Effects Of L-dopa Methyl Ester On The Strabismic Amblyopia Cats

Objective: Amblyopia is one of the major diseases affecting children vission, it can lead to lifelong poor vision if not treated in time. The formation of amblyopia involves a variety of factors, and its pathogenesis is still under exploration. From the 90s onwards, domestic and foreign scholars have done many clinical studies on the use of drug treatment of amblyopia. At present, clinically L-dopa is often used for the treatment of amblyopia and achieved some success. However, the L-dopa has some disadvantages such as side effects, more poor tolerance, and effects of instability. We obtained L-dopa methyl ester (LDME) through the structural transformation of L-dopa. The main purpose of this study was to observe the effects of LDME on the treatment of strabismic amblyopic cats. Effects of the LDME were evaluated and the possible mechanisms were discussed.Methods: 30 normal kittens were randomly divided into 6 groups: low dose of L-dopa methyl ester (20mg/kg), medium dose of L-dopa methyl ester (40mg/kg), and high dose of L-dopa methyl ester (80mg/kg,), positive control (L-dopa 40mg/kg), normal control, and model control groups. The surgery for producing iatrogenic convergent strabismus was performed on 4 week old kittens (normal control group excluded). After the confirmation of the development of amblyopia by pattern visual evoked potential, L-dopa methyl ester, L-dopa and normal saline were given to the corresponding animals respectively, once daily continuously for 30 days.In order to study the impact of levodopa methyl ester on the of function of cat visual cortex, we observed the pattern visual evoked potential (PVEP) change with visual electrophysiology instrument. The morphological change of the visual cortex was investigated by the HE stain, nissl stain and transmission electron microscope. The c-fos mRNA expression of visual cortex area 17 was detected with the technology of in situ hybridization. C-fos protein expression in the visual cortex area 17 was detected by immunohistochemistry. We had observed nerve growth factor (NGF) expression in the visual cortex area 17 by immunohistochemical technique. NOS immunocytochemstry staining method was applied accordingly to observe the changes of distribution and cells density across the visual cortex under the light microscope.Results:(1) The visual electrophysiology of strabismic amblyopia cats.The change in model control group (MC) was characterized by P100 amplitude reduction and P100 latency extension. P100 latency delayed in MC, latency was more longer than the contralateral eyes and the eyes in NC, the difference was significant (P<0.05). Compared with the same self normal eyes and the ipsilateral eye in NC, P100 amplitude in MC decreased significantly (P<0.01). After treatment, compared with the MC, P100 latency was reduced to varying degrees, and P100 latency of high-dose group was the most significant, which was statistically significant (p<0.01). After treatment with LDME, the amplitude increased, and there was no significant difference (p>0.05) when LDMEH was compared with the normal control group, suggesting that L-dopa methyl ester can significantly improve conduction and sensory function in model of strabismic amblyopia cats. (2) The morphological findings of strabismic amblyopia cats.In the MC, the neurons in the visual cortex area 17 were showed dysfunctionly, but the neurons of LDMEH, LDMEM, LDMEL and PC showed recovering signs, the restoration of LDMEH was more well than PC, LDMEM, and LDMEL. The inner particles and particle outer were recovered.(3) The influence of LDME on c-fos mRNA expression in the visual cortex area 17 of strabismic amblyopia cats.In the 17 area of the visual cortex of NC, the c-fos mRNA positive cells were widely distributed, and expressed actively. the distribution of hybrid cells: the densities ofâ…¡/â…¢andâ…¤/â…¥layer were higher, the density ofâ…£layer lowest. Compared with the normal control group, in the 17 area of the visual cortex of MC, the c-fos mRNA positive neurons were decreased in all layers, and the difference was significant (P<0.01). Compared with the model group, in the treatment group (including the PC, LDMEL, LDMEM, LDMEH) c-fosmRNA positive cells in the visual cortexâ…¡/â…¢,â…£, andâ…¤/â…¥layers were increased. Except inâ…£, there was a significant (P<0.05 or P<0.01) in the other differences between the groups.(4) The influence of LDME on Fos expression in the visual cortex area 17 of strabismic amblyopia cats.The Fos positive cells, which nucleus showed dark brown, were seen in the whole floor of visual cortex area 17 of the NC cat. The density of the positive cells in layerâ…¡,â…¢andâ…¥was higher than that in layerâ…£andâ…¤. The distribution patterns of Fos positive cells between layers were similar to the NC, but the density of positive cell in each layer was significantly lower than that in NC, and the difference was statistically significant (P<0.01). In the treatment group (including the positive control group, L-dopa methyl ester of high, medium and low-dose group) the distribution of positive cells was basically in layer II, III, V and VI, but the density in each layer of the treatment group was more higher than that in model control group, and the difference was significant (P<0.05). Compared with PC, the density of Fos positive cells in each layers of LDME treatment group was more hiher than that in PC respectively, and some layers had statistical significance.(5) The influence of LDME on NGF expression in he visual cortex area 17 of strabismic amblyopia cats.Compared with the NC, the number density of NGF-immunoreactive cells in MC was reduced, and there was a significant difference between the two groups (p<0.01). Compared with MC, the number density of NGF-immunoreactive cells in each layers of the treatment group (including the PC, L-dopa methyl ester of high, medium and low-dose groups) was increased correspondingly, and the differences were statistically significant (p<0.01 or p<0.05) in addition to LDMEL inâ…¢,â…¤andâ…¥. Compared with the positive control group, the number density of NGF-immunoreactive cells in each layer of L-dopa methyl ester group was in the upward trend, and the differences in layerâ…¢andâ…¥of the LDMEH were statistically significant (p<0.01 or p< 0.05). Compared with NC, the number density of NGF-immunoreactive cells in LDMEH was less than that in the normal group, but there was no statistical difference between the two groups (p>0.05).(6) The influence of LDME on NOS expression in the visual cortex area 17 of strabismic amblyopia cats.The NOS positive cells in the NC were mainly distributed in the gray matter of theâ…¡,â…¢,â…£,â…¤andâ…¥layers and white matter, the number of the NOS positive cells inâ…¡/â…¢was highest,â…¤/â…¥next, andâ…£at least. The distribution pattern of NOS-positive cells in visual cortex area 17 of MC cats was similar to that of the normal control group, however, within each layer and interlayer the NOS-positive fibers were significantly reduced, and the NOS-positive cell density was also significantly decreased (P<0.05). In the treatment group (including the positive control group, high low-dose L-dopa methyl ester groups) the NOS-positive cell density in each layers was increased, and the LDMEH was most obvious.Conclusions:(1) LDME can shorten the peak latency of P100 and increase P100 amplitude in strabismic amblyopia cats, and make them to recover to normal level, which can improve the function of conduction and perception in strabismic amblyopia cats eyes.(2) LDME has good therapeutic effect on the injured neuron in strabismic amblyopic cats, and can restore the neuron morphology. It also can promote the replication, transcription process, protein synthesis, protuberance of the neurons, and the synaptic transmission.(3). LDME can promote c-fos mRNA expression in area 17 of visual cortex of strabismic amblyopia cat.(4) LDME can promote Fos protein expression in area 17 of visual cortex in strabismic amblyopia cat.(5) LDME ester can increase NGF synthesis and release in area 17 of visual cortex in strabismic amblyopia cat. NGP expression coincides with of the change the PVEP. It is proved that LDME can improve the visual function of strabismic amblyopia cats eyes in certain degree. As a regulatory factor in cell function NGP may be play an important role on developmental plasticity in the visual system. (6) LDME can increase the nitric oxide levels in area 17 of visual cortex of strabismic amblyopia cats to some extent.(7) LDME has therapeutic effects on the strabismic amblyopic cats, and its mechanism may be related to the increases in NGF synthesis and release and the nitric oxide level in visual cortex area 17 of strabismic amblyopic cats.