Abstract
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.
Highlights
Preterm babies have an increased risk of neurodevelopmental delay, disability, sensory and learning deficits and cerebral palsy (Beck et al, 2010; Doyle et al, 2010), and deficits are more pronounced the earlier that infants are born (Saigal and Doyle, 2008)
We have previously shown that recombinant humanEPO is protective against LPS-induced brain injury in the fetal sheep cerebrum, reducing cerebral white matter (WM) injury, apoptosis, microgliosis, astrogliosis and blood-brain barrier leakage (Rees et al, 2010). recombinant human EPO (rhEPO) is currently being tested in clinical trials for its neuroprotective effects in preterm infants, with initial results suggesting that rhEPO improves neurological outcomes (Neubauer et al, 2010) and reduces cerebral WM damage (O’Gorman et al, 2015)
Our major findings were that rhEPO treatment reduced: (1) microgliosis in the lobular WM; (2) the increase in thickness of the proliferative and post-mitotic zones of the external granule layer (EGL); and (3) the increase in granule cell density in the internal granule layer (IGL), induced by LPS
Summary
Preterm babies have an increased risk of neurodevelopmental delay, disability, sensory and learning deficits and cerebral palsy (Beck et al, 2010; Doyle et al, 2010), and deficits are more pronounced the earlier that infants are born (Saigal and Doyle, 2008). Advances in perinatal care have resulted in an increasing number of very preterm babies surviving and the number of infants with potential perinatal brain injury and disability is increasing. Infants who have been exposed to maternal or intrauterine infection and inflammation have an increased risk of diffuse cerebral white matter (WM) injury, intraventricular hemeorrhage, periventricular leukomalacia and cerebral palsy (Grether and Nelson, 1997; Goldenberg et al, 2000; Wu and Colford, 2000; Leviton et al, 2005; Romero et al, 2006). The effects of inflammation on the human cerebrum are well established, far less attention has been paid to any injury and adverse development sustained by the cerebellum
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