Rainald Schmidt-Kastner

Selective vulnerability of the hippocampus in brain ischemia

*Department of Neurophysiology, Institute of Physiology, Medical Faculty, Ruhr-Universitlt Bochum, Universitiitsstr. 150, D-4630 Bochum 1, F.R.G. SDepartment of Functional Neuroanatomy, Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O. Box 67. H-1450 Budapest, Hungary, and MRC Anatomical Neuropharmacology Unit, _ Department of Pha-rmacology, University of Oxford, Oxford, U.K.

NeuN: a useful neuronal marker for diagnostic histopathology.:

The monoclonal antibody A60 specifically recognizes the DNA-binding, neuron-specific protein NeuN, which is present in most neuronal cell types of vertebrates. In this study we demonstrate the potential use of NeuN as a diagnostic neuronal marker using a wide range of formalin-fixed, paraffin-embedded human surgical and autopsy specimens from the central and peripheral nervous system. After microwave antigen retrieval, almost all neuronal populations revealed strong immunoreactivity for NeuN in nuclei, perikarya, and some proximal neuronal processes, whereas more distal axon cylinders and dendritic ramifications were not stained. The stain greatly enhanced the gray matter architecture. NeuN immunoreactivity was not detected in Purkinje cells, most neurons of the internal nuclear layer of the retina, and in sympathetic chain ganglia. We examined nine gangliogliomas and 14 dysembryoplastic neuroepithelial tumors, one ganglioneuroma, and one dysplastic cerebellar gangliocytoma. The neuronal component of all of these lesions showed marked immunoreactivity for NeuN. In addition, NeuN immunoreactivity was focally seen in one of seven medulloblastomas with prominent neuronal differentiation. There was no staining of non-neuronal structures. The results indicate that NeuN immunoreactivity is a sensitive and specific neuronal marker in formalin-fixed, paraffin-embedded tissues, and may be useful in diagnostic histopathology.

Lesion-induced transient suppression of inhibitory function in rat neocortex in vitro

The structural and functional consequences of a local thermolesion were examined in rat neocortex with electrophysiological in vitro techniques and immunocytochemistry. Age-matched untreated and sham-operated animals served as controls and were analysed in the same way. The lesions consisted of a core of coagulated tissue 2-3 mm in diameter and reached ventrally into the deep cortical layers. After two days reactive astrocytes and after nine days a dense gliosis were observed in the immediate vicinity. Modifications in the intrinsic membrane characteristics and the synaptic network properties were investigated with intra- and extracellular recording techniques after survival times of one to eight days. Neurons recorded in the surrounding of lesions in neocortical slices revealed a significantly more depolarized resting membrane potential and a higher neuronal input resistance. In comparison to cells in control slices, maximal discharge rates to injection of depolarizing current pulses of neurons close to a focal lesion were not significantly altered and intrinsic burst firing was never observed. However, between postlesion days 1 and 5, neurons in the surroundings of lesions showed a transient increase in synaptic excitability. This hyperactivity was most clearly pronounced at a distance of 2-3 mm from the centre of the lesion (i.e. about 1-1.5 mm away from the lesion border) and characterized by long-duration field potential responses and multiphasic long-lasting excitatory postsynaptic potentials to orthodromic stimulation of the afferent input. This lesion-induced hyperexcitability was associated with a significant reduction in the peak conductance of the Cl(-)-dependent fast inhibitory postsynaptic potential and the K(+)-dependent long-latency inhibitory postsynaptic potential, suggesting that the intracortical GABAergic system was functionally impaired. The decrease in synaptic inhibition was associated with prolonged N-methyl-D-aspartate receptor-mediated activity, which could be reversibly blocked by D-amino-phosphonovaleric acid. In addition, neurons recorded in the vicinity of the lesion responded to an orthodromic synaptic stimulus with a long-lasting burst. The lesion-induced disturbance in the balance between the excitatory and inhibitory system may not only have profound influences on the mechanisms of intracortical information processing, but may also lead to the expression of epileptiform activity and long-term functional deficits.

Improvement in neuronal survival after ischemic preconditioning in hippocampal slice cultures

The main goals of the current study were to assess: (a) whether a sublethal ischemic insult could protect the CA1 subregion of the hippocampus in organotypic slices against a lethal ischemic insult; and (b) whether this protection is long lasting as determined with an accurate immunohistochemical neuronal marker, NeuN. Hippocampal slice cultures were grown for 12-14 days in vitro. Slices were exposed either to oxygen/glucose deprivation (OGD) for 45 min (ischemia), or OGD for 15 min (ischemic preconditioning), 48 h prior to 45 min OGD, or were untreated (sham). Cell death was estimated by propidium iodide fluorescence 1 day after OGD and by NeuN immunohistochemistry 7 days after OGD. Image analysis was employed to measure the relative optical density of the NeuN-signal in all groups. After ischemia, damaged neurons were shrunken or lost and NeuN immunoreactivity was reduced. Relative optical density of NeuN (ROD [NeuN]) was 0.193+/-0.015 in control (sham) (n=9). In slices that underwent ischemia, ROD [NeuN] declined to 0.108+/-0.018 (n=5) in CA1 (*P<0.05 ROD [NeuN] in preconditioned slice cultures was 0.190+/-0.037 (76% higher than the ischemia group). Similar results were found after measuring PI fluorescence. In the CA1 sub-region, PI fluorescence was about 13, 47 and 17% in the sham, ischemic and IPC groups, respectively. We suggest that the immunohistochemical approach validates the dye uptake method used in slice cultures and yields quantitative data specific for neurons. We also conclude that the organotypic hippocampal slice model is useful for studying delayed ischemic preconditioning that is maintained for hours or days after the preconditioning event.

Nuclear localization of the hypoxia-regulated pro-apoptotic protein BNIP3 after global brain ischemia in the rat hippocampus.

The 19 kD interacting protein 3, Nip3/BNIP3, is a pro-apoptotic member of the Bcl-2 family induced during hypoxia via the hypoxia-inducible factor (HIF) 1. BNIP3 has been linked to both apoptotic and necrotic cell death involving mitochondrial permeability transition. Since apoptotic and necrotic mechanisms may occur in brain ischemia, immunohistochemical changes of BNIP3 were studied at 1, 2, 3 and 7 days after transient global brain ischemia (12.5 min) in ventilated normothermic rats. In control brains, BNIP3-like immunoreactivity was moderately strong in neuronal processes or cytoplasm and absent in the nucleus. In the ischemia-vulnerable CA1 neurons, BNIP3-positive granules were seen in the nucleus at 1 and 2 days, and these neurons were damaged at 3 and 7 days. The resistant CA3 neurons showed nuclear BNIP3 labeling by 1 day and then returned to the normal state. BNIP3-positive granules did not overlap with the nucleolus. Constitutively expressed BNIP3 may participate in apoptotic and necrotic processes after brain ischemia. Nuclear location of BNIP3 after brain ischemia indicates a novel role for the regulation of cell survival in neurons or a general disturbance of the nuclear envelope.

Immunohistochemical changes of neuronal calcium-binding proteins parvalbumin and calbindin-D-28k following unilateral deafferentation in the rat visual system

The neuron-specific calcium-binding proteins, parvalbumin and calbindin-D-28k, were studied in the subcortical visual system of normal and unilaterally deafferented albino rats. Immunohistochemistry with monoclonal antibodies was used on vibratome sections through optic tract (OT), dorsal lateral geniculate nucleus (dLGN), olivary pretectal nucleus (OPN), and superior colliculus (SC). In controls, OT stained strongly for parvalbumin and weakly for calbindin-D-28k. The dLGN contained a plexus of parvalbumin-positive fibers. In dLGN, calbindin-D-28k-antibodies showed strong labeling of some neurons with long dendrites and weak staining of the cytoplasm in other neurons. In OPN, parvalbumin stained a ring of neurons and terminals in the shell region, whereas calbindin-D-28k was contained in medial cell populations. In SC, parvalbumin was contained in fibers, terminals, and neurons throughout the visual layer. Calbindin-D-28k showed a laminar distribution of neurons with a predominance in deep portions of superficial grey matter and in ventral portions of stratum opticum. Following unilateral deafferentation induced by optic nerve section, retinal axons showed immunohistochemical changes related to Wallerian degeneration and target neurons reacted by changes of calcium-binding proteins. Parvalbumin and calbindin-D-28k immunostaining decreased during Wallerian degeneration of OT. In the deafferented dLGN, immunohistochemical labeling for calbindin-D-28k declined in strongly stained neurons from 4 to 21 days after lesion. Measurement of dendritic length per number of cells or per area of dLGN showed a significant decline for the contralateral side at 4, 8, and 21 days (ANOVA, P less than 0.05). In deafferented OPN, terminal-like staining for parvalbumin decreased and neuronal labeling was enhanced. In deafferented SC, the neuronal and dendritic staining for parvalbumin increased beginning from Day 1 on and persisting at Day 21, whereas fibers and terminal-like elements decreased in staining. Measurement of parvalbumin-positive neurons per area of SC showed a significant increase of labeling in the contralateral side from Day 1 to Day 21 (ANOVA, P less than 0.05). These studies show that cellular responses to deafferentation of visual neurons involve a regulation of calcium-binding proteins. The decline in staining for calbindin-D-28k in dLGN may relate to reduced retinal afferent activity. The progressive cellular changes in parvalbumin staining may be related to unmasking of intrinsic neurons after removal of parvalbumin-containing, afferent fibers and terminals. Additionally, the changes of parvalbumin labeling in SC neurons may reflect a plastic reorganization of local circuits known to occur in rat SC in response to deafferentation.

Immunohistochemical staining for glial fibrillary acidic protein (GFAP) after deafferentation or ischemic infarction in rat visual system: Features of reactive and damaged astrocytes

Immunohistochemical staining for glial fibrillary acidic protein (GFAP) is standard for visualization of reactive astrocytes in tissue sections, whereas various forms of astrocytic damage remain to be described in detail. In this study we tested differences in GFAP labeling in reactive astrocytes and in glial cells damaged by ischemia and edema. Studies were performed in the anatomically well defined visual system of rat. Basic staining patterns for GFAP were established in subcortical visual nuclei and visual cortex. In the first model, deafferentation of visual centers was performed by unilateral optic nerve lesion, and characteristic changes of GFAP labeling in reactive astrocytes were studied at 0.5,1,1.5,2,4,8 and 21 days after lesion. Initial changes were seen in the deafferented superior colliculus at 1 day after deafferentation with a diffuse increase and stellate types of reactive cells formed at 2–8 days. In the second model, small ischemie infarcts were produced in the visual cortex of rats using the method of photochemically‐induced thrombosis. GFAP labeling with a polyclonal antiserum was massively enhanced in the infarct at 4 hr. Characteristic morphological changes in damaged astrocytes were seen which were also identified in experiments with simulated global ischemia. In the surround of the infarct, swelling of astrocytes also caused increased labeling. At 3–4 days infarction typical reactive astrocytes surrounded the lesioned area. In conclusion, these immunohistochemical studies on GFAP in rat visual system allow for the following classifications, (a) Normal astrocytes vary in labeling at different anatomical localizations, (b) Reactive astrocytes show enhanced labeling and larger cell‐size within an interval of 1–2 days after lesion (c) Astrocytes damaged by ischemia reveal increased labeling of disintegrating cellular elements within hours after a lesion (d) Swollen astrocytes undergo enhanced labeling in areas with vasogenic edema.

Neuroglobin mRNA expression after transient global brain ischemia and prolonged hypoxia in cell culture.

Neuroglobin is a nerve-specific respiratory protein that has been proposed to play an important role in the protection of brain neurons from ischemic and hypoxic injuries. Here, we investigated the regulation of neuroglobin expression after transient global ischemia in the rat brain using mRNA in situ hybridization and under hypoxic stress in cultured neuronal cell lines (PC12, HN33) by quantitative RT-PCR. While neuroglobin mRNA expression was significantly enhanced in cell culture after severe prolonged hypoxia (0-1% O2 for 24 h), we did not find any significant increases in neuroglobin mRNA levels in the rat brain after transient global ischemia. Vegf and Glut1 mRNAs showed increases in the hippocampus as expected. Therefore, it is unlikely that neuroglobin is instrumental in the acute response of neurons to hypoxic or ischemic insults, for which the mammalian brain is not adapted.

Hypoxia and hypoxia-inducible factor modulated gene expression in brain: involvement in neuroprotection and cell death.

Abstract Hypoxia, due to impaired cerebral blood flow, has hazardous effects on brain structure and function. Therefore, mechanisms should exist to meet the needs for hypoxic adaptation via regulation of gene expression. Signaling between the O2 sensor and the regulator(s) of transcription is only partially characterized and requires regulatory transcription factors. Among these regulatory proteins, hypoxia-inducible factor–1 (HIF-1) appears to have a key role. HIF-1 modulates gene activity in response to low O2 tensions in the developing and in the adult brain. Moderate hypoxia may elicit autoprotective mechanisms or hypoxia-induced regulators can contribute to mechanisms leading to cell death. Moreover, reactivation of embryonic gene expression may occur after injury-induced hypoxia. Thus, analyses of embryonic and pathogenic models should help to understand how hypoxia-mediated proliferative / cell death processes are involved in brain development and in the pathogenesis of acute or chronic neurodegenerative brain diseases.

Astrocytes react to oligemia in the forebrain induced by chronic bilateral common carotid artery occlusion in rats.

The effects of oligemia (moderate ischemia) on the brain need to be explored because of the potential role of subtle microvascular changes in vascular cognitive impairment and dementia. Chronic bilateral common carotid artery occlusion (BCCAO) in adult rats has been used to study effects of oligemia (hypoperfusion) using neuropathological and neurochemical analysis as well as behavioral tests. In this study, BCCAO was induced for 1 week, or 2, 4, and 6 months. Sensitive immunohistochemistry with marker proteins was used to study reactions of astrocytes (GFAP, nestin), and lectin binding to study microglial cells during BCCAO. Overt neuronal loss was visualized with NeuN antibodies. Astrocytes reacted to changes in the optic tract at all time points, and strong glial reactions also occurred in the target areas of retinal fibers, indicating damage to the retina and optic nerve. Astrocytes indicated a change in the corpus callosum from early to late time points. Diffuse increases in GFAP labeling occurred in parts of the neocortex after 1 week of BCCAO, in the absence of focal changes of neuronal marker proteins. No significant differences emerged in the cortex at longer time points. Nestin labeling was elevated in the optic tract. Reactions of microglia cells were seen in the cortex after 1 week. Measurements of the basilar artery indicated a considerable hypertrophy, indicative of macrovascular compensation in the chronic occlusion model. These results indicate that chronic BCCAO and, by inference, oligemia have a transient effect on the neocortex and a long-lasting effect on white matter structures.