Ca2+-ATPase cytochemistry frequently uses the incubation medium of Ando et al. that was introduced in 1981. Some studies, however, have suggested that this medium localizes ecto-ATPase in addition to Ca2+-ATPase and that Ca2+-ATPase is sensitive to fixation. Strong activity of the enzyme on the luminal surface of the blood-brain barrier (BBB) also is considered indicative of immature or pathological microvessels. We address here five questions. 1) Is the incubation medium of Ando et al. specific for BBB Ca2+-ATPase or does it also localize ecto-ATPase? 2) How are the two enzymes distributed in the BBB? 3) How would data interpretation be prone to error if the cytochemical study does not use controls identifying ecto-ATPase? 4) Does the amount of reaction product of both enzymes vary significantly when the cortical tissue is exposed to different fixatives? 5) Does the presence of Ca2+-ATPase on the luminal membrane of the BBB necessarily indicate immature or abnormal brain endothelial cells? Adult male Sprague-Dawley rats were perfused with one of two different fixatives and vibratome slices of the brain cortex were incubated in the medium of Ando et al. The controls used were those demonstrating the ecto-ATPase and those that do not. The results indicate that the incubation medium is not specific for Ca2+-ATPase, because it also localizes the ecto-ATPase. Ca2+-ATPase appears to be localized primarily on the luminal surface of the BBB, while ecto-ATPase is localized on both the luminal and abluminal surfaces. The portion of the reaction product contributed by Ca2+-ATPase would not have been identified if the controls uniquely identifying the ecto-ATPase had not been used. The amount of reaction product formed by Ca2+-ATPase is strongly dependent on the type of fixative used. The strong localization of Ca2+-ATPase on the luminal surface of the BBB is not only normal, but also better accounts for the physiological homeostasis of Ca2+ across the blood-brain interface and should not be interpreted as indicative of immature or pathological microvessels.

Therapeutic angiogenesis by vascular endothelial growth factor (VEGF) is considered a promising treatment strategy for brain ischemic stroke. However, data in the literature demonstrating the benefit of therapeutic angiogenesis are contradictory. Thus, we explored the effects of non-angiogenic and angiogenic doses of VEGF165 on microanatomy of normal and ischemic brain cortices of adult Sprague Dawley rats. VEGF165 was continuously and slowly infused through the internal carotid artery for 7 days by an indwelling miniosmotic pump. Ischemia was induced by a 4-hour occlusion of the middle cerebral artery. In normal brains, the non-angiogenic doses of VEGF165 did not alter the cortical microanatomy in contrast to the angiogenic dose which was associated with neuronal pyknosis and damage of the neuropil. In ischemic brains, the non-angiogenic doses significantly promoted neuroprotection in contrast to the angiogenic dose which showed no therapeutic effect. These results suggest that VEGF-based therapeutic angiogenesis injures the nervous tissue, and neuroprotection of ischemic brains by VEGF monotherapy is confined to the non-angiogenic doses. The relationship between angiogenesis and neuroprotection by exogenous VEGF appears inversely related. We recommend angiogenesis by VEGF monotherapy for the treatment of ischemic stroke be viewed with caution, or avoided.

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.

Journal Article Nissl Staining of Cat Spinal Cord is Significantly Influenced by Concentrations of Glutaraldehyde in Fixative Get access J Chavez, J Chavez Neural Engineering Program, Huntington Medical Research Institutes, Pasadena CA 91105 Search for other works by this author on: Oxford Academic Google Scholar C Graham, C Graham Neural Engineering Program, Huntington Medical Research Institutes, Pasadena CA 91105 Search for other works by this author on: Oxford Academic Google Scholar AS Lossinsky, AS Lossinsky Neural Engineering Program, Huntington Medical Research Institutes, Pasadena CA 91105 Search for other works by this author on: Oxford Academic Google Scholar RL Schultz, RL Schultz Division of Human Anatomy, Loma Linda University, Loma Linda CA 92507 Search for other works by this author on: Oxford Academic Google Scholar PS Manoonkitiwongsa PS Manoonkitiwongsa Neural Engineering Program, Huntington Medical Research Institutes, Pasadena CA 91105 Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 9, Issue S02, 1 August 2003, Pages 1500–1501, https://doi.org/10.1017/S1431927603447508 Published: 18 July 2003

Brain cells manufacture and secrete angiogenic peptides after focal cerebral ischemia, but the purpose of this angiogenic response is unknown. Because the maximum possible regional cerebral blood flow is determined by the quantity of microvessels in each unit volume, it is possible that angiogenic peptides are secreted to generate new collateral channels; other possibilities include neuroprotection, recovery/regeneration, and removal of necrotic debris. If the brain attempts to create new collaterals, microvessel density should increase significantly after ischemia. Conversely, if angiogenic-signaling molecules serve some other purpose, microvessel densities may increase slightly or not at all. To clarify, the authors measured microvessel densities with quantitative morphometry. Left middle cerebral arteries of adult male Sprague–Dawley rats were occluded with intraluminal nylon suture for 4 hours followed by 7, 14, 19, or 30 days of reperfusion. Controls received no surgery or suture occlusion. Changes in microvessel density and macrophage numbers were measured by light microscopic morphometry using semiautomated stereologic methods. Microvessel density increased only in the ischemic margin adjacent to areas of pannecrosis and was always associated with increased numbers of macrophages. Ischemic brain areas without macrophages displayed no vascularity changes compared with normal animals. These data suggest that ischemia-induced microvessels are formed to facilitate macrophage infiltration and removal of necrotic brain.

P95 INTRODUCTION: Stimulation of angiogenesis in ischemic brains may be of major value in stroke therapy. Administration of vascular endothelial growth factor (VEGF) is considered particularly effective as an angiogenic factor for hypoxia-related injuries. We therefore quantitatively compared the effect of VEGF on microvessels of normal and ischemic brain cortices. Vascular parameters of microvessels measured included changes in numerical (N A ), volume (V v ), surface (S v ) and length (L v ) densities along with diameter. METHODS: VEGF 165 protein at 1μg/ml was infused into the common carotid artery of normal and ischemic adult male Sprague-Dawley rats with a miniosmotic pump for 7 days at 1 μl/hr (VEGF infusion therefore 1 ηg/hr). Animals (n = 3 or 4 per group) were sacrificed at 7 days post-surgery. Ischemia was induced by occlusion of the middle cerebral artery for 4 hours. Vascular parameters were quantitated by light microscopic morphometry. Normal and ischemic brains not infused by VEGF served as controls. RESULTS: Mean and S.D. of the vascular parameters (N A [# of vessels/mm 2 ]; V v [%]; S v [mm 2 /mm 3 ]; L v [mm/mm 3 ]; D [μm]) of the different groups are as follows: A) Normal brains (462 ± 63; 2.00 ± 0.2; 13.9 ± 1.7; 925 ± 127; 5.48 ± 0.5) B) Normal brains (480 ± 38; 2.25 ± 0.3; 14.9 ± 0.8; 961 ± 76; 4.98 ± 0.3) + VEGF C) Ischemic brains (471 ± 47; 2.06 ± 0.2; 14.3 ± 1.4; 942 ± 94; 5.11 ± 0.8) D) Ischemic brains (579 ± 102; 2.04 ± 0.3; 16.2 ± 2.7; 1159 ± 205; 4.64 ± 0.4) + VEGF There are no significant differences between the groups. CONCLUSIONS: Intra-arterial infusion of VEGF 165 protein for 7 days at 1 ηg/hr has no effect on the vascular parameters of normal or ischemic cortices. Infusion of VEGF alone may not be sufficient for inducing neovascularization in intact brain. Further studies specifically testing the appropriate concentration, duration, VEGF isoform(s), manner of administration (protein, plasmids or viral vectors), use of combinational angiogenic factors along with permeability or other side effects are critical to ascertain the angiogenic merits of VEGF for stroke therapy.

Previous methods for determining morphological features of vascular networks in cerebral cortex were subject to arbitrary variation and bias. Unbiased estimates of vessel number, volume, surface area and length can be obtained using stereology but these techniques tend to be tedious and time-consuming. Stereologic protocols generally require micrographs that have to be analyzed manually for intersections of vessels on grid points or lines. In this report, we provide a simpler and more precise method for measuring morphological features of cerebral cortical microvessels. Images of microvessels in 1 μm toluidine blue stained sections were captured using a popular image analysis software package. Luminal surfaces of endothelial cells were automatically traced using commonly available features; the two-dimensional data of vessels (diameter, area, perimeter and number of vessels) were automatically computed and transferred to a spreadsheet. Three-dimensional features were then determined using basic stereologic equations. The method eliminates the need for manual measurements and is particularly time- and cost-effective for quantitative studies where numerous images have to be evaluated.

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.

It is presently believed that sodium, potassium-activated adenosine triphosphatase (Na+, K+-ATPase) is localized on the abluminal plasma membrane of brain endothelial cells. But there have been contrary reports from some cytochemical studies. We examined the localization of the enzyme in rat cerebral microvessel endothelium using the in situ model originally employed to establish the abluminal polarity concept. Alterations in fixation and incubation media from the original reports were conducted to determine the effect on localization pattern. With the Ernst indirect incubation method as originally used, three types of localization patterns were obtained: abluminal only, luminal only, and on both surfaces of endothelial cells. With the direct incubation method of Mayahara, reaction product was seen on both surfaces. Reduction in fixation time followed by the use of the indirect incubation method resulted in a complete loss of the reaction product. The same reduction in fixation time followed by the use of the direct method did not alter the localization pattern of the enzyme. Our results demonstrated that Na+, K+-ATPase is localized on both surfaces of brain endothelial cells. The localization pattern of Na+, K+-ATPase is significantly dependent upon fixation and the incubation medium used in the in situ model. Data discrepancies for the enzyme as reported in the literature appear to be caused by differences in cytochemical protocols, rather than the biological reasons advocated by other investigators. We conclude that past cytochemical reports of blood-brain barrier (BBB) Na+, K+-ATPase abluminal localization were incomplete. The currently held abluminal polarity theory of the enzyme needs to be reexamined. Past basic and clinical cytochemical studies of BBB Na+, K+-ATPase should be viewed and interpreted 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.

Pineal "synaptic" ribbons are a heterogeneous population of organelles. "Synaptic" ribbons (SR) sensu stricto, "synaptic" spherules (SS), and intermediate forms (IMF) are present. Their function and origin are unknown, and a knowledge of their prenatal development is lacking. Thus the pineal glands of prenatal, neonatal, and adult guinea pigs were prepared for electron microscopy. "Synaptic" ribbons were studied morphologically and quantitatively. The three categories of "synaptic" ribbons reported in adult pineal glands were also present in prenatal pineal glands. Their structural features, distribution, grouping, and composition patterns are similar to those in adults. "Synaptic" ribbons were first detected in pinealocytes of the distal region of a 42-day postcoitus (PC) pineal gland and were comparable with those in adults. They increased in number with age and reached a peak at 63 days PC, followed by a steep decline at 66 and 67 days PC. By day 69 PC, the numbers increased again and showed a dramatic increase after birth. Several true ribbon synapses were seen at day 63 PC between pinealocyte cell processes or between pinealocyte cell process and pinealocyte cell body. Since true ribbon synapses have not been found in adult guinea pig pinealocytes, their synaptic nature could have been lost during development. No precursors for the "synaptic" ribbons were found. The endoplasmic reticulum cisternae may be the origin for the ribbon vesicles because of their close association with the "synaptic" ribbons.

The ultrastructural localization of acetylcholinesterase (AChE) activity in guinea pig pineal gland was studied using the copper-glycine procedure. A small number of pinealocytes and bundles of unmyelinated nerve fibers were labeled by the AChE reaction. The AChE-positive pinealocytes were located near blood vessels and distributed in small groups. The AChE reaction product was localized in the perinuclear cistern, in the cisternae of the endoplasmic reticulum (ER), and in the saccules of the Golgi apparatus. These findings suggest that the AChE-positive pinealocytes synthesize AChE. The AChE reaction product was also seen in the intercellular space between pinealocyte processes. Besides pinealocytes, AChE activity was localized on the axolemma of myelinated and unmyelinated nerve fibers and in the basement membrane surrounding unmyelinated nerve fibers. Pseudocholinesterase activity was confined to Schwann cells, which showed the reaction product in their perinuclear cistern, in the cisternae of the ER, and on the plasmalemma.