Alterations of Calbindin-D_28k Localization in Cerebellar Purkinje Cells in the Hypoxic Guinea Pig Fetus at Term

Abstract 2049 Neurology Platform, Sunday, 5/2

Excessive activation of the N-methyl-D-aspartate (NMDA)receptor leading to increase in intracellular Ca²⁺ and generation of free radicals is a proposed mechanism of hypoxia-induced neuronal damage. Calbindin-D_28k, an intracellular Ca²⁺-binding protein, is a Ca²⁺ buffer in cerebellar Purkinje cells. We hypothesize that hypoxia reduces the calbindin-D_28k concentration in Purkinje cells, thereby reducing their buffering capacity and resulting in increased intracellular Ca²⁺. Studies were performed on fetuses obtained from 4 normoxic (Nx) and 10 hypoxic (Hx) pregnant guinea pigs at term. Hypoxia was induced by decreasing the inspired O₂ to 7% for 1 hour. The pregnant guinea pigs were allowed to recover for 0 hr (Hx0, n=2), 24 hr (Hx24h, n=3) or 72 hr (Hx72h, n=5) after hypoxia. Fetal brain tissue was obtained and the cerebella were fixed in 10% phosphate-buffered formalin and embedded in paraffin. Immunohistochemistry was performed according to the avidin-biotin complex, method, using mAb CL-300 against calbindin-D_28k. Mid-sagittal sections were analyzed and 276 to 881 (median: 576) Purkinje cells were counted per fetal brain. Purkinje cells from normoxic animals exhibited diffuse, panneuronal somatodendritic and axonal localization of calbindin-D_28k (total positive Purkinje cells: 88% ± 8%).Compared to Nx fetuses, a significant somatodendritic reduction of calbindin-D_28k staining in Purkinje cells was detected in Hx0hr (total positive Purkinje cells 65% ± 5%; p=0.02 vs Nx), Hx24hr (total positive Purkinje cells: 66% ± 9%; p=0.02 vs Nx) and Hx72hr (total positive Purkinje cells: 59% ± 9%; p=0.002 vs Nx) fetuses respectively. Cellular alterations of calbindin-D_28k in Purkinje cells in all three post-hypoxic groups included lack of staining in the cell bodies (somata), weak or punctuate somatic localization and paucity of dendritic staining. Certain Purkinje cells exhibited solely a nuclear compartmentalization of calbindin-D_28k at all time points after hypoxia, but was significant only in Hx0hr fetuses (p=0.02 vs Nx, p=0.3 Nx vs Hx24hr, p=0.6 Nx vs Hx72hr). Abnormalities in dendritic distribution included either total absence of diminution of staining, with preservation of immunoreactivity in the proximal segments of primary dendries, but loss of staining in the secondary or tertiary arbors and spines. These alterations were present in the apical dendrites of Hx0hr (p=0.01 vs Nx), Hx24hr (p=0.02 vs Nx) and Hx72hr (p=0.002 vs Nx) fetuses. The data show a reduction in immunoreactivity together with altered patterns of cellular localization of calbindin-D_28k in Purkinje cells during fetal hypoxia. We conclude that the decrease in calbindin-D_28k expression will lead to increase of intracellular Ca²⁺. We speculate that the hypoxia-induced reduction of calbindin-D_28k expression will limit the neuronal Ca²⁺ buffering capacity, resulting in activation of CA²⁺-mediated pathways of free medical generation.