STUDIES OF MICROGLIAL CELLS IN PRE-AND POSTNATAL RAT BRAIN

Abstract

Microglial cells are one of the three major types of neuroglial cells in the central nervous system (CNS). It is widely accepted that the microglia in the adult brain are derived from transformation of amoeboid microglial cells, which appear transiently in the developing CNS. These cells play important roles in normal CNS development and in many neuropathological conditions, e.g. brain damage which results from hypoxia in the fetus and newborn infant. Microglial cells can be identified by various staining methods. In the present study, amoeboid microglial cells in fetal brains were identified by haematoxylin and eosin (H & E) staining, immunohistochemistry and lectin histochemistry. These cells were labelled by rhodamine B isothiocyanate (Rhlc) when injected intravenously or intraperitoneally into mother rats at late stage of pregnancy. Rhlc-labelled amoeboid microglia were first observed in the cavum septum pellucidum and subependymal cysts associated with the cerebral aqueduct as well as the fourth ventricle, and subsequently at other sites including the corpus callosum and other subcortical white matter. The intensity of fluorescence increased with time after Rhlc administration, so that 1 day after injection the cells were brightly labelled. The majority of the labelled cells were round and amoeboidic, with some elongated ones bearing a few processes in the fetal brain. All fluorescent cells were double labelled with OX-42 and OX-18 antibodies that recognize complement type 3 (CR3) receptors and major histocompatibility complex (MHC) class I surface antigen, respectively. Amoeboid microglia appeared to display a more intense fluorescence than the ramified cells; their reactivity with OX-42, OX- 18 and the isolectin Griffonia simplicifolia (GSA I-B4) was also stronger, In the present study, neuropathological changes were also examined in the brain of fetal and postnatal rats after transient exposure of mother rats in advanced pregnancy to hypoxia. Light and electron microscopic studies were carried out to study the cellular changes in the regions of cerebral cortex (sensorimotor and cingulate cortex), hippocampus, corpus callosum and brainstem at different time intervals following maternal hypoxia. Furthermore, cell count was carried out to evaluate the alteration of neuron density after hypoxia. Microglia responded vigorously to the hypoxic stress, as determined by the monoclonal antibody OX-42 and the isolectin GSA I-B4. Hypertrophied microglial cells which exhibited intense immunoreaction with OX-42 were particularly evident in the cingulate cortex and the corpus callosum between 3 hours and 14 days after exposure to hypoxia; thereafter, the rnicroglial immunoreactivity was comparable to that of the control rats. The immunoreactivity of microtubule associated protein II (MAP2) was transiently reduced between 3 hours and 3 days after the hypoxic insult, but was comparable to that of the controls thereafter. Results of cell count and electron microscopy showed massive cellular injury, mainly necrosis, and significant neuronal loss in the cingulate cortex but not in the hippocampus at 1 and 3 days after hypoxic insults. Only occasional apoptotic cells were identified by tenninal deoxynucleotidyl transferase mediated dUTP- fluorescein nick end labelling (TUNEL) and electron microscopy. In rats killed at 7 and 14 days after hypoxia, the neuronal density in all regions examined was comparable to that of the controls. The data derived from the present study indicated that when introduced into the maternal circulation, Rhlc could readily gain access into the fetal brain through the inefficient placental, blood-brain and blood-cerebrospinal-fluid barriers. Other than H & E staining, immunohistochemistry and lectin histochemistry, the labelling of cells by Rhlc through maternal route offers a rapid method for study of microglial cells in fetal brain. The avid uptake of Rhlc in circulation by amoeboid microglial cells indicates that they are active phagocytes which may serve as an effective and functional barrier to remove any foreign materials that may have entered the fetal brain tissue from the maternal blood circulation. Microglia can respond swiftly to imminent or ongoing hypoxic brain injury as manifested by their morphologic and immunophenotypic changes. Besides being involved in the clearance of cellular debris by active phagocytosis, microglial cells may have neurotrophic functions by producing some cytokines. Present results suggest that the cingulate cortex is most vulnerable to the hypoxic injury, and this may be attributable to the redistribution of the cerebral blood flow in favour of posterior regions by an autoregulation mechanism.