How to use the optical fractionator: an example based on the estimation of neurons in the hippocampal CA1 and CA3 regions of tree shrews.

Until recently, exposure of the hippocampus to prolonged elevated glucocorticoid levels was thought to result in damage and loss of pyramidal neurons. Most of the earlier studies were based on measures of neuronal density and used assumptions-based counting methods. Using a stereological technique, the optical fractionator, which eliminates potential biases inherent in the assumption-based techniques, we were able to demonstrate that chronic psychosocial stress in tree shrews has no effect on neuronal number in the hippocampal CA1 and CA3 regions. The present report will focus on the practical aspects of the optical fractionator, by describing in detail how to estimate the total number of neurons in the hippocampal CA1 and CA3 regions of tree shrews. In this example the group sizes have been increased over those used in the earlier study. The present study supports our previous conclusion that stress does not affect the number of hippocampal neurons in the CA1 and CA3 areas as suggested by other authors. The results obtained with the optical fractionator can be used to estimate the precision of the data.

Calcium antagonists decrease capillary wall damage in aging hypertensive rat brain.

Chronic hypertension during aging is a serious threat to the cerebral vasculature. The larger brain arteries can react to hypertension with an abnormal wall thickening, a loss of elasticity and a narrowed lumen. However, little is known about the hypertension-induced alterations of cerebral capillaries. The present study describes ultrastructural alterations of the cerebrocortical capillary wall, such as thickening and collagen accumulation in the basement membrane of aging spontaneously hypertensive stroke-prone rats. The ratio of cortical capillaries with such vascular pathology occurred significantly more frequently in hypertensive animals. Nimodipine and nifedipine are potential drugs to decrease blood pressure in hypertension but their beneficial effects in experimental studies reach beyond the control of blood pressure. Nimodipine and nifedipine can alleviate ischemia-related symptoms and improve cognition. These drugs differ in that nifedipine, but not nimodipine reduces blood pressure at the here-used concentration while both drugs can penetrate the blood-brain barrier. Here we show that chronic treatment of aging hypertensive stroke-prone rats with nimodipine or nifedipine could preserve microvascular integrity in the cerebral cortex.

Capillary changes in hippocampal CA1 and CA3 areas of the aging rhesus monkey.

The rhesus monkey is considered a useful animal model for studying human aging, because non-human primates show many of the neurobiological alterations that have been reported in aging humans. Cognitive impairment that accompanies normal aging may, at least partially, originate from capillary changes in the hippocampus, known to be involved in learning and memory. Age-related effects on the cerebral capillaries in the non-human primate hippocampus have not yet been studied. Therefore, we investigated age-related microvascular changes in the hippocampus of the aged non-human primate. We examined by electron microscopy the microvascular ultrastructure in the CAI and CA3 areas of 14 male rhesus monkeys (Macaca mulatta), ranging from 1 to 31 years of age. The percentages of capillaries showing basement membrane thickening and deposits of collagen in the basement membrane were determined semiquantitatively in 4 young (1-6 years), 6 middle-aged (17-24 years), and 4 aged (29-31 years) monkeys. Aberrations in the basement membrane are few in young subjects (28 +/- 6% of capillaries), and occur with increasing frequency during the aging process in rhesus monkeys (aged animals: 71 +/- 5% of capillaries). This could be ascribed to an aging-associated increasing number of capillaries showing depositions of collagen fibrils, rather than local thickenings of the basement membrane. The observed changes in microvascular integrity are very similar to those seen in humans, supporting the view of rhesus monkeys as a model for human aging. The slow but steady progression of these changes could be detrimental for an efficient nutrient supply of the neuropil, and might therefore contribute to decreased cognitive functioning during normal aging.

Hyperglycemia-exaggerated ischemic brain damage following 30 min of middle cerebral artery occlusion is not due to capillary obstruction.

Transient focal ischemia of brief duration (15-30 min) gives rise to brain damage. In normoglycemic animals this damage usually consists of selective neuronal necrosis (SNN), and is largely confined to the lateral caudoputamen. In hyperglycemic subjects damage occurs more rapidly, involves also neocortical areas, and is often of the pan-necrotic type ('infarction'). Since experiments on forebrain ischemia of 30 min duration suggest that microcirculatory compromise develops during recirculation, we studied whether focal ischemia of the same duration, followed by reperfusion for 1, 2 or 4 h, leads to microcirculatory dysfunction. To test this possibility, we fixed the tissue by perfusion and counted the number of formed elements (leukocytes, macrophages and erythrocytes) in capillaries and postcapillary venules. Furthermore, capillary patency was evaluated following in vivo injection of Evan's blue. Histopathological examination of tissue fixed by perfusion after 1, 2 and 4 h of recirculation showed an increasing density of SNN in the caudoputamen of normoglycemic animals. Hyperglycemic, but not normoglycemic, animals showed pan-necrotic lesions ('infarction') after 4 h of recirculation. As a result, the total volume of tissue damage (SNN plus infarction) was larger in hyper- than in normoglycemic animals at 2 and 4 h of recirculation. In addition, hyperglycemic animals showed involvement of neocortex which increased with the time of reperfusion. In the ischemic hemisphere, between 5 and 10% of counted capillaries contained formed elements. However, since hyperglycemic animals contained an equal (or smaller) amount of cells the results did not suggest that capillary 'plugging' could explain the aggravated damage. Moreover, both normo- and hyperglycemic animals showed close to 100% capillary patency. The results thus fail to support the notion that the aggravation of focal ischemic damage by hyperglycemia is due to obstruction of microvessel by swelling or leukocyte adherence.