Abstract: Two mechanisms of brain cell death coexist, necrosis and apoptosis. We investigated the correlation between the apoptotic index and the expression of apoptosis modulators and stress response in an ultraviolet-induced cortical microinfarct. Adult rat neocortex was exposed to an ultraviolet beam and brains removed at different intervals after injury were paraffin-embedded and processed for TUNEL assay and immunohistochemistry against apoptotic modulators Bax and Bcl-2, and stress protein HSP70. During the 12-72h postirradiation period, apoptotic nuclei decreased from 11% to 4% in the infarcted area whereas only 1.2% of such nuclei was seen in the perilesional area. While Bcl-2 was always negative in the lesion focus, Bax was positive at all survival times, mainly in glial cells. HSP70 was expressed over a broad area of the ipsilateral hemisphere from 3h after brain injury, firstly in neurons and progressively in glial cells and finally in endothelium. At longer survival times, positive cells could be also seen in the contralateral hemisphere. Apoptosis seems to play only a quantitatively modest role in the progression of brain damage in penumbra areas despite the wide expression of pro-apoptotic factors. On the other hand HSP70 appears to be one of the main protective responses to injury stress.

Abstract: Objectives The aim was to investigate, qualitatively and quantitatively, the regional cortical effects of intracortical infusion and neutralization of VEGF on the light trauma induced by the implantation of an osmotic minipump during the first days of the critical visual period in rats, when peak levels of endogeneous VEGF secretion are reached. Methodology Recombinant human VEGF165 was administered intracortically (25 ng/ml) using intracerebral osmotic minipumps with a delivery rate of 1 µl/h which were placed in middle cortical layers of 18-day-post-natal Long Evans rats. To study the effects of endogenous VEGF inhibition, we administered anti-VEGF (25 µg/ml) in another group. Infusion of PBS at the same diffusion rate was used as a control. Minipumps were maintained for one week and afterwards brains were removed and samples were paraffin embedded or cryopreserved to obtain sequential slices. Various immunopathological analyses were performed: GFAP for the study of the astroglial response, Endothelial Barrier Antigen (EBA) for the BBB integrity and HSP70 and NeuN for the study of the neuroprotective response. Results Although VEGF-infused rats showed protein deposits in the neuropil around the cannula placement suggesting an increase in vascular permeability, immunopositivity for EBA remained constant indicating a natural, physiological way of BBB opening. The neutralization of VEGF seems to induce a lower degree of barrier opening with lower protein deposits in the neuropil and a reduced glial reaction, however it also shows a fall in the number of EBA-positive vessels. This could be interpreted as a breakdown of the structural substract of BBB, with more damaging consequences for the surrounding tissue. The apparently contradictory lower NeuN density found in the VEGF group could be due to the preservation of the damaged tissue surrounding the lesion, a sector which is lost in other experimental groups, and to the relatively high amounts of the other components of the neuropil, such as vessels, astrocytes or even proteins. Conclusion The increase of permeability induced by VEGF can be considered as a form of physiological BBB opening which acts as a protective factor against the necrosis of the tissue, a fact which is supported by the positivity of the EBA. The neutralization of VEGF has more deleterious consequences for the surrounding tissue. These effects are also present in the contralateral cortex, suggesting that the role of VEGF in the critical period goes beyond vascularization. Although the repair mechanisms are quite similar to those in adults, further studies should focus on the functional consequences of VEGF in the evolution of injuries in immature brains.

Abstract: VEGF is a major regulator of angiogenesis and vascular permeability in development and injury. The involvement of one of its receptors, Flk-1 in angiogenesis has been widely demonstrated, but few studies elucidate its role as a mediator of the BBB permeability and none displays its distribution following a cortical micronecrosis. A microvascular marker (LEA lectin), two BBB markers (EBA, GluT-1) and the VEGFR2 receptor were studied in adult rats after a minimal brain injury. Immunohistochemistry shows an increase of positive vessels, somata and processes around the micronecrosis from 6 to 72 hours after injury. Flk-1 was overexpressed mainly in endothelial cells, but also in astrocytes, neuronal somata and processes adjacent to the damage. This increase correlates to the lose of positivity for EBA. After injury, VEGFR-2 expression increases and its distribution corresponds to VEGF one. The whole system seems to play a role in the disruption of the BBB.

Abstract: The development of the cortical vascular tree depends on functional development. External inputs are an essential requirement in the modeling of the visual cortex, mainly during the critical period, when congruous blood supply is needed. The blood brain barrier (BBB) function regulates the passage of substances between the blood and the brain parenchyma, which is one of the main differential features of central nervous system (CNS) microvessels. The endothelial barrier antigen (EBA) has been reported as a specific marker for the BBB physiological function in rats. We studied the postnatal development of EBA expression in the visual cortex of rats reared under opposite paradigms of visual experience, e.g., standard laboratory conditions, dark rearing, and enriched environment at 14, 21, 28, 35, 42, 49, 56, and 63 days postnatal (dpn). Parallel sections were immunohistochemically processed for endothelial barrier antigen (EBA) and glucose transporter-1 (GluT-1). Total vasculature was quantified by Lycopersicon esculentum (LEA) lectin histochemistry. No differences in EBA expression were found between groups, although quantitative differences were recorded paralleling differences in vascular density. Paradoxically, there was no expression in certain cortical vessels which were GluT-1 immunopositive and positivity was consistent in non-barrier areas such as the pineal gland. These findings were completely independent of age or experimental conditions. Therefore, the role of the EBA antigen in the BBB remains unclear: it has been undeniably linked to vascular permeability, but its presence in non-barrier vessels suggests another vascular function. Although visual experience modifies vascular density in the visual cortex, it has not been shown to have an influence on the maturation of the BBB function.

Abstract: After birth, exposure to visual inputs modulates cortical development, inducing numerous changes of all components of the visual cortex. Most of the cortical changes thus induced occur during what is called the critical period. Astrocytes play an important role in the development, maintenance and plasticity of the cortex, as well as in the structure and function of the vascular network. Dark-reared Sprague-Dawley rats and age-matched controls sampled at 14, 21, 28, 35, 42, 49, 56 and 63 days postnatal (dpn) were studied in order to elucidate quantitative differences in the number of positive cells in the striate cortex. The astrocytic population was estimated by immunohistochemistry for S-100beta protein. The same quantification was also performed in a nonsensory area, the retrosplenial granular cortex. S-100beta positive cells had adult morphology in the visual cortex at 14 dpn and their numbers were not significantly different in light-exposed and nonexposed rats up to 35 dpn, and were even higher in dark-reared rats at 21 dpn. However, significant quantitative changes were recorded after the beginning of the critical period. The main finding of the present study was the significantly lower astroglial density estimated in the visual cortex of dark-reared rats over 35 dpn as well as the lack of difference at previous ages. Our results also showed that there were no differences when comparing the measurements from a nonsensory area between both groups. This led us to postulate that the astrocytic population in the visual cortex is downregulated by the lack of visual experience.

Abstract: OBJECTIVE: To report the current state of understanding of the basis of post natal development of microvascularization and the influence of the environment. DEVELOPMENT: Postnatal development of sensory systems occurs in two stages: one genetically predetermined and the other modulated by experience, which is especially important during a so called critical period. Increased demand during this period leads to adaptive changes, particularly vascular changes. The cerebral cortical vascularization is mainly composed of a dense capillary network which extends over the entire cortex and whose density corresponds to the metabolic demand. The development of cortical vascularization starts with perpendicular trunks which branch according to demand and form a cortical capillary network, mediated by factors such as endothelial vascular growth factor (VEGF). Many investigations into the effect of external experience have been done on the visual cortex, both for increase and deprivation. CONCLUSIONS: Post natal development with enriched visual environments induces increased vascular density. Absence of visual experience leads to a reduction in cortical activity which induces reduced neurone and vascular density, together with delay in maduration of the cortical angio architectural pattern. The relation between the neurone population and superficial vascular density is unchanged. These changes appear after the critical period of maximal synatic reorganization.

Abstract: Cerebral vascular density is correlated with metabolic demands, which increase in highly active brain areas. External inputs are an essential requirement in the modeling of the visual cortex. Experience-mediated development is very active during the first postnatal month, when congruous blood supply is needed. We studied the development of visual cortex vascularization in relation to experience, comparing rats raised in darkness with rats reared in normal conditions. Vascular density, vascular area and their ratio vs. neuronal density were calculated. Conventionally stained semi-thin sections were used to measure the vascular area by computer assisted morphometry. Animals from both groups were sampled at 14, 21, and 60 days postnatal (dpn). We found a significantly lower density of vessels and neurons as well as a smaller vascular area in dark-reared adult rats while no differences were founded at the other ages. Our results also show no differences between the ratio of vessels/neuron, and vascular area/neuron, between both groups. The absence of visual experience causes decrease of cortical activity which correlates with lower vessels density and vascular area, without their ratio/neuron being affected.

Abstract: Cerebral vascular density corresponds to metabolic demand, which increases in highly active areas. External inputs play an important role in the modeling and development of the visual cortex. Experience-mediated development is very active during the first postnatal month, when accurate simultaneous blood supply is needed to satisfy increased demand. We studied the development of visual cortex vascularization in relation to experience, comparing rats raised in darkness with rats raised in standard conditions. The parameters measured were cortical thickness, vascular density and number of perpendicular vessels, constituting the first stage of cortical vascular development. Vessels were stained using butyryl cholinesterase histochemistry, which labels some neurons and microvascularization (vessels from 5 to 50 microns). Animals from both groups were sampled at 0, 7, 14, 21 and 60 days postnatal. Vascularization of the brain starts with vertically oriented intracortical vascular trunks whose density decreases notably after birth in rats reared in standard laboratory conditions. The most striking finding of our work is the significantly lower decrease in the number of these vessels in dark-reared rats. Our results also show that cortex thickness and vessel density are significantly lower in dark-reared rats. These results suggest that the absence of visual stimuli retards the maturation of the visual cortex including its vascular bed.

Abstract: Autologous serum proteins have proved to be suitable tracer to evaluate vascular permeability. The dynamic behaviour of anti-HRP immunoglobulins was studied in ultraviolet (UV) irradiation induced brain edema. Cerebral cortex of 36 anaesthetized adult rats was irradiated following a 2 x 2 mm parietal craniotomy. Immunization was carried out by 3 subcutaneous injections of 10 mg HRP in 0.5 ml complete freund adjuvant (CFA), 6, 4 and 2 weeks before the injury. Control animals were immunized only with CFA; further control animals were operated and irradiated without any previous immunization. After survival times ranging from 30 min to 24 hours, postoperation animals were transcardially perfused with 4% fresh paraformaldehyde solution in phosphate buffered saline. After postfixation at 4 degrees C, 20 microns vibratome sections were prepared for incubation with a solution of 0.05% HRP, washed and developed by the DAB reaction. The reactions showed a remarkable exsudation and spreading of anti-HRP antibodies in the edematous brain. The antigen-antibody reaction was conspicuous in animals with shorter survival periods in the necrotic area and near the lesion (1-2 mm). After a longer survival time extravasation involved the whole hemisphere. In animals with the longest survival period labeled serum proteins were found even in the white matter of the hemisphere contralateral to the injury. Endogenous tracer of BBB function is useful to study the spreading of brain edema in a delayed time after the edematous lesion.

Abstract: The mechanism involved in the relative preservation of the subcortical U-fibers in the arcuate zone was studied in a post infarct edema after sagittal superior sinus occlusion. Superior sagittal sinus (SSS) of 36 mongrel cats were occluded by polymer injection. Immediately before the occlusion Evans-blue (EB) was administered intravenously. The cats were killed 1, 2, 3, 6, 12, 24, 72 and 120 hours after sinus occlusion. In 20 cats in which cortical veins were occluded, in addition to the SSS, EB was extravasated. In 9 of these cats, which had moderate edema, EB-staining was present only in the cortex. In 11 cats with severe edema, massive EB extravasation was observed also in the white matter. The U-fiber layer was free of EB, suggesting that the extension of edema was blocked by this zone. Our findings demonstrated that the U-fibers act not only as a resistance against extension of edema from white to gray matter, but also in a reverse direction. The characteristics of the spread of brain edema is not yet completely understood; both anatomical and biochemical peculiarities from its basis. Different morphological patterns in the astrocytic reaction as well as the U-fibers sector vascularization are important. To evaluate the role of each one of these factors in the preservation of subcortical U-fiber layer in brain edema further investigations should be done.