Ultrastructural evidence for bouton proliferation in the partially deafferented dentate gyrus of the adult rat

A quantitative morphological study of the changes in the dentate gyrus molecular layer in response to the removal of perforant path afferents was made utilizing electron microscopic techniques. Alterations in 1. the population of remaining afferents, 2. glial cells, and 3. granule cell dendrites are reported. The major observation was an increase in intact bouton density in the region of denervation which began at 5 days post-lesion and continued through 11 days post-lesion, the longest post-lesion survival time studied.     

Expression of vascular endothelial growth factor (VEGF) and its receptors (Flt-1 and Flk-1) following permanent and transient occlusion of the middle cerebral artery in the rat

Vascular endothelial growth factor (VEGF) is a known endothelial mitogen and a potent enhancer of vascular permeability although its role in focal cerebral ischemia is still not completely understood. The present report describes the immunohistochemical distribution of VEGF and its 2 receptors, Flt-1 and Flk-1 at day 1 and 3 following permanent and transient middle cerebral artery occlusion (MCAO) in the rat. A bilateral increase in VEGF immunoreactivity, particularly in neurons and blood vessels, was seen in both the experimental designs by day 1. By day 3, the immunoreactivity was restricted chiefly to the lesion side, where reaction was most prominent in the border zones of the infarcts. Immunoreaction to VEGF was more pronounced in cases of permanent MCAO than in transient MCAO. Flt-1 reaction was increased in neurons, glial and endothelial cells after both transient and permanent MCAO. Immunoreactivity to Flk-1 was prominent in glial cells and was present to some extent in endothelial cells. These findings indicate an early upregulation of VEGF and its receptors after permanent as well as transient focal cerebral ischemia in the rat.     

Age-induced hypertrophy of astrocytes in rat supraoptic nucleus: a cytological, morphometric, and immunocytochemical study

BACKGROUND: In the adult rat, neuron-astroglia interactions in the supraoptic nucleus (SON) are characterized by the structural and functional plasticity of astrocytes in response to several physiological and experimental conditions. This study has analyzed the plasticity of the supraoptic nucleus astrocytes in response to the age-induced changes in neuronal activity. METHODS: The study was performed in 5-, 12-, 18- and 24-month-old rats. The cytology and organization of astrocytes in the SON were examined using glial fibrillary acidic and vimentin immunocytochemistry and ultrastructural and morphometric analysis. RESULTS: No significant age-related variations in the total number of neurons and astrocytes in the SON were detected, although a few degenerating neurons were found in old rats. An age-dependent increase in GFAP immunoreactivity was observed at the ventral glial lamina, perivascularly and between neuronal perikarya. Vimentin overexpression was also detected in ventral lamina astrocytes with advancing age. At the cell nucleus level, we observed an age-associated increase in nuclear size and in the number of coiled bodies, nuclear bodies, and "cleared" nucleoplasmic areas, as well as changes in the nucleolar organization. At the cytoplasmic level, characteristic ultrastructural features in astrocytes of old rats were the hypertrophy of intermediate filament bundles and the formation of an extensive network of Golgi stacks interlinked by tubulovesicular elements. Glial filaments were often associated with the nuclear envelope and polyribosomes. CONCLUSIONS: The increased GFAP and vimentin immunoreactivity and the morphometric and cytological changes in rat SON astrocytes may reflect a sustained upregulation of cellular activity with age, resulting in hypertrophy of glial perikarya and cell processes. Several factors that are known to influence the expression of the astrocytic phenotype, such as signals produced by degenerating neurons and activated microglia, as well as variations in neuronal activity are considered possible causes of the age-associated changes in SON astrocytes.     

Circadian rhythms of arginine vasopressin and vasoactive intestinal polypeptide do not depend on cytoarchitecture of dispersed cell culture of rat suprachiasmatic nucleus

Dispersed cells of rat suprachiasmatic nucleus were cultured for more than a month with chemically defined medium. Arginine vasopressin and vasoactive intestinal polypeptide in the culture medium showed robust circadian rhythms starting 24 h after the cell dissociation. The two rhythms had similar periods, with a phase-lead of the vasoactive intestinal polypeptide peaks to the arginine vasopressin peak of about 1 h. The two rhythms remained two weeks later, with both peaks appearing at almost the same time, suggesting the synchronization of the two rhythms. Significant differences in cell architecture were detected depending on precoating matrices of culture dishes, which did not affect the circadian rhythms of arginine vasopressin and vasoactive intestinal polypeptide. Antimitotic treatment at the beginning of the culture not only reduced the number, but also changed the type of glial cells developed. The treatment did not interrupt the synchronized arginine vasopressin and vasoactive intestinal polypeptide rhythms until day 31. Early appearance of circadian rhythms indicates that neural networks in the suprachiasmatic nucleus are not necessary for the synchronous release of arginine vasopressin and vasoactive intestinal polypeptide. Glial proliferation is not essential for the generation, expression and synchronization of arginine vasopressin and vasoactive intestinal polypeptide rhythms in the dispersed suprachiasmatic nucleus cell culture.     

Development and differentiation of glial precursor cells in the rat cerebellum

The development and differentiation of bipotential glial precursor cells has been studied extensively in tissue culture, but little is known about the distribution and fate of these cells within intact animals. To analyze the development of glial progenitor cells in the developing rat cerebellum, we utilized immunofluorescent, immunocytochemical, and autoradiographic techniques. Glial progenitor cells were identified with antibodies against the NG2 chondroitin-sulfate proteoglycan, a cell-surface antigen of 02A progenitor cells in vitro, and the distribution of this marker antigen was compared to that of marker antigens that identify immature astrocytes, mature astrocytes, oligodendrocyte precursors, and mature oligodendrocytes. Cells expressing the NG2 antigen appeared in the cerebellum during the last 3-4 days of embryonic life. Over the first 10 days of postnatal life, the NG2-labeled cells incorporated 3H-thymidine into their nuclei and their total number increased. At all ages examined, the NG2-labeled cells did not contain either vimentin-like or glial fibrillary acidic protein (GFAP)-like immunoreactivity, suggesting that they do not develop along an astrocytic pathway. NG2-labeled cells of embryonic animals expressed GD3 ganglioside antigens, a property of oligodendrocyte precursors, whereas NG2-positive cells of postnatal animals did not express GD3 immunoreactivity. Nevertheless, the NG2-labeled cells of the nascent white matter expressed oligodendrocyte-specific marker antigens. Cells lying outside of the white matter continued to express the NG2 antigen. In adult animals, the NG2-labeled cells incorporated 3H-thymidine. Glial cells isolated from adult animals and grown in tissue culture express the NG2 antigen and display the phenotypic plasticity characteristic of 02A progenitor cells. These findings demonstrate that a population of glial progenitor cells is extensive within both young and adult animals.     

Conformational changes of the smooth endoplasmic reticulum are facilitated by L-glutamate and its receptors in rat Purkinje cells

Following a unilateral lesion of the visual cortex (cortical areas 17, 18, and 18a) in adult rats, neurons in the ipsilateral dorsal lateral geniculate nucleus (LGN) are axotomized, which leads to their atrophy and death. The time course of this neuronal degeneration was studied quantitatively, and the astroglial response was examined with glial fibrillary acidic protein immunohistochemistry. More than 95% of the neurons in the ipsilateral LGN survive during the first 3 days following a lesion of the visual cortex. However, in the next 4 days, massive neuronal death ensues, reducing the number of surviving neurons to approximately 33% of normal by the end of the first postoperative week. Between 2 weeks and 24 weeks postoperatively, the number of neurons present in the LGN declines very gradually from 34% to 17% of normal. Three days after a lesion of the visual cortex, the mean cross-sectional areas of ipsilateral LGN neurons are 13% smaller than normal (87%). By 1 week after the operation, surviving LGN neurons have atrophied to 66% of their normal area. Subsequently, the size of surviving neurons declines slowly to approximately 50% of normal at 24 weeks after the cortical lesion. Astrocytes in the ipsilateral LGN also react to cortical damage. At 1 day after a lesion of the visual cortex, glial fibrillary acidic protein immunoreactivity in the LGN is almost undetectable, but a distinct increase in immunoreactivity is seen at 3 days. Immunoreactivity peaks between 1 week and 2 weeks postoperatively and, thereafter, remains intense for at least 24 weeks. Thus, following a lesion of the visual cortex, the somata of neurons in the LGN remain essentially normal morphologically for about 3 days before the onset of rapid atrophy and death. Moreover, most of the neural cell death that occurs in the LGN after axotomy takes place in the last half of the first postoperative week.     

Astrocytes are the major source of nerve growth factor upregulation following traumatic brain injury in the rat

A between-side comparison of GABAA receptor subunit expression levels in the globus pallidus and anterior-pole motor thalamic nuclei of rats with an ibotenate lesion of the striatum, and rats receiving a fetal striatal graft in the lesioned area was made by using immunocytochemistry with subunit-specific antibodies, at different times post-lesion or different times post-grafting. At 10 days post-lesion, there was already an increase in the labeling of the alpha 1- and beta 2/3-subunits in the globus pallidus, entopeduncular nucleus and ventrolateral nucleus ipsilateral to the lesion when compared with the contralateral side, while there were no significant changes at the level of the ventromedial nucleus. Labeling of the alpha 2-subunit showed a clear increase in the entopeduncular nucleus compared with the contralateral side at 10 days post-lesion. Similar changes were also observed for the different subunits studied at 30 and 120 days after lesioning. Rats with 20-day old transplants of fetal striatal neurons that were implanted in the ibotenate lesioned striatum at 10 days post-lesioning, continued to show changes in the expression of GABAA receptor subunits, albeit at a lower level than those of ibotenate lesioned rats at similar age post-lesion. However, when examining rats with 70-day old transplants, the ibotenate-lesion induced between-side changes were almost completely compensated. These findings suggest a correlation between the maturation of the grafts and their capability to function in reestablishing neuronal circuits as shown by the reduction of changes in GABAergic transmission induced by ibotenate lesions, as indicated by the reversal of changes in GABAA receptor subunit in several areas of the basal ganglia circuit.     

Application of infrared spectroscopy to development of stable lyophilized protein formulations

Global cerebral ischemia selectively damages neurons, but its contribution to glial cell death is uncertain. Accordingly, adult male rats were sacrificed by perfusion fixation at 1, 2, 3, 5, and 14 days following 10 minutes of global ischemia. This insult produces CA1 hippocampal neuronal death at post-ischemic (PI) day 3, but minor or no damage to neurons in other regions. In situ end labeling (ISEL) and immunohistochemistry identified fragmented DNA of dead or dying glia and distinguished glial subtypes. Rare ISEL-positive oligodendroglia, astrocytes, and microglia were present in control brain. Apoptotic bodies and ISEL-positive glia significantly increased at PI day 1 in cortex and thalamus (p < 0.05), but were similar to controls in other regions and at other PI intervals. Most were oligodendroglia, although ISEL-positive microglia and astrocytes were also observed. These results show that oligodendroglia die rapidly after brief global ischemia and are more sensitive than neurons in certain brain regions. Their selective vulnerability to ischemia may be responsible for the delayed white matter damage following anoxia or CO poisoning or that associated with white matter arteriopathies. Glial apoptosis could contribute to the DNA ladders of apoptotic oligonucleosomes that have been found in post-ischemic brain.