Critical questions for preclinical trials on safety and efficacy of vascular endothelial growth factor-based therapeutic angiogenesis for ischemic stroke.

Therapeutic angiogenesis is a novel treatment for ischemic stroke, and vascular endothelial growth factor (VEGF) is a key angiogenic and neuroprotective pharmacological candidate for therapy. However, the greatest challenge of preclinical studies is demonstrating that VEGF-based therapeutic angiogenesis is safe and effective for ischemic stroke patients. This review presents the following crucial questions which must first be answered by preclinical studies before VEGF-based therapeutic angiogenesis advances to human stroke trials, (1) Does angiogenesis induced by VEGF monotherapy promote neuroprotection or further damage the nervous tissue? (2) Does angiogenesis by VEGF in combination with other agents (combination therapy) promote greater neuroprotection than monotherapy, and without additional side effects? (3) Which exogenous VEGF isoform best promotes angiogenesis and neuroprotection, with least adverse effects on other organs? (4) Does angiogenesis induced by exogenous VEGF produce similar results in different animal models of ischemic stroke, including variations in age, gender and coexisting chronic diseases? (5) Can angiogenesis be induced by exogenous VEGF without clinically-significant alterations of systemic hemodynamics? (6) Are gene therapy and stem cells more beneficial than recombinant protein for VEGF-based therapeutic angiogenesis? (7) What are the best routes, timing and duration for administering VEGF, and how do these parameters influence inflammation? (8) Does exogenous VEGF exacerbate inflammation when traumatic or other injuries are present with ischemia? (9) Are VEGF doses not causing tissue alterations at the light microscopy level associated with clinically-significant ultrastructural damages of the neurovascular unit? Both published and unpublished preclinical data from the author's laboratory are presented.

Blood-brain barrier Ca2+-ATPase cytochemistry: incubation media and fixation methods for differentiating Ca2+-specific ATPase from ecto-ATPase.

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.

Use of image analysis for estimation of the numerical densities of neurons and synapses in cerebral cortex.

Relating to the protocol by Mikki et al. [Brain Res. Protocols 2 (1997) 9-16], the use of an image analysis system is recommended in place of micrographs and photoprints for the counting and measuring of neuronal nuclei.

Angiogenesis after stroke is correlated with increased numbers of macrophages: the clean-up hypothesis.

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.

A simple stereologic method for analysis of cerebral cortical microvessels using image analysis.

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 microm 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.

Calcium-dependent ATPase unlike ecto-ATPase is located primarily on the luminal surface of brain endothelial cells.

Numerous cytochemical studies have reported that calcium-activated adenosine triphosphatase (Ca2+-ATPase) is localized on the abluminal plasma membrane of mature brain endothelial cells. Since the effects of fixation and co-localization of ecto-ATPase have never been properly addressed, we investigated the influence of these parameters on Ca2+-ATPase localization in rat cerebral microvessel endothelium. Formaldehyde at 2% resulted in only abluminal staining while both luminal and abluminal surfaces were equally stained following 4% formaldehyde. Fixation with 2% formaldehyde plus 0.25% glutaraldehyde revealed more abluminal staining than luminal while 2% formaldehyde plus 0.5% glutaraldehyde produced vessels with staining similar to 4% and 2% formaldehyde plus 0.25% glutaraldehyde. The abluminal reaction appeared unaltered when ATP was replaced by GTP, CTP, UTP, ADP or when Ca2+ was replaced by Mg2+ or Mn2+ or p-chloromercuribenzoate included as inhibitor. But the luminal reaction was diminished. Contrary to previous reports, our results showed that Ca2+-specific ATPase is located more on the luminal surface while the abluminal reaction is primarily due to ecto-ATPase. The strong Ca2+-specific-ATPase luminal localization explains the stable Ca2+ gradient between blood and brain, and is not necessarily indicative of immature or pathological vessels as interpreted in the past.

Luminal localization of blood-brain barrier sodium, potassium adenosine triphosphatase is dependent on fixation.

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.

An in situ cytochemical evaluation of blood-brain barrier sodium, potassium-activated adenosine triphosphatase polarity.

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.