Characterization of spinal cord glial cells in a model of hindlimb unloading in mice

Abstract

Exposure to microgravity has been shown to result in damaging alterations to skeletal muscle, bones, and inner organs. In this study, we investigated the effects of microgravity by using a hindlimb unloading model (HUM) in mice. The characteristics of the lumbar spinal cords of HUM mice 30days after hindlimb unloading were examined. Morphometric analysis showed reductions of the total area, gray matter, and white matter by 17%, 20%, and 12%, respectively. Myelinated fibers in the white matter showed prominent myelin destruction. Analysis of the number of glial fibrillary acidic protein (GFAP+)/S100 calcium-binding protein B (S100B−), GFAP+/S100B+, and GFAP−/S100B+ astrocytes in the ventral horn (VH), central channel area (CC), dorsal root entry zone (DREZ), main corticospinal tract (CST), and ventral funiculi (VF) showed that the number of GFAP+/S100B− astrocytes was increased in the DREZ and CST of HUM mice. Additionally, GFAP+/S100B+ cell numbers were significantly decreased in the VH and CST but did not differ in the CC or DREZ of HUM mice, as compared with the control. The numbers of GFAP−/S100B+ cells were significantly reduced only in the VH of HUM mice. Moreover, the number of ionized calcium-binding adaptor molecule 1 (Iba1+) microglia cells was significantly increased in the CC and DREZ of HUM mice. In control mice, homeobox protein HoxB8 (HoxB8+) cells were found only in the CC; in contrast, HoxB8+ cells were observed in all studied areas in HUM mice, with the greatest number found in the CC. Genome-wide transcriptome analysis of the lumbar spinal cords of HUM mice showed decreased expression of genes encoding myelin, extracellular matrix, cytoskeleton, and cell adhesion proteins. Real-time polymerase chain reaction (PCR) confirmed reductions in the expression of mpz, pmp2, pmp22, and prx genes, which are involved in myelination, as well as decreases in the levels of genes encoding extracellular matrix molecules, including glycoproteins (matrix gla protein (MGP), osteoglycin (OGN), microfibrillar associated protein 5 (MFAP), and collagen, type IV, alpha 1 (COL4A)), proteoglycans (perlecan (heparan sulfate proteoglycan) (HSPG)), and metalloproteinases (lysyl oxidase (LOX)). Thus, our results showed that hindlimb unloading caused decreases in gray and white matter areas, changes in gene expression, alterations in myelination, and phenotypic modifications in glial cells in the lumbar spinal cords of mice.