ABSTRACT
Background Reasearches showed that α-melanocyte-stimulating hormone (α-MSH) inhibits inflammation and ameliorates the ocular surface abnormalities in a scopolamine-induced dry eye rat model,and the managing effect of sodium carboxymethylcellulose (CMC) on dry eyes also has been determined.However,whether α-MSH can enhance the therapeutic effects of CMC remains to be investigated.Objective This study was to investigate the protective effects of α-MSH combined with CMC on ocular surface in a scopolamine-induced dry eye rat model.Methods Sixty clean female Wistar rats were randomly divided into normal control group,model control group,NaCl group,CMC group,α-MSH group and α-MSH+CMC group,and 10 rats for each group.The dry eye models were established by subcutaneous injection of scopolamine hydrobromide at 9:00,12:00,15:00 and 18:00 per day for 28 days.0.9％ NaCl solution,1×10 3 mg/ml α-MSH solution,0.5％ CMC eye drop,and 1 ×10-3 mg/ml α-MSH+0.5％ CMC solution were topically administered twice a day (8:00,17:00) since the initial day of modeling according to grouping.Shirmer Ⅰ test (S Ⅰ t),breakup time of tear film (BUT) and corneal fluorescence staining were performed before and 7,14,21,28 days after the application of drugs.At 28 days following the administration of drugs,the eyeballs of the rats were collected.Hemotoxylin and eosin staining was employed to examine the morphology of corneas,and periodic acid schiff (PAS) staining was used to count the conjunctival goblet cells.This study protocol was approved by Experimental Animal Ethic Committee of Tianjin Medical University (SYXK 2009-0001),and the use and care of the rats complied with ARVO Statement.Results The S Ⅰ t and BUT values were significantly reduced,and the corneal fluorescence staining scores were significantly increased over time following modeling in the model control group (all at P＜0.01).No significant differences were found in the S Ⅰ t,BUT and corneal fluorescence staining scores between model control group and NaCl group at various time points (all at P＞0.05).At 7,14 and 21 days after intervention,the S Ⅰ t values were (4.800±0.789),(4.100±0.516) and (4.300±0.856) mm in the α-MSH+CMC group,which were considerably higher than (2.875 ±0.719),(2.375 ±0.619) and (2.532±0.957)mm in the NaCl group (all at P＜0.01).At 7 days after intervention,the BUT values were (4.938± 1.843) seconds and (5.000±1.491) seconds in the α-MSH group and α-MSH+CMC group,which were significantly higher than (3.250±1.000) seconds in the NaCl group (both at P＜0.01).The corneal fluorescence staining scores in the CMC group,α-MSH group and α-MSH+CMC group were significantly lower than that in the NaCl group,with the lowest score in the α-MSH +CMC group (all at P＜0.05).The thickening of corneal epithelial layer,corneal edema and arrangement disorder of corneal stroma were found in the model control group and NaCl group;while slight corneal edema and epithelial cell proliferation were exhibited in the α-MSH+CMC group by hemotoxylin and eosin staining.PAS staining showed that the number of goblet cells was much more in the CMC group,α-MSH group and α-MSH+ CMC group than that in the model control group and NaCl group (all at P ＜ 0.01).Conclusions The sole application of α-MSH or CMC alleviates ocular surface damage and morphological abnormality to certain extent,and the combination of α-MSH and CMC generates more effective protection in comparison with sole administration of α-MSH or CMC.The early application of the drugs plays an improvement role in tear secretion and tear film stability in dry eyes.
ABSTRACT
Background Mesenchymal stem cells (MSCs) have been applied in basic and clinical researches of organ transplantation.Our previous study showed that intravenous injection of MSCs prolonged corneal allograft survival in rat.However,the effect of local administration of MSCs on corneal allograft rejection remains unclear.Objective The aim of this study was to investigate the effect of subconjunctival injection of MSCs on corneal allograft rejection in rat model of keratoplasty.Methods MSCs were isolated and cultured from femur and tibia bone marrow of clean Wistar rats,and then the cells were identified by induced differentiation of osteoblast and adipocyte.The third generation of MSCs was used in subsequent experiment.Allogenic penetrating keratoplasty was performed with the bilateral corneas of 20 Wistar rats as donor grafts and the right eyes of 40 Lewis rats as recipients.PBS 0.1 ml containing 2 × 106 MSCs and 0.1 ml PBS only was subconjunctivally injected immediately and postoperative 3 days respectively in randomized two groups,and another 6 normal Lewis rats served as the normal control group.Corneal rejection response was evaluated under the slit lamp after surgery based corneal opacity,edema and neovascularization,and the grafts were scored according to the criteria of Larkin.The corneal samples were extracted from 12 rats of the PBS control group and the MSCs group separately 10 days after surgery.The relative expressions of Th1 cytokines (interferon-γ [IFN-γ] mRNA and interleukin-2 [IL-2] mRNA) and Th2 cytokines (IL-4 mRNA and IL-10 mRNA) were detected by real-time quantitative PCR.Protein levels of IL-4 and IL-10 proteins in the corneas were assayed by ELISA.All experimental protocols involving rats were approved by the laboratory animal care and use committee of the Tianjin Medical University and treated with the ARVO statement for the use of animals in ophthalmic and vision research.Results The cells grew well with the orange stain for alizarin red in differentiated the osteoblasts and red stain for Oil red O in differentiated adipocytes.The survival time of corneal graft in the MSCs group was (11.8±1.6) days,it was significantly longer than (9.6±1.4) days in the PBS control group (P=0.004).The levels of IL-4 mRNA and IL-10 mRNA in the MSCs group were significantly higher than those in the PBS control group (both at P =0.00);while the levels of IFN-γ mRNA and IL-2 mRNA were not significantly different between the groups (both at P＞0.05).The IL-10 protein contents were (22.74 ±7.06),(68.40±12.83) and (215.41 ±44.66)pg/ml in the normal control group,PBS control group and MSCs group,showing significant difference among the three groups (F =55.06,P =0.00) and a significant increase in the MSCs group compared with the PBS control group and the normal control group (both at P ＜ 0.05).Conclusions Subconjunctival injection of MSCs prolongs the survival time of cornea allograft in penetrating keratoplasty probably by modulating the balance between Th1 and Th2 cytokines,especially by up-regulating Th2 cytokines.