Effects Of Minocycline On Expression Of Activated In Retinal Ganglion GDNF And VEGF Cells Of The Optic Nerve Injured Rats

Objective: Optic nerve injury is a common ocular trauma type, can leadto severe visual impairment and even complete loss of vision, is a difficultproblem to be solved urgently in the field of Department of Ophthalmology.Past that no regeneration after optic nerve injury, and in recent years, studiesshow, about50%with optic nerve injury were different degrees of recovery ofvisual acuity after optic nerve injury, show that under certain conditions can berecycled. Therefore, the development of imminent to find a safe and effectivemedicine to delay the damage to the optic nerve lesion. Minocycline is asecond generation semi-synthetic tetracycline antibiotics, recent studies haveshown, minocycline (Minocycline, MC) has a protective effect on thedamaged neurons is strong. The study of the central nervous system, it hasanti-inflammatory, anti-apoptotic role in degeneration and other diseases ofthe nervous system, through inhibition of microglial cells and proapoptotic,neurotoxic effects of glutamate antagonists, so as to improve the survival rateof neurons, play a role in protecting neurons. Animal model studies haveshown, the peak plasma concentration of minocycline can reach50%in thebrain, so in many nervous system diseases, such as cerebral ischemia,traumatic brain injury, amyotrophic lateral sclerosis, Huntington’s disease andother cultured retinal diseases have neuroprotective effect of certain showedthe model of central nervous system diseases and in vitro. It is proved bypractice, long-term oral minocycline without obvious adverse reaction, safeand effective. Although minocycline in central nerve protection research isincreasing, but after optic nerve injury, minocycline could play aneuroprotective effect and action mechanism is not clear, and in theDepartment of Ophthalmology, optic nerve protection aspects of the report isvery rare. This experiment by using optic nerve crush model of optic nerve injury model of rats, and intraperitoneal injection of a certain dose ofminocycline to intervene, the application of computer image analysis andimmunohistochemistry on retinal ganglion cells and glial derived neurotrophicfactor (Glial cell line-derived neurotrophic factor, GDNF) and vascularendothelial growth factor (vascular endothelial growth factor, VEGF) weremeasured by semi quantitative expression, protective effect of minocycline onretinal ganglion cells, so as to provide experimental basis for the clinicaltreatment of optic nerve after injury.Methods: Male SD rats of clean grade50(provided by the experimentalanimal research center of Hebei Medical University), body weight (200±10)g, adaptive feeding5D check eyes, refractive interstitial Qing, pupil big, round,acute to reflection, fundus no abnormalities were randomly divided into threegroups, groups: group I,10rats in the blank group, II group normal salinecontrol group20, group III, minocycline treatment group with20rats. II, IIIgroup were treated with optic nerve crush the left optic nerve injury modelwas made (referred to as group II model eye, group Ⅲmodel eye, right eye)for its3(referred to as group II control eyes, group Ⅲcontrol eyes), afterdirect ophthalmoscopy, fundus red light reflection is not normal, ischemia, noeye outstanding, eyelid closure completely is success model, the right not toprocessing. Experimental animal10%chloral hydrate (35mg/kg)intraperitoneal injection of anesthesia and routine aseptic processing, cut openthe temporal bulbar conjunctiva sclera edge ring, blunt separation of the opticnerve was exposed in the orbit, with forceps clip the ball after calibration forcein2mm by optic nerve, injury time is20s with l0-0nylon suture, conjunctiva2needles, conjunctival sac with ofloxacin eye ointment. The minocycline group,saline group in optic nerve crush1h before and after crush injury of each Dwas intraperitoneally injected with Mino ring45mg/kg, normal saline, normalcontrol group without special treatment. After injury,1d,3d,7d,14d,28dparaformaldehyde perfusion of rats, removed immediately left eye, serratededge0.5mm place cut open the eye wall, anterior segment and vitreousremoval. With the volume fraction of4%paraformaldehyde fixation,4 ℃r efrigerator overnight, eye cup the whole layer of paraffin embedding,preparation of5~7μm retinal slice, microscope image acquisition andsynchronization. To observe the histological changes of the retina HE staining.Application of image analysis system (*400) to analyze the results (eachsection4view, at a distance of papillary1.5mm retina were beginning to takea view on all sides of the1eyeballs, each2sections). Methods GDNF wasdetected by immunohistochemistry semiquantitative, computer image analysissystem was analyzed, and the positive reaction in the retina of parts withmultifunction true color cell image analysis, obtains the average opticaldensity value. Application of SPSS13.0statistical software, the average opticaldensity and the average number of positive cells was expressed by±s, singlefactor variance analysis with the data at different time points within a group(One-way ANOVA), at the same time, the data of two sample t test betweendifferent groups, P<0.05had significant difference, P<0.01differencestatistical significance.Results:1retinal ganglion cells hematoxylin-eosin staining results: Change1.1retinal ganglion cell morphology: blank control group rat retina wasroughly3layer are arranged in parallel, divided into the ganglion cell layer,bipolar cell layer and photoreceptor, retinal ganglion cells in the inner retina,cell monolayer, dense, cells arranged in uniform. Normal saline control groupwith time after injury, rat retinal ganglion cell layer nuclei were arranged indisorder, vacuolization degree enhancement, bipolar cell layer nucleus numberdecreased in different degree. In comparison, the same time point ofminocycline treatment group rat retinal ganglion cells arranged relatively neat,vacuolization degree is relatively weak, bipolar cell layer saturated relativequantities.1.2the number of retinal ganglion cells change: normal saline control grouprats were1,3,7,14,28sky mirror every x400visual field mean retinalganglion cell number were:23.6±2.07,18.2±0.84,12.2±1.30,7.4±2.19,3.6±1.51, Meno Soji therapy rats in the1groups,3,7,14,28sky mirror every x 400visual field mean retinal ganglion cell number were:26.4±2.07,21.8±2.59,15.4±1.14,11.8±1.64,7.6±1.81, at the same time the number ofretinal ganglion cells in treatment group was higher than that of the controlgroup, there was statistically significant difference in3days time points (P <0.05), there was significant at7,14,28days time difference (P <0.01).2The detection results of semi-quantitative retinal GDNF and VEGF immunegroup2.1GDNF expression in retina of rat retina: GDNF mainly expressed in thecell body, brownish yellow. The saline control group, GDNF expression ofretinal ganglion cell layer of the average optical density of1,3,7,14,28daysrespectively:0.2323±0.0024,0.2804±0.0040,0.2962±0.0022,0.3401±0.0021,0.2830±0.0027, minocycline treatment group of retinal ganglion celllayer of the expression of GDNF1, the average optical density value3,7,14,28days respectively:0.2348±0.0036,0.2870±0.0041,0.3016±0.0022,0.3506±0.0060,0.2882±0.0023, after optic nerve crush in two experimentalgroups were GDNF positive expression, physiological saline control groupshowed weak positive; minocycline treatment group, the expression of GDNFis rising,14d expression was strongly positive; saline control group was alsoincreased with time, but14d expression were the treatment group is notobvious; the same time points for the positive rate of group GDNF was higherthan that of the control group, the difference had statistical significance.2.2VEGF expression in retina: the retina of VEGF rats in each layer of cells,cytoplasmic expression, brownish yellow. The saline control group, VEGFexpression of retinal ganglion cell layer of the average optical density of1days,3days,7days,14days,28days respectively:0.2266±0.0013,0.2276±0.0011,0.2909±0.0044,0.3209±0.0033,0.3427±0.0011; the averageoptical density of minocycline treatment group by the expression of retinalGod festival the cell layer of VEGF values of1days,3days,7days,14days,28days respectively:0.2271±0.0017,0.2287±0.0007,0.3090±0.0105,0.3256±0.0021,0.3488±0.0010. Minocycline treatment group and normalsaline control group, the expression of VEGF showed an upward trend, first,3, 7days of saline group compared with minocycline treatment group theaverage optical density values were not significant (P>0.05), startminocycline treatment group the average optical density of VEGF value ishigh from fourteenth normal saline control group day, the difference wasstatistically significant (P<0.05),28days time, average optical density ofvascular endothelial growth factor expression values than the saline controlgroup, the difference was statistically significant (P<0.01).Conclusion:1minocycline could reduce the optic nerve and the apoptosis of retinalganglion cells after optic nerve injury, protective effect.2minocycline can enhance after optic nerve injury early retinal glial cellline-derived neurotrophic factor (GDNF) expression, has the strongprotective effect on injured retinal ganglion cells.3minocycline can enhance the retinal optic nerve injury of vascularendothelial growth factor (VEGF) expression, and thus play a protective effectof retinal ganglion cells.