Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain.

Programmed cell death (apoptosis) occurs during normal development of the central nervous system. However, the mechanisms that determine which neurons will succumb to apoptosis are poorly understood. Blockade of N-methyl-D-aspartate (NMDA) glutamate receptors for only a few hours during late fetal or early neonatal life triggered widespread apoptotic neurodegeneration in the developing rat brain, suggesting that the excitatory neurotransmitter glutamate, acting at NMDA receptors, controls neuronal survival. These findings may have relevance to human neurodevelopmental disorders involving prenatal (drug-abusing mothers) or postnatal (pediatric anesthesia) exposure to drugs that block NMDA receptors.

Distinguishing excitotoxic from apoptotic neurodegeneration in the developing rat brain.

Much confusion has arisen recently over the question of whether excitotoxic neuronal degeneration can be considered an apoptotic phenomenon. Here, we addressed this question by using ultrastructural methods and DNA fragmentation analysis to compare a prototypic apoptotic in vivo central nervous system cell death process (physiologic cell death in the developing rat brain) with several central nervous system cell death processes in the in vivo infant rat brain that are generally considered excitotoxic (degeneration of hypothalamic neurons after subcutaneous administration of glutamate and acute neurodegeneration induced by hypoxia/ischemia or by concussive head trauma). We found by ultrastructural analysis that glutamate induces neurodegenerative changes in the hypothalamus that are identical to acute changes induced in the infant rat brain by either hypoxia/ischemia or head trauma, and that these changes are fundamentally different both in type and sequence from those associated with physiologic cell death (apoptosis). In addition, we show by ultrastructural analysis that concussive head trauma induces both excitotoxic and apoptotic neurodegeneration, the excitotoxic degeneration being very acute and localized to the impact site, and the apoptotic degeneration being delayed and occurring in regions distant from the impact site. Thus, in the head trauma model, excitotoxic and apoptotic degeneration can be distinguished not only by ultrastructural criteria but by their temporal and spatial patterns of expression. Whereas ultrastructural analysis provided an unambiguous means of distinguishing between excitotoxic and apoptotic neurodegeneration in each example analysed in this study, DNA fragmentation analysis (TUNEL staining or gel electrophoresis) was of no value because these tests were positive for both processes.

Multifocal brain damage induced by phencyclidine is augmented by pilocarpine.

Phencyclidine and other antagonists of the N-methyl-D-aspartate subtype of glutamate receptor cause psychosis in humans. In low doses these agents induce a reversible neurotoxic reaction in the rat brain that is limited to the retrosplenial granular cortex. Some investigators have reported that phencyclidine at higher doses or by more prolonged treatment causes a more disseminated pattern of damage. However, it has not been clearly demonstrated whether the disseminated damage is reversible or irreversible and whether it is consistently reproducible, nor is it known how many and which neurons are at risk. In the present study we addressed these questions using several histological approaches (plastic-embedded thin sections for light microscopy and ultrathin plastic sections for electron microscopy, paraffin-embedded haematoxylin and eosin sections, 72 kDa heat shock protein immunocytochemistry and de Olmos silver impregnation) to study the lesions induced in rat brain by phencyclidine (alone or when augmented with pilocarpine). We found that phencyclidine can kill a relatively large number of neurons distributed over many cerebrocortical and limbic brain regions, but the multifocal pattern of damage occurred in only a small percentage of treated rats. The addition of a low dose of pilocarpine to phencyclidine caused the widespread pattern of damage to manifest on a much more consistent basis. Available evidence suggests that disinhibition of multiple converging excitatory pathways is the mechanism by which phencyclidine triggers widespread neuronal degeneration; however, the specific combination of excitatory inputs that contributes to the pathological process may differ from region to region.

The shape and distribution of lysosomes and endocytosis in the ciliary epithelial cells of rats.

The shape and distribution of lysosomes in the ciliary epithelium of rat eyes were examined by electron microscopy combined with acid phosphatase (ACPase) cytochemistry and three-dimensional observation of 2 microns-thick sections. ACPase activity was cytochemically localized in lysosomes and trans Golgi cisternae in the non-pigmented epithelial (NPE) and pigmented epithelial (PE) cells. In NPE cells, it was shown three-dimensionally, that most lysosomes had an elongate form, up to 5 microns in length, and a diameter of 70-100 nm. These elongate lysosomes (nematolysosomes) were predominantly located in the basal region of the cells. In contrast, PE cells had spherical lysosomes distributed at random throughout the cytoplasm. However, no nematolysosomes were seen in the PE cells. When the isolated ciliary processes were incubated in a medium containing horseradish peroxidase (HRP), HRP was incorporated into the nematolysosome-like structures by pinocytosis from the basal surface of the NPE cells. These findings suggest that nematolysosomes are associated with the pinocytotic activity of NPE cells. The pinocytosis-nematolysosomal route may be involved in the uptake and degradation of macromolecules from the aqueous humor in the posterior chamber.

Wall cytoarchitecture of the rat ciliary process microvasculature revealed with scanning electron microscopy.

Ciliary process vasculature has an important role in aqueous humor production. There have been, however, few reports describing the overall cytoarchitecture of ciliary process vasculature. The wall cytoarchitecture of microvessels in the rat ciliary process was elucidated by scanning electron microscopy after removal of ciliary epithelia and connective tissue components with HCl hydrolysis. Utilizing characteristics of cellular morphology and vessel diameters, several vascular components were identified along the vascular tree: 1) arterial iridociliary circles (30-60 microm in outer diameter), containing a compact layer of circularly oriented spindle-shaped smooth muscle cells; 2) the proximal part of the radial ciliary arteriole (10-25 microm), containing a less compact layer of circularly oriented branched-smooth muscle cells and spindle-shaped smooth muscle cells; 3) a middle part of the radial ciliary arteriole (20-35 pm), with circularly oriented branched-smooth muscle cells and irregularly oriented stellate cells with ramifying projections; 4) a distal part of the radial ciliary arteriole (10-20 microm), possessing irregularly oriented stellate cells with ramifying projections; 5) marginal venules (15-20 microm), with spidery pericytes possessing highly ramifying and overlapped projections; 6) capillaries in the ciliary process (4-7 microm), with widely scattered pericytes having longitudinal and several circular projections; 7) venules in the posterior basal region of the ciliary process (greater than 5 microm), with widely scattered pericytes having a few thin projections. From arterial iridociliary circles to venules in the basal region of ciliary process, seven parts could be recognized by wall cytoarchitecture, which was discussed in relation with the function.