Immunohistochemical evaluation of calbindin expression in mouse brain at different times after rabies virus inoculation.

Abstract

Introduction: The calcium-binding protein calbindin (CB) plays a critical role in intracellular calcium metabolism. In a previous study we demonstrated that the infection with fixed rabies virus caused loss of CB expression in mouse brain during terminal state of the disease, 7-8 days after intramuscular inoculation. Objectives: To determine loss of CB expression also appears in early stages of the infection and if it is a consequence immediate to the appearance of viral antigens in the brain. Materials and methods: Mice were inoculated with fixed rabies virus, by intramuscular route, in the left hind limbs. Daily, to sixth day, 4-6 animals and their respective controls were killed and fixed by intracardiac perfusion with paraformaldehyde and their brains were extracted to obtain coronal sections using a vibratome. Free-floating sections were treated by immunohistochemical procedures to evaluate the presence of rabies viral antigens and the expression of CB. The last one was quantified by optical densitometry in a microscope with a system of image analysis. Results: Rabies immunoreactivity was observed in the brain three days after virus inoculation, beginning in pyramidal neurons of the frontal cortex whereas the immunostaining for CB did not undergo any qualitative nor quantitative changes neither in the cerebral cortex or striatum during the six days post-inoculation. Conclusions: These results agree with other studies in which it has been settled down that the rabies molecular or metabolic effects on the nervous system are well-known only in the final stage of the disease.