Erythropoietin Protects Against Lipopolysaccharide-Induced Microgliosis and Abnormal Granule Cell Development in the Ovine Fetal Cerebellum

Annie R. A. McDougall, Nadia Hale, Sandra Rees, Richard Harding, Robert De Matteo, Stuart B. Hooper, Mary Tolcos

Erythropoietin (EPO) ameliorates inflammation-induced injury in cerebral white matter (WM). However, effects of inflammation on the cerebellum and neuroprotective effects of EPO are unknown. Our aims were to determine: (i) whether lipopolysaccharide (LPS)-induced intrauterine inflammation causes injury to, and/or impairs development of the cerebellum; and (ii) whether recombinant human EPO (rhEPO) mitigates these changes. At 107 ± 1 days gestational age (DGA; ~0.7 of term), fetal sheep received LPS (~0.9 μg/kg; i.v.) or an equivalent volume of saline, followed 1 h later with 5000 IU/kg rhEPO (i.v.) or an equivalent volume of saline (i.v.). This generated the following experimental groups: control (saline + saline; n = 6), LPS (LPS + saline, n = 8) and LPS + rhEPO (n = 8). At necropsy (116 ± 1 DGA; ~0.8 of term) the brain was perfusion-fixed and stained histologically (H&E) and immunostained to identify granule cells (Neuronal Nuclei, NeuN), granule cell proliferation (Ki67), Bergmann glia (glial fibrillary acidic protein, GFAP), astrogliosis (GFAP) and microgliosis (Iba-1). In comparison to controls, LPS fetuses had an increased density of Iba-1-positive microglia (p < 0.005) in the lobular WM; rhEPO prevented this increase (p < 0.05). The thickness of both the proliferative (Ki67-positive) and post-mitotic zones (Ki67-negative) of the EGL were increased in LPS-exposed fetuses compared to controls (p < 0.05), but were not different between controls and LPS + rhEPO fetuses. LPS also increased (p < 0.001) the density of granule cells (NeuN-positive) in the internal granule layer (IGL); rhEPO prevented the increase (p < 0.01). There was no difference between groups in the areas of the vermis (total cross-section), molecular layer (ML), IGL or WM, the density of NeuN-positive granule cells in the ML, the linear density of Bergmann glial fibers, the areal density or somal area of the Purkinje cells, the areal coverage of GFAP-positive astrocytes in the lobular and deep WM, the density of Iba-1-positive microglia in the deep WM or the density of apopotic cells in the cerebellum. LPS-induced intrauterine inflammation caused microgliosis and abnormal development of granule cells. rhEPO ameliorated these changes, suggesting that it is neuroprotective against LPS-induced inflammatory effects in the cerebellum.