Photodynamic therapy combined with anti-vascular endothelial growth factor therapy for pachychoroid neovasculopathy.

This multicenter retrospective study was conducted to evaluate the 1-year treatment outcome of photodynamic therapy (PDT) combined with anti-vascular endothelial growth factor (VEGF) therapy for pachychoroid neovasculopathy (PNV). A total of 42 eyes of 42 patients with treatment-naïve PNV who were treated with PDT combined with intravitreal injections of an anti-VEGF agent (ranibizumab or aflibercept) for 1 year. All eyes showed exudative and/or hemorrhagic changes that affected the fovea at baseline. After the initial combination therapy, subfoveal choroidal thickness (SCT) and central retinal thickness (CRT) were significantly reduced and were maintained as such for 12 months (P < 0.01 in SCT and CRT). The best-corrected visual acuity (BCVA) (0.19 ± 0.30 at baseline) significantly improved at 3 months (0.15 ± 0.29, P < 0.05) and further improved at 12 months (0.10 ± 0.30, P < 0.01) when compared to that at baseline. After the initial combination therapy, 32 eyes (76.2%) required no additional treatments for 12 months. The mean number of additional PDT and intravitreal injections of anti-VEGF agents was 0.1 ± 0.3 and 0.9 ± 1.9, respectively. Of the 42 eyes included in this study, 22 eyes (52.4%) had polypoidal lesions at baseline. No significant differences in SCT, CRT, or BCVA were observed at any time points between eyes with and without polypoidal lesions. Of 20 eyes without polypoidal lesions, only 1 eye (5.0%) needed additional treatments. PNV, especially without polypoidal lesions, can be treated effectively with PDT combined with anti-VEGF therapy with few sessions.

Results of switchback from ranibizumab to aflibercept in patients with exudative age-related macular degeneration.

PURPOSE: Intravitreal injection of anti-VEGF drugs has become standard therapy for patients with exudative age-related macular degeneration (AMD). However, some patients do not exhibit sufficient response to the drugs for suppression of choroidal neovascularization activity. We investigated the efficacy of switchback from ranibizumab to aflibercept in patients with AMD who could not achieve further benefit beyond initial therapy of aflibercept injection. METHODS: Eleven eyes of eleven patients were included in this study. Two patients were nonresponders, and nine exhibited tachyphylaxis to aflibercept. All patients received three monthly injections of ranibizumab as an initial phase of switching and received aflibercept as a switchback drug. We investigated changes in injection interval, visual acuity, and central retinal thickness. RESULTS: In four patients (36.4%), injection interval was extended. The interval was 6.73 weeks before switch and 9.27 weeks after switchback (P=0.96). LogMAR visual acuity was 0.22 before switch and 0.24 after switchback (P=0.62). Central retinal thickness was 306.8 µm before switch and 256.1 after switchback (P=0.13). In all patients who were nonresponders to aflibercept, injection interval could not be extended. CONCLUSION: A switchback from ranibizumab to aflibercept may be beneficial in some patients with AMD who exhibit tachyphylaxis to aflibercept.

A modified treat-and-extend regimen of aflibercept for treatment-naïve patients with neovascular age-related macular degeneration.

PURPOSE: To evaluate a modified treat-and-extend (TAE) regimen of intravitreal aflibercept injection (IAI) for treatment-naïve patients with neovascular age-related macular degeneration (AMD). METHODS: Thirty-six eyes (36 patients) treated with the modified TAE regimen were evaluated at 12 months retrospectively. The modified TAE regimen consisted of three steps: 1) an induction phase, during which patients were treated with ≥ 3-monthly IAIs until exudative activity disappeared, 2) an observation phase, during which patients were monitored until exudative activity appeared, and 3) a TAE phase, for which the initial treatment interval was determined based on the disease recurrence interval, followed by treatment intervals changing by 2 weeks. RESULTS: Mean logMAR BCVA improved significantly from 0.48 ± 0.51 at baseline to 0.40 ± 0.53 at 12 months (P < 0.01), and was maintained (losing <0.3 logMAR units) in 35 eyes (97.2 %). Mean central retinal thickness and central choroidal thickness decreased significantly after 12 months. In the TAE phase, the distribution of treatment intervals was ≥8 weeks in 64.7 % (11 eyes) at 12 months. The mean number of injections was 4.53. CONCLUSION: A modified TAE regimen of IAI for neovascular AMD produced good functional outcomes over 12 months with the small number of injections.

Long-term expression of glial cell line-derived neurotrophic factor slows, but does not stop retinal degeneration in a model of retinitis pigmentosa.

Retinitis pigmentosa is a group of diseases in which one of hundreds of mutations causes death of rod photoreceptor cells and then cones gradually die from oxidative damage. As different mutations cause rod cell death by different mechanisms, mutation-specific treatments are needed. Another approach is to use a neurotrophic factor to promote photoreceptor survival regardless of the mechanism of cell death, and previous studies have demonstrated encouraging short-term results with gene transfer of glial cell line-derived neurotrophic factor (GDNF). We generated rd10 mice with doxycycline-inducible expression of GDNF in photoreceptors (Tet/IRBP/GDNF-rd10 mice) or retinal pigmented epithelial cells (Tet/VMD2/GDNF-rd10 mice). In doxycycline-treated Tet/IRBP/GDNF-rd10 mice, there was a 9.3 × 10(4) -fold increase in Gdnf mRNA at P35 and although it decreased over time, it was still increased by 9.4 × 10(3) -fold at P70. Gdnf mRNA was increased 4.5 × 10(2) -fold in doxycycline-treated Tet/VMD2/GDMF-rd10 mice at P35 and was not significantly decreased at P70. GDNF protein levels were increased about 2.3-fold at P35 and 30% at P70 in Tet/IRBP/GDNF-rd10 mice, and in Tet/VMD2/GDNF-rd10 mice they were increased 30% at P35 and not significantly increased at P70. Despite the difference in expression, Tet/IRBP/GDNF-rd10 and Tet/VMD2/GDNF-rd10 mice had comparable significant increases in outer nuclear layer thickness and mean photopic and scotopic ERG b-wave amplitudes compared with rd10 mice at P35 which decreased, but was still significant at P70. Compared with rd10 mice, Tet/IRBP/GDNF-rd10 and Tet/VMD2/GDNF-rd10 mice had comparable significant improvements in cone density at P50 that decreased, but were still significant at P70. These data indicate that despite a large difference in expression of GDNF, Tet/IRBP/GDNF-rd10 and Tet/VMD2/GDNF-rd10 provide comparable slowing of photoreceptor degeneration, but cannot stop the degeneration.

Injury-independent induction of reactive gliosis in retina by loss of function of the LIM homeodomain transcription factor Lhx2.

Müller glia are the primary glial subtype in the retina and perform a wide range of physiological tasks in support of retinal function, but little is known about the transcriptional network that maintains these cells in their differentiated state. We report that selective deletion of the LIM homeodomain transcription factor Lhx2 from mature Müller glia leads to the induction of reactive retinal gliosis in the absence of injury. Furthermore, Lhx2 expression is also down-regulated in Prph2(Rd2/Rd2) animals immediately before the onset of reactive gliosis. Analysis of conditional Lhx2 knockouts showed that gliosis was hypertrophic but not proliferative. Aging of experimental animals demonstrated that constitutive reactive gliosis induced by deletion of Lhx2 reduced rates of ongoing apoptosis and compromised both rod and cone photoreceptor function. Additionally, these animals showed a dramatically reduced ability to induce expression of secreted neuroprotective factors and displayed enhanced rates of apoptosis in light-damage assays. We provide in vivo evidence that Lhx2 actively maintains mature Müller glia in a nonreactive state, with loss of function initiating a specific program of nonproliferative hypertrophic gliosis.

Prolonged blockade of VEGF receptors does not damage retinal photoreceptors or ganglion cells.

It has recently been reported that relatively short-term inhibition of vascular endothelial growth factor (VEGF) signaling can cause photoreceptor cell death, a potentially clinically important finding since VEGF blockade has become an important modality of treatment of ocular neovascularization and macular edema. However, in a set of studies in which we achieved extended and complete blockage of VEGF-induced vascular leakage through retinal expression of a VEGF binding protein, we did not observe any toxicity to retinal neurons. To follow-up on these apparently discrepant findings, we designed a set of experiments with the kinase inhibitor SU4312, which blocks phosphorylation of VEGF receptors, to look directly for evidence of VEGF inhibition-related retinal toxicity. Using transgenic mice with sustained expression of VEGF in photoreceptors, we determined that periocular injection of 3 microg of SU4312 every 5 days markedly suppressed subretinal neovascularization, indicating effective blockade of VEGF signaling. Wild-type mice given periocular injections of 5 microg of SU4312 every 5 days for up to 12 weeks showed normal scotopic and photopic electroretinograms (ERGs), no TUNEL stained cells in the retina, and no reduction in outer nuclear layer thickness. Incubation of cultured ganglion cells or retinal cultures containing photoreceptors with high doses of SU4312 did not reduce cell viability. These data suggest that blocking VEGF signaling in the retina for up to 12 weeks does not damage photoreceptors nor alter ERG function and should reassure patients who are receiving frequent injections of VEGF antagonists for choroidal and retinal vascular diseases.

Effects of intraocular ranibizumab and bevacizumab in transgenic mice expressing human vascular endothelial growth factor.

OBJECTIVE: This study compared the effects of intraocular injections of ranibizumab (RBZ) and bevacizumab (BVZ) in transgenic mouse models in which human vascular endothelial growth factor (VEGF) causes subretinal neovascularization (NV) or exudative retinal detachment. DESIGN: Randomized trials in animal models. PARTICIPANTS: Transgenic mice in which the rhodopsin promoter drives expression of human VEGF in photoreceptors (rho/VEGF mice) and double transgenic mice with doxycycline-inducible expression of human VEGF in photoreceptors (Tet/opsin/VEGF mice). METHODS: Rho/VEGF mice received intraocular injections of RBZ, BVZ, or vehicle, and after various time periods the area of subretinal NV was measured. Tet/opsin/VEGF mice were given an intraocular injection of RBZ, BVZ, or vehicle, and after 5 days of doxycycline treatment the presence or absence of retinal detachment was determined. MAIN OUTCOME MEASURES: Area of subretinal NV per retina in rho/VEGF mice and the occurrence of retinal detachment in Tet/opsin/VEGF mice. RESULTS: In rho/VEGF mice, intraocular injections of RBZ or BVZ strongly suppressed subretinal NV, but the duration of effect was greater for BVZ. Three injections of 10 microg of BVZ over the course of 2 weeks not only suppressed subretinal NV in the injected eye but also caused significant suppression in the fellow eye, indicating a systemic effect. In doxycycline-treated Tet/opsin/VEGF mice, intraocular injection of 10 microg of BVZ significantly reduced the incidence of exudative retinal detachment compared with injection of 10 microg of RBZ. Injection of 25 microg of BVZ reduced the incidence of retinal detachment in both eyes. CONCLUSIONS: Intraocular injections of RBZ and BVZ had similar efficacy in rho/VEGF mice, but the duration of effect was greater for BVZ. In Tet/opsin/VEGF mice, in which expression levels of human VEGF are very high and the phenotype is severe, BVZ showed greater efficacy than RBZ. In both models, higher doses or repeated injections of BVZ, but not RBZ, resulted in a systemic effect. These data suggest that BVZ is not inferior to RBZ for treatment of subretinal NV in mice and is superior in a severe model. The systemic effects of BVZ after intraocular injection deserve further study and consideration of their potential consequences. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

NADPH oxidase plays a central role in cone cell death in retinitis pigmentosa.

Retinitis pigmentosa (RP) is a collection of diseases in which rod photoreceptors die from a variety of mutations. After rods die, the level of tissue oxygen in the outer retina becomes elevated and there is progressive oxidative damage to cones that ultimately triggers apoptosis. In this study, we investigated the hypothesis that NADPH oxidase (Nox) and/or xanthine oxidase serve as critical intermediaries between increased tissue oxygen and the generation of excessive reactive oxygen species that cause oxidative damage to cones. Apocynin, a blocker of Nox, but not allopurinol, a blocker of xanthine oxidase, markedly reduced the superoxide radicals visualized by hydroethidine in the outer retina in the retinal degeneration-1 (rd1(+/+)) model of RP. Compared to rd1(+/+) mice treated with vehicle, those treated with apocynin, but not those treated with allopurinol, had significantly less oxidative damage in the retina measured by ELISA for carbonyl adducts. Apocynin-treated, but not allopurinol-treated, rd1(+/+) mice had preservation of cone cell density, increased mRNA levels for m- and s-cone opsin, and increased mean photopic b-wave amplitude. In Q344ter mice, a model of dominant RP in which mutant rhodopsin is expressed, apocynin treatment preserved photopic electroretinogram b-wave amplitude compared to vehicle-treated controls. These data indicate that Nox, but not xanthine oxidase, plays a critical role in generation of the oxidative stress that leads to cone cell death in RP and inhibition of Nox provides a new treatment strategy.

PARP inhibitors accelerate N-methyl-N-nitrosourea-induced cataractogenesis in Sprague-Dawley rats.

BACKGROUND: There have been no previous studies of the effects of poly(ADP-ribose) polymerase (PARP) inhibitors on N-methyl-N-nitrosourea (MNU)-induced cataractogenesis in rats. MATERIALS AND METHODS: A single intraperitoneal injection of 70 mg/kg MNU was administered to 15-day-old male and female Sprague-Dawley rats. In Experiment 1, rats were then subcutaneously injected with 1000 mg/kg nicotinamide either once or 3 times at 1-week intervals. In Experiment 2, rats were subcutaneously injected once with 50 mg/kg 3-aminobenzamide. For comparison, the following age-matched controls were included: MNU-untreated nicotinamide-injected rats, MNU-untreated 3-aminobenzamide-injected rats, and MNU-untreated PARP-inhibitor-untreated rats. Rats were examined for lens opacity. At 28 days after MNU injection, 10 to 20 rats per group were sacrificed In Experiment 1, at 3 days after MNU injection, 10 rats per group were sacrificed for apoptosis and cell proliferation detection. RESULTS: MNU caused lens epithelial cell apoptosis in the germinative zone, as indicated by TUNEL staining. However, regardless of MNU treatment lens epithelial cell proliferation was consistently seen in the germinative zone and sporadically seen in the central zone. At 28 days after MNU, mature cataracts were observed Nicotinamide significantly accelerated lens opacity and cataractogenesis, as indicated by a cataract index 3-Aminobenzamide significantly accelerated the development of lens opacity and tended to accelerate cataractogenesis. CONCLUSION: The PARP inhibitors nicotinamide and 3-aminobenzamide accelerated MNU-induced cataractogenesis.

An autopsy case of pseudoxanthoma elasticum: histochemical characteristics.

An autopsy case of pseudoxanthoma elasticum is reported. A Japanese female patient complained of yellow papules on the neck, precordium, and axilla, beginning at 54 years of age. When the patient was 58 years old, in response to her visual disturbance a funduscopic examination was performed, revealing angioid streaks, and skin biopsy identified a characteristic pseudoxanthoma elasticum (PXE) lesion. The patient developed congestive heart failure, and following mitral valve prolapse and regurgitation flow into the left atrium, mitral valve replacement with a prosthetic valve was performed when the patient was 65 years old. Soon afterward, the patient complained of gait disturbance, and she died of congestive heart failure at 68 years of age. Autopsy specimen revealed fragmented, granular, and calcified elastic fibers in the middle to deep dermis and in the thickened subendocardium, and small to medium-sized muscular arteries revealed fragmented, laminated, and calcified elastic lamina; vascular changes were seen in the heart, lung, kidney, gastrointestinal tract, and iliac artery. Disrupted elastic fibers were visualized using the Weigert resorcin fuchsin method and were stained positive by antielastin and antifibronectin antibodies. Calcification was confirmed by von Kossa staining. Affected areas were PAS-positive after diastase digestion, indicating the presence of glycoprotein. Affected areas were colloidal iron-positive, indicating the presence of proteoglycan matrix.

N-methyl-N-nitrosourea-induced mammary carcinogenesis is promoted by short-term treatment with estrogen and progesterone mimicking pregnancy in aged female Lewis rats.

We examined the effects of short-term estrogen and progesterone treatment mimicking pregnancy in aged female Lewis rats on the development of N-methyl-N-nitrosourea (MNU)-induced mammary carcinoma. Rats were administered a single intraperitoneal injection of 20 mg/kg MNU at 7 weeks of age and half of those rats were administered a subcutaneously implanted 21-day release pellet containing 0.5 mg 17beta-estradiol and 32.5 mg progesterone (E/P) at 24 weeks of age. The rats were then monitored for the occurrence of mammary tumors. Rats were sacrificed when the largest mammary tumor became > or = 1 cm in diameter, or when the rat reached 48 weeks of age. Development of MNU-induced mammary carcinomas was accelerated after short-term E/P treatment, compared with E/P-untreated rats: the incidence of > or = 1-cm mammary carcinomas tended to increase (60 vs. 44%); the latency tended to shorten (28.7 vs. 34.6 weeks); and cancer multiplicity (number of all-sized carcinomas per rat) significantly increased (1.8 vs. 0.8). In E/P-treated rats, comedo necrosis was frequently seen and the incidence of estrogen receptor and/or progesterone receptor-negative mammary carcinomas was significantly increased. Early age at full-term pregnancy or short-term hormone treatment mimicking pregnancy may suppress the risk of breast cancer, but the age of hormone exposure is a crucial factor, because hormone exposure mimicking pregnancy in aged individuals may exert effects opposite of those exerted in younger individuals.

Autopsy case of desminopathy involving skeletal and cardiac muscle.

Desminopathy is a familial or sporadic skeletal and cardiac muscular dystrophy caused by mutation in the desmin gene. Desmin-reactive deposits in the affected muscles are the morphological hallmarks of this disease. Herein is reported an autopsy case of a 57-year-old Japanese man with adult-onset skeletal muscle weakness and atrioventricular (A-V) conducting block, with a missense A337P mutation in exon 5 of the desmin gene. Disease onset occurred when the patient was 45 years old. The initial presentation was lower limb weakness, and the weakness progressed to the upper limbs. When the patient was 51 years old, a cardiac pacemaker was implanted due to complete A-V block. When the patient was 53 years old, respiratory insufficiency occurred due to weakness of respiratory muscles, and the patient died at the age of 57 years. On autopsy, intrasarcoplasmic desmin-immunoreactive deposits were identified in the skeletal and cardiac muscle, and abnormal accumulations of granulofilamentous material were identified at the ultrastructural level. In the cardiac conducting system, calcification was observed at the bundle of His, and sporadic calcium deposits were observed at the left and right bundle branches.

Poly (ADP-ribose) polymerase inhibitor 3-aminobenzamide rescues N-methyl-N-nitrosourea-induced photoreceptor cell apoptosis in Sprague-Dawley rats through preservation of nuclear factor-kappaB activity.

The activation of poly (ADP-ribose) polymerase (PARP) plays a pivotal role in mediating N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell apoptosis. We examined the retinoprotective effects of the PARP inhibitor 3-aminobenzamide (3-AB) against MNU-induced retinal damage in relation to dose and timing of prescription, and the involvement of the transcription factor nuclear factor (NF)-kappaB. Female Sprague-Dawley rats were intraperitoneally injected with 60 mg/kg MNU at 50 days of age, and were then immediately given a subcutaneous injection of 0, 1, 5, 10, 30 or 50 mg/kg of 3-AB, or were injected with 50 mg/kg 3-AB 12h before, concurrently, or 4, 6 or 12h after MNU. Rats were killed 3 and 7 days after MNU, and MNU-treated and 3-AB-injected retinas were compared with MNU-untreated control retinas or MNU-treated/3-AB-uninjected retinas. Apoptosis in photoreceptor cells was detected by performing formamide-induced DNA denaturation and staining with anti-single-stranded DNA antibody. Retinal morphologies were compared and evaluated morphometrically using the photoreceptor cell ratio and retinal damage ratio as indices to evaluate the efficacy of 3-AB. We examined expression of the phosphorylated form of NF-kappaB and IkappaBalpha (p-NF-kappaB and p-IkappaBalpha, respectively) in retinas of MNU-treated rats concurrently treated with or without 50mg/kg 3-AB, compared with MNU-untreated control retinas. 3-AB dose-dependently suppressed photoreceptor cell apoptosis: 50mg/kg 3-AB injected concurrently with MNU completely rescued photoreceptor cell damage; 30 mg/kg 3-AB significantly reduced photoreceptor cell damage; 10 mg/kg 3-AB tended to suppress photoreceptor cell damage; or=4h after MNU, it did not exert a retinoprotective effect. p-NF-kappaB levels of MNU-treated rat retinas were significantly lower than those of MNU-untreated control retinas, while 50 mg/kg 3-AB injected concurrently with MNU preserved the p-NF-kappaB levels; p-IkappaBalpha levels tended to decrease after MNU injection, compared with untreated control retinas, but the difference was not significant. Thus, 3-AB dose-dependently suppressed MNU-induced retinal damage, and 50mg/kg 3-AB injected concurrently with MNU completely rescued photoreceptor cell apoptosis via preservation of NF-kappaB activity.

Biphasic effects of zeranol on the growth of estrogen receptor-positive human breast carcinoma cells.

Zeranol (alpha-zearalanol) is a metabolite of the mycoestrogen zearalenone, and is used as a growth promoter of livestock due to its strong estrogenic activity. In the present study, we investigated the effects of zeranol on the growth of estrogen receptor (ER)-positive and -negative human breast carcinoma cells in vitro, and the molecules involved. At low concentrations, zeranol accelerated the growth of ER-positive MCF-7 and KPL-1 human breast carcinoma cells, but did not affect the growth of ER-negative MDA-MB-231 cells. At high concentrations, zeranol suppressed the growth of both ER-positive and -negative human breast carcinoma cells. The acceleration of ER-positive cell growth by low-dose zeranol involved the down-regulation of p21Cip1 (a cyclin-dependent kinase inhibitor), which resulted in cell cycle progression. High-dose zeranol induced the formation of a sub-G1 fraction and the up-regulation of the apoptosis stimulator p53, suggesting the induction of apoptosis. Thus, zeranol exerted dose-dependent biphasic effects on ER-positive human breast carcinoma cells, accelerating cell growth at low concentrations and inducing apoptosis at high concentrations.

An autopsy case of primary mixed choriocarcinoma and mature teratoma located in the thymic region associated with elevated human chorionic gonadotropin levels and characteristic testicular changes.

An autopsy case of a 19-year-old male Japanese student with a primary mixed choriocarcinoma and mature teratoma in the thymic region is reported. The patient died 7 days after he first noticed fever and dyspnea. On autopsy, an anterior mediastinal mass was found to be in contact with the thymic gland. The mass weighed 270 g and measured 12.5 cm x 10 cm x 5 cm. The left thoracic cavity contained 2200 ml bloody pleural effusion and 200 g coagula due to hemorrhage from the tumor. Metastasized choriocarcinoma was seen in both lungs and the liver. High serum levels of human chorionic gonadotropin (HCG, 1 634 000 mIU/ml) and a decreased weight of the testes (2.0 g each) with Leydig cell hyperplasia/hypertrophy and the seminiferous tubules with hyaline ghost tubules or Sertoli cell only tubules were seen; other male reproductive organs were histologically normal. Although the serum testosterone level was within the normal range (5.75 ng/ml), luteinizing hormone (LH, 0.1 mIU/ml) and follicle-stimulating hormone (FSH, 0.3 mIU/ml) levels were decreased. High serum levels of HCG and characteristic testicular changes are drscribed.

Effects of gender and gonad status on N-methyl-N-nitrosourea-induced cataractogenesis and retinopathy in Lewis rats.

BACKGROUND: The effects of differences in gender and gonadal status on the occurrence of N-methyl-N-nitrosourea (MNU)-induced cataract and retinopathy were studied using ovary-intact, ovariectomized, testis-intact and testectomized Lewis rats. MATERIALS AND METHODS: Castration was performed at 36 days of age, 50 mg/kg MNU was administered intraperitoneally at 50 days of age, and lens and retinal changes were evaluated at 260 days of age (210 days after MNU injection). RESULTS: Although there was little difference in the incidence of cataract and retinopathy among the groups, ovary-intact rats had a significantly higher cataract index and retinal damage ratio (both are indicators of disease severity) than ovariectomized rats and testis-intact rats, respectively. However, the cataract index and retinal damage ratio did not correlate with the serum 17beta-estradiol and progesterone levels, respectively. CONCLUSION: The presence of ovaries and female gender appear to be associated with greater severity of cataracts and retinopathy, respectively, but the severity of these diseases did not correlate with the serum hormone levels.

Nicotinamide blocks N-methyl-N-nitrosourea-induced photoreceptor cell apoptosis in rats through poly (ADP-ribose) polymerase activity and Jun N-terminal kinase/activator protein-1 pathway inhibition.

Nicotinamide (NAM) blocks N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell apoptosis in rats, though the underlying molecular mechanisms remain unclear. Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) in response to DNA damage plays a pivotal role in apoptosis. Thus, the role of NAM in the regulation of PARP and Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) was investigated by Western blot analyses. During 7 days after the intraperitoneal injection of MNU (60 mg/kg), rat retinas exhibited DNA fragmentation characteristic of apoptosis and activation of PARP, phosphorylation of JNK and c-Jun, induction of AP-1 (c-Jun and c-Fos) and Bax, as well as photoreceptor cell loss. However, when NAM (1000 mg/kg, subcutaneously) was given immediately after MNU, it was found that PARP activation was diminished, the phosphorylation of JNK and c-Jun was suppressed, and the induction of c-Jun, c-Fos and Bax was suppressed. This resulted in the retinal structure being protected. Therefore, NAM blocked MNU-induced photoreceptor cell apoptosis by inhibiting both PARP activity and the JNK/AP-1 signalling pathway.

The semi-quantitative comparison of oxidative stress mediated DNA single and double strand breaks using terminal deoxynucleotidyl transferase mediated end labeling combined with a slot blot technique.

The accumulation of DNA damages by environmental stresses is represented by the steady state level of single strand breaks (SSBs) and double strand breaks (DSBs). Terminal deoxynucleotidyl transferase (TdT) mediated end labeling is suitable in detecting DSBs, but is unsuitable for SSBs due to its catalyzing characteristics. However, the sensitivity of TdT to detect SSBs may be significantly improved by first denaturing the double strands and expose all the DNA nicks as potential substrates for TdT. By coupling DNA denaturation to slot blot southern hybridization, the authors demonstrate the sensitive detection of SSBs as well as DSBs in 20 ng DNA samples derived from a retinal pigment epithelial cell line treated with tert-butyl hydroperoxide. The signal intensity of denatured and TdT-treated DNA in slot blot hybridization correlated to the amount of SSBs calculated in an S1 nuclease digestion assay. The signal ratio between denatured and non-denatured DNA likely approximates the SSBs/DSBs ratio in genomic DNA. The combination of DNA denaturing, TdT treatment and slot blot hybridization could be a useful method to assess oxidative stress-induced DNA strand damages.

The effects on the intraocular pressure and visual field resulting from a switch in the treatment from timolol to betaxolol.

Patients with primary open-angle glaucoma (POAG) were dosed twice-daily with 0.5% timolol maleate for 2 years, followed by 2 years of twice-daily dosing with 0.5% betaxolol hydrochloride. The changes in intraocular pressure (IOP) and effects on visual field by each treatment were monitored over this 4-year period. Both the timolol and betaxolol treatments controlled IOP. The mean MD (mean deviation in visual field) values at 24 months of the timolol treatment were -6.18 dB (decibels) and -5.32 dB. Mean MD baseline values following 24 months of the timolol treatment produced 2.31 dB and 0.95 dB reductions in sensitivity in the right and left eyes, and the reduction in the right eye was statistically significant (p < 0.01). The mean MD values after 24 months of the betaxolol treatment were -3.82 dB in the right eyes, an increase of 2.36 dB, and -4.05 dB in the left eyes, up by 1.28 dB. The results from this clinical trial demonstrated that betaxolol was superior over timolol in improving mean retinal sensitivity.