Study On The Mitigation Effect Of Chitosan Derivatives On Rat Evaporative Dry Eye

Purpose：In recent years, the user of the computers are stimulated by electromagneticradiation and glare in the long time, so blink rate decreases, the area exposed theocular surface increases, that result in tear film instability and ocular surface damageand other symptoms of dry eyes. At present, the basic dry eye therapy is to useartificial tears. In recent years, artificial tears is developing from moisturizing andwetting ocular surface of mild dry eyes to the repairing of damaged ocular surfacecells of moderate-severe dry eyes.Chitosan is an ideal biomedical material due to its good biocompatibility,biodegradability and non-toxic degradation products. The research of chitosan and itsderivatives in the field of ophthalmology shows that they can promote the healing ofthe damaged corneal epithelium, promote the growth of conjunctival epithelial cells invitro, and can be used as tissue engineering materials of corneal epithelial cells,stromal cells and endothelial cells. It can be seen that chitosan and its derivatives haspotential application prospect in the complex treatment of ocular surface disease.The present study mainly using macromolecular chitosan as raw materials tosynthesise carboxymethyl chitosan(CM-CTS) and hydroxyethyl chitosan(HE-CTS). Arat model of dry eyes is established and the effect of carboxymethyl chitosan andhydroxyethyl chitosan on the model is evaluated.Methods:1. Carboxymethyl chitosan and hydroxyethyl chitosan were prepared andpurified. Their location and degree of deacetylation, molecular weight weremeasured.2. Establishment of dry eye rat model, the cornea and conjunctiva were exposedto air by sutured upper and lower eyelids of the rats for six days in20℃roomtemperature and20%relative air humidity. The model was evaluated by Schirmer I test, breakup time of tear film(BUT), sodium fluorescein staining and pathologicaltissue section.3. To evaluate the the relaxation effect of carboxymethyl chitosan andhydroxyethyl chitosan on the successful rat dry eyes model. The eye drops weremade up from carboxymethyl chitosan and hydroxyethyl chitosan with appropriate pHand osmotic pressure. They were droped to the rat eyes three times a day. Variousindicators were inspected on the2nd day,4th day,7th day and14th day and evaluatedby Schirmer I test, breakup time of tear film(BUT), sodium fluorescein staining andpathological tissue section.4. Preliminary exploration of primary culture of rat conjunctival goblet cells: Theconjunctiva was stripped from eye bolls, and then the tissues were cultured in highsugar DMEM culture with10%fetal bovine serum. Cell emigration was observed andimmunofluorescence staining was used to identify the growth of conjunctival gobletcells.Results:1. The basic physical and chemical properties showed that the substitution ofcarboxymethyl chitosan and hydroxyethyl chitosan were both on C6-OH and thesubstitution degrees were0.98and3.11, the molecular weight were196kD and124kD.These provided basis for ophthalmology application of the two materials.2. The dry eyes model group which their cornea and conjunctiva were exposed toair by sutured upper and lower eyelids, compared with normal group, tear secretionwas decreased, tear film break-up time was shortened, and corneal epithelial cells hadpartial deletion. In the model group, the tissue section showed that the number ofgoblet cells on the the bulbar conjunctiva, fornical conjunctiva and palpebralconjunctiva reduced drastically. We could see that the dry eyes model was successful.It laid foundation for the research of chitosan derivatives on the effect of dry eyes.3. Two kinds of eye drops prepared with carboxymethyl chitosan andhydroxyethyl chitosan were in line with the basic requirements of eyes medication,their pH were7.31and7.39, osmotic pressure were319mOsm/kg and317mOsm/kg.With the continue use of of eye drops, changes of the tear buffer group in the ocular tear index saline was not significantly negative when compared with model group,corneal injury remained positive in14days, despite the tear secretion increase butonly about40%section recovered. The corneal injury in the sodium hyaluronategroup had been completely restored in14days, which tear secretion increasedsignificantly from the fourth day onwards, and had been restored to85%in7days.The effects of carboxylase chitosan group and hydroxyethyl chitosan group weresimilar to the effect of sodium hyaluronate group to improve dry eyes. Ocular tissuesections by HE staining, AB-PAS and MUC5AC immunofluorescence stainingshowed that sodium hyaluronate, carboxymethyl chitosan and hydroxyethyl chitosancould promote the repairment of corneal cells, as time goes on, the number of thegoblet cells in the bulbar conjunctiva, fornical conjunctiva and palpebral conjunctivaalso increased. These results indicated that carboxymethyl chitosan and hydroxyethylchitosan could relieve the symptoms of exposed dry eyes in rats, also hinted that thesesubstances could come into play by promoting the repairment of conjunctival gobletcells.4. From the primitive culture of rat conjunctival goblet cells, we could see asmall number of cells were emigrated from the edge of tissue after2days theyadhered and they increased gradually after4days. They were polygonal or fusiform.The change was not obvious5days later and some suspended dead cells appeared.Immunofluorescence staining of emigrated cells from the2nd day to7th day ofprimitive culture showed that with the passage of time, the growth of the goblet cellsshowed a trend of first increase and then decrease.Conclusions: Carboxymethyl chitosan and hydroxyethyl chitosan play an importantrole to ease dry eyes and it laid a foundation for the medical research of VDT-led dryeyes.