| Optic nerve of adult mammal is one part of central nervous system. Glaucoma, eye trauma and so on can cause optic nerve trauma. Absence of essential inner surround may cause that optic nerve cann't regenerate itself and loss of vision function. Regeneration of optic nerve after trauma is always a problem for all over the world. To make optic nerve regenerate is communicate with several problem, such as regeneration central nervous system and principle process of nerve. The research of reparation of optic nerve is important to treatment of optic nerve trauma and central nerve system trauma. Researchers have found that len trauma may cause the elevation of expression of growth associated protein in retinal ganglion cells. It may be caused by assemble of macrophages near the len. Macrophage derived factors may cause the activation of retinal ganglion cells and densenss of growth cone of retinal ganglion cells. It confirms that macrophages might be important for the survival and axon regeneration of retinal ganglion cells.Nasal-eye disease is important for both opthalmologist and otolaryngologist. Professor demin Han and jiqun Wang have made improvement of the domain.Zymosan A is ingredient of cell wall of fungi. It can activate powerfully macrophages.Three labs reported the gene of neurite growth inhibitor, Nogo gene. Nogo protein coded by Nogo gene has three kinds of isomers, etc NogoA, B, C. NogoA is most important for restriction of axon regeneration. Discovery of Nogo gene is of extremely importance for treatment of trauma of central nerve system, including optic nerve.Our research include five parts: to establish a optic nerve crush injury model in Wistar rats and interpret the methods of surgery in detail; to investigate effect of factors derived by activated macrophage on RGC survival of retinal ganglion cells after optic nerve crush; to investigate the effect of macrophage conditioned medium on the survival and axon growth of retinal ganglion cells; to investigate effect of activated macrophage on axon regeneration after optic nerve crush; to investigate the expression and distribution of Nogo-A mRNA in retina and optic nerve of normal,injuried and regenerated adult rat.Part â… To Establish a Model of Optic Nerve Crush Injury in Rats1.PurposeTo establish a optic nerve crush injury model in Wistar rats and interpret the methods of surgery in detail.2. Methods2.1 Ninety male Wistar rats were randomly divided into two groups.Fifteen rats were in normal control group.Experimental group,partial optic nerve injury group were divided into five groups according to the sacrificing date.2.2 Partial optic nerve injury model was induced in the left eyes by a special designed optic nerve clip with 40gram power for 9 seconds at optic nerve 2mm behind the eyeball.The right eyes served as the control.All the rats received 3% fluorogold injected into superior colliculi to label the retinal ganglion cells(RGCs) 7 days before sacrifice. The labeled RGCs photos were counted by a computerized image analyzer.2.3 The optic nerve sections were longitudely cut and investigated under microscope.3. Results3.1 In control group, the labeled RGCs of left eyes were 195.76+36.12,the right eyes' werel97.52±39.25, r=0.823,/?>0.05.3.2 In experimental group, 1 day after injury the labeled RGCs of left eyes were 165.12 + 26.36, the right eyes' were 191.21 +35.26, t=3.125,p<0.01;3.3 the labeled RGCs of left eyes were 152.26+25.12,the right eyes' were 192.16 + 32.12 in 3 days after injury ,t=3.157,p<0.01;3.4 7 days after injury the labeled RGCs of left eyes were 135.19 + 21.32,the right eyes' were 189.26+26.16,/=^.2/6,jp<0.07;3.5 15 days after injury the labeled RGCs of left eyes were 123.96+27.19, the right eyes' were 191.76 + 25.29,f=5J52,/?0.05) .3.9 As time past ,it is revealed that optic nerve injury is gradually evident.4. Conclusion4.1 We find that there is not significance between right eyes and left eyes.4.2 Using 40 gram power to clip the optic nerve can create optic nerve crush injury model in rats.4.3 Results show that RGCs may decrease as survival time lasts. There is notable significance between that of crush group and control group. It may be useful to study the pathological changes of optic nerve injury and how to repair and regenerate it.Part II Effect of activated macrophage on protection of retinalganglion cells after optic nerve crush1. PurposeTo investigate effect of factors derived by activated macrophage on RGC survival of retinal ganglion cells after optic nerve crush.2. Methods2.2 Adult male Wistar rats were randomly divided into normal control,crush control,medium treatment and zymosan treatment groups,and 60 .Right eye of each rat served as experimental group,while left eye as control group.2.3 All the rats were retrogradly by injecting fluorogold into superior colliculi of both sides.2.4 Partial optic nerve injury model was induced in the left eyes by a special designed optic nerve clip with 40gram power for 9 seconds at optic nerve 2mm behind the eyeball except for normal control group.2.5 The labeled RGCs photos were counted by a computerized image analyzer.2.6 Experimental group received intravitreal injections of Zymosan ,while medium group received intravitreal injections of PBS.3. Results3.1 The rates of RGC of crush group were 92.6%,82.9%,72.6% and 57.6% of normal controls on the third,7 th, 15 th and 21 day, respectively.3.2 The numbers of RGC labeled with fluorogold of zymosan group and PBS group decrease as time past (^=25.73.29.47, P<0. 01) .3.3 In the group with zymosan injection, the numbers of RGC were much higher than that of controls on the third,7 th,15 th and 21 day( (^4.85,5.08,18.29,8.31, P<0. 01) .3. 4 In the group with zymosan injection, the numbers of RGC were much higher than that of crush group (^=28.32,49.91,42.25,49.55, P<0. 01) . In the group withzymosan injection, the numbers of RGC were much higher than that of PBS group(^=29.42,48.39,41.93,48.12, P<0. 01)3.6 In the group with PBS injection, the numbers of RGC were much lower than that of control group (<7=15.42,31.77,37.24,29.74, P<0. 01), and similar to that of crush group (9=2.29,1.40,2.31,1.54, P>0. 05)4. Conclusion4.1 There is notable significance between RGCs labeled with fluorogold of crush group and control group.4.2 There is notable significance between RGCs labeled with fluorogold of zymosan group and PBS group.4.3 Intravitreal injections of Zymosan can activate macrophage.4.4 RGCs of crush group, zymosan group and PBS group decrease as time past.4.5 Macrophage dereived factor might protect RGC after optic nerve crush and enhance their survival.Part IE Effect of macrophage conditioned medium on the survival and axon growth of retinal ganglion cells1. PurposeTo investigate the effect of macrophage conditioned medium on the survival and axon growth of retinal ganglion cells.2. Methods2.1 Macrophage conditioned medium was centrifugaled for ten minutes after the filtrated zymosan was added into the medium when macrophages was cultured for one hour. The percentage is 25 pi macrophage conditioned medium per 100 u 1 mediumof retinal ganglion cells.Half of the medium of retinal ganglion cell was replaced after cultured twenty hours, and Brdu which was added into can restrain the growth of non-nervous cells.The cells were investigated under phase-contrast microscope everyday.2.2 The cells cultured were immunostained with Thy 1.1 antibody after they were fixated with 4% paraformaldehyde.MTT were added into medium to evaluate A numerical value.2.3 The axon growth of retinal ganglion cells was analyzed with image analyse system.3. Result3.1 The growth of macrophages:Zymosan can be seen in the macrophages after zymosan was added into the medium of macrophage for twenty-four hours.3.2 The survival and growth of retinal ganglion cellsRetinal ganglion cells begin to adhere to the wall, and all of them adhere to the wall after they were cultured for twenty-four hours.The cells which were round or ellipse arrange with single layer.The axon begin to grow after retinal ganglion cells were cultured for twenty-four hours, and connect with each other after cultured for three days, and a few cells live after cultured for seven days.There is notable significance of RGCs between experimental and control groups (F=39.55,46.72,JP<0.01)3.3 The survival of retinal ganglion cellsThere is significant influence between the axon length cultured for seven days and that of three days^ <0.01).There is significant influence between experimental group and control group(P <0.01)o3.4 The length of axon of retinal ganglion cells was analyzed with image analyzer system.There is significant influence between the axon length after cultured for three days and that of one day(P <0.01).There is significant influence between the axon length cultured for seven days and that of three days(P <0.01).3.5 To evaluate the purity of retinal ganglion cellsThe retinal ganlion cells often fields which immunostained were selected randomly to investigate under phase-contrast microsope.The ratio of immunostained positive cellswas94.7%±5.62%o4. Conclusion4.1 We establish the system of cultured retinal ganglion cells successfully. It is possible to purify the cultured retinal ganglion cell for long term. It is important to make foundation for research of physiology and pathology of cultured retinal ganglion cells.4.2 Zymosan can activate cultured macrophages.4.3 Macrophage conditioned medium can improve survival and axon growth of cultured retinal ganglion cells. There is notable significance between experimental and control group.It is important for establishment of cultured system of retinal ganglion cells to explore the principle of traumatc diseases of retinal ganglion cells.Part IV The effect of activated macrophage on optic nerve regeneration after optic nerve crushl.PurposeTo investigate effect of activated macrophage on axon regeneration after optic nerve crush.2. Methods2.1 Adult male Wistar rats were randomly divided into groups according to the sacrificing date. Right eye of each rat served as experimental group, while left eye as control group. Partial optic nerve injury model was induced by a special designed optic nerve clip with 40gram power for 9 seconds at optic nerve 2mm behind the eyeball. Experimental group received intravitreal injections of Zymosan , whilecontrol group received intravitreal injections of PBS.2.2 Frozen section were immunostained to investigate GAP-43 and ED-1 byimmunofluoroscence.3. Result3.1 Numerous ED-1-positive macrophages were seen in the vitreous and along the inner retinal surface in every case both in experimental and control group.3.2 RGCs and optic nerve showed an intense upregulation of GAP-43 in their somata in experimental group, while few GAP-43 in control group.3.3 There is notable significance of area of positive stained cells in retina among experimental and control groups ( F=23. 64, 13. 98, P<0. 01) . Expression of GAP-43 in retina may be the most three days after crush, then decrease as time past.3.4 Much expression of Gi\P~43 can be found along optic nerve of experimental group. There is notable significance of area of positive stained cells in optic nerve among experimental and control groups (f=49. 21, 36. 90, P<0. 01) . Expression of GAP-43 in optic nerve may be the most three days after crush, then decrease as time past.4. Conclusion4.1 Macrophage can be activated by intravitreal injections of zymosan.4.2 Activated macrophages might enhance GAP-43 expression after optic nerve trauma.4.3 Our experiment show that activated macrophages might enhance axon regeneration after optic nerve crush.Part V Expression of Nogo-A mRNA in retina and optic nerve of normal, injuried and regenerated adult rat1. PurposeTo investigate the expression and distribution of Nogo-A mRNA in retina and optic nerve of normal,injuried and regenerated adult rat.2. Methods2.1 Ninety male Wistar rats were divided into two groups. Right eyes of first group were control group. Left of them were crush group and were crushed with optic nerve crush forcep for 9 seconds 2mm behind the eyeball. Right eyes of the second group were injected PBS into vitreous after crush. Left of them were injected zymosan into bitreous after crush. All groups was divided into three subgroups according to sacrificing time.2.2 To seperate the eyeball and optic nerve.2.3 The frozen sections of retina were longitudely cut. To detect the the expression and distribution of Nogo-A mRNA in retina of ninety healthy adult ratwith in situ hybridization. Sections of retina were randomly selected and photos were counted by a computerized image analyzer.3. Result3.1 The expression of Nogo-A mRNA can be seen in retinal ganglion cells layer ^ inner nuclear layer and outer nuclear layer.3.2 The area and A value of positively stained cells for Nogo-A mRNA expression in retina and optic nerve was increased during 1 day to 3 days after optic nerve crush and decreased during 3 days to 7 days after optic nerve crush.There is notable significance of area of positive stained cell between experimental group and control group(/=3.82,4.21,4.07; PO.01) , so as A value (/=3.90,4.52,3.78; PO.01).3.3 Both of Nogo-A mRNA expression in retina and optic nerve of zymosan group and PBS group was increased during 1 day to 3 days and decreased during 3 days to 7 days.There is notable significance of area of positive stained cell between IVa ,Va, Via group and IVb,Vband VIb group, respectively (f=3.58,4.93,3.72; p<0.01), soas A value(/=4.85,4.19,4.81; P<0.01 ). The area and A value of positively stained cells for Nogo-A mRNA expression in retina and optic nerve of regenerated adult rat was decreased compared with that of injected PBS group.There is notable significance between zymosan group and PBS group( PO.01).4. Conclusion4.1 Nogo-A which can be detected in retina and optic nerve after the trauma of optic nerve much more than that of normal rat.4.2 Expression of Nogo-A of retina and optic nerve after axon regneration decreases compared with that of PBS group.4.3 Nogo-A may be of important effect in the mechanism of inhibiting the regeneration after the trauma of optic nerve.
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