Increased Number of Cerebellar Granule Cells and Astrocytes in the Internal Granule Layer in Sheep Following Prenatal Intra-amniotic Injection of Lipopolysaccharide

Abstract

Chorioamnionitis is an important problem in perinatology today, leading to brain injury and neurological handicaps. However, there are almost no data available regarding chorioamnionitis and a specific damage of the cerebellum. Therefore, this study aimed at determining if chorioamnionitis causes cerebellar morphological alterations. Chorioamnionitis was induced in sheep by the intra-amniotic injection of lipopolysaccharide (LPS) at a gestational age (GA) of 110 days. At a GA of 140 days, we assessed the mean total and layer-specific volume and the mean total granule cell (GCs) and Purkinje cell (PC) number in the cerebelli of LPS-exposed and control animals using high-precision design-based stereology. Astrogliosis was assessed in the gray and white matter (WM) using a glial fibrillary acidic protein staining combined with gray value image analysis. The present study showed an unchanged volume of the total cerebellum as well as the molecular layer, outer and inner granular cell layers (OGL and IGL, respectively), and WM. Interestingly, compared with controls, the LPS-exposed brains showed a statistically significant increase (+20.4%) in the mean total number of GCs, whereas the number of PCs did not show any difference between the two groups. In addition, LPS-exposed animals showed signs of astrogliosis specifically affecting the IGL. Intra-amniotic injection of LPS causes morphological changes in the cerebellum of fetal sheep still detectable at full-term birth. In this study, changes were restricted to the inner granule layer. These cerebellar changes might correspond to some of the motor or non-motor deficits seen in neonates from compromised pregnancies.

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

“…The significant increase in the density of granule cells in the IGL of LPS-exposed animals was unexpected but is in accordance with the findings of Strackx et al ( 2012 ) who reported an increase in total granule cell number in the IGL 30 days after LPS exposure. Although we were not able to calculate total neuronal numbers, the finding that the cross-sectional area of the IGL was not different from controls suggests that the increase in density might have resulted in an increase in total granule cell numbers.…”

“…In accordance with the only study to examine the effects of chorioamnionitis in the human infant cerebellum (Limperopoulos et al, 2005 ), we did not find any gross injury such as focal infarcts or hemeorrhage in the LPS-exposed cerebellar cortex or WM in this, or our previous study (Duncan et al, 2002 ). Strackx et al ( 2012 ) also reported no overt cerebellar damage in a similar sheep model of inflammation although Dean et al ( 2009 ) found some focal lesions in animals exposed to LPS at an earlier gestational age (93-96 DGA). Studies that have characterized LPS-induced WM injury at the cellular level report finding microgliosis, astrogliosis, increased apoptosis, decreased maturation of oligodendrocytes, and compromised blood-brain barrier integrity (Hutton et al, 2007 ; Dean et al, 2009 ; Gavilanes et al, 2009 ; Strackx et al, 2012 ).…”

“…Strackx et al ( 2012 ) also reported no overt cerebellar damage in a similar sheep model of inflammation although Dean et al ( 2009 ) found some focal lesions in animals exposed to LPS at an earlier gestational age (93-96 DGA). Studies that have characterized LPS-induced WM injury at the cellular level report finding microgliosis, astrogliosis, increased apoptosis, decreased maturation of oligodendrocytes, and compromised blood-brain barrier integrity (Hutton et al, 2007 ; Dean et al, 2009 ; Gavilanes et al, 2009 ; Strackx et al, 2012 ). In the present study, we found that LPS-induced microgliosis but not astrogliosis in the cerebellar WM, and that there was no evidence of an increase in apoptosis in any cerebellar region.…”

“…There is evidence, however, from animal studies, largely in sheep, that the fetal cerebellum is vulnerable to inflammation induced by lipopolysaccharide (LPS). Inflammatory damage has been reported to include: cerebellar WM injury and focal WM lesions (Dean et al, 2009 ), increased expression of pro-inflammatory cytokines (Strackx et al, 2015 ), apoptosis (Hutton et al, 2007 ; Gavilanes et al, 2009 ), increased activation of microglia (Hutton et al, 2007 ; Gavilanes et al, 2009 ), infiltration of macrophages (Hutton et al, 2007 ), astrogliosis (Hutton et al, 2007 ; Gavilanes et al, 2009 ; Strackx et al, 2012 ), an increase in the number of granule neurons in the internal granule layer (IGL; Strackx et al, 2012 ), a reduction in the proportion of mature oligodendrocytes (Dean et al, 2009 ; Gavilanes et al, 2009 ) and breakdown of the blood-brain barrier (Hutton et al, 2007 ). In a previous study on the effects of LPS on the fetal sheep brain (Duncan et al, 2002 ), we found significant injury in the cerebrum but no gross cerebellar damage; as we recognize that damage could have occurred at the cellular level, we have addressed this possibility in the present study.…”

Effects of in utero endotoxemia on the ovine fetal brain A model for schizophrenia

“…To test this hypothesis, we used a sheep model in which lipopolysaccharide (LPS) was injected intravenously in instrumented fetal lambs. Our group has developed several well established sheep models to study both the effects of chorioamnionitis and endotoxemia/sepsis on the fetal brain (20)(21)(22)(23). In this study, the (cyto-) architecture of the cingulate cortex (CC) was examined using immunohistochemistry with focus on the different subsets of GABA-ergic interneurons which were shown to play a major role in the pathogenesis of schizophrenia.…”