptosis in 40 medulloblastomas by in situ end labeling (ISEL) of DNA strand breaks. In situ end labeling was performed on paraffin-embedded material from classical and desmoplastic medulloblastomas and medulloblastomas with glial and combined differentiation. The number of labeled apoptotic cells varied considerably from tumor to tumor as well as between different areas in the same tumor. However, there was no significant difference in the averaged numbers or in the pattern of apoptotic tumor cells between the different differentiations of medulloblastoma. Unlabeled tumor cells displaying features of apoptosis were found in small numbers, indicating that tumor cells may also be able to undergo cell death different from classical apoptosis. A significantly higher number of apoptotic tumor cells in male patients could be demonstrated corresponding to the reported difference in survival rates between male and female patients suffering from medulloblastoma. A clear cut trend of a negative relation between apoptosis and age by operation was found.

Degenerative changes in unmyelinated nerve fibers in late-infantile neuronal ceroidlipofuscinosis. A morphometric study of conjunctival biopsy specimens.

Late-infantile neuronal ceroidlipofuscinosis (LINCL) is an autosomal recessive disease involving rapidly progressive myoclonic epilepsy, mental and motor regression and progressive visual failure. Neurodegeneration and deposition of fluorescent lipid bodies are the neuropathological hallmarks of this disease. In this study we examined the conjunctival biopsy (CB) specimens of three siblings and two unrelated patients with LINCL. At the time of examination each of three siblings presented a different stage of the disease. The unrelated patients were examined at an advanced stage of LINCL. The findings in these patients are compared to the normal data derived from analysis of seven age-matched 9- to 41-month-old healthy subjects. In young children with LINCL the distribution of unmyelinated fiber (UF) diameter is unimodal. In advanced disease there is a bimodal distribution and a significant reduction of UF density and of relative UF area. As the disease progresses, degenerative changes can be demonstrated: at first a diffuse UF swelling, followed by a decrease of UF density and finally the increase of regenerates (microaxons). These changes, however, seem to reflect an unspecific reaction to nerve injury. They can be demonstrated in a variety of conditions of different pathophysiology such as diabetes mellitus, crush injury and normal aging. This is the first morphometric study of CB specimens. Normal data of UF distribution (unimodal, mode at 0.4-0.6 micron) and UF density (1447,760 +/- 19,347/mm2) in CB specimens correspond well to age-specific data published on the sural nerve.

Adult neuronal ceroid lipofuscinosis with clinical findings consistent with a butterfly glioma. Case report.

The authors report a case of neuronal ceroid lipofuscinosis (Kufs' disease) confirmed by stereotactically obtained brain biopsy findings and initially diagnosed as a butterfly glioma. The presenting symptoms in the 64-year-old patient were mental alterations with progressive dementia, followed by muscular atrophy and myoclonia with distal preponderance. The mild initial disturbances of coordination increased, and the patient developed a markedly ataxic gait. Computerized tomography (CT) scanning and magnetic resonance imaging revealed generalized cerebral atrophy and a bifrontal space-occupying lesion involving the callosum. The original "clearcut" diagnosis of glioblastoma multiforme, based on CT scans, was unexpectedly disproved by examination of stereotactically obtained brain biopsy specimens, which revealed a neuronal ceroid lipofuscinosis (Kufs' disease). To the authors' knowledge, this is the first report of a case presenting with both diffuse brain atrophy and localized accumulation of neuronal lipofuscin, mimicking a mass lesion on radiological studies.

Lectinhistochemistry of mixed gliomas demonstrating an intermediate cell type.

18 cases of low-graded mixed gliomas were studied using the two lectins Concanavalin A (Con A) and Peanut lectin (PNA). Con A stained cytoplasm and processes of tumoral astrocytes, whereas PNA stained cell membranes of tumoral oligodendrocytes. Con A and PNA are reliable markers for astrocyte and oligodendrocyte areas of mixed gliomas, respectively. A part of cells were overlappingly positive for both lectins. They expressed an oligosaccharide pattern of both glioma types and represented a third, intermediate cell type of mixed gliomas. The existence of intermediate cells close to astrocytic and oligodendroglial cell types in mixed gliomas could result from transformation processes of neoplastic glial cells or from the malignant transformation of a common glial precursor cell.

NGF content in the cerebral cortex of non-demented patients with amyloid-plaques and in symptomatic Alzheimer's disease.

There is increasing evidence that in Alzheimer's disease nerve growth factor (NGF) protein and NGF mRNA content in postmortem cortex is not decreased, but may even be elevated although the NGF-sensitive cholinergic basal forebrain neurons are preferentially affected. However, only little is known about the early pathophysiological events leading to Alzheimer's disease. We therefore measured the post-mortem NGF concentrations in temporal and frontal cortex of Alzheimer's disease patients, non-demented controls without Alzheimer's disease-related pathology, as well as non-demented patients with beta A4 plaques who might be classified as 'preclinical' cases. In the Alzheimer's disease group we found up to 43% increase in NGF concentrations in the frontal and temporal cortex as compared to the two other groups. In a subgroup analysis of the non-demented patients with plaques, NGF concentrations were lower in the frontal cortex when beta A4 plaques were present (46% of the control temporal area) than in patients without evidence of frontal plaques (81% of the control temporal area). This NGF decrease was paralleled to a similar decrease of choline acetyltransferase activity, which is regulated by NGF in the cholinergic basal forebrain. These findings support the hypothesis of lower cortical NGF content at the onset of plaque formation and of elevated NGF levels in the clinically manifest and neuropathologically advanced stage of the disease.