Vascular endothelial growth factor (VEGF) is currently considered a potential pharmacologic agent for stroke therapy because of its strong neuroprotective and angiogenic capacities. Nonetheless, it is unclear how neuroprotection and angiogenesis by exogenous VEGF are related and whether they are concurrent events. In this study, the authors evaluated by stereology the effect of VEGF on neuronal and vascular volume densities of normal and ischemic brain cortices of adult male Sprague-Dawley rats. Ischemia was induced by a 4-hour occlusion of the middle cerebral artery. Low, intermediate, and high doses of VEGF165 were infused through the internal carotid artery for 7 days by an indwelling osmotic pump. The low and intermediate doses, which did not induce angiogenesis, significantly promoted neuroprotection of ischemic brains and did not damage neurons of normal brains. In contrast, the high dose that induced angiogenesis showed no neuroprotection of ischemic brains and damaged neurons of normal brains. These findings suggest that in vivo neuroprotection of ischemic brains by exogenous VEGF does not necessarily occur simultaneously with angiogenesis. Instead, neuroprotection may be greatly compromised by doses of VEGF capable of inducing angiogenesis. Stroke intervention efforts attempting to induce neuroprotection and angiogenesis concurrently through VEGF monotherapy should be approached with caution.

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In the present study the "interstitial" cells of the superficial pineal gland and the nonparenchymal cells of the pineal stalk in Sprague-Dawley rats were examined ultrastructurally with the aim of defining the cells more closely. The "interstitial" cells of the superficial pineal gland do not represent a homogeneous cell population. The most abundant cell type is the mononuclear phagocyte, most easily recognized by its dark appearance and its content of primary and conspicuous secondary lysosomes. Astrocytes can be distinguished by the typical appearance of their nuclei (i.e., a thin continuous rim of heterochromatin adjacent to the nuclear membrane), identical to that of astrocytes in the CNS. Depending on the absence or presence of glial filaments and their amount, a spectrum of astrocytic cells is present. Mature astrocytes with filaments throughout their cytoplasm are rare. Immature glial cells with few or no filaments predominate. In the vicinity of blood vessels pericytes are present. In view of the fact that the "interstitial" cells could generally be identified it is suggested to abandon the term interstitial for the cells in question. In the pineal stalk mature astrocytes predominate; they have some features in common with pinealocytes, i.e., the presence of intergrade endoplasmic reticulum and grumose bodies (lysosomes). Other unusual features are a relative abundance of coated pits and vesicles. Oligodendrocytes are restricted to the proximal part of the stalk, near the deep pineal, where myelinated axons are abundant. More distally a few Schwann cells were seen.

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In view of the increasing interest in the central innervation of the mammalian pineal gland, this aspect was studied in depth in the rat. This species is especially suited since the nerve fibers in question form a distinct bundle running from the deep to the superficial pineal gland through the pineal stalk. The axons were counted and analysed ultrastructurally in the pineal stalks cut transversely at three levels (proximal, intermediate, and distal) relative to the neural axis and in longitudinal sections. The number of nerve fibers was highly variable, ranging from 551 to 1,132 proximally and from 110 to 448 distally, indicating that many fibers terminate in the stalk or leave the stalk after forming a loop. Large myelinated axons, which are abundant proximally, appear to lose their sheaths along their course through the stalk. Most of the axons were small and unmyelinated. A few of these had the appearance of sympathetic fibers and disappeared after sympathectomy. Others contained abundant neurosecretory granules, and, according to the literature, may originate in the hypothalamic paraventricular nuclei. The majority of the small axons which are apparently devoid of granules and dense-cored vesicles may come from the habenular nuclei and the stria medullaris. In addition to axons, the stalk contains astrocytes, a few oligodendrocytes and Schwann cells, as well as pinealocytes identical to those of the superficial pineal gland.

Myelin/oligodendrocyte specific protein was compared to glial fibrillary acidic protein and 2'3'-cyclic nucleotide 3'-phosphodiesterase expression in normal rat brains and following stab wounds to the cerebral cortex, corpus callosum and hippocampus. Animals with stab wounds were allowed to recover for 5, 15, 28, 45 and 70 days post-operation before fixation by perfusion. Sections were reacted with antibodies against myelin/oligodendrocyte specific protein, glial fibrillary acidic protein and 2'3'-cyclic nucleotide 3'-phosphodiesterase, and observed by light and electron microscopy. Normal cerebral cortex had very few myelin/oligodendrocyte specific protein-positive and 2'3'-cyclic nucleotide 3'-phosphodiesterase-positive cells, but some glial fibrillary acidic protein-positive cells. The myelinated fibres of the corpus callosum were heavily stained for myelin/oligodendrocyte specific protein but unstained by glial fibrillary acidic protein or 2'3'-cyclic nucleotide 3'-phosphodiesterase antibodies. Some immunopositive cells were present in the corpus callosum and hippocampus with all three antibodies. After stab wound myelin/oligodendrocyte specific protein-positive reactive cells had more and longer processes and stained more intensely than equivalent cells in normal brain. These cells were distributed along the wound track, including within the cerebral cortex. The numbers of these cells increased until 28 days post-operation and then decreased so that very few were found at 70 days post-operation except in the corpus callosum. Where demyelination occurred myelin/oligodendrocyte specific protein-staining was lost. Staining for 2'3-cyclic nucleotide 3'-phosphodiesterase revealed a similar pattern. Glial fibrillary acidic protein-positive reactive cells, which were also more robust than the normal cells, were more widely distributed. They increased in number throughout the time periods studied and gliosis was evident on the contralateral side. The glial fibrillary acidic protein-positive astrocytes were also different from the myelin/oligodendrocyte specific protein-positive and 2'3'-cyclic nucleotide 3'-phosphodiesterase-positive oligodendrocytes in terms of cell shape. With electron microscopy myelin/oligodendrocyte specific protein-positive cells showed features typical of immature oligodendrocytes. We conclude that the injury caused a numerical increase in oligodendrocytes and that myelin/oligodendrocyte specific protein is a good marker for the oligodendroglial response and demyelination in pathological conditions.

Cytochemical data in the literature reporting localization of sodium, potassium adenosine triphosphatase (Na(+), K(+)-ATPase) in the blood-brain barrier (BBB) have been contradictory. Whereas some studies showed the enzyme to be located exclusively on the abluminal endothelial plasma membrane, others demonstrated it on both the luminal and abluminal membranes. The influence of fixation on localization of the enzyme was not considered a critical factor, but our preliminary studies showed data to the contrary. We therefore quantitatively investigated the effect of commonly used fixatives on the localization pattern of the enzyme in adult rat cerebral microvessels. Fixation with 1%, 2%, and 4% formaldehyde allowed deposition of reaction product on both the luminal and abluminal plasma membranes. The luminal reaction was reduced with increasing concentration of formaldehyde. Glutaraldehyde at 0.1%, 0.25%, 0.5%, in combination with 2% formaldehyde, drastically inhibited the luminal reaction. The abluminal reaction was not significantly altered in all groups. These results show that luminal localization of BBB Na(+), K(+)-ATPase is strongly dependent on fixation. The lack of luminal localization, as reported in the literature, may have been the result of fixation. The currently accepted abluminal polarity of the enzyme should be viewed with caution.

Thiamine pyrophosphatase (TPPase), nucleoside diphosphatase (NDPase), and glucose-6-phosphatase (G-6-Pase) were localized by the cerium technique in guinea pig pinealocytes and compared with the corresponding lead technique. NDPase and TPPase were also compared at different pH values using the cerium technique. Vibratome sections of perfusion-fixed tissue were incubated with cerium chloride or lead nitrate. Substrates used were thiamine pyrophosphate (for TPPase), sodium inosine diphosphate (NDPase), and disodium glucose-6-phosphate (G-6-Pase). The 1-2 trans saccules of the Golgi apparatus showed TPPase and NDPase activity but none for G-6-Pase. The endoplasmic reticulum (ER) cisternae and perinuclear space had NDPase and G-6-Pase activity but not TPPase. The abluminal plasmalemma of endothelial cells and the plasmalemma of Schwann cells demonstrated TPPase and NDPase activity but the luminal plasmalemma of the endothelial cells and the plasmalemma of pinealocyte processes showed only NDPase activity. TPPase was active at all pH values tested, but NDPase was most active at pH values of 6.5 and 7.0. Lead phosphate precipitate was frequently seen in nuclei, perinuclear space, ER cisternae, and "synaptic" vesicles when lead was used as the capturing agent. These sites were usually not labeled when cerium was used.