Induction of NADPH-diaphorase activity in the forebrain in a model of intracerebral hemorrhage and its inhibition by the traditional Chinese medicine complex Nao Yi An

Induction of NADPH-diaphorase (NDP) activity in the rat cerebral cortex was studied after autologous blood injection into the internal capsule as experimental model of intracerebral hemorrhage. The potential inhibitory effect on NDP induction by Nao Yi An (NYA), a complex derived from materials of animal and plant origin used in the treatment of intracerebral hemorrhage in traditional Chinese medicine, was also investigated. in animals without therapeutic treatment 2 and 4 days after injection of autologous blood, NDP activity was highly induced in pyramidal neurons in the neocortex, piriform, and entorhinal cortices, in astrocytes and in phagocytes in the hematoma and the area surrounding it, as well as in the subcortical white matter, and in endothelial cells in both the cortex and subcortical white matter bilaterally. Oral administration of NYA failed to inhibit NDP induction in endothelial cells but demonstrated a strong inhibitory effect on NDP activity induced in pyramidal neurons and astrocytes. NDP induction in phagocytes was also inhibited by the administration of NYA. Altogether the present results suggest that intracerebral hemorrhage in the internal capsule may induce nitric oxide synthase activity in different cell populations in the cortex and that administration of NYA can selectively inhibit such induction and, thus, potentially play a neuroprotective role.     

Morphologische Befunde am Rattengehirn nach lokaler Vereisung

Summary In continuation of our investigations of the wound healing process after cryonecrosis in the liver, kidney, spleen and stomach, the wound healing in the brain was studied under the same conditions. Through a trepanation of about 3 mm diameter, the cortex of the parietal region of the cerebrum was frozen by a cryoprobe applied directly through the intact dura mater (–196° C/30 s). The animals were killed at definite time intervals between 12 h and 21 days after freezing.Twelve hours after freezing, the necrosis of the brain cortex is complete. It extends wedgeshaped into the subcortical white matter. The central parts of the necrosis become colliquative and are demarcated by leucocytes. Two days after freezing, the necrotic area is almost totally invaded by leucocytes. Three days p. op. the brain wound is infiltrated by microglial cells, later on the mesenchymal cell proliferation extends to the meninges. There is new capillary formation and partially a wall like proliferation of the perivascular connective tissue cells. Ten days and later, an uni- or multiloculated pseudocyst develops, which can be considered as the final stage of the reparative wound healing of the cryonecrosis.Similar as in the other investigated organs (liver, kidneys, spleen) these investigations underline the rapid wound healing of the cryonecrosis in the brain. The advantages of cryosurgical manipulations with the well known good wound healing process of other organs, justifies therefore the short term tissue freezing on the cerebrum.

Mit Unterstützung des Landesamtes für Forschung Nordrhein-Westfalen Düsseldorf     

Disruption of postnatal progenitor migration and consequent abnormal pattern of glial distribution in the cerebrum following administration of methylmercury

Transplacental administration of methylmercury (MeHg) induces disruption of neuronal migration in the developing cerebral cortex. However, the effects of MeHg on glial progenitor migration remain unclear. To understand this, we performed double administration of MeHg and 5-bromo-2-deoxyuridine (BrdU) to neonatal rat pups on postnatal day 2 (P2), when glial cells are generated from progenitors in the subventricular zone (SVZ). Histopathological examination of a proportion of the MeHg-treated rats on P28 revealed no apparent abnormalities of cytoarchitecture or neuron count in either the primary motor or primary somatosensory cortex of the cerebrum. BrdU immunohistochemistry revealed abnormal accumulation of the labeled cells in the deeper layers of the cortices and underlying white matter of both areas, where an excessive number of astrocytes (glial fibrillary acidic protein- or S-100beta-immunolabeled cells) and oligodendrocytes (2',3'-cyclic-nucleotide 3'-phosphohydrolase-labeled cells) were located. Next, to investigate the migration of individual progenitors from the forebrain SVZ of P2 neonates, we labeled them in vivo with a retrovirus encoding green fluorescent protein (GFP), following administration of MeHg, and then examined the distribution pattern of the GFP-labeled cells in the P28 cerebrum. We found that the labeled cells developed into astrocytes and oligodendrocytes and were accumulated abnormally in the lateral white matter as well as in the adjacent deeper layer of the lateral cortex and lateral side of the striatum. Thus, exposure to MeHg in the gliogenic period induced irregular distribution of glia as a consequence of abnormal migration of the postnatal progenitors.     

Differentiation and migration of astrocytes in the spinal cord following dorsal root injury in the adult rat

Nerve fibre degeneration in the spinal cord is accompanied by astroglial proliferation. It is not known whether these cells proliferate in situ or are recruited from specific regions harbouring astroglial precursors. We found cells expressing nestin, characteristic of astroglial precursors, at the dorsal surface of the spinal cord on the operated side from 30 h after dorsal root injury. Nestin-expressing cells dispersed to deeper areas of the dorsal funiculus and dorsal horn on the operated side during the first few days after injury. Injection of bromodeoxyuridine (BrdU) 2 h before the end of the experiment, at 30 h after injury, revealed numerous BrdU-labelled, nestin-positive cells in the dorsal superficial region. In animals surviving 20 h after BrdU injection at 28 h postlesion, cells double-labelled with BrdU and nestin were also found in deeper areas. Labeling with BrdU 2 h before perfusion showed proliferation of microglia and radial astrocytes in the ventral and lateral funiculi on both sides of the spinal cord 30 h after injury. Nestin-positive cells coexpressed the calcium-binding protein Mts1, a marker for white matter astrocytes, in the dorsal funiculus, and were positive for glial fibrillary acidic protein (GFAP), but negative for Mts1 in the dorsal horn. One week after injury the level of nestin expression decreased and was undetectable after 3 months. Taken together, our data indicate that after dorsal root injury newly formed astrocytes in the degenerating white and grey matter first appear at the dorsal surface of the spinal cord from where some of them subsequently migrate ventrally, and differentiate into white- or grey-matter astrocytes.     

Control of astrocyte migration in the developing cerebral cortex

Development of the vertebrate central nervous system is characterized by significant long distance cell migration. While the radial migration of neuronal precursors to their final location is well established the migration of glial cells and their precursors is less well understood. To define the pathways of migration and dissect the cell and molecular mechanisms mediating such migration requires the development of appropriate models. Here we show that purified neonatal astrocytes injected into organotypic slice cultures of developing cerebral cortex migrate in defined patterns depending on where they are placed within the tissue. Injection into gray matter resulted in radially oriented migration either towards the pial or ventricular surface. By contrast injection into developing white matter resulted in largely longitudinal migration along developing axon tracts. While the cytoarchitecture of the tissue influenced the pattern of migration, the extent of migration appeared to be regulated primarily by the age of the host tissue. Homochronic injections performed prior to postnatal day 4 resulted in extensive migration while after day 7 migration was relatively limited. Heterochronic injections indicated that while astrocytes within the 1st postnatal week retained the capacity to migrate extensively, older tissue failed to support extensive migration of either young or old astrocytes. These data suggest the existence of distinct migrational cues in the CNS and that environmental, not cell intrinsic properties primarily regulate astrocyte migration through the developing cortex.     

Edema of the cortical gray matter of the human cerebrum.

Five cases of brain tumor are reported in which an extracellular edema of the cerebral cortex was recognized in the form of lakes of eosinophilic, PAS positive material, presumably serum proteins, permeating among the neuronal and glial cells and processes. In three, the astrocytes and their processes were stained in replicate paraffin sections by a gold sublimate technique. The astrocytic changes were variable. Most often, the astrocytes changed from protoplasmic to fibrillary, with long deeply stained processes, the cells sometimes becoming large and distorted. Many became enlarged and smudgy, with short stubby processes. Only a few showed the fragmentation and disintegration of processes. Only a few showed the fragmentation and disintegration of processes which are regularly observed in edematous white matter. The neuronal processes remained essentially normal. In comparison with edema of white matter, edema of the cortex is rare, involves only small portions of tissue, and differs in its effects on astrocytes. The edema in both cortex and white matter is extracellular. We have not been able to recognize a specific intracellular form of edema, grossly or with light microscopic techniques.     

Immunohistochemical studies on the proliferation of reactive astrocytes and the expression of cytoskeletal proteins following brain injury in rats

The appearance of reactive astrocytes following brain injury was investigated in 4-week-old rats with special reference to their proliferation and chronological changes in the cytoskeletal proteins. Two days after the injury, glial fibrillary acidic protein (GFAP)-positive cells had increased in number around the lesion and spread to the entire ipsilateral cortex by 3 days after the injury. To investigate the distribution of mitotic cells and its chronological change, immunohistochemical staining with monoclonal antibody to bromodeoxyuridine (BrdU) was performed. BrdU-positive cells began to appear around the lesion and spread to the entire ipsilateral cortex by 3 days and their distribution was the same as that of GFAP-positive cells. To investigate the association of GFAP-positive cells with cell division, double labeling experiments using [3H]thymidine autoradiography and immunohistochemical staining with antiserum to GFAP were performed. Cells doubly labeled with GFAP and [3H]thymidine were localized in the area adjacent to the lesion, in the molecular layer of the cortex and in the white matter. By contrast, none of the cells were doubly labeled in the IInd to VIth layers of the cortex. Furthermore, only astrocytes in the former areas expressed vimentin transiently from 2 to 10 days after the injury. In the rats administered vincristine, cells arrested during mitosis were found in the regions which express vimentin. From these results, it was suggested that astrocytes in the molecular layer of the cortex and the white matter adjacent to the lesion proliferated in response to the injury and expressed vimentin transiently, then acquired GFAP, and that astrocytes in the IInd to VIth layers of the cortex became reactive astrocytes without mitosis.     

Matrix metalloproteases and their inhibitors are produced by overlapping populations of activated astrocytes

Matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteases (TIMPs) are involved in many cell migration phenomena and produced by many cell types, including neurons and glia. To assess their possible roles in brain injury and regeneration, we investigate their production by glial cells, after brain injury and in tissue culture, and we investigate whether they are capable of digesting known axon-inhibitory proteoglycans. To determine the action of MMPs, we incubated astrocyte conditioned medium with activated MMPs, then did western blots for several chondroitin sulphate proteoglycans. MMP-3 digested all five proteoglycans tested, whereas MMP-2 digested only two and MMP-9 none. To determine whether MMPs or TIMPs are produced by astrocytes in vitro, we tested both primary cultures and astrocyte cell lines by western blotting, and compared them with Schwann cells. All cultures produced at least some MMPs and TIMPs, with no obvious correlation with the ability of axons to grow on those cells. Both MMP-9 and TIMP-3 were regulated by various cytokines. To determine which cells produce MMPs and TIMPs after brain injury, we made lesions of adult rat cortex, and did immunohistochemistry. MMP-2 was seen to be induced in activated astrocytes through the whole thickness of the cortex but not deeper, but MMP-3 was not seen in the injured brain. TIMP-2 and TIMP-3 immunoreactivities were induced in activated astrocytes in deep cortex and the underlying white matter. In situ hybridisation confirmed induction of TIMP-2 in glia as well as neurons, but showed no expression of TIMP-4. These results show that both MMPs and TIMPs are produced by some astrocytes, but TIMP production is particularly strong, especially in deep cortex and white matter which is more inhibitory for axon regeneration. Conversely the MMPs produced may not be adequate to promote migration of cells and axons within the glial scar.     

Double encephalitis with herpes simplex virus and cytomegalovirus in an adult

A 51-year-old housewife developed symptoms of a cold followed by high fever, delirium, coma, rigidity of extremity muscles, positive Babinski sign and generalized convulsions, while complement-fixing antibody titre to herpes simplex virus in the sera raised over 128 X and declined to 8 X in the course. She finally expired of bronchopneumonia following status epilepticus after 94 days of illness. Severe necrosis with extensive hemorrhage in the white matter was predominant in the temporal, insular and orbitofrontal cortex, thalamus and globus pallidus. Focal rarefaction of the cerebral cortex with a very few eosinophilic intranuclear inclusions in the oligodendroglia and nerve cells, nerve cell destruction in the substantia nigra with glial nodules and perivascular inflammatory cell cuffs were observed. Abundant cytomegalic inclusion cells, originating from hypertrophic astrocytes, were present in the necrotic areas of cerebrum as well as in the rarefied tissue in the subependymal layers of the brainstem and cerebellum. Electron-microscopic study of the cytomegalic cells demonstrated the presence of numerous virions in both nucleus and cytoplasm. Fortuitour infection of the brain by cytomegalovirus with necrotizing encephalitis by herpes simplex virus is unique. The cause of double viral infections and severe lesions by less virulent strains is discussed.     

A case of simple form of sudanophilic leukodystrophy of a child which showed a marked loss of cerebral white matter and fatty liver

A sporadic case of sudanophilic leukodystrophy of the simple form (Peiffer) was reported. The patient was three-year-old girl who had suffered from progressive developmental retardation and neurological disorders such as ataxia, cortical blindness and spastic paralysis of the extremities for eighteen months after she had showed normal development till one and a half years old and died from respiratory insufficiency. On admission, computerized tomogram scan demonstrated diffuse low density lesions of the cerebral white matter extending subsequently to the subcortical white matter. Examination of cerebrospinal fluid revealed only slight increase of protein. Lysosomal enzyme activities such as arylsulfatase and beta-galactosidase in the white blood cells were normal except for distinctly low activity of a-mannosidase without any clinical symptoms suggesting a-mannosidase deficiency. Amino acids in blood were normal. The brain weighed 900 gm. On the coronal sections most part of the cerebral white matter was so strongly degenerated and disappeared that the lateral ventricular structure was not discernible. Histologically, a diffuse and symmetrical demylination, loss of axons including U fibers and moderate gliosis were observed in the residual white matter in the cerebrum and pons. There was no inflammatory cells and metachromatic substances. Large amount of sudanophilic droplets showing polarizing cross and needle like crystals were found in the intra- and/or extracytoplasm of macrophages. Demyelinated lesions with little tissue reaction were also found in the cerebellum, medulla oblongata and in pyramidal tracts through midbrain to cervical spinal cord. There were slight loss of neurons and moderate astrocytosis in the cerebral cortex and basal ganglia. There were no Rosenthal fibers and no sparing of islets of myelin.(ABSTRACT TRUNCATED AT 250 WORDS)