José Vicente Lafuente

Effects of dark-rearing on the vascularization of the developmental rat visual cortex

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.

Effects of Visual Experience on Vascular Endothelial Growth Factor Expression during the Postnatal Development of the Rat Visual Cortex

The development of the cortical vascular network depends on functional maturation. External inputs are an essential requirement in the modeling of the visual cortex, mainly during the critical period, when the functional and structural properties of visual cortical neurons are particularly susceptible to alterations. Vascular endothelial growth factor (VEGF) is the major angiogenic factor, a key signal in the induction of vessel growth. Our study focused on the role of visual stimuli on the development of the vascular pattern correlated with VEGF levels. Vascular density and the expression of VEGF were examined in the primary visual cortex of rats reared under different visual environments (dark rearing, dark-rearing in conditions of enriched environment, enriched environment, and laboratory standard conditions) during postnatal development (before, during, and after the critical period). Our results show a restricted VEGF cellular expression to astroglial cells. Quantitative differences appeared during the critical period: higher vascular density and VEGF protein levels were found in the enriched environment group; both dark-reared groups showed lower vascular density and VEGF levels, which means that enriched environment without the physical exercise component does not exert effects in dark-reared rats.

VEGFR-2 expression in brain injury : its distribution related to brain-blood barrier markers

Summary.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.

Increased antiparkinson efficacy of the combined administration of VegF- and gDNF-loaded nanospheres in a partial lesion model of Parkinson's disease

Current research efforts are focused on the application of growth factors, such as glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), as neuroregenerative approaches that will prevent the neurodegenerative process in Parkinson’s disease. Continuing a previous work published by our research group, and with the aim to overcome different limitations related to growth factor administration, VEGF and GDNF were encapsulated in poly(lactic-co-glycolic acid) nanospheres (NS). This strategy facilitates the combined administration of the VEGF and GDNF into the brain of 6-hydroxydopamine (6-OHDA) partially lesioned rats, resulting in a continuous and simultaneous drug release. The NS particle size was about 200 nm and the simultaneous addition of VEGF NS and GDNF NS resulted in significant protection of the PC-12 cell line against 6-OHDA in vitro. Once the poly(lactic-co-glycolic acid) NS were implanted into the striatum of 6-OHDA partially lesioned rats, the amphetamine rotation behavior test was carried out over 10 weeks, in order to check for in vivo efficacy. The results showed that VEGF NS and GDNF NS significantly decreased the number of amphetamine-induced rotations at the end of the study. In addition, tyrosine hydroxylase immunohistochemical analysis in the striatum and the external substantia nigra confirmed a significant enhancement of neurons in the VEGF NS and GDNF NS treatment group. The synergistic effect of VEGF NS and GDNF NS allows for a reduction of the dose by half, and may be a valuable neurogenerative/neuroreparative approach for treating Parkinson’s disease.

Influence of visual experience deprivation on the postnatal development of the microvascular bed in layer IV of the rat visual cortex.

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.

Enriched and Deprived Sensory Experience Induces Structural Changes and Rewires Connectivity during the Postnatal Development of the Brain

During postnatal development, sensory experience modulates cortical development, inducing numerous changes in all of the components of the cortex. Most of the cortical changes thus induced occur during the critical period, when the functional and structural properties of cortical neurons are particularly susceptible to alterations. Although the time course for experience-mediated sensory development is specific for each system, postnatal development acts as a whole, and if one cortical area is deprived of its normal sensory inputs during early stages, it will be reorganized by the nondeprived senses in a process of cross-modal plasticity that not only increases performance in the remaining senses when one is deprived, but also rewires the brain allowing the deprived cortex to process inputs from other senses and cortices, maintaining the modular configuration. This paper summarizes our current understanding of sensory systems development, focused specially in the visual system. It delineates sensory enhancement and sensory deprivation effects at both physiological and anatomical levels and describes the use of enriched environment as a tool to rewire loss of brain areas to enhance other active senses. Finally, strategies to apply restorative features in human-deprived senses are studied, discussing the beneficial and detrimental effects of cross-modal plasticity in prostheses and sensory substitution devices implantation.

In vivo administration of VEGF- and GDNF-releasing biodegradable polymeric microspheres in a severe lesion model of Parkinson’s disease

In this work, the neuroregenerative potentials of microencapsulated VEGF, GDNF and their combination on a severely lesioned rat model were compared with the aim of developing a new strategy to treat advanced stages of Parkinsons disease. Both neurotrophic factors were separately encapsulated into polymeric microspheres (MSs) to obtain a continuous drug release over time. The regenerative effects of these growth factors were evaluated using a rotation behaviour test and quantified by the number of surviving TH+cells. The biological activities of encapsulated vascular endothelial growth factor (VEGF) and glial cell line-derived neurotrophic factor (GDNF) were investigated in HUVEC and PC12 cells, respectively. The treatment of 6-OHDA-lesioned rats with GDNF microspheres and with both VEGF and GDNF microspheres resulted in improved results in the rotation behaviour test. Both groups also showed higher levels of neuroregeneration/neuroreparation in the substantia nigra than the control group did. These results were confirmed by the pronounced TH+neuron recovery in the group receiving VEGF+GDNF-MS, demonstrating regenerative effects.

Vascular Endothelial Growth Factor and Other Angioglioneurins: Key Molecules in Brain Development and Restoration

Angioneurines are a family of molecules that include vascular growth factors such as VEGF, neurotrophins such as BDNF, IGF-I, and Erythropoietin, among others. They affect both neural and vascular processes. Due to the fact that all of them act over glia, we propose the term angioglioneurins to name them. They play a key role in the neurogliovascular unit that represents the functional core maintaining BBB. Although delivery to CNS is still an unsolved problem nowadays, exogenous angioglioneurin administration represents a promising therapeutic strategy for many neurological pathologies due to their neurotrophic and neurogenic role. In brains, VEGF is produced by neurons and astrocytes in different stages and situation, binding to tyrosine kinase receptors and also to neuropilin family. This fact reinforces its key role in the cross talk between neural and vascular development and activity. Angioglioneurins described in this report might become an important therapeutic resource in CNS restoration, especially in pathologies as stroke or traumatic brain injury.

Combination of intracortically administered VEGF and environmental enrichment enhances brain protection in developing rats.

Postnatal development of the visual cortex is modulated by experience, especially during the critical period. In rats, a stable neuronal population is only acquired after this relatively prolonged period. Vascular endothelial growth factor (VEGF) is the most important angiogenic factor and also has strong neuroprotective, neurotrophic and neurogenic properties. Similar effects have been described for rearing in enriched environments. Our aim is to investigate the vascular and neuronal effects of combining VEGF infusion and environmental enrichment on the visual cortex during the initial days of the critical period. Results showed that a small percentage of Cleaved Caspase-3 positive cells colocalized with neuronal markers. The lesion produced by the cannula implantation resulted in decreased vascular, neuronal and Caspase-3 positive cell densities. Rearing under enriched environment was unable to reverse these effects in any group, whereas VEGF infusion alone partially corrected those effects. A higher effectiveness was reached by combining both the procedures, the most effective combination being when enriched-environment rearing was introduced only after minipump implantation. In addition to the angiogenic effect of VEGF, applied strategies also had synergic neuroprotective effects, and the combination of the two strategies had more remarkable effects than those achieved by each strategy applied individually.

Role of VEGF in an experimental model of cortical micronecrosis.

Summary. Vascular endothelial growth factor (VEGF) is a major mediator in angiogenesis and vascular permeability. In central nervous system (CNS) it plays a pivotal role as: 1. inductor of endothelial cell proliferation, migration and inhibition of apoptosis, and 2. mediator of vascular permeability and subsequently of brain edema. This ubiquitous epiphenomenon is a major complication in several CNS pathologies, including head trauma and stroke.After brain injury the expression of VEGF is increased contributing to disruption of the blood brain barrier (BBB). VEGF increase the permeability of BBB via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. The immunohistochemistry shows an increase of stained astrocytes and endothelial cells around cortical micronecrosis. VEGF immunopositivity distribution shows some correspondence with the blood brain barrier breakdown following a cortical micronecrosis.