| ObjectiveDry eye is one of the most common ocular surface diseases,mainly manifested as eye discomfort,decreased visual quality,accompanied by increased osmotic pressure and ocular surface inflammation.Inflammation,hormonal imbalances,neurological dysfunction,apoptosis and oxidative stress take part in the mechanism of dry eye.Currently,artificial tear eye-replacement therapies in the treatment of dry eye have a certain effect,but long term use has side effects,easily to relapse after stopping.Anti-inflammatory treatments relieve symptoms of dry eye.Topical application of glucocorticoid,cyclosporine and non-steroidal anti-inflammatory eye drops has obvious effects,but toxic side effect manifested for long term use.How to effectively control and even reverse dry eye syndromes,finding a safe and effective drug is critical to the situation.Studies have found that hydrogen can selectively resist oxidative damage and inhibit the release of inflammatory cytokines.Hydrogen could be dissolved in normal saline under high pressure of 0.4MPa and the product is called hydrogen-rich saline(HRS)with the saturation concentration of 0.6mmol/L.HRS has significant antioxidant and anti-inflammatory effects in various animal model of ischemia-reperfusion,degenerative diseases and so on.This experiment observed HRS eye drop and intraperitoneal injection of HRS to intervene dry eye model induced by scopolamine,and tested clinical indicators,corneal and conjunctival histopathological changes,cell apoptosis,squamous metaplasia degree and the expression of inflammatory factor nuclear factor-kB(NF-kB)of rats in each group and investigated whether there was protective effect of HRS on the ocular surface damage of dry eye rats.Methods1.Thirty female Wistar rats about six weeks old were randomly divided into normal group(A),dry eye group(B),HRS eye drops group(C),normal saline eye drops group(NS)(D),HRS intraperitoneal injection group(E)and NS intraperitoneal injection group(F),with 5 rats in each group.The dry eye rat model was induced by subcutaneous injection of scopolamine hydrobromide in the latter five groups.The eye drops of HRS and NS were respectively applied to the groups of C and D(one time per hour,nine times per day).The HRS and NS were respectively peritoneal injected to the groups of C and D(5ml·kg-1 per time,one time per day)for 28 days.2.The clinical tests of dry eye including tear volume(SⅠt),tear break-up time(BUT)and corneal epithelial fluorescein staining scores were evaluated on day 0,7,14,21 and 28.3.On the 28 th day,all the rats were sacrificed by cervical dislocation method,then the whole eyeballs were taken and paraffin sections were made followed by HE staining.4.Paraffin sections were stained with PAS,the morphologic changes of goblet cells were observed and goblet cells counting per high-power field under the light microsco-pe was calculated.5.The apoptosis of cornea and conjunctiva of each group were detected under fluorescence microscopy by in situ terminal transferase labeling.6.Immunohistochemical staining was used to observe the expression of cytokeratin 10 in the corneal and conjunctival tissue of rats under light microscope.7.The corneal and conjunctival tissues of rats in each group were taken,using Western blotting to detect the expression of NF-kB in corneal and conjunctival tissue.Results1.SⅠt: On the 7th day,there was significant difference of SⅠt values between the groups(F=2.897,P<0.05),the SⅠt value of dry eye group decreased compared with the normal group(P<0.05).On the 14 th day,there was significant difference of SⅠt values between the groups(F=5.194,P<0.05).SⅠt of dry eye group decreased significantly while compared with normal control.SⅠt of HRS eyedrop group and HRS intraperitoneal injection group increased respectively compared with NS eyedrop group and NS intraperitoneal injection group(P<0.05).There was no significance between dry eye group,NS eyedrop group and NS intraperitoneal injection group(P>0.05).SⅠt remained stable until the 28 th day.2.BUT: On the 7th day,there was significant difference of BUT values between the groups(F=2.910,P<0.05),the BUT value of dry eye group decreased compared with the normal group(P<0.05).On the 14 th day,there was significant difference of BUT values between the groups(F=3.894,P<0.05).BUT of dry eye group decreased significantly while compared with normal control.BUT of HRS eyedrop group and HRS intraperitoneal injection group increased respectively compared with NS eyedrop group and NS intraperitoneal injection group(P<0.05).There was no significance between dry eye group,NS eyedrop group and NS intraperitoneal injection group(P>0.05).BUT remained stable until the 28 th day.3.Corneal epithelial sodium fluorescein staining score: On the 7th day,there was significant difference of scores between the groups(F=2.424,P<0.05),the score of dry eye group increased compared with the normal group(P<0.05).On the 14 th day,there was significant difference of scores between the groups(F=16.487,P<0.05).Score of dry eye group increased significantly while compared with normal control.Scores of HRS eyedrop group and HRS intraperitoneal injection group decreased respectively compared with NS eyedrop group and NS intraperitoneal injection group(P<0.05).There was no significance between dry eye group,NS eyedrop group and NS intraperitoneal injection group(P>0.05).Scores remained stable until the 28 th day.4.HE staining under light microscopy showed the corneal epithelial cells of dry eye group had hierarchical fuzzy cell boundary,greater cell volume.The basal layer cells partially exfoliated,and the squamous epithelium was closer to the surface of the cornea compared with the normal control.In the eyes of HRS group and HRS intraperitoneal injection group,the conjunctival epithelium of rats was smoother and much more regular.The cell layers were decreased while the cellular boundary was much clearer with decreased edema.And the tissue proliferation was reduced.5.PAS staining under light microscopy showed the conjunctival goblet cells were not uniform,and less goblet cells was detected at the closer part under the basement in dry eye group.HRS eye drop group and HRS intraperitoneal injection group showed layers and size of goblet cells tended to be normal,and the number of goblet cells increased.There was no significant difference between the two groups(P>0.05).6.Apoptosis of corneal and conjunctival epithelium cells significantly increased in dry eye group whiel it significantly decreased in HRS eye group and HRS intraperitoneal injection group.There was no significant difference between HRS eye group and HRS intraperitoneal injection group(P>0.05).7.There was no expression of K10 in the corneal and palpebral conjunctival epithelium of the normal group while its expression was weak in the fornix conjunctiva under immunohistochemical staining.The expression of K10 in the corneal and fornix conjunctival epithelium of the dry eye group was obviously positive.However,there was no expression of K10 in the palpebral conjunctiva in the dry eye group.The expression of K10 in the corneal and fornix conjunctival epithelium of HRS eye drop group and HRS intraperitoneal injection group significantly decreased.8.Western blot showed the NF-kB expression of the corneal and conjunctival tissue of dry eye group increased significantly than it in the normal group.NF-kB expression in HRS eye drop group and intraperitoneal injection group decreased simutaneously than dry eye group and NS intervention groups.And the difference between the two HRS groups was not statistically significant(P>0.05).ConclusionThis experiment selected two ways of HRS intervention(eye drop and intraperitoneal injection)to treat dry eye model induced by scopolamine.Clinical dry eye tests,histopathological manifestation,and cell apoptosis showed that HRS eye drops and intraperitoneal injection can relieve the signs and histological changes of dry eye.The corneal and conjunctival epithelial hyperplasia decreased,the morphology of goblet cells tended to be normal while squamous metaplasia relieved,the apoptosis of ocular surface decreased too.Meanwhile,the expression of inflammatory factor NF-kB decreased in corneal and conjunctival tissues when intervened by HRS.In conclusion,HRS may alleviate the ocular surface injury of dry eye rats induced by scopolamine probably through inhibiting the inflammatory response.And HRS may be a new drug for the treatment of dry eye. |
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