ABSTRACT
One of the most important natural extracellular constituents is hyaluronic acid (HA) with the potential to develop a highly organized microenvironment. In the present study, we enriched HA hydrogel with tenascin-C (TN-C) and examined the viability and survival of mouse neural stem cells (NSCs) using different biological assays. Following NSCs isolation and expansion, their phenotype was identified using flow cytometry analysis. Cell survival was measured using MTT assay and DAPI staining after exposure to various concentrations of 50, 100, 200, and 400 nM TN-C. Using acridine orange/ethidium bromide staining, we measured the number of live and necrotic cells after exposure to the combination of HA and TN-C. MTT assay revealed the highest NSCs viability rate in the group exposed to 100 nM TN-C compared to other groups, and a combination of 1% HA + 100 nM TN-C increased the viability of NSCs compared to the HA group after 24 hours. Electron scanning microscopy revealed the higher attachment of these cells to the HA + 100 nM TN-C substrate relative to the HA substrate. Epifluorescence imaging and DAPI staining of loaded cells on HA + 100 nM TN-C substrate significantly increased the number of NSCs per field over 72 hours compared to the HA group (P < 0.05). Live and dead assay revealed that the number of live NSCs significantly increased in the HA + 100 TN-C group compared to HA and control groups. The enrichment of HA substrate with TN-C promoted viability and survival of NSCs and could be applied in neural tissue engineering approaches and regenerative medicine.
Subject(s)
Biocompatible Materials/toxicity , Cell Survival/drug effects , Cells, Cultured/drug effects , Cytotoxins/toxicity , Hyaluronic Acid/toxicity , Neural Stem Cells/drug effects , Tenascin/toxicity , Animals , Disease Models, Animal , Dose-Response Relationship, Drug , Male , MiceABSTRACT
Tenascin-C (TNC), a matricellular protein, is induced in association with cerebral vasospasm after subarachnoid hemorrhage. The aim of this study was to assess the vasoconstrictive effects of TNC and its mechanisms of action on cerebral arteries in vivo. Two dosages (1 and 10µg) of TNC were administered intracisternally to healthy rats, and the effects were evaluated by neurobehavioral tests and India-ink angiography at 24, 48, and 72h after the administration. Western blotting and immunohistochemistry were performed to explore the underlying mechanisms on constricted cerebral arteries after 24h. The effects of toll-like receptor 4 (TLR4) antagonists (LPS-RS), c-Jun N-terminal kinase (JNK), and p38 inhibitors (SP600125 and SB203580) on TNC-induced vasoconstriction were evaluated at 24h. Higher dosages of TNC induced more severe cerebral arterial constriction, which continued for more than 72h. TNC administration also upregulated TLR4, and activated JNK and p38 in the smooth muscle cell layer of the constricted cerebral artery. LPS-RS blocked TNC-induced TLR4 upregulation, JNK and p38 activation, and vasoconstrictive effects. SP600125 and SB203580 abolished TNC-induced TLR4 upregulation and vasoconstrictive effects. TNC may cause prolonged cerebral arterial constriction via TLR4 and activation of JNK and p38, which may upregulate TLR4. These findings suggest that TNC causes cerebral vasospasm and provides a novel therapeutic approach against it.
Subject(s)
Cerebral Arteries/drug effects , Tenascin/toxicity , Vasospasm, Intracranial/chemically induced , Angiography , Animals , Disease Models, Animal , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , MAP Kinase Kinase 4/metabolism , Male , Nervous System Diseases/etiology , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Statistics, Nonparametric , Time Factors , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Up-Regulation/drug effects , Vasospasm, Intracranial/complications , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The aim of this study was to investigate the effect of cholecystokinin (CCK) receptor antagonist on the abnormal behavior and dopamine (DA) transmission of tenascin (TN)-gene knockout mice. Recently, we demonstrated that TN-gene deficient mice show hyperlocomotion that is related to reduced DA transmission and tyrosine hydroxylase (TH) activities in the brain. In this report, we show that the intraperitoneal administration of a CCK-B receptor antagonist, PD135158 (0.1 mg/kg), but not a CCK-A receptor antagonist, lorglumide, inhibited hyperlocomotion. Moreover, PD135158 reversed the low levels of DA turnover rate and TH activities in the striatum of TN-gene knockout mouse brain. These results suggest that CCK-B receptor is involved in the behavior of TN-gene knockout mouse through striatal DA transmission.
