The cellular components of the satellite cell niche participate in the regulation of skeletal muscle regeneration. Beside myogenic cells at different developmental stages, this niche is formed by cells of the immune system, the interstitial connective tissue and the vascular ystem. Unambiguous determination of the origin of these cell types could contribute to optimization of the cell-based therapy of skeletal muscle disorders. In our work, we intravenously transplanted mouse GFP+ unseparated bone marrow cells into whole-body lethally irradiated immunocom-petent mice four weeks before cardiotoxin-induced injury of the recipients’ skeletal muscles. Seven and 28 days after the toxin injection, the injured regenerating and contralateral intact muscles were examined for identification of GFP+ bone marrow-derived cells by direct fluorescence, protein immunohistochemistry and immunogold transmission electron microscopy. In both the intact and injured muscles, GFP positivity was determined in immune cells, mainly in macrophages, and in interstitial spindle-shaped cells. Moreover, in the injured muscles, rare GFP+ endothelial cells of the blood vessels and newly formed myotubes and muscle fibres were present. Our results confirmed the ability of bone marrow-derived cells to contribute to the cellular component of the satellite cell niche in the intact and regenerating skeletal muscle. These cells originated not only from haematopoietic stem cells, but obviously also from other stem or progenitor cells residing in the bone marrow, such as multipotent mesenchymal stromal cells and endothelial progenitors. and Corresponding author: Dana Čížková
The heat shock protein 70 (HSP70) is a key component of the stress response induced by various noxious conditions such as heat, oxygen stress, trauma and infection. In present study we have assessed the consequences of the compression of lower lumbar and sacral nerve roots caused by a multiple cauda equina constrictions (MCEC) on HSP70 immunoreactivity (HSP70-IR) in the dog. Our data indicate that constriction of central processes evokes HSP70 up-regulation in the spinal cord (L7, S1-Co3) as well as in the corresponding dorsal root ganglion cells (DRGs) (L7-S1) two days following injury. A limited number of bipolar or triangular HSP-IR neurons were found in the lateral collateral pathway (LCP) as well as in the pericentral region (lamina X) of the spinal cord. In contrast, a high number of HSP70 exhibiting motoneurons with fine processes appeared in the ventral horn (laminae VIII-IX) of lumbosacral segments.
Concomitantly, close to them a few lightly HSP70-positive neuronal somata or cell bodies lacking the HSP70-IR occurred. In the DRGs, HSP70 expression was mildly up-regulated in small and medium-sized neurons and in satellite cells. On the contrary, DRGs from intact or sham-operated dogs did not reveal HSP70 specific neuronal staining. In conclusion, we have demonstrated that the MCEC in dogs mimicking the cauda equina syndrome in clinical settings evokes expression of HSP70 synthesis in specific neurons of the lumbo-sacro-coccygeal spinal cord segments and in small and medium sized neurons of corresponding DRGs. This suggests that HSP70 may play an active role in neuroprotective processes partly by maintaining intracellular protein integrity and preventing the neuronal degeneration in this experimental paradigm.
Here we describe a comparative study of phenotypic properties of hepatic cells in situ and in vitro. We analyzed the expression levels and distribution patterns of ABC transporters MRP2 and MDR1, pan-cytokeratin, cytokeratin 18, albumin, alpha-fetoprotein and the specific hepatocyte marker OCH1E5 in the fetal and adult rat as well as human liver tissue and in human fetal hepatocytes of WRL 68 cell line using peroxidase immunohistochemistry or immunofluorescence. Transporters MRP2 and MDR1 were expressed in all examined liver tissues, except rat ED13 embryo. The immunopositivity of these proteins was localized to the canalicular membrane of differentiating and mature hepatocytes but in the later developmental stages and in the adult liver tissues it was also found in the apical membrane of cholangiocytes. In WRL 68 cells, MRP2 and MDR1 immunoreactivity appeared after 5-6 days of cultivation and both transporters were fully expressed in the plasmalemma and in the cytoplasm 9 days after the passage. In conclusion, we observed only moderate variances reflecting diverse ontogenetic phases between the fetal and adult liver tissue. To study functions of hepatocytes in vitro, WRL 68 cells have to differentiate prior to the examination. Our findings indicate that WRL 68 cells can undergo differentiation in vitro and their antigenic profile closely resembles hepatocytes in the human liver.
In the present study we investigated the effect of a two-stage bilateral lesion of the olfactory bulb (OB) in rats on the regeneration ability of peripheral olfactory neurons and their reinnervation capacity in the spared OB. The outgrowth of newly-generated olfactory axons as well as the maturation of their terminal synaptic field was detected by immunohistochemistry of the growth-associated phosphoprotein B-50/GAP-43. In addition, the glial response to the surgery was monitored by an immunohistochemical marker for astrocytes, glial fibrillary acidic protein (GFAP). In neonatal rats (P3-P5), the right OB was removed, then three months later the contralateral side was ablated. Six days after the second operation the animals were transcardially perfused. Their brains were embedded in paraplast, serially sectioned and processed for histological and immunohistochemical observations. After neonatal OB ablation, homogeneous B-50-immunoreactivity (BIR) was found in the forebrain, olfactory axons and ectopic glomeruli localized in the small OB remnant-like structures and in the regenerated neuroepithelium. A strong GFAP response was revealed in the brain cortex as well as in the newly-formed olfactory axons and glomeruli-like structures of the OB remnants. After adult OB ablation strong BIR was observed in olfactory axons, while remaining glomerular structures were only faintly stained. The neuroepithelium revealed signs of massive degenerative processes with a substantial decrease in BIR. The GFAP-positive astrocytes were scattered throughout the entire OB remnant and were prominent in the glomeruli-like structures and adjacent frontal cortex. In the present study, we applied GAP-43 and GFAP immunohistochemistry to characterize the responses of individual olfactory components after two-stage olfactory bulbectomy. Furthermore, this model of OB ablation characterized by two immunohistochemical markers could elucidate certain molecular mechanisms involved in the regeneration and/or plasticity of the olfactory system.