Kainic acid and 3-Nitropropionic acid induced expression of laminin in vascular elements of the rat brain

Laminin is a glycoprotein component of the basement membrane and has been reported to be found in different areas of the nervous system including brain endothelial cells, Schwann cells and peripheral nerves. Although the in-vitro studies suggest that laminin plays an important role in growth and neurite extension of cultured neurons, localization of laminin in the brain has been controversial and inconsistent results have been reported. Recently, laminin immunoreactivity has been used as a marker for vascular elements in the brain. In this study, we have investigated the effect of two mechanistically different neurotoxins, kainic acid (KA), an NMDA agonist and 3-Nitropropionic acid (3-NPA), an inhibitor of mitochondrial respiration, on brain vascular elements revealed by laminin immunolabeling. We also explored whether administration of these two neurotoxic drugs correlate with the neuronal degeneration observed after neurotoxic insult by staining with Fluoro-Jade C dye. We have employed single immunolabeling to localize laminin in the brains. In KA treated rats, most of the laminin immunoreactivity is present in the piriform cortex, corpus callosum (myelinated tracts) amygdala, hippocampus, ventral thalamus and tenia tacta. In 3-NPA treated animals, laminin immunoreactivity was confined mostly to the striatum. In contrast, saline treated rats showed very little laminin immunolabeling around capillaries, arteries and in the meningeal membranes. To determine the effects of these neurotoxins on the integrity of the blood brain barrier (BBB), endothelial brain barrier antigen (EBA) immunolabeling was also performed. In addition, we performed CD11b immunolabeling to evaluate the effect of 3-NPA and KA on the activation of microglia in the brain. CD11b was dramatically increased in KA and 3-NPA treated animals. We have also combined laminin immunolabeling with Fluoro-Jade C labeling to evaluate the spatio-temporal association of degenerating neurons and the expression of laminin containing microvessels. Areas which showed intense laminin immunolabeling following KA or 3-NPA exposure correlated with those exhibiting the greatest number of degenerating neurons observed after Fluoro-Jade C staining. EBA-laminin double immunolabeling demonstrated that the expressions of laminin were predominantly localized in the areas (cortex, thalamus and hippocampus) where EBA has been either reduced or is absent. Our results from these experiments demonstrate that vascular laminin expression increases after treatment with KA or 3-NPA, suggesting the occurrence of neovascularization. Microglia may also contribute to the neurotoxic induced neovascularization and neurodegeneration.

Research highlightsThe present study reveals:
► Minimal laminin immunoreactivity was detected in the largest blood vessels in the brain of control animals.
► Conspicuous laminin expression was seen in the blood vessels especially in thalamus, cortex and hippocampus following kainic acid exposure.
► Conspicuous laminin expression was seen in the blood vessels especially in stratum and occasionally in the hippocampus following 3-NPA exposure.
► Regions expressing laminin immunoreactivity correlated with the areas expressing conspicuous Fluoro-Jade C labeled degenerating neurons.