Correspondence should be addressed to Jung-Kai Tseng;

Transcription

1 Interntionl Journl of Endocrinology, Article ID , 7 pges Reserch Article Investigtion of the Protective Effects of Turine ginst Alloxn-Induced Dibetic Retinl Chnges vi Electroretinogrm nd Retinl Histology with New Zelnd White Rbbits Smuel Tung-Hsing Ching, 1 Shng-Min Yeh, 2 Yi-Chen Chen, 3 Shiun-Long Lin, 4 nd Jung-Ki Tseng 2,5 1 Deprtment of Optometry nd Vision Science, Fculty of Medicl nd Helth Science, The University of Aucklnd, 85 Prk Rod, Grfton, Aucklnd 123, New Zelnd 2 School of Optometry, Chung Shn Medicl University, No. 11, Section 1, Jinguo N. Rod, Tichung 421, Tiwn 3 Deprtment of Animl Science nd Technology, Ntionl Tiwn University, No. 5, Lne 155, Section 3, Keelung Rod, Tipei 1154, Tiwn 4 Deprtment of Veterinry Medicine, Ntionl Chung Hsing University, No. 25, Kuo-Kung Rod, Tichung 4227, Tiwn 5 Deprtment of Ophthlmology, Chung Shn Medicl University Hospitl, No. 11, Section 1, Jinguo N. Rod, Tichung 421, Tiwn Correspondence should be ddressed to Jung-Ki Tseng; hki42@gmil.com Received 31 July 214; Revised 31 August 214; Accepted 4 September 214; Published 14 September 214 Acdemic Editor: Ilis Migdlis Copyright 214 Smuel Tung-Hsing Ching et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. The purpose of this study ws to investigte the protective role of orlly dministered turine ginst dibetic retinl chnges vi electroretinogrm (ERG) nd retinl histology on rbbits. Rbbits were rndomly ssigned into groups: (vehicle dministrtion only); (dibetes: induced by 1 mg/kg lloxn injection); (dibetes nd fed with 2 mg/kg turine); nd V (dibetes nd fed with 4 mg/kg turine). The body weight nd blood glucose levels of the rbbits were monitored weekly. The ERG ws mesured on weeks 5 nd 15. Retinl histology ws nlyzed in the end of the experiment. Results reveled tht turine supplement significntly meliortes the lloxn-induced hyperglycemi nd protects the retin from electrophysiologicl chnges. showed significnt (P <.5) chnge in the men scotopic b-wve mplitude when compred to tht of, wheres the dibetic rbbits treted with turine ( nd IV) were nlogous to. Histologiclly, the mount of Bipolr nd Müller cells showed no difference (P >.5) between ll groups nd when compred with those of. Our study provides solid evidences tht turine possesses n ntidibetic ctivity, reduced loss of body weight, nd less electrophysiologicl chnges of the dibetic retin. 1. Introduction Dibetes mellitus is one of the most serious medicl issues round the world. Untreted dibetes ultimtely leds to vriety of secondry complictions, such s neuropthy, hert disese, kidney filure, nd retinopthy [1]. In the United Sttes, mong those dults ged between 2 nd 74 yers, dibetic retinopthy hs been shown to be the leding cuse of new cses of blindness [2, 3]. Fong nd collegues [4] described tht the prevlence of ny signs of retinopthy ws s high s 8% t 15 yers of hving dibetes. The clinicl signs of dibetic retinopthy within the retinl circultion include microneurysms, hemorrhges, intrretinl microvsculr bnormlities, nd neovsculriztion [5, 6]. Microneurysms re usully the first cliniclly detectble lesion of dibetic retinopthy; they representwekeningofthecpillrywllsndmybessocited with retinl oedem due to serum lekge from

2 2 Interntionl Journl of Endocrinology the vessels. Hemorrhges re lso n erly sign of dibetic dmge to blood vessels. They my include dot nd blot hemorrhges tht occur deeper in the retin nd shllow flme-shped hemorrhges tht follow the retinl nerve fibre lyer. Intrretinl microvsculr bnormlities (IRMA) my lso be present nd cused by poor functioning or nonperfusion of cpillries which prevent norml blood flow. Neovsculriztion cn occur nywhere within the retin s response to ischemi nd is the hllmrk of the dvnced nd prolifertive stge of dibetic retinopthy. The occurrence of neovsculriztion increses the risk of vision loss in the dibetic ptient. In ddition to the clinicl signs in the retin tht cn be visulized vi ophthlmoscopic view, evidence from previous studies lso suggests tht choroidl ngiopthy my coexist long with retinl vsculr dmge [7, 8]. Other thn the clinicl signs mentioned bove in detection of dibetic retinopthy, severl studies hve found tht dibetes ffects the electrophysiologicl spects of vision. Electroretinogrm (ERG) is one of the tests tht hve been well described in the detection of erly functionl chnges in dibetic retins. In fct, previous studies hve demonstrted tht ERG bnormlities (i.e., chnges in b-wve mplitude) occur before ny signs of structurl bnormlities cn be detected by fundus photogrphy [9], fluorescein ngiogrphy [1], nd morphologicl exmintions [11]. Turine (2-minoethnesulfonic cid) is conditionlly essentil mino cid tht is present in the retin in high concentrtion nd is widely distributed in mmmlin tissues.theminsourceofturinein vivo is from dietry intke of met or sefood nd biosynthesis tht is derived from methionine nd cysteine metbolism. However, previous study reported tht biosynthetic cpcity of turine in humns is very low nd bsent in cts [12]. Turine hs mny biologicl roles nd is involved in severl physiologicl ctions, such s the formtion of bile cid, osmoregultion, ntioxidtion, mintining the structurl integrity of the membrne, nd modultion of clcium binding nd trnsport [13 15]. In vrious experimentl models, turine hs been shown to protect ginst lloxn-induced hyperglycemi in type I dibetes [16] nd to inhibit ctrctogenesis in rbbit lenses exposed to 3 mm glctose [17]. Previous studies hve estblished tht turine is essentil for visul development nd those deficiencies re ssocited with retinl degenertion [18]. The physiologicl role of turine hs been pid ttention since reports of cts developing centrl retinl degenertion when they hve been fed to induce chronic deficiency of turine, which is similr to the retinitis pigmentos in humns [19, 2]. Since turine hs been demonstrted to hve such excellent bioctivity properties, we hypothesized tht turine dministrtion cn protect rbbits from lloxn-induced dibetic retinl chnges. The extent of lloxn-induced dibetic retinl chnges nd protective effects of turine were mesured by electroretinogrm (ERG) nd histologicl observtions. 2. Methods 2.1. Animls. Twenty-two mle New Zelnd White Rbbits (1 weeks old) from T Tsung Frm (Chnghu City, Tiwn) were used in this study. The nimls were qurntined nd llowed to cclimtize for one week prior to the experiment phse. The nimls were housed one rbbit per cge under stndrd lbortory conditions with 12-hour light/drk cycle. The temperture of the niml room ws mintined t 25 ± 2 Cwithreltivehumidityof55 ± 5%. The irhndling units were set to provide pproximtely 12 fresh ir chnges per hour. Food nd wter were vilble d libitum. The experimentl protocols for this study were pproved by the Institutionl Animl Cre nd Use Committee (IACUC), Chung Shn Medicl University Experimentl Animl Center (pprovl number: 684), nd the nimls were cred for in ccordnce with the institutionl ethicl guidelines. All procedures were performed ccording to the ARVO Sttement for the Use of Animls in Ophthlmic nd Vision Reserch Experimentl Design. The rbbits were rndomly ssigned into groups. served s the control (n =6). The experimentl groups (Groups II, III, nd IV) received n intrvenous injection of lloxn [21, 22] in.9%sodium chloride t dosge of 1 mg/kg body weight to induce dibetes. The blood glucose levels were monitored weekly. Those nimls with blood glucose levels >2 mg/dl in consecutive 3-week period were included in the experiment ndweredistributedintothefollowinggroups:groupii dibetes (untreted dibetic rbbits, n = 5); (DT2; dibetic rbbits treted with 2 mg/kg turine in the drinking wter, n = 5); V (DT4; dibetic rbbits treted with 4 mg/kg turine in the drinking wter, n=6). The food nd wter intke were checked dily, nd body weight ws mesured weekly Blood Glucose Mesurement. The blood ws collected weekly during 15 weeks of experiment from the mrginl er vein to mesure the blood glucose levels (Accu-Chek Active bloodglucosemeter;rochedignosticsgmbh,germny) Electroretinogrm (ERG) Anlyses. The electroretinogrm ws mesured in both photopic (35 Lux of mbient light) nd scotopic (fter 5 minutes of drk dpttion) conditions. The instruments used in this study were Eickemeyer ERG, HP Compq 623 Notebook, ERG-jet, nd ERG-probe by Universo Plstique. The instruments were tested prior to the nimls preprtion to ensure tht the electrodes were in good order nd then the rbbits were prepred in the mbient light with the following steps. (1) One to two drops of tropicmide 1% mydritic were instilled into rbbits eyes for t lest 3 min prior to ERG. (2) Subcutneously tropine sulphte ws injected t.5 mg/kg prior to nesthesi to reduce the slivry nd bronchil secretions. Pupils were checked to

3 Interntionl Journl of Endocrinology Body weight (kg) Blood glucose (mg/dl) Weeks Weeks V V () (b) Figure 1: The effect of the turine on body weight () nd blood glucose levels (b) in lloxn-induced dibetic rbbits over the 15 weeks of experimenttion. confirm pupil diltion, nd mydritic drops were repeted t this stge if necessry. (3) Zoletil 5 nesthetics were injected into rbbit s thigh muscles, nd then 1 to 2 drops of proprcine hydrochloride.5% locl nesthetics were instilled into the rbbits eyes. Both were done 5 minutes prior to the ERG mesurements. (4) Electrodes were plced. Artificil ters were used with contct lens electrode to ensure better contcts of electrode nd corne. (5) Light stimulus ws plced 1 cm wy from the corne with stndrd flsh of 2-3 cd/m 2 /s. (6) The electroretinogrm ws mesured with mbient light nd fter 5 minutes of drk dpttion. (7) Results of ERG including -wve nd b-wve were recorded Histologicl Evlution. The rbbits were scrificed t the end of the experiment with 1 15 mg/kg pentobrbitl injection nd CO 2 ; the eyes were removed, weighed, nd fixed in Dvidson s fixtive. The eyes were processed for prffin embedding following stndrd microtechniques. Four- to five-micron sections of the eyes tissue were stined with hemtoxylin nd eosin to estimte the retinl dmge nd were observed under microscope (IX71S8F-2, Olympus, Tokyo, Jpn) Sttisticl Anlysis. All results re expressed s men ± SD. The comprison between ny two groups ws performed using one-wy nlysis of vrince (ANOVA), followed by Tukey s multiple comprison tests using the sttisticl softwre SPSS (Drmrketing Co., Ltd., New Tipei City, Tiwn). Sttisticlly significnt differences between groups were defined s P < Results 3.1. Effects of Turine on Body Weights. The body weight losses re well dignosed in lloxn-induced dibetic niml studies. The body weights were recorded weekly over the 15 weeks of study, nd the results re shown in Figure 1(). The trend of the body weight gin reveled n increse in the weights of,, nd V over time. Over the 15 weeks of study, the men increse in body weight for ws.33 kg, incresed by.45 kg, nd V incresed by.47 kg. In contrst, lost on verge.8 kg over the course of the experiment. Although the finl body weight of the turine treted groups ws still lower thn, they were 24.6% () nd 25.6% (V) hevier thn Effects of Turine on Blood Glucose Levels. Blood glucose levels re commonly used s n indictor for lloxn-induced dibetes in experimentl nimls. They indicte whether or not dibetes ws successfully induced. The results of blood glucose levels over the 15 weeks re shown in Figure 1(b). As shown in Figure 1(b), themenglucoselevelsofgroup I,, nd V remined constnt over time, wheres s men blood glucose levels incresed over time. This indicted tht lloxn is very effective (P <.1) inelevtingthebloodglucoselevelsincomprison to. This is lso s n indiction of successfully induced dibetes in rbbits. Our results lso demonstrted tht turine tretments llevite increses of blood glucose levels effectively (P <.5). On verge t 15th week of the study, the glucose levels were lowered by 145 mg/dl

4 4 Interntionl Journl of Endocrinology Amplitude 15 1 Amplitude T1 T2 T2-T1 Photopic condition T1 T2 T2-T1 Scotopic condition V V () (b) Figure 2: The electroretinogrm (ERG) ws performed on the control (), dibetes (), nd dibetes + turine (, V) groups on week 5 (T1) nd week 15 (T2). The verged -wve mplitude under the photopic () nd scotopic (b) conditions ws recorded nd nlyzed Amplitude T1 T2 T2-T1 Photopic condition b Amplitude b b T1 T2 T2-T1 Scotopic condition V V () (b) Figure 3: The electroretinogrm (ERG) ws performed on the control (), dibetes (), nd dibetes + turine (, V) groups on week 5 (T1) nd week 15 (T2). The verged b-wve mplitude under the photopic () nd scotopic (b) conditions ws recorded nd nlyzed., b: brs with different lphbetic letters differ (P <.5). (3.9%)inGroupIIInd151mg/dL(32.1%)inGroupIV when compred to the men glucose levels of. These findings suggested tht the hyperglycemi ws induced successfully by lloxn nd ws effectively meliorted by turine tretments Electroretinogrm. The electroretinogrm (ERG) ws performed for ll groups on weeks 5 nd 15 to investigte the possible electrophysiologicl chnges of the dibetic retin. The men mplitude of -wve nd b-wve ws nlyzed nd is shown in Figures 2() nd 2(b) (-wve) nd Figures 3() nd 3(b) (b-wve). Over the 15 weeks of study, themenmplitudechngesinbothphotopicndscotopic -wve were insignificnt (P >.5). In regrd to the b- wve, the men mplitude under photopic condition ws lso insignificnt (P >.5). However, the men mplitude of scotopic b-wve in showed significntly higher chnge over time during the experimentl period (P <.5), whilst nd V s scotopic chnges were not significntly different from nimls in the control condition. showed slightly greter chnge in scotopic b- wve thn V, which my men tht V is more superior in minimizing the electrophysiologicl chnges of the retin over time.

5 Interntionl Journl of Endocrinology 5 Cell numbers Bipolr cells Muller cells Totl cells V Figure 4: The comprison of Bipolr nd Müller cells number mong ll groups. The cross-section of the retins ws prepred nd exmined under the microscope for histologicl evlution Histologicl Evlution. The cross-section of the retins ws prepred nd exmined under the microscope for histologicl evlution. The Bipolr nd Müller cells were counted nd results re shown in Figure 4. Ourresultsreveledtht there re not significnt differences (P >.5) in Bipolr nd Müller cells count mong ll groups when compred with control group. We then further dded the Bipolr nd Müller cells count ll together to see if there were ny clerer trends of cell counts. However, the results still reveled sttisticlly insignificnt difference (P >.5). These results indicted tht there were no obvious cell losses over the 15 weeks of dibetes in rbbits. 4. Discussion Turine is n orgnic cid tht hs mny fundmentl biologicl roles in our humn body nd in nimls. Lombrdini [23] found tht turine hs severl functions in the retin, which includes the regultion of C 2+ trnsport, protection of the photoreceptor, nd regultion of signl trnsduction. A previous study demonstrted tht turine tretments ttenute the induction of retinl VEGF tht ssocites with vsculriztion in STZ-dibetic rts, suggesting tht turine my normlize the retinl vsculr function in dibetes [24]. While elevtion of glutmte in the retin is ssocited with the development of dibetic retinopthy, turine ppered to be ble to regulte Müller cells glutmte uptke nd degrdtion under dibetic conditions vi its ntioxidnt mechnism [25]. Our study showed tht turine hs significnt hypoglycemic properties, which is in line with previous work. For exmple, Gvrovsky et l. [16] found decrese in glucose concentrtion, together with the protection of b-cells of the islets of Lngerhns in experimentl insulin dependent dibetes mellitus, nd other reserches hve demonstrted the effectiveness of turine tretments ginst both insulin dependent nd non-insulin dependent dibetes mellitus [26, 27]. In electroretinogrm (ERG), the -wve is negtive, mximl combined response tht is believed to reflect the membrne potentil in photoreceptors. The b-wve is positive, mximl combined response tht is thought to originte from the Bipolr nd Müller cells tht postsynpse to the photoreceptors [28]. However, in vrious niml nd humn studies, ERG mesured under scotopic conditions showed tht the lrge b-wve ppered to be directly generted by the Bipolr cells. Previous studies investigted the reltionship between the ERG chnges nd dibetic eyes. Holopigin et l. [29] found tht the b-wve ctivity in electroretinogrm might indicte retinl chnges in erly dibetic retinopthy. Couplnd [3] nd Hrdy et l. [31] hve demonstrted tht the mplitude of the scotopic b-wve reflects the bnorml ctivity of the Bipolr cells in dibetes even with the bsence of visible fundus signs of retinopthy. Kern et l. [32] looked t the streptozotocin-induced dibetes in different species of rts nd observed tht ll strins tended to show dibetesinduced impirment of the drk-dpted (scotopic) b-wve mplitude. The ERG results from our study reveled tht the chnges in men b-wve mplitude of re significntly higher in comprison to, wheres the rbbits with turine tretments (both nd V) revel no sttisticl difference ginst control. Therefore, bsed on our ERG results, one cn ssume the following. Firstly, Bipolr cells re probbly more susceptible to the lloxn-induced dibetic dmges, s the scotopic b-wve ppers to be directlygenertedbythebipolrcells.secondly,turinehs protective effects on the dibetic retins, s it minimizes the retinl electrophysiologicl chnges in the dibetic rbbits. We hve lso noticed tht the rbbits fed dily with 4 mg/kg of turine hd fewer electrophysiologicl chnges over time thn the nimls ssigned to 2 mg/kg of turine intke. Therefore, the 4 mg/kg turine supplement my be more superior thn the 2 mg/kg of turine. In regrd to the Bipolr nd Müller cells counts in retinl histologicl evlution, our results reveled no significnt difference in Bipolr nd Müller cells counts mong ll groups (P >.5). This is probbly due to the short experimentl period which ws unble to observe the retinl histology chnges cused by dibetes. This finding confirms previous reserch findings tht the electroretinogrm is ble to detect bnormlities before ny structurl chnges. The body weight losses in lloxn-induced dibetes nimls were well demonstrted in our study s well. On verge, the dibetes group exhibited 31.7% weight loss when compred with the control group t the end of the 15-week experiment. However, those rbbits treted with 2 mg/kg nd 4 mg/kg of turine dily showed similr trend of weight gin s the control group over the study period. This lso implies tht nd V end up with much less weight loss compred to those in.

6 6 Interntionl Journl of Endocrinology Therefore, we cn conclude tht tretment of turine protects the lloxn-induced dibetes rbbits from body weight losses. There re number of limittions to this study. Firstly, we were unble to determine whether the protective effect of turine on the retin in dibetic rbbits cted directly on the retin or the effect ws indirect nd due to hypoglycemic. Secondly, we found no significnt differences between the groups for the histologicl evlution. This my be becuse period of 15 weeks is too short to cuse the rbbits retinl tissues chnge. Future studies could extend the experimentl period to further investigte histologicl differences over time including fluorescein ngiogrphy nd opticl coherence tomogrphy (OCT), prticulrly for the dibetic group. In conclusion, our study demonstrted tht turine possesses ntidibetic ctivities, especilly in the hypoglycemic effect, reduces body weight loss, nd menwhile directly or indirectly minimizes the electrophysiologicl chnges in dibetic retin. Future studies re needed to determine whether these results cn be replicted in other niml models. If these results hold for humn models, turine could be chep nd effective wy to prevent the dmge to retin cused by dibetes. Conflict of Interests The uthors declre tht there is no conflict of interests regrding the publiction of this pper. Acknowledgments The uthors re grteful to Drs. J. C. Ju nd P. Intwich of Ntionl Chung Hsing University, Tiwn, for the technicl ssistnce in the dibetic rbbit model. This work ws supported by Grnt from the Ntionl Science Council (NSC B-4-3-MY3). References [1] UK Prospective Dibetes Study VIII: study design, progress nd performnce, Dibetologi, vol. 34, pp , [2] M. I. 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7 Interntionl Journl of Endocrinology 7 [26] F. Frnconi, M. A. Di Leo, F. Bennrdini, nd G. Ghirlnd, Is turine beneficil in reducing risk fctors for dibetes mellitus? Neurochemicl Reserch,vol.29,no.1,pp ,24. [27] S. W. Schffer, J. Azum, nd M. Mozffri, Role of ntioxidnt ctivity of turine in dibetes, Cndin Journl of Physiology nd Phrmcology,vol.87,no.2,pp.91 99,29. [28] I. Perlmn, The Electroretinogrm: ERG. Webvision: The Orgniztion of the Retin nd Visul System, Online Textbook of the Visul System, University of Uth, Slt Lke City, Uth, USA, 211. [29] K. Holopigin, W. Seiple, M. Lorenzo, nd R. Crr, A comprison of photopic nd scotopic electroretinogrphic chnges in erly dibetic retinopthy, Investigtive Ophthlmology nd Visul Science, vol. 33, no. 1, pp , [3] S. G. Couplnd, A comprison of oscilltory potentil nd pttern electroretinogrm mesures in dibetic retinopthy, Document Ophthlmologic,vol.66,no.3,pp ,1987. [31]K.J.Hrdy,C.Fisher,P.Heth,D.H.Foster,ndJ.H.B. Scrpello, Comprison of colour discrimintion nd electroretinogrphy in evlution of visul pthwy dysfunction in retinopthic IDDM ptients, The British Journl of Ophthlmology,vol.79,no.1,pp.35 37,1995. [32]T.S.Kern,C.M.Miller,J.Tng,Y.Du,S.L.Bll,ndL. Berti-Mter, Comprison of three strins of dibetic rts with respect to the rte t which retinopthy nd tctile llodyni develop, Moleculr Vision,vol.16,pp ,21.

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