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The Improved Effects of L-dopa Methyl Ester on Monocular Deprivation Amblyopic Cat

Author: LiRong
Tutor: HuangRenBin
School: Guangxi Medical University
Course: Pharmacology
Keywords: Monocular deprivation amblyopia cat visual cortex area 17 visual evoked potential apoptosis nerve growth factor c-fos c-Fos protein neural regeneration
CLC: R777.44
Type: Master's thesis
Year: 2012
Downloads: 33
Quote: 1
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Abstract


Objective: Amblyopia occurs commonly in children. Its onset is the severedisorder of spatial vision and visual loss during the visual sensitive period.Progress in amblyopia will lead to lifelong functional dysfunction in visualcortex without timely or properly treating. Some clinical studies havedemonstrated that L-dopa treatment of amblyopia achieved some success.However, it has severe side-effects and poor patient tolerance. We havepreviously synthesized a novel L-dopa derivate namely L-dopa methyl ester(LDME), which has superior physicochemical property to L-dopa and can easilypenetrate the blood-brain barrier, ultimately accumulating dopamine in visualpathway. The purpose of this study is to observe the effects of different doses ofLDME on deprivation amblyopia cat model, and to evaluate the treatment ofamblyopia pharmacodynamic effects, as well as to explore the possiblemechanism of drug action of the LDME.Methods: A total of thirty kittens, at age of 2-4 weeks old and weighingabout 200-300 g regardless their genders were purchased from the MedicalLaboratory Animal Center of Guangxi Medical University and used in the study.Routine eye examinations were performed to confirm normal extraocular areas,refracting media, and fundus of the eyes. Cats were randomly assigned intonormal control group, positive control group and low, moderate and high dose LDME treatment groups. The lids of the left eyes of all cats except those in thenormal control group were sutured to induce amblyopia according to the classicexperimental method of Hubel. 12 weeks later, as the pattern visual evokedpotentials (P-VEP) being used to determine the formation of amblyopia, the catsin the LDME treatment groups were intragastrically perfused with 20, 40, and80 mg·kg-1LDEM dissolved in physiological saline daily for 30 consecutivedays, respectively; cats in the positive control group was similarly given 40mg·kg-1L-dopa; and cats in the normal and model control groups were givensame volume of normal saline. The experimental animals were treated accordingto the Guidance Suggestions for the Care and Use of Laboratory Animals issuedby the Ministry of Science and Technology of the People’s Republic of China.①The effects of L-dopa and L-dopa methyl groups onelectrophysiology of feline visual cortex was detected by patternvisual evoked potentials (P-VEP);②The anatomic and physiologiceffects of L-dopa methyl ester at different doses on visual cortex area17 in feline model with amblyopia induced by monocular visiondeprivation were assessed;③As revealed by TUNEL assay, bothL-dopa and LDME treatments resulted in inhibited apoptosis ofneurons in LGN of amblyopic cats;④Immunohistochemical stainingand western blot were used to examine the expressions of nervegrowth factor (NGF) and c-Fos in visual cortex area 17, respectively.⑤The expression of c-fos mRNA in visual cortex area 17 were detected by the fluorescent quantitative PCR (FQ-PCR).②Results:1、The study of visual electrophysiologyAs shown in P-VEP results, deprived eye in amblyopic control group (AC)significantly exhibited the reduction of amplitude values and extension of P100values. Compared to the same side of the normal control eyes, the differenceswere statistically significant (P<0.01). Compared to the amblyopic control group,P100latency of the amblyopic eyes in treatment groups was gradually shortened,which LDMEH displayed obviously, the differences were statistically significant(p<0.05 and p<0.01); while amplitude of the amblyopic eyes in treatment groupsincreased in various degree, the differences were statistically significant(p<0.05 and p<0.01). It indicated that LDME could improve the function ofsignal transduction in the amblyopic eye of deprivation amblyopia cat.2、Histological study of the visual cortex area 17The anatomic and physiologic on neuronal morphology of visual cortexarea 17 in amblyopia control group (AC) exhibited damage. After the treatmentsof L-dopa and LDME, the neurons of visual cortex area 17 in groups graduallyrepair and regenerate, showing increased the number of Nissl bodies and nervecells.3、Effect of LDME on the expression of apoptosis in visual cortex area 17of amblyopic catThe neurons in the amblyopic group triggered severe DNA strand breaks,displaying a larger number of TUNEL-positive cells. The neuronal apoptosiscell counts in visual cortex area 17 of the administration groups reduced invarious degrees. And LDMEHshowed significant anti-apoptotic effect, the difference was statistically significant (P <0.01). The nerve cells of theamblyopic eye visual cortex area 17 in treatment groups had a certain degree ofreversal of apoptosis, the differences were statistically significant (P <0.05 andP <0.01).4、Effect of LDME on NGF expression of visual cortex area 17 inamblyopia cat modelCompared to amblyopic model group, NGF-immunoreactive cells amonglayers in treatment groups (including the L-dopa group and L-dopa methyl estergroups) gradually increased, the differences were statistically significant (P<0.01 and P <0.05). As compared with L-dopa control group, those numbers ofNGF-immunoreactive cells in LDMEMwere more than that in L-dopa controlgroup, implied LDME playing a better therapeutic effect, but those cells inLDMEMlower than that in LDMEHand normal control group, the differenceswere not statistically significant. Western blot analysis showed that the NGFprotein level in amblyopic model group was evidently less than other treatmentgroups (P <0.01). And NGF protein expression in treatment groups graduallyincreased.5、Effect of LDME on c-fos mRNA expression of visual cortex area 17 inamblyopic cat modelFQ-PCR exhibited that c-fos mRNA expression in cat visual cortex of thenormal group was active. Compared with normal control group, c-fos mRNAexpression level in visual cortex in amblyopic control group distinctly reduced,the differences were significant (P <0.01). Compared to amblyopic model group,c-fos mRNA levels the treatment groups (including L-dopa and LDME)gradually elevated, the difference was statistically significant (P <0.01), whichthose expression was obvious in LDMEH(P <0.01). 6、Effect of LDME on the c-Fos protein expression in visual cortex area 17of amblyopic catThe c-Fos positive cells in visual cortex area 17 of amblyopic control groupwere less expressed, and its density was significantly reduced compared withnormal group, the differences were statistically significant (P <0.01 and P <0.05).And positive cell density in LDMEH was higher than that in amblyopic controlgroup, the difference was statistically significant (P <0.01). However, comparedwith normal control group, c-Fos immunoreactive cell density in LDMEH wereless than that in the normal control groups in the layers , there was no significantdifference between the two groups. Western blot analysis displayed that thec-Fos protein expression in amblyopic model group was apparently less thanother treatment groups (P <0.01). And c-Fos protein expression in treatmentgroups gradually augmented.Conclusions:①L-dopa methyl ester notably shortens P100peak latency and enhances P100amplitude of deprivation amblyopia cat. Therefore, it has role of restore visualfunction by improving the signaling transduction of deprivation amblyopia cat.②Treatment of L-dopa methyl ester has the capacities of recovery of visualcortical neurons repairment and regeneration in deprivation amblyopia, andpromotes myelin sheath of myelin synthesis thus enhances the transmission ofnerve impulses for better protecting the myelin sheath.③L-dopa methyl ester effectively suppresses the neuronic apoptoticprocess of visual cortex area 17 of amblyopic cat.④Increased NGF expression in nerve cells in visual cortex area 17 ofdeprivation amblyopia cat via L-dopa methyl ester treatment is consistent withaugmented number of nerve cells (increase in the number of Nissl bodies) and improved P-VEP values of the waves.⑤L-dopa methyl ester effectively promotes the upregulation of c-fosmRNA level in visual cortex area 17 of deprivation amblyopia cat.⑥L-dopa methyl ester effectively increases the expression of c-Fos proteinin visual cortex area 17 of deprivation amblyopia cat.⑦L-dopa methyl ester effectively restores the function of the visual cortexarea 17 and it can serve as a potential drug for amblyopia treatment. LDMEtreating for amblyopia may be through the mechanisms that regulates NGF、c-fos expressions in visual cortex area 17 to inhibit apoptotic process andpromote neuronal repairment or regeneration.

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CLC: > Medicine, health > Ophthalmology > Ocular adnexal organ disease > Ocular disease and strabismus > Amblyopia
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