Protein profiling of anastomosed facial nerve treated with mesenchymal stromal cells.

BACKGROUND AIMS: The types of proteins released from mesenchymal stromal cells (MSC) are still unclear. Our aim was to compare apoptosis scores and the expression of myelin-associated glycoprotein (MAG), myelin basic protein (MBP), neural cell adhesion molecule (NCAM)-1,matrix metalloproteinase (MMP)-1A, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-1/MMP-1A ratio, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophin (NT)-3, NT-4, glial cell-derived neurotropic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (FGF)-2, insulin-like growth factor (IGF)-1, platelet-derived growth factor (PDGF)-α and transforming growth factor (TGF)-ß1 in anastomosed facial nerves that had been treated with or without MSC. METHODS: In seven rats, the buccal branch of the right facial nerve was transected, anastomosed and treated with MSC (anastomosed + MSC group). The left buccal branch was anastomosed only (anastomosed-only group). The left mandibular branch served as an intact nerve group. On days 18-20, the distal segments of the branches were examined in terms of expression of the mentioned proteins and apoptosis scores using polymerase chain reaction (PCR) and terminal deoxynucleotidyl transferase-mediated digoxigenin-UTP nick end labeling (TUNEL) assays. RESULTS: MSC application significantly increased CNTF, PDGF-α, LIF, TGF-ß1, BDNF and NT-3 expression (P < 0.05). MAG expression slightly decreased whereas NCAM-1, MMP-1A and FGF-2 slightly increased(P > 0.05). Changes in other proteins and apoptosis scores were not significant. CONCLUSIONS: These results suggest that MSC increases expression of CNTF, PDGF-α, LIF,TGF-ß1, BDNF and NT-3. MAG, NCAM-1, MMP-1A and FGF-2 expressions were slightly changed in this stage of nerve regeneration. The comparison of apoptotic activity was not conclusive. Overall, it appears that MSC might have differential effects on the mentioned tissue-related proteins and trophic/growth factors.

Immunohistochemical investigation of inducible nitric oxide synthase, osteopontin, and calcium-sensing receptor in a myringosclerosis/tympanosclerosis model.

HYPOTHESIS: To investigate roles of types of inflammation, inducible nitric oxide synthase (iNOS), osteopontin (OPN), and calcium sensing receptor (CaSR) in the tympanic membrane and middle ear in etiopathogenesis of myringosclerosis/tympanosclerosis (MT). BACKGROUND: Etiopathogenesis of myringosclerosis/tympanosclerosis is still unclear. Clinical and experimental observations demonstrate that hyperoxygenation might induce tympanosclerosis. METHODS: Seventy-five rats were divided into 3 groups: ventilation tube (VT) insertion, the Eustachian tube (ET) obliteration, and both procedures. Right ears were selected for mentioned interventions. Left ears served as controls. Then, histopathologic and immunohistochemical investigations were performed in tympanic bulla. MT and inflammation in tympanic membrane and middle ear space were investigated. Immunohistochemical investigation included staining with iNOS, OPN, and CaSR. RESULTS: Overall 42.7% of all rats developed MT. There was no significant difference in MT incidence among the groups (ET + VT group: 56%; ET group: 44%; VT group: 28%; p > 0.017). iNOS expression occurred in 30.6% of the intervention groups with insignificant differences (ET + VT group: 40%; ET group:36%; VT group:16%; p > 0.05). There was no significant difference in iNOS expression between tympanosclerotic (25%) and non tympanosclerotic ears (34.9%) (p = 0.359). OPN was expressed in 82.6% overall. It was the highest for ET group and ET + VT group (92% for each) followed by VT group (64%). There was a marginal significance in comparison of OPN staining between VT group and ET group and also between VT group and ET + VT group (p = 0.017). There was a significant difference in OPN expression between tympanosclerotic (100%) and nontympanosclerotic ears (69.8%) (p = 0.001). Neither control ears nor intervention groups showed CaSR expression. Comparisons of inflammation of the tympanic membrane and middle ear space between tympanosclerotic and non-tympanosclerotic ears yielded significant differences (p = 0.003, p = 0.002, respectively). Tympanosclerotic ears had a tendency to show chronic or mixed inflammation in contrast to non-tympanosclerotic ears (p < 0.017). Filled-middle ear space was seen in 25% of the intervention groups with no significant difference (p > 0.017). There was a significant difference in the incidence between tympanosclerotic (46.8%) and non-tympanosclerotic ears (7%) (p < 0.017). CONCLUSION: Based on these findings, iNOS may not be evident in stage of MT. OPN staining is strongly associated with the development of MT. CaSR has no role in formation of MT. The results proved roles of mixed or chronic inflammation and the presence of the filled-middle ear in development of MT.

Repair of transected facial nerve with mesenchymal stromal cells: histopathologic evidence of superior outcome.

OBJECTIVES/HYPOTHESIS: Despite advanced surgical techniques, clinical results of the transected facial nerve are still far from the desired outcome. Mesenchymal stromal cells (MSCs) were shown to transdifferentiate into Schwann cells and express some growth factors beneficial in peripheral nerve injury. We aimed to document histopathological improvement obtained from application of the homograft bone marrow-derived MSCs immediately after conventional anastomosis of a transected facial nerve branch in rats, and to compare the results with those nerves anastomosed only. STUDY DESIGN: Animal, prospective, and controlled study. METHODS: The study was performed in 15 rats. The right buccal branch was completely transected and repaired with epineural sutures. The right-side anastomosis was additionally treated with MSCs thereafter. The right marginal mandibular branch was kept intact, but in contact with MSCs. The left buccal branch was transected and repaired in a similar fashion except for MSC application. The left-side marginal mandibular branch was left intact. Rats were sacrificed at month 1, 3, and 6. Four branches of each rat were sampled, and nerve segments distal to the anastomosis were histopathologically examined. RESULTS: The examination revealed that intact nerve segments and nerve segments in contact with MSCs had completely normal appearance regardless of the time interval. Samples from the nerves anastomosed and treated with MSCs did better than those nerves anastomosed only in terms of axonal organization and myelin thickness. CONCLUSIONS: This preliminary report witnessed beneficial effects of MSCs application onto the injured facial nerve as evidenced by the histopathological examination.