New Type Of Optic Nerve Protection Drug Experimental Research Of Local Drug Delivery System

Background Glaucoma is a group of threat and damage to the opticnerve visual function and optic nerve atrophy and visual fielddefect is a common feature of the disease, is one of the mainblinding eye disease, its dangerous, dangerous, quick, the worldhealth organization has put it as a second blinding eye disease[1],the treatment of glaucoma, including medicines, laser and operation,its main goal is to reduce the intraocular pressure, reduce theincreased intraocular pressure and the intraocular pressurefluctuation caused by visual impairment, but simply loweringintraocular pressure is not enough to prevent glaucoma optic nervecaused by progressive damage and other pathological mechanism arealso likely to lead to retinal ganglion cells (retinal ganglioncells, RGCs) and optic nerve injury. Glaucoma in traditionalChinese medicine belongs to the "five winds cataract" range, thelate may also belong to the "green blind","visual deep faint toescape", etc. Attack on its basic pathogenesis is due to wind fireleader, or qi depression, spleen wet fire ShengTan caused liverdepression, phlegm, fire attack, courage lively, phlegm YuHua fire fire, Yin and Yang sheng, the disorder of qi activity, etc. Variouskinds of reasons, led to estrangement of qi and blood, meridians,xuan fu in the eyes closed, and qi stagnation and blood stasis, thegod of water stasis, alongwith the disease. Disease mainly in theliver, spleen, kidney. Treating glaucoma mainly by lowering, XieYu,invigorate the circulation of traditional Chinese medicine, water,wind, begin to understand, and qi, t2dm, late to nourishing liverand kidney, and liver XieYu method such as[2].With modern medicinethinks because of mechanical damage, ischemic injury causeoxidative damage of retinal ganglion cells death and optic nerveaxons declining almost the same[3]. With drugs or other means so thatthose who has not been damaged, only partly damaged, even is in atoxic environment near death in RGCs are alive and continue toextend the survival time, the new research focus in the worldglaucoma medication-optic nerve protection; However the existingoptic nerve protection drugs in clinical treatment effect is notideal, the reason may be the local effective drug in the retina,optic nerve concentration is low, leading to poor curative effect.Current drug dosing mode optic nerve protection, including oral,intravenous injection and muscle injection, etc., are all throughthe systemic circulation before they can reach the eye tissues, notonly unable to therapeutic effective concentrations are achieved,also increase the systemic side effects from drugs. Thereforesought to make a neuroprotective drugs more effectively, localapplication in the new dosing method of RGCs and optic nerve, becomethe urgent needs of clinical therapy.Objective Retinal ganglion cell apoptosis lead to optic nervedamage, is the core of today’s glaucoma treatment has yet to conquer the difficulties. Current dosing way of clinical treatment effectwas not significant and systemic side effects of big faults.Ultrasound microbubble is a new technology, targeted release drugsby drug in the body positioning release technology, not only cansignificantly reduce drug dosage, reduce drug adverse always use,at the same time can increase the drug concentration in lesion areaand delay the drug release, reduce dosing frequency, to enhance theefficacy of treatment. Research has confirmed that the whitethistle saponins (gross saponins from Tribulus terrestris L, GSTT)with injectable Mouse Nerve Growth Factor (Mouse Nerve GrowthFactor for Injection, mNGF) can significantly reduce the apoptosisof retinal ganglion cells enhance their survival, have the effectof treatment of optic Nerve damage. This topic proposed for thefirst time to develop a new type of ultrasound mediated microbubbleeye local drug delivery system, and studies the ginseng saponinsRg1(Ginsenoside Rg1) effects on retinal ganglion cells. This studythrough experiments in vitro safety and is suitable for ultra soundintensity, irradiation time, concentration of microvesicles andultrasound microbubble mediated drug antiapoptotic mechanisms ofretinal ganglion cells. The drug delivery system and three kindsof single dose of drug application, can effectively improve theretinal local drug concentration, reduce the systemic side effects,delay or prevent the occurrence/development of glaucoma optic nervedamage, brings more effective treatments for glaucoma patients toreduce the blindness rate, comes and brings new hope for patients.Methods:1.Newborn SD rat retinal ganglion cells in vitro primaryculture and identification.2.The N-methyl-D-aspartate (NMDA) on the original generation of retinal ganglion cell apoptosis of RGCs cell lines. Bytetrazolium salt colorimetry to detect cell survival rate：①Determination of different concentrations of NMDA (1000μM、100μM、10μM) effects on RGCs cell survival rate，select NMDA buildingconcentration.②According to the step①the selected NMDAconcentration by tetrazolium salt colorimetry to detect cell in30min,24h,48h survival at three different time points.3.Screening of GSTT (10-5μg/ml、10-4μg/ml、10-3μg/ml、10-2μg/ml),injectable mNGF (6×10-2μg/ml、8×10-2μg/ml、10×10-2μg/ml、12×10-2μg/ml), Ginsenoside Rg1(100μg/ml、101μg/ml、102μg/ml、103μg/ml) of three drugs against RGCs apoptosis in the besteffective concentration.Calculation of cell survival rate at thesame time to determine these three drugs to protect RGCs optimalmass concentration (experiment cell survival rate relatively thehighest of the three kinds of drug concentration).④Determinedby MTT colorimetric determination of different concentrations ofmicrovesicles (22.5μg/ml、45μg/ml、90μg/ml), different volumeratio of microvesicles solution (1%,5%,10%) of RGCs cell survivalrate, to observe different concentration and volume ofmicrovesicles on RGCs cell growth activity.4. Normal rabbit eye glass body cavity injection Rg1ginsenosidesin the retina, optic nerve safety studies.12only healthy NewZealand white rabbit, randomly divided into4groups, each group3(6eyes). In experimental group is as follows: A group of normalcontrol group0.1ml glass cavity of0.9%saline injection; GroupB glass body cavity injection of low dose (0.05mg/kg) Rg1ginsenosides, group C glass body cavity injection dose (0.5mg/kg)in Rg1ginsenosides, group D glass body cavity injection of high dose (2.5mg/kg) Rg1ginsenosides. Within1week,2weeks after theinjection medicine,4weeks respectively behavior observation,Accupen handheld tonometer daily measuring intraocular pressure,with slit lamp microscope and direct ophthalmoscope, eye Bultrasonic, anterior segment photography, fundus photography, OCTvivo observation section and fundus changes in rabbit eyes eyes,air embolism executed after4weeks of New Zealand white rabbit,retina, optic nerve line of optical microscope and transmissionelectron microscope morphology and ultrastructure of the retina,optic nerve. Glass body cavity injection medicine treat once every3days, a total of28days,28d executed rabbits out.5. Glass body cavity injection Rg1ginsenosides in rabbit eyes afterobserved by ultrasound irradiation micro bubble intervention andwithout ultrasonic irradiation micro bubble intervention, at thesame time point comparison of different eye tissues drugconcentration and different time points in the same eye tissueconcentration of drug.48only healthy New Zealand white rabbit,the experimental group are as follows: left eye glass body cavityinjection (2.5mg/kg) ginseng saponin Rg10.1ml, right eye glassbody cavity injection (2.5mg/kg) ginseng saponin Rg10.1ml afterinjection (45mu g/ml) sound novy0.1ml, closed after the injectionof rabbit eyes with coupling agent, the ultrasonic probe in the eyewith a frequency of1MHZ, immediately above the sound intensityis0.5W/cm2illuminate eye60s, each time point6New Zealand whiterabbit, at30min after drug infusion,1h,2h,3h,4h,6h,12h,24h by air embolism executed rabbit, respectively in aqueoushumor and vitreous body, retina detached, optic nerve tissue forhigh-performance liquid chromatography (HPLC) testing. 6. Targeted ultrasound break the cell mediated optic protectionexperimental study drug in treatment of glaucoma optic nerve damage.Anterior chamber injection of0.3%compound carbomer solution isadopted to establish the rabbit animal model of chronic highintraocular pressure optic nerve damage. Healthy New Zealand whiterabbit24, were randomly divided into8groups: blank control group1(0.1ml glass cavity of0.9%saline injection),2high intraocularpressure model group (do not do processing),3high intraocularpressure model group+glass body cavity injection of ginsengsaponin Rg1groups (0.1ml glass cavity injection of ginseng saponinRg1solution),4high intraocular pressure model mNGF+glass bodycavity injection group (0.1mlmNGF solution glass cavity injection),5high intraocular pressure model group+glass body cavityinjection of ginseng saponin Rg1+ultrasound group (0.1ml glasscavity injection of ginseng saponin Rg1solution after giving soundintensity is0.5W/cm2irradiation60s),6high intraocularpressure model+glass body cavity injection mNGF+ultrasound group(0.1mlmNGF glass cavity injection solution after giving soundintensity is0.5W/cm2irradiation60s),7high intraocularpressure model group+glass body cavity injection GSTT+microbubble ultrasound group (glass cavity injection respectively0.1and0.1ml Rg1ginsenosides solution ml cell suspension liquid,then give the sound intensity is0.5W/cm2irradiation60s),8highintraocular pressure model group+glass body cavity injection mNGF+microbubble ultrasound group (glass cavity injection respectively0.1and0.1ml mlmNGF solution cell suspension liquid, then givethe sound intensity is0.5W/cm2irradiation60s). Each processingonce every three days, a total of28days,1week,2weeks after the injection drug,4weeks respectively behavior observation,Accupen handheld tonometer intraocular pressure measurement, withslit lamp microscope, eye B ultrasonic, anterior segment, liveobservation of rabbit eyes at present, the change of the section,air embolism executed after4weeks of rabbit, rabbit retina andoptic nerve, line of rabbit retina pathological section, usingmeasuring retinal thickness, retina and optic nerve transmissionelectron microscopy (sem) test, test the content of NO, MDA, SODretina.Result:1. Got more purified rat retinal ganglion cells, and by cellimmunochemical examination, the purity of more than90%.2. NMDA cells for in vitro culture of rat RGCs excitatory toxiceffect is dose dependent, add10μM、100μM、1000μM NMDA after30min, RGCs survival rates were91.46%,64.74%and50.02%respectively. Concentration with the normal control group of100microns or more the RGCs survival rate have obvious difference (P<0.05). Join100mu M NMDA role respectively for30min,24h,48h survival rates are63.3%,55.19%and63.3%respectively, as theextension of time, the RGCs survival rate decreased significantly;With increasing doses, prolonged, RGCs of survival. Therefore,select100microns effect30min to follow-up experiments.3.Determined by MTT assay showed that30min after NMDA processingof RGCs cell survival rate were decreased, cell survival rate plusdrug group were increased the NMDA group, including10-4μg/ml GSTT+NMDA group,8×10-2μg/ml mNGF+NMDA group,102μg/ml of GinsenosideRg1groups, compared to other dose group could increase the cellsurvival rate, selected the corresponding concentration forsubsequent experiments. 4.Rg1ginsenosides can suppress the apoptosis of retinal ganglioncells induced by NMDA, promote the survival of the in vitro culturedRGCs, protect retinal neurons. Tetrazolium salt (determined by MTTcolorimetric test showed that30min after NMDA processing of RGCscell survival rate were decreased, and cell survival medicine groupwere improved the NMDA injury group, including10-4mu g/ml GSTT+NMDA group,8x10-2mu g/ml mNGF+NMDA group,102mu g/ml Rg1ginsenosides+NMDA group, relative to other dose group couldimprove the cell survival rate. Three kinds of drugs can promotethe survival of retinal ganglion cells in vitro, has significantoptic nerve protective effect.5. Pure vision medicine group and the glass body cavity medicineinjection by ultrasound irradiation after micro bubbleintervention group, two different methods of rabbit eyes visionwithin24h after injection of Rg1ginsenosides, in addition to thegroups in the vitreous Rg1ginsenosides content gradient descent,the peak time for15min, the rest of the eye tissues (aqueous humor,retina, choroid, optic nerve) ginseng saponin content of Rg1correlation curve changes. Its up to the peak time for aqueous humorand internal organization with retinal choroid at first peak, bothfor3h, and the outer wall of eye tissue of peak time late, theoptic nerve is4h. Will simply body cavity medicine injection groupand the glass body cavity medicine injection by ultrasoundirradiation after micro bubble intervention group organizationsRg1ginsenosides in rabbit eyes of peak levels, by ultrasoundirradiation microbubble intervention group organizations Rg1ginsenosides peak levels were significantly higher than that ofpure glass body cavity medicine injection group, with statistical significance (P <0.05or P <0.01), suggesting the glass body cavityinject drugs by ultrasound irradiation micro bubble after theintervention is relatively simple glass body cavity medicineinjection can reach high drug concentration in each organizationin the eye. Move each each peak QiuBi organization Rg1ginsenosidescontent comparison, found that the glass body cavity injection Rg1eye organizations ginsenosides content distribution for vitreous> retina detached> optic> aqueous humor. Except vitreous internalcause direct injection Rg1ginsenosides result in high drugconcentration, the retina and optic nerve is the most can obtainideal drug content organization, so the glass body cavity injectionof ginseng saponin Rg1section is to ensure that eye after treatment,especially the retina nerve protective effect of effective drugconcentration method. Ultrasonic irradiation micro bubble on thebasis of further improve the drug use.6. Rabbit anterior chamber injection of0.3%compound carbomerchronic high intraocular pressure model is a better solution method,successful glaucoma optic nerve retinal damage model is established.Rabbit retina using morphological observation:1set of rabbitretinal ganglion cells in monolayer, cell sizes and irregularcontour, nuclear hyperchromatism, cell borders is clear, neat cellsclosely, stratified clear; Number2group model of rabbit retinalganglion cells decreased significantly, the layers of loosestructure, disordered arrangement, is no clear boundary betweenlayers and retinal ganglion cell layer in a large number of vacuoleschange, some cells serious degeneration and karyolysis, shallowlight nuclei and cytoplasm staining shallow light, nuclearstructure sparse, nucleolus pyknosis and offset, cytoplasmic chromatin edge set, endoplasmic reticulum swelling, mitochondriaswelling and cristae disappear, vacuoles degeneration.3,4,5,6groups of rabbit retinal ganglion cells number less, the rabbitretina to be relatively complete, mild disorder, layered relativelyclear, visible in cavity of cell;7,8groups of retinal layersstructure is complete, the arrangement is neat, roughly normalmorphology, inner and outer nuclear layer appear a small amount ofcell loss, partial sample had mild vacuoles change. Measuring thethickness of the rabbit retina, each group of rabbit retinalthickness differences were statistically significant (P <0.05);2rabbit retinal thickness significantly thinner than1group, thedifference was statistically significant (P <0.05);3,4,5,6groups of rabbit retinal thickness thickness obviously in twogroups, the difference was statistically significant (P <0.05);7,8groups of rabbit retinal thickness is very apparent thicknessfrom3,4,5,6groups, the difference was statistically significant(P <0.05). Rabbit retina ultrastructure observation: group1normal retinal ganglion cell nuclei were round, clear RGC cellnuclear membrane, dyeing uniformity, nucleolus, inner and outernuclear layer of nuclear chromatin color clear, uniformdistribution, endoplasmic reticulum, mitochondria and otherorganelles normal form, the cell membrane integrity, neatphotoreceptor cells.2model group photoreceptor cellsmitochondria swelling, bubble and different level; Inner and outernuclear layer structure disorder; Reduced Numbers of RGC, swellingcolor light.3,4,5,6groups of RGC within the nucleus ofuniformity, color pale euchromatin, photoreceptor cells arrangeddisorder, slightly not seen obvious swelling of mitochondria and vacuoles of samples, a few cell chromatin edge set, the kernel layerpart slightly deformed, nuclear chromatin is shown slightly thin,the color is a bit light, mild mitochondria vacuoles degeneration,cristae mitochondria, endoplasmic reticulum only mild swelling.7,8group not seen obvious necrosis and apoptosis of retinal neurons,RGC chromatin distributed equably, obvious nucleolus, basic normalrough endoplasmic reticulum, mitochondria, change is not obvious,the photoreceptor outer segment order plate membrane, no obviousdissolved. Rabbit optic nerve ultrastructure observation:1set ofnormal rabbit optic fiber thicknesses, myelin integrity, closelypacked, the rules,"hair" shaped, axons surface smooth, plate ofthe axon myelin layer density, optic nerve myelin integrity,axoplasmic evenly, can be seen in the axoplasm clear normalmicrotubule, microfilament and oval mitochondria;2model groupcommon lesions, nerve fibers visible axon disappear, the disorderedarrangement of myelin sheath, segmental release, dissolved,shrinking clumps or pulp degeneration, gliosis, microtubules andneural structure of microfilament disappeared, swollenmitochondria and vacuoles degeneration;3,4,5,6groups of opticnerve myelin is relatively uniform, part of the myelin sheath thin,loose "onion skin" shaped, axons within microtubules and actinfilaments swollen, but not disappeared, some mitochondria vacuolesdegeneration;7,8groups of optic nerve myelin is arrangedrelatively dense, myelin integrity, rules, no demyelinatingphenomenon, visible microtubule, microfilament, axon structureclose to normal. Rabbit retina tissues of NO, MDA, SOD contentdetection: two groups of rabbit retina of NO, MDA content increasedsignificantly in the1group, SOD activity significantly reduced in the1group, are statistically significant differences (P <0.05).3,4,5,6,7,8groups of NO, MDA content in two groups decreasedobviously, SOD activity is significantly increased in the2groups,with significant statistical difference (P <0.05).Conclusion RGCs conclusion SD rats in vitro, and RGCs is purified,it is a kind of ideal model of the cell culture experiments. NMDAof RGCs excitatory toxic effect, can be induced RGCs apoptosis;White thistle saponins, injectable mouse nerve growth factor andginseng saponin Rg1against the RGCs apoptosis induced by NMDA,protect retinal ganglion cells, enhance the role of the active;Glass body cavity injection of2.5mg/kg of Rg1ginsenosides, noobvious side effects on rabbit retina, optic nerve, is safe. Rabbitanterior chamber injection of compound carbomer solution is a kindof ideal rabbit high intraocular pressure model method. Compoundcarbomer induced rabbit model of glaucoma has cause elevatedintraocular pressure medium, stability, long duration and methodof Jane Sheet, the advantages of easy operation and control, canbe used for the study of glaucoma optic nerve sex retinal damageand glaucoma drugs research, is a kind of ideal of the rabbit modelof glaucoma. Targeted ultrasound can damage the cell targetedrelease drugs, strengthen drug to the concentration of the eye,ultrasonic break the cell mediated optic protection drug Rg1ginsenosides and mNGF therapy high intraocular pressure of rabbitretina, optic nerve damage is effective. Ultrasound microbubblemediated optic protection drugs by changing the retina in the amountof the expression of NO, MDA and SOD in play a role of protectionof high intraocular pressure rabbit optic nerve injury.