We have previously shown that somatostatin (SS) immunoreactive (-i) neurons, located in the rat dentate hilus, are vulnerable to cerebral ischemia (Johansen et al., 1987). Within 40 h after ischemia, the cells show clear signs of cell death. At the same time, we observed that dying cells, located in the projection field of the mossy fibers (dentate hilus and CA3 mossy fiber layer), accumulate free zinc. We now demonstrate that the hilar cells, accumulating zinc after ischemia, are SS-i cells. Since it is known that hypothermia can ameliorate ischemic brain damage, we furthermore studied whether hypothermia (29 degrees C) protects the vulnerable SS-i neurons in hilus from zinc accumulation and ischemic cell death. We found that hypothermia both prevented ischemia-induced neuronal zinc accumulation and cell death. We speculate that hilar SS-i cells are highly vulnerable to ischemia, and develop rapid ischemic cell death, because they accumulate zinc shortly after ischemia.

Animals↗

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Memory impaired aged rats: no loss of principal hippocampal and subicular neurons.

A group of 52 male, 2-year-old Ico:WIST rats were tested on the spatial reference memory version of the Morris water maze. Their performance was rated by comparisons with the performance of 2.5-month-old control rats. Unbiased stereological estimates were made of the number of neurons in the major subdivisions of the hippocampus proper and the subiculum of the 5 aged rats with the most impaired performance, the 5 aged rats with the best performance, and 5 young control rats. There were no significant differences between the mean numbers of neurons in the various subdivisions of the hippocampal region of the impaired and nonimpaired aged groups and similarly no decreases in neuron numbers in the pooled group of aged rats relative to the control rats. The results indicate that, in rats, the structural correlates of age-related deficits in spatial memory are to be found in parameters other than the number of neurons in the hippocampus proper and the subiculum.

Age Factors↗

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Induction of microglial immunomolecules by anterogradely degenerating mossy fibres in the rat hippocampal formation.

Degeneration of myelinated axonal connections is generally held to provide a strong stimulus for microglial expression of major histocompatibility complex (MHC) class II antigen. The present study demonstrates that strong microglial reactions also are induced by axonal and terminal degeneration of the unmyelinated hippocampal mossy fibres. After destruction of dentate granule cells by focal injections of colchicine (or transection of the mossy fibres) in adult rats, immunocytochemical analysis of the mossy fibre terminal fields in the dentate hilus and regio inferior of hippocampus proper (CA3) revealed profound changes in microglial cells with increased expression of the complement receptor type 3 and induction of MHC class I antigen, leukocyte common antigen, lymphocyte function-associated antigen-1 and MHC class II antigen. The microglial reaction, first detectable 4 days after the lesion, became maximal during the third postlesional week, and had almost vanished 6 weeks after the lesion. From recent studies we know that anterograde degeneration of myelinated Schaffer-collaterals from CA3 to regio superior of hippocampus proper and myelinated entorhinal perforant path fibres to fascia dentata is accompanied by microglial expression of MHC class I antigen, but not class II. Together with the present findings, this demonstrates that myelin debris is neither necessary nor sufficient to induce expression of microglial MHC class II antigen within the hippocampus.

Animals↗

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Neurotoxic and neuroprotective effects of the glutamate transporter inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA) during physiological and ischemia-like conditions.

Maintenance of low extracellular glutamate ([Glu](O)) preventing excitotoxic cell death requires fast removal of glutamate from the synaptic cleft. This clearance is mainly provided by high affinity sodium-dependent glutamate transporters. These transporters can, however, also be reversed and release glutamate to the extracellular space in situations with energy failure. In this study the cellular localisation of the glutamate transporters GLAST and GLT-1 in organotypic hippocampal slice cultures was studied by immunofluorescence confocal microscopy, under normal culture conditions, and after a simulated ischemic insult, achieved by oxygen and glucose deprivation (OGD). In accordance with in vivo findings, GLAST and GLT-1 were primarily expressed by astrocytes under normal culture conditions, but after OGD some damaged neurons also expressed GLAST and GLT-1. The potential damaging effect of inhibition of the glutamate transporters by DL-threo-beta-benzyloxyaspartate (DL-TBOA) was studied using cellular uptake of propidium iodide (PI) as a quantitative marker for the cell death. Addition of DL-TBOA for 48 h was found to induce significant cell death in all hippocampal regions, with EC(50) values ranging from 38 to 48 microM for the different hippocampal subregions. The cell death was prevented by addition of the glutamate receptor antagonists NBQX and MK-801, together with an otherwise saturating concentration of DL-TBOA (100 microM). Finally, the effect of inhibition of glutamate release, via reverse operating transporters during OGD, was investigated. Addition of a sub-toxic (10 microM) dose of DL-TBOA during OGD, but not during the subsequent 48 h recovery period, significantly reduced the OGD-induced PI uptake. It is concluded: (1) that the cellular expression of the glutamate transporters GLAST and GLT-1 in hippocampal slice cultures in general corresponds to the expression in vivo, (2) that inhibition of the glutamate transporters induces cell death in the slice cultures, and (3) that partial inhibition during simulation of ischemia by OGD protects against the induced PI uptake, most likely by blocking the reverse operating transporters otherwise triggered by the energy failure.

Amino Acid Transport System X-AG↗

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GDNF pre-treatment aggravates neuronal cell loss in oxygen-glucose deprived hippocampal slice cultures: a possible effect of glutamate transporter up-regulation.

Besides its neurotrophic and neuroprotective effects on dopaminergic neurons and spinal motoneurons, glial cell line-derived neurotrophic factor (GDNF) has potent neuroprotective effects in cerebral ischemia. The protective effect has so far been related to reduced activation of N-methyl-D-aspartate receptors (NMDAr). This study tested the effects of GDNF on glutamate transporter expression, with the hypothesis that modulation of glutamate transporter activity would affect the outcome of cerebral ischemia. Organotypic hippocampal slice cultures, derived from 1-week-old rats, were treated with 100 ng/ml GDNF for either 2 or 5 days, followed by Western blot analysis of NMDAr subunit 1 (NR1) and two glutamate transporter subtypes, GLAST and GLT-1. After 5-day exposure to GDNF, expression of GLAST and GLT-1 was up-regulated to 169 and 181% of control values, respectively, whereas NR1 was down-regulated to 64% of control. However, despite these changes that potentially would support neuronal resistance to excitotoxicity, the long-term treatment with GDNF was found to aggravate the neuronal damage induced by oxygen-glucose deprivation (OGD). The increased cell death, assessed by propidium iodide (PI) uptake, occurred not only among the most susceptible CA1 pyramidal cells, but also in CA3 and fascia dentata. Given that glutamate transporters are able to release glutamate by reversed action during energy failure, it is suggested that the observed increase in OGD-induced cell death in the GDNF-pretreated cultures was caused by the build-up of excitotoxic concentrations of extracellular glutamate released through the glutamate transporters, which were up-regulated by GDNF. Although the extent and consequences of glutamate release via reversal of GLAST and GLT-1 transporters seem to vary in different energy failure models, the present findings should be taken into account in clinical trials of GDNF.

Amino Acid Transport System X-AG↗

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Brain grafts can restore irradiation-damaged neuronal connections in newborn rats.

Immature rat brain tissue grafted to the brain of other immature and adult rats can survive and establish nerve connections with the host brains. In addition to facilitating the study of factors involved in the formation of central neural connections, brain grafts may also be used to substitute damaged or maldeveloped neurones. With exceptions in the visual system, the restoration of specific central neural connections has to date involved grafts of cholinergic and monoaminergic neurones, which have good regenerative capacity. In the present study, rat hippocampal neurones were damaged by neonatal X-ray irradiation and replaced by transplantation of normal, developing neurones of the same type. The grafted neurones (dentate granule cells) are not cholinergic or monoaminergic, but when appropriately located in the host hippocampal region they established specific and highly ordered afferent and efferent connections with the damaged host brain. Moreover, simultaneous demonstration of afferent and efferent transplant pathways showed that serial host-transplant-host connections had formed, restoring the normal neuronal circuitry initially disrupted by the irradiation.

Animals↗

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Kindling induces transient changes in neuronal expression of somatostatin, neuropeptide Y, and calbindin in adult rat hippocampus and fascia dentata.

Fully hippocampus-kindled rats were examined 1 day and 1 month after the last stimulation for changes in somatostatin (SS)-, neuropeptide Y (NPY)-, and calbindin (CaBP)-immunoreactivity (ir) and SS- and NPY-mRNA in situ hybridization (ISH). One day after the last stimulation, there was marked, bilateral increase in SS- and NPY-ir in the outer part of the dentate molecular layer. The cell bodies of dentate hilar SS- and NPY-containing neurons, known to project to this area, also appeared to display increased immunoreactivity as well as an increased ISH signal for SS and NPY mRNA. Bilateral de novo expression of NPY-ir in dentate mossy fiber projection to dentate hilus and CA3 was also evident, but we noted no corresponding NPY-mRNA signal in the parent cell bodies, the dentate granule cells. After 1 month, the levels of NPY-ir and ISH signal appeared essentially normal. In contrast, the levels of SS apparently were decreased, although not yet normal. CaBP-ir was markedly and selectively reduced in dentate granule cell bodies, dendrites, and mossy fibers 1 day after the last stimulation, but after 1 month CaBP-ir appeared essentially normal. Because kindling, once established, is a permanent phenomenon, the observed transient changes in SS, NPY, and CaBP in specific hippocampal terminal fields and neuronal populations cannot be associated specifically with kindling. Rather, they relate to the repeated high-frequency stimulations and may serve as protective measures against deleterious effects of such stimulations.

Animals↗

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Direct assay of vitamins B1, B2, and B6 in hospitalized patients: relationship to level of intake.

High-performance liquid chromatography assays for vitamins B1 by erythrocyte thiamine pyrophosphate, B2 by plasma and urinary riboflavin, and B6 by plasma pyridoxal phosphate and urinary pyridoxic acid were used to evaluate the B vitamin status of hospitalized patients. Over an intake range of up to 3.4 mg of thiamine per day and up to 4.1 mg of riboflavin per day, erythrocyte thiamine pyrophosphate and urine and plasma riboflavin increased proportionately with intake. There was no relationship between B6 intake and blood levels. Rather, a constant blood level was maintained with an intake range of 0.5 to 4 mg/d, and urinary pyridoxic acid showed a linear increase proportionate to intake. There were extremely variable blood and urine concentrations of B vitamins noted in our patient population.

Adult↗

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Novel advanced in vitro methods for long-term toxicity testing: the report and recommendations of ECVAM workshop 45. European Centre for the Validation of Alternative Methods.

Explore the source record for details and available documents.

Animal Testing Alternatives↗

PubMedDate not supplied

The DaNeX study of embryonic mesencephalic, dopaminergic tissue grafted to a minipig model of Parkinson's disease: preliminary findings of effect of MPTP poisoning on striatal dopaminergic markers.

A multicenter study is under way to investigate the efficacy of allografting of embryonic mesencephalic neurons in a pig model of Parkinson's disease. We have first established that a stable parkinsonian syndrome can be established by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication of adult male Göttingen minipigs. We are now using positron emission tomography (PET) methods for testing the physiological responses to MPTP intoxication and the time course of the response to several treatment strategies. We now report preliminary results obtained in 11 pigs employed in the initial phase of the study; the completed study shall ultimately include 30 pigs. Animals were randomly assigned to one of five groups: 1) Control, 2) MPTP intoxication, 3) MPTP intoxication followed by allograft, 4) MPTP intoxication followed by allograft with immunosuppression, and 5) MPTP intoxication followed by allograft with immunosuppression and co-grafting of immortalized HiB5 cells, which had been manipulated to secrete glia cell line-derived neurotrophic factor (GDNF) (approximately 2 ng GDNF/h/10(5) cells). MPTP was administered (1 mg/kg/day, SC) for 7-10 days until the pigs had developed mild parkinsonian symptoms of muscle rigidity, hypokinesia, and impaired coordination, especially of the hind limbs. Approximately 2 weeks after the last MPTP dose, animals received a T1-weighted magnetic resonance imaging (MRI) scan, and a series of dynamic PET recordings. After the first series of PET scans, four grafts of porcine embryonic mesencephalic tissue (E28 days) were placed in each striatum of some MPTP-intoxicated pigs, using MRI-based stereotactic techniques. Immunosuppression of some animals with cyclosporin and prednisolone began just prior to surgery. Two more series of PET scans were performed at 4-month intervals after surgery. After the last scans, pigs were killed and the brains were perfused for unbiased stereological examination of cytological and histochemical markers in striatum and substantial nigra. The behavioral impairment of the animals (the "Parkinson's score") had been evaluated throughout the 8-month period. Kinetic analysis of the first set of PET scans has indicated that the rate constant for the decarboxylation of FDOPA in catecholamine fibers was reduced by 33% in striatum of the mildly parkinsonian pigs. The rate of association of [11C]NS-2214 to catecholamine uptake sites was reduced by 62% in the same groups of pigs. No significant difference was found in the binding potential of [11C]raclopride to the dopamine D2-like receptors in striatum of the MPTP-intoxicated versus control pigs. These preliminary results are suggestive that the activity of DOPA decarboxylase may be upregulated in the partially denervated pig striatum.

Animals↗