Differential hippocampal neuron density between inbred Roman high- (low anxious) and low-avoidance (high anxious) rats.

The inbred Roman low- (RLA-I) and high-avoidance (RHA-I) rats used in this study were initially selected and bred for extremely poor vs. rapid acquisition of active two-way avoidance behavior in the shuttle box. As a result of the selection for divergent avoidance acquisition, clear behavioral differences have been found between RHA and RLA rats in a variety of tasks related to anxiety and conflict. In rats of these two strains/lines previous brain studies have been performed, specifically in the striatum, the mesencephalic dopaminergic areas and the prefrontal cortex, as these brain areas are the classical ones for their critical role in sensitization and may play a role in the well-characterized anxiety response. In this study we analyzed, in RHA and RLA groups (N=5 each), the density of NeuN neurons counterstained with toluidine blue in the cingulate cortex (subdivision 1) and the hippocampus (CA1, CA2 and CA3). A statistical difference was found in the density of neurons of CA1 and CA2 (p=0.047 in both) and in the total density of the hippocampus (p=0.009). Contrary to our expectations, significant strain differences for the density of neurons in the cingulate cortex were not found. The relationship between those differences in the hippocampus and the between-strain differences in anxiety and in learning processes depending on anxiety are discussed.

Increased levels of proinflammatory cytokines in the aged rat brain attenuate injury-induced cytokine response after excitotoxic damage.

In order to evaluate proinflammatory cytokine levels and their producing cell types in the control aged rat brain and after acute excitotoxic damage, both adult and aged male Wistar rats were injected with N-methyl-D-aspartate in the striatum. At different survival times between 6 hr and 7 days after lesioning, interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) were analyzed by enzyme-linked immunosorbent assay and by double immunofluorescence of cryostat sections by using cell-specific markers. Basal cytokine expression was attributed to astrocytes and was increased in the normal aged brain showing region specificity: TNF-alpha and IL-6 displayed age-dependent higher levels in the aged cortex, and IL-1beta and IL-6 in the aged striatum. After excitotoxic striatal damage, notable age-dependent differences in cytokine induction in the aged vs. the adult were seen. The adult injured striatum exhibited a rapid induction of all cytokines analyzed, but the aged injured striatum showed a weak induction of cytokine expression: IL-1beta showed no injury-induced changes at any time, TNF-alpha presented a late induction at 5 days after lesioning, and IL-6 was only induced at 6 hr after lesioning. At both ages, in the lesion core, all cytokines were early expressed by neurons and astrocytes, and by microglia/macrophages later on. However, in the adjacent lesion border, cytokines were found in reactive astrocytes. This study highlights the particular inflammatory response of the aged brain and suggests an important role of increased basal levels of proinflammatory cytokines in the reduced ability to induce their expression after damage.

Distinct pattern of microglial response, cyclooxygenase-2, and inducible nitric oxide synthase expression in the aged rat brain after excitotoxic damage.

Microglial and inflammatory responses to acute damage in aging are still poorly understood, although the aged brain responds differently to injury, showing poor lesion outcome. In this study, excitotoxicity was induced by intrastriatal injection of N-methyl-D-aspartate in adult (3-4 months) and aged (22-24 months) rats. Cryostat brain sections were processed for the analysis of microglial response by lectin histochemistry and cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS) expression by immunohistochemistry and confocal analysis. Aged injured animals showed more widespread area of microglial response at 12 hr postlesion (hpl) and greater microglia/macrophage density at 3 days postlesion (dpl). However, aged reactive microglia showed prevalence of ramified morphologies and fewer amoeboid/round forms. Aged injured animals presented a diminished area of COX2 expression, but a significantly larger density of COX2(+) cells, with higher numbers of COX2(+) neurons during the first 24 hpl and COX2(+) microglia/macrophages later. In contrast, the amount of COX2(+) neutrophils was diminished in the aged. iNOS was more rapidly induced in the aged injured striatum, with higher cell density at 12 hpl, when expression was mainly neuronal. From 1 dpl, both the iNOS(+) area and the density of iNOS(+) cells were reduced in the aged, with lower numbers of iNOS(+) neurons, microglia/macrophages, neutrophils, and astrocytes. In conclusion, excitotoxic damage in aging induces a distinct pattern of microglia/macrophage response and expression of inflammatory enzymes, which may account for the changes in lesion outcome in the aged, and highlight the importance of using aged animals for the study of acute age-related insults.

Delayed neurodegeneration and early astrogliosis after excitotoxicity to the aged brain.

Excitotoxicity is well recognised as a mechanism underlying neuronal cell death in several brain injuries. To investigate age-dependent differences in neurodegeneration, edema formation and astrogliosis, intrastriatal N-methyl-d-aspartate injections were performed in young (3 months) and aged (22-24 months) male Wistar rats. Animals were sacrificed at different times between 12h and 14 days post-lesion (DPL) and cryostat sections were processed for Toluidine blue, Fluoro-Jade B staining, NeuN and GFAP immunohistochemistry. Our results show that both size of tissue injury and edema were reduced in the old subjects only up to 1DPL, correlating with a slower progression of neurodegeneration with peak numbers of degenerating neurons at 3DPL in the aged, contrasting with maximum neurodegeneration at 1DPL in the young. However, old animals showed an earlier onset of astroglial response, seen at 1DPL, and a larger area of astrogliosis at all time-points studied, including a greater glial scar. In conclusion, after excitotoxic striatal damage, progression of neurodegeneration is delayed in the aged but the astroglial response is earlier and exacerbated. Our results emphasize the importance of using aged animals and several survival times for the study of acute age-related brain insults.