| BACKGROUNDCentral nerve system diseases, diabetic retinopathy, glaucoma, trauma and age-related macular degeneration (AMD) are the most common causes of visual impairment and legal blindness in many countries. Most important thing of them is the impairment of retinal ganglion cells (RGCs).RGC damage occurs in two steps:initial insult and subsequent degeneration through apoptosis. Current treatments target the first step; however, the continuous degeneration occurs even when the initial insult is well controlled. So the first aim is to establish the ideal animal model, seeking the key apoptosis factors and interrupt the continuous degeneration.The current animal models for RGCs apoptosis are variety, including qualitative and quantitative, mechanical, high-ocular-tension, ischemic, toxic and diabetic et al. Because of the variety of optic nerve diseases, it is too hard to judge experimental results with a uniform standard. Previous optic nerve transection model is to cut off the nerve at any part behind the eyeball, which resulted in eyeball insufficiency. We used a micro scissors cutting off optic nerve 2mm behind the eyeball after peeling the outer membrane of the nerve, which can create an optic nerve transaction model successfully in rats.β-amyloid protein (Aβ) is one of the most important apoptotic factors which is formed after sequential cleavage of the amyloid precursor protein (APP), a transmembrane glycoprotein of undetermined function. APP can be processed byα-,β-and y-secretases; Aβprotein is generated by successive action of theβandγsecretases. Deposition of Aβpeptides in the brain and retina tissues results in neuroinflammation and neurovascular inflammation. Loss of the normal physiological functions of Aβis also thought to contribute to neuronal dysfunction, including Alzheimer’s disease (AD) and optic nerve disease. In Alzheimer’s disease, some experiment findings indicated that the significant increase in the amount of Caspase-3 (proapoptotic) and decrease in Bcl-2/Bax ratio (Bcl-2:anti-apoptotic; Bax:proapoptotic) is one of the most important apoptotic avenues induced by Aβ. But there is still no demonstration of similar nerve damage induced by Aβin optic nerve diseases. We studied the expression of Aβprotein in optic nerve transection rats.Retinal ganglion cells are usually not able to regenerate their axons after optic nerve injury or degenerative disorders, resulting in lifelong visual loss. This situation can be partially reversed by activating the intrinsic growth state of retinal ganglion cells, maintaining their viability, and counteracting inhibitory signals in the extracellular environment. Such as:1. Preventing continous apoptosis of Ganglion cells:glutamate receptor antagonist agent, calcium channel blockers, nitric oxide (NO) and its synthase (NOS) inhibitor, gene regulation of apoptosis, free radical scavengers and antioxidants, heat shock protein, zinc and so on.2. Promoting the regeneration of new ganglion cells:the supplement of exogenous neurotrophic factor, optic nerve stem cell transplantation, Schwann cells transplanted in the vitreous cavity and so on.3. Repairing ganglion cell apoptosis:the supplement of exogenous neurotrophic factor, optic nerve stem cell transplantation, intravitreal transplantation of Schwann cells and a variety of Chinese medicine. Though advances during the past few years continue to extend the amount of regeneration that can be achieved in animal models, there are still limited medicines to use in clinic. In order to enrich the medicines, it is very necessary to take advantage of Chinese medicine. BFP has long been recognized to have estrogen-like activities, such as its ability to increase the expression of cystic fibrosis transmembrane-conductance regulator, reduce blood pressure, increase vasorelaxation, reduce serum triglyceride, and even its antiplatelet activity and stimulating effects on dopamine release of the brain[8-11]. Although BFP contains phytoestrogens such as ginseng that may act similarly to estrogen, which has been shown to prevent neuronal degeneration caused by increased oxidative stress[18] and apoptosis[19]; other component herbs of BFP may act through different mechanisms, the details of which remain to be elucidated. Xie JX et al studied that the BFP could inhibit neuronal apoptosis. Few reports were made in the field of optic nerve diseases, so we examined the expression of some of the apoptotic factors such as Caspase-3, Bax, and Bcl-2 that we expected to get impact from BFP.OBJECTIVETo establish a complete optic nerve transection injury model in rats for investigating the expression level of P-amyloid in retinas of optic nerve transaction rats and its mechanism. Then investigate the effect of Bak Foong Pills (BFP) on the expression of P-amyloid (Aβ) in retinas with optic nerve transection rats.METHODS1. Establishing optic nerve transaction rat model:Fifty-four healthy Sptague-Dawley (SD) rats were randomly divided into three groups. Six rats were in normal control group. Twenty-four rats were in model group, model of optic nerve transaction was induced in the left eye by a micro scissors at optic nerve 2mm behind the eyeball followed by peeling the outer membrane and cutting off the nerve. The right eyes served as a control. Another 24 rats served as sham group. According to the sacrificing date of the rats, four subgroups in groups in injury group were further divided for three hours, fourty-eight hours, seven days and fourteen days later. Therefore, six rats were in each subgroup.5% fluorogold was injected into both superior colliculi bilaterally to each subgroup and normal group. The eyes were enucleated seven days later, flat mounts of the retina from both eyes were prepared on a slide and observed under a fluorescence microscope. The retina was sectioned and colored by hematine-eosine for counting the cells in the ganglionic layer of the optic nerve, quantitating optic nerve lesion and observing change of retina-morph. Leptonomorphological change was observed by coloring retinal blade using hematoxylin-eosine after optic nerve injury.2. Expression level of P-amyloid in the retinas of optic nerve transection rats and its mechanism:Eighty-one healthy Sptague-Dawley (SD) rats were randomly divided into three groups. Night rats were in normal control group. Thirty-six rats were in model group, model of optic nerve transaction was induced in the left eye by a micro scissors at optic nerve 2mm behind the eyeball followed by peeling the outer membrane and cutting off the nerve. The right eyes served as a control. Another 36 rats served as sham group. According to the sacrificing date of the rats, four subgroups in groups in injury group were further divided for three hours, fourty-eight hours, seven days and fourteen days later. Therefore, 12 rats were in each subgroup. The eyes were enucleated at the four different time.①The expression level ofβ-amyloid was detected by Western-blotting in all the subgroups.②The expression level ofβ-amyloid was detected by immunohistochemistry in all the subgroups.③The expression level of Bcl-2 mRNA、Bax mRNA and Caspase-3 mRNA were detected by RT-PCR in all the subgroups.3. Protective effect of BFP on RGCs:Fourty-eight healthy Sptague-Dawley (SD) rats were randomly divided into four groups. Twelve rats were in each group. The right eyes served as a control. Twelve rats in A group were induced in the left eye with model of optic nerve transaction, followed by intravitreal injection of BFP (100ug/ml) 10ul. Twelve rats in B group were only induced in the left eye with model of optic nerve transaction. Twelve rats in C group were induced in the left eye with model of optic nerve transaction, followed by intravitreal injection of PBS lOul. Twelve rats were in normal control group.①The expression level of P-amyloid was detected by Western-blotting in all the subgroups.②The expression level of Bcl-2 mRNA、Bax mRNA and Caspase-3 mRNA were detected by RT-PCR in all the subgroups.RESULTS1. Establishing optic nerve transaction rat model:①In model group, the retinal ganglion cells (3h:152.06±15.21,48h:103.19±10.55,7d: 61.89±27.38,14d:52.10±20.56) factorial analysis of variance showed significant diferences compared with the control group (3h: 195.16±22.12,48h:188.26±16.16,7d:187.76±21.19,14d:199.46±13.67) (P<0.05). There were no significant differences between sham gtoup and control group (P>0.05).±Pathological change:After optic nerve injury, there was karyopycnosis on ganglion cell layer of retina, thinningz on each layer of retina, derangement of cell and decrease in retinal ganglion cells. There was different degree of the above change in different time after optic nerve injury. There were the swelling, the hemorrhage, derangement of spongiocyte and the denaturation like vacuole in the spot of optic nerve injury. Moreover, they were aggrava-ting with increases of the time after optic nerve injury. There were no pathological changes in sham group and control group.2. Expression level ofβ-amyloid in the retinas of optic nerve transaction rats and its mechanism:①Western-blotting:The expression level ofβ-amyloid in model group was higher than sham group and control group, showing significant differences (P<0.05). There were no significant differences between sham group and control group (P>0.05).②Immunohistochemistry:Light microscope:there was positive expression ofβ-amyloid protein in retinal ganglion cell layer, inner nuclear layer and inner plexiform layer of model group. Coloration: yellow-brown granular distribution. At 48h, the coloration was deepest and the number was largest. There was no positive coloration in the sham group and control group.③RT-PCR:In model group, the expression level of Bcl-2 mRNA (anti-apoptotic) was down-regulated, while the expression level of Bax mRNA and Caspase-3 mRNA (proapoptotic) were up-regulated. Comparing with sham group and control group, there were significant differences (P<0.05). There was no significant difference in the sham group and control group.3. Protective effect of BFP on RGCs:①Western-blotting:The expression level of (3-amyloid in A group was lower than B group and C group, showing significant differences (P<0.05). However, there was still significant difference between A group and D group (P<0.05). And there was no significant difference between B group and C group (P>0.05).②RT-PCR:In A group, the expression level of Bcl-2 mRNA (anti-apoptotic) was up-regulated, while the expression level of Bax mRNA and Caspase-3 mRNA (proapoptotic) were down-regulated. Comparing with B group and C group, there were significant differences (P<0.05). However, there was still significant difference between A group and D group (P<0.05).And there was no significant difference in the B group and C group (P>0.05).CONCLUSION1. Using a micro scissors cutting off optic nerve 2mm behind the eyeball after peeling the outer membrane of the nerve can create an optic nerve transaction model successfully in rats. And the model may be applied to researching mechanism and treatment of the optic nerve diseases.2. The expression level ofβ-amyloid protein was significantly up-regulated in the retinas of optic nerve transaction rats, which demonstratedβ-amyloid protein is one of the most important factors in RGCs apoptosis. The expression level of Bcl-2 mRNA (anti-apoptotic) was significantly down-regulated, meanwhile, Bax mRNA and Caspase-3 mRNA (proapoptotic) was significantly up-regulated, which shows thatβ-amyloid plays proapoptotic role in optic nerve transection by enhancing Bcl-2/Bax and Caspase apoptosis mechanism.3. Intravitreal injection of Bak Foong Pills can down-regulate the expression ofβ-amyloid in retina, which may play protecting roles in optic nerve damage. And it may work by inhibiting Bcl-2/Bax and Caspase apoptosis mechanism. |
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