The Mechanisms Of Photoreceptor Apoptosis In SD Rats Induced By NNU And The Transient Protection By Minocycline

PrefaceRetinitis pigmentosa(RP) is a human hereditary disorder characterized by photoreceptors loss leading to visual disturbance and eventually to blindness.Main symptoms include progressive loss of visual functions with night blindness, narrowing of the visual field,reduced central vision and increasing sensitivity to glare. RP has been the main cause of legal blindness in the developed countries.Disease prevalence is about 0.3%(Bird,1995;Kellner et al.,2004).The mechanism of RP is complex.The mode of inheritance may be autosomal dominant or recessive or X-linked manner,as well as in digenic,mitochondrial,and simplex modes.So called simplex cases mostly are transmitted by autosomal-recessive or x-chromosomal inheritance with no other affected family members known.Despite the nearly indistinguishable clinical characteristics of RP with different modes of inheritance,a broad genetic heterogeneity exists.At least over 50 genes abnormal have been known,and additional loci are yet to be mapped. Although multiple genes encoding photoreceptor-specific proteins have been implicated in retinitis pigmentosa,the final common pathway of the disease is apoptotic cell death of photoreceptor cells.Therefore,apoptosis has been a potential therapeutic target for retinitis pigmentosa.Animal models are indispensable for analysis of this disease.Varieties of animal models with retinal degeneration have been used for many years to study disease progression and pathology.Well established animal models of retinal degeneration are very important for human researches and have led to a better understanding of the disease and to the development of possible therapeutic strategies.Herrold(1967) was the first to report retinal degeneration in Syrian golden hamsters after systemic administration of N-methyl-N-nitrosourea(MNU),an alkylating agent.Later,Nakajima et al.confirmed that MNU-induced retinal degeneration in rats was due to photoreceptor cell apoptosis,and found that MNU-induced photoreceptor cell apoptosis can be induced in a variety of animals (Tsubura et al.,2003).Because MNU-induced photoreceptor damage is highly reproducible,and because the disease progression ends within 1 week,MNU-induced photoreceptor apoptosis in animals provide useful models for evaluation of therapeutic strategies for human retinitis pigmentosa.For example,the following compounds exhibit retinoprotecive effects in MNU-induced retinal damage models in rats:Ac-DEVD-CHO,a caspase-3 inhibitor(Yoshizawa et al.,2000); docosahexaenoic acid(Moriguchi et al.,2003,2004),an essential fatty acid; tetramethylpyrazine,an alkaloid present in Ligusticum wallichii Franch(L.wallichii) (Yang et al.,2005);and nicotinamide,a water-soluble B-group vitamin(vitamin B3) (Kiuchi et al.,2002,2003),and nicotinamide suppresses MNU-induced photoreceptor cell apoptosis via inhibition of poly(ADP-ribose) polymerase(PARP) activation (Uehara et al.,2006).Minocycline is a highly lipophilic semisynthetic derivative of tetracycline that is capable of crossing the blood-brain barrier.Minocycline has been shown to be therapeutically effective in various experimental models of central nervous system (CNS) injury and disease that comprise inflammatory and apoptotic components,and these actions are distinct from its ability to inhibit bacterial protein synthesis. Minocycline treatment conferred neuroprotection following spinal cord injury, excitotoxicity and ischemic injury,also prevented neurodegeneration in the 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine(MPTP) mouse model of Parkinson's disease,and delayed mortality in the R6/2 mouse model of Huntington's disease as well as in amyotrophic lateral sclerosis mice.Although the mechanisms of actions of minocycline treatments have not been fully elucidated,its beneficial effects appear to be associated with inhibition of microglial activation and proliferation(Fan et al.,2005;He et al.,2001;Krady et al., 2005;Tikka et al.,2001;Tikka and Koistinaho,2001;Wu et al.,2002;Yrj(?)nheikki et al.,1998) and downregulation of inducible nitric oxide synthase transcription (Yrj(?)nheikki et al.,1998).Neuroprotection by minocycline may also be the result of direct anti-apoptotic effects,including inhibition of caspase-1(Chen et al.,2000; Sanchez Mejia et al.,2001) and caspase-3(Chen et al.,2000;Krady et al.,2005),and blockade of the permeability transition pore to prevent cytochrome c release from the mitochondria(Teng et al.,2004;Zhu et al.,2002).These findings indicate that minocycline is able to influence multiple cascades of the secondary degenerative responses that occur in the injured brain,suggesting that it may be a valuable therapeutic agent for the treatment of RP.Recent studies observed that minocycline delayed photoreceptor apoptosis in rds mice.The possibility that minocycline may exert its protective effect through iNOS-suppressive or microglial-suppressive mechanisms.In the present study,we investigated the mechanism of MNU induced photoreceptor apoptosis in Sprague-Dawley rats with the help of microarrays and examined whether the treatment with minocycline is protective in this experimental model of RP.The volume of tissue damage,as well as the amount of apoptotic cell death,the level of apoptosis related mRNAs and proteins expression,was assessed for up to 7 days post-injury in minocycline and saline-treated rats.We attempted to discover the intracellular targets involved in the anti-apoptotic actions of minocycline by determining whether the mechanism of the neuroprotection involves,at least in part,inhibition caspase cascade upstream signals in the cell suicide sequence.PartⅠPhotoreceptor Apoptosis Induced by a Single Systemic Administration of N-methyl-N-nitrosourea in the SD RatsPURPOSE.To verify the sequential retinal changes in Sprague-Dawley(SD) rats induced by N-methyl-N-nitrosourea(MNU).METHODS.Female SD rats received a single intraperitoneal injection of 60 mg/kg MNU at 7 weeks of age,and the retina was examined light and electron microscopically,by the TdT-mediated dUTP-digoxigenin nick end labeling(TUNEL) method and flow-cytometer at 12,24,48,72 hours and 7 days after the treatment.RESULTS.The MNU injection caused apoptosis only could be found in the retinal outer nuclear layer.Photoreceptor apoptosis occurred 12 hours after the treatment and resulted in photoreceptor cells loss at day 7.The apoptotic indexes of the photoreceptor cells in the retinal outer nuclear layer 12,24,48,72 hours and 7 days after MNU treatment calculated by TUNEL labeling were(16.3±2.1)%,(31.5±3.4)%,(19.3±1.5)%,(17.±1.2)%,and(3.8±1.5)%.The ratio of apoptosis cells in the whole retina amounted by flow-cytometer were(6.88±0.81)%,(7.98±1.45)%, (12.2±4.44)%,(9.63±2.78)%,(10.15±1.56)%.CONCLUSIONS.MNU intraperitoneal injection specifically cause photoreceptor apoptosis.The primary event and the course of this disease closely resemble those of retinitis pigmentosa in humans.This is a well established animal models of retinal degeneration that could be very helpful to human researches and lead to a better understanding of the disease and to the development of possible therapeutic strategies.PartⅡThe Mechanisms of N-methyl-N-nitrosourea-indueed Apoptosis of Photoreceptor Cells in SD RatsSectionⅠMicroarray Based Analysis of Up-regulated Apoptosis GenesPURPOSE.To apply microarray based technology to detect up-regulated genes related to photoreceptor apoptosis 12 hours after MNU intraperitoneal injection.METHODS.A rat apoptosis microarray(ORN-012,Superarray) profiles the expression of 112 genes involved in apoptosis was used to examine the changes in RNA profiles of retinas 12 hours after MNU intraperitoneal injection.Total RNA was extracted from retinas of both eyes of 4 SD rats.RNA was then reverse transcribed into cDNA.Radiolabeled cDNA was then hybridized to the microarray.The results were assessed by autoradiography and densitometric analysis.The study was repeated three times.RESULTS.Apoptotic genes in the retina up-regulated more than 2 folds compared to control 12 hours after MNU intraperitoneal injection including:TNFR1 (3.3 folds),FADD(7.7 folds),CASPASE 8(9.0 folds),CASPASE 3(2.4 folds),BAK 1 (6.5 folds),BAX(2.8 folds).Classified according to their functions,these genes belonged to TNF receptors(TNFR1),Death effector domain family members(FADD), CASPASES(CASPASE 8,CASPASE 3) and members of the BCL-2 gene family(BAK 1,BAX),respectively.CONCLUSIONS.Up-regulation of TNFR1,FADD,CASPASE 8,CASPASE 3,BAK 1,and BAX may be responsible to the SD rats photoreceptor apoptosis induced by MNU.SectionⅡVerification of the Results of the MicroarrayPURPOSE.To verify the mRNA and protein expression of the results of the microarray.METHODS.Because TNFR1,FADD may act as the CASPASES cascade upstream signals in the cell suicide sequence.The up-regulation of TNFR1,FADD were chosen to be confirmed.Their mRNA levels were studied by Real time PCR. Expression of the corresponding proteins were evaluated by immunohistochemistry method,and Western blot analysis.RESULTS.The mRNA levels of TNFR1,FADD were significantly up-regulated in the SD rats retina 12 hours after MNU intraperitoneal injection. Immunohistochemistry method shown both TNFR1 and FADD specifically expressed in photoreceptors.Western blot analysis confirmed the up-regulation of TNFR1, FADD proteins.CONCLUSIONS.Since the TNFR1 and FADD may be the CASPASES cascade upstream signals in the cell apoptosis,the up-regulation of TNFR1,FADD both in mRNA and protein levels indicates that TNF R1/FADD/CASPASE signal pathway may be responsible,at least in part,to MNU induced photoreceptor apoptosis.PartⅢThe Study on Protective Effects of Minocycline on MNU Induced Photoreceptor Apoptosis and its MechanismsPURPOSE.To investigated whether minocycline is neuroprotective against MNU-induced photoreceptor apoptosis and the possible mechanisms.METHODS.Apoptosis of photoreceptors was induced in SD rats by MNU intraperitoneal injection.Minocycline(50 mg/kg) was administered 12 hours before and every 12 hours after MNU injection in rats.Minocycline and vehicle-treated retinas subjected to MNU injury were compared with respect to quantitative counts of TUNEL stains,morphometry of the outer nuclear layer(ONL) thickness and histopathological examination.Expression of TNF R1 and FADD was studied using real time PCR,and Western blot.RESULTS.The minocycline treatment not only reduced the TUNEL index through the 7 days after MNU treatment,but also attenuated the ONL thickness reduction 3 days after MNU injection.The increase of mRNA level of TNF R1 and FADD in the retinas that underwent apoptosis was significantly reduced in minocycline treated group.Intraperitoneal administration of minocycline before and after MNU injection also resulted in less TNF R1 and FADD expression,as assessed by the Western blot.The decrease were statistically significant.CONCLUSIONS.The use of a minocycline was effective in the suppression of MNU-induced photoreceptor apoptosis and may be a therapeutic intervention in human retinitis pigmentosa.Although the mechanisms of actions of minocycline treatments have not been fully elucidated,its beneficial effects appear to be associated with inhibition of TNF R1 and FADD expressed in photoreceptor cells.