Hiroyuki Nambu

MORPHOLOGIC CHARACTERISTICS OF N-METHYL-N-NITROSOUREA-INDUCED RETINAL DEGENERATION IN C57BL MICE

Morphologic characteristics of retinal degeneration induced by a single systemic administration of Methyl‐Knitro‐sourea (MNU) in mice was Investigated. The aim was to characterize the MNU‐Induced retinal lesions In mice and compare them with human retinitis pigmentosa. A dose of 60 mg/kg body weight MNU, Injected intraperttoneally into male and female C57BL mice, evoked progressive retinal degeneration in all treated mice, while control mice re mined normal. An early change was photoreceptor apoptosis followed by Infiltration of macrophages and swelling of the pigment epithellal cells with phagosomal inclusions for apoptotic photoreceptor cell removal. Loss of the majority of photoreceptor cells occurred within a week Then, Feulgenpositive corpuscies, indicative of an aggregation of degenerative photoreceptor elements, vitread the outer limiting membrane were surrounded by Müller cell processes, and the duplication of the pigment epithelial cells sciurid the outer limiting membrane were seen 2 and 3 weeks after the treatment. Finally, the Feulgen‐positive corpuscles disappeared and Müller cell processes were in direct contact with the continuous lining of the single layer of pigment epithellal cells. As in retinitis pigmentosa in humans, the primary event was loss of photoreceptor cells by apoptosis, but the migration of the pigment epithellal cells within the retina was not seen in the present model.

Retinal degeneration induced by N-methyl-N-nitrosourea in Syrian golden hamsters

Abstract · Background: The sequential retinal changes in Syrian golden hamsters induced by N-methyl-N-nitrosourea (MNU) have not been studied. · Methods: Female hamsters received a single intraperitoneal injection of 90 mg/kg MNU at 50 days of age, and the retina was examined light and electron microscopically, immunohistochemically and by the TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL) method until 20 weeks after the treatment. · Results: The retinal changes were as follows: (1) Photoreceptor apoptosis occurred 1 day after the treatment and resulted in photoreceptor loss at day 7. During the degeneration, Müller cell proliferation was conspicuous at day 5. (2) After the photoreceptor cell loss, migration of the pigment epithelial cells in all layers of the retina which were in contact with blood vessels occurred. Due to the Müller cell proliferation, gliosis was prominent at the later stage. · Conclusions: The MNU injection caused photoreceptor apoptosis followed by pigment epithelial cell migration around the blood vessels, accompanied by gliosis. The primary event and the course of this disease closely resemble those of retinitis pigmentosa in humans.

Inhibition of tumor growth and metastasis by angiogenesis inhibitor TNP-470 on breast cancer cell lines in vitro and in vivo

Antitumor and antimetastatic activity of the angiogenesis inhibitor O-(chloroacetyl-carbamoyl) fumagillol (TNP-470), a semisynthetic analogue offumagillin, was evaluated in breast cancer cell lines. In an in vitro MTTassay, after 72 hrs continuous exposure to TNP-470, growth inhibition wasobserved in all seven cell lines of murine (JYG-A, JYG-B, DD-762, andBALB/c-MC) or human (KPL-1, MDA-MB-231, and MKL-F) origin, in which the50% inhibitory concentrations (IC50) at 72 hrstreatment were 4.6, 4.4, 4.6, 10.1, 35.0, 25.3, and 33.4 µg/ml,respectively. In an in vivo assay using JYG-A, JYG-B, KPL-1, and MDA-MB-231cells by orthotopic (right thoracic mammary fat pad) transplantation infemale nude mice, TNP-470 at 30 or 50 mg/kg body weight was injected s.c.every other day from the day of tumor cell inoculation until the end of theexperiment. The inhibitory effect on primary tumor growth was obtained inall four cell lines in a dose-dependent manner. In the 50 mg/kgTNP-470-treated group, the reductions in tumor weight of the JYG-A, JYG-B,KPL-1, and MDA-MB-231 cells with respect to the controls were 50%,30%, 4%, and 49%, respectively. Metastasis was seen inthe JYG-A, JYG-B, and KPL-1 cells. The numbers of mice bearing pulmonarymetastases of JYG-A and JYG-B cells and regional axillary lymph nodemetastases of KPL-1 cells were reduced, and TNP-470 at the 50 mg/kg dose toKPL-1 cells significantly reduced lymph node metastases compared with thecontrol. Although the weight gain was retarded in the TNP-470-treated mice,weight loss was not seen. TNP-470 was highly effective in the treatment ofbreast cancer cells. These results suggest that the clinical use of TNP-470may be a promising treatment for breast cancer patients.

A comparison of optic disc topographic parameters in patients with primary open angle glaucoma, normal tension glaucoma, and ocular hypertension

BackgroundHeidelberg Retina Tomograph (HRT) findings have been employed to quantitatively assess the topography of optic discs. We measured topographic parameters of optic discs in patients with primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and ocular hypertension (OH) using an HRT in order to determine whether HRT topographic parameters can be used to differentiate those conditions.MethodsSeventeen eyes in 17 patients with POAG, 23 eyes in 23 patients with NTG, and 15 eyes in 15 patients with OH were examined using an HRT, and the results were analyzed by age, refractive error, and topographic parameters.ResultsAmong the HRT parameters, the mean values for rim area, rim volume, cup disk area ratio, and classification showed significant differences among POAG, NTG, and OH eyes. The mean values for cup area, cup volume, mean RNFL thickness, and RNFL cross section area showed significant differences between POAG and NTG eyes, and NTG and OH eyes, however, not between POAG and OH eyes. Cup shape measure showed significant differences between POAG and OH, and NTG and OH eyes, but not between POAG and NTG eyes.ConclusionsOur results suggest that POAG is distinguishable from NTG and OH based on evaluations of rim area and rim volume. Patients with NTG tend to have larger cupping, smaller rims, and thinner retinal nerve fiber layers as compared to POAG and OH patients. Thus, HRT topographic parameters are useful to differentiate patients with POAG, NTG, and OH.

Pigmentary degeneration induced by N-methyl-N-nitrosourea and the fate of pigment epithelial cells in the rat retina

Pigmentary degeneration of the retina was induced by a single intraperitoneal Injection of 75mgkg of N‐methyl‐N‐nitrosourea (MNU) In female Brown‐Norway colored rats at 50 days of age, which were then observed at 24, 48 and 72 h and 7, 21,35 and 150 days after the treatment. MNU‐treated rats showed selective destruction of the photoreceptor cells by an apoptotic mechanlsm 24 h after the treatment, and the destruction was completed by day 7. During the photoreceptor cell degeneration, proliferation of Miller cells and infiltratlon of macrophages was prominent 72h and 21 days aRttr the treatment, respectively. Müller cell proliferation and macrophage infiltratbn corresponded to degenerative photo‐receptor cell phagocytosis, and prollferating Müller cell processes responded to stabilize the damaged retina. Pigment epithelial cell detachment from the Bruchs membrane was seen 72 h after the treatment, and migration within all layers of the retina was seen at day 7 when photoreceptor Cells were lost. At 21, 35 and 150 days after the treatment, lack of photoreceptor cells and deposition of pigment epithelial cells within the retina but not in contact to vascular endothe‐lial cells were characteristic. MNU‐induced photoreceptor apoptosis followed by Miiller cell and macrophage reaction then pigment epithellal cells deposition withln the retina partially resembles retinitis pigmentosa in humans.

Time‐specific action of N‐methyl‐N‐nitrosourea in the occurrence of retinal dysplasia and retinal degeneration in neonatal mice

The morphologic response of neonatal mouse retina to the alkylating agent N‐methyl‐N‐nitrosourea (MNU) was examined at different periods of retinal development. A dose of 60 mg/kg N‐methyl‐N‐nitrosourea was injected intraperitoneally to neonatal C57BL mice at 0, 3, 5, 8, 11, 14, 17, and 20 days of age and to C3H mice at 0 days of age, and the retinas were examined sequentially. In the C67BL mice, MNU evoked a time‐dependent occurrence of retinal dysplasia and retinal degeneration. With MNU treatment at day 0 and day 3 (the stage of retinal cell proliferation), retinal dysplasia characterized by the progressive disorganization of neuroblasts, which led to the formation of rosettes, was found in the outer neuroblastic/nuclear layer above the normal pigment epithelial cells during days 8–20, but decreased at day 50. The rosettes were surrounded by photoreceptor segments and Muller cell processes, and by photoreceptor nuclei. The MNU response was related to retinal differentiation; following MNU treatment at day 5 or 8 (the stage of retinal cell differentiation) the cells were much less sensitive (i.e. no retinal response was found). However, with MNU treatment at days 11, 14, 17, and 20 (after cellular differentiation), retinal degeneration characterized by selective photoreceptor apoptosis was seen. These results suggest that there is a critical period for the time of MNU administration in the development of mouse retinal lesions. In C3H (rd/rd) mice, MNU treatment at day 0 resulted in retinal degeneration with only slight rosette formation at the peripheral retina.

Cataractogenesis in neonatal Sprague-Dawley rats by N-methyl-N-nitrosourea.

Cataract was induced by a single intraperitoneal injection of 100 mg/kg N-methyl-N-nitrosourea (MNU) to 0-, 5-, 10-, 15-, or 20-day-old male and female Sprague-Dawley rats. In day 0, 5, 10, and 15 MNU-treated rats, mature cataracts were constantly seen 7, 14, 14, and 30 days after dosing, respectively. In the day 20 MNU-treated rats, only subcapsular cataract was seen 30 days after dosing. Therefore, the rats exposed to MNU at an earlier age caused cataract more rapidly and severely. In the day 0 MNU-treated rats, 7-methyldeoxyguanosine DNA adduct was detected in the lens epithelial nuclei 12 hours after MNU dosing, followed by apoptosis, which was confirmed by morphology, by TUNEL signals, and by DNA ladder and peaked 3 days after MNU dosing. In the apoptosis cascade, upregulation of Bax, downregulation of Bcl-2, and increased CPP32 protease (caspase-3) activity were seen 12 hours after MNU dosing. Therefore, the pathogenesis of MNU-induced cataract was associated with DNA adduct formation in the lens epithelial cell nuclei leading to apoptosis by upregulation of Bax protein, downmodulation of Bcl-2 protein, and activation of caspase-3.

Intraocular Pressure (IOP) Reduction by Latanoprost in Japanese Normal Tension Glaucoma Patients Over a Five-Year Period Stratified by Presenting IOP

PURPOSE We examined the effectiveness of latanoprost for reducing intraocular pressure (IOP) in Japanese patients with normal tension glaucoma (NTG) over a 5-year period. DESIGN Prospective interventional case series. The patients were classified into 2 groups based on mean IOP. METHODS A total of 38 patients with NTG were studied after being classified into the high-tension (mean IOP 16 mmHg or greater, n = 27) and low-tension (mean IOP lower than 15 mmHg, n = 11) groups. IOP was measured and Humphrey Field Analyzer (HFA) examinations were conducted at 6, 12, 24, 36, 48, and 60 months after beginning a daily administration of latanoprost. RESULTS Mean IOP before administration was 17.6 mmHg in the high-tension group, which was reduced to 13.9, 14.6, 14.4, 14.1, 13.6, and 14.6 mmHg at 6, 12, 24, 36, 48, and 60 months, respectively, after beginning administration. That in the low-tension group was 13.6 mmHg before administration, and then was reduced to 12.2, 11.4, 11.5, 12.5, 10.5, and 11.5 mmHg, respectively, after beginning administration was noted. Mean deviation (MD) values in the HFA examinations were reduced by -4.27 and -1.49 dB after 5 years in the high- and low-tension groups, respectively. CONCLUSIONS Latanoprost administration was effective in reducing IOP over a 5-year period in a range of 3.1-4.1 and 1.3-3.6 mmHg in NTG patients with high- and low-tension levels, respectively. In addition, our results indicate that latanoprost helped to prevent a decrease in MD values in both groups, as shown by the results of HFA examinations.

Time-specific occurrence of alopecia in neonatal C57BL mice treated with N-methyl-N-nitrosourea and the therapeutic efficacy of tacrolimus hydrate

Alopecia was induced in male and female neonatal C57BL mice by a single intraperitoneal injection of 60 mg/kg N‐methyl‐N‐nitrosourea (MNU). MNU administration was most effective in the 8‐day‐old mice and less effective in the 5‐day‐old mice (at active and early anagen stages of the first hair cycle, respectively). No alopecia was seen in the day 14 MNU‐treated animals (at telogen stage of the first hair cycle). MNU effectively induced hair follicular cell apoptosis at the anagen stage by up‐regulation of Bax protein without down‐modulation of Bcl‐2 protein. In day 8 MNU‐treated mice, the immunosuppressive agent 0.01% tacrolimus hydrate (FK506), when topically applied for 5 days from 1 day after MNU treatment (before the occurrence of alopecia), decreased the severity of alopecia. However, it did not stimulate hair growth when applied for 5 days from 20 days of age (after occurrence of alopecia).

Morphological detection of cell kinetics and progesterone receptor expression during growth and regression of pregnancy-dependent mammary tumors of GRS/A mice

Cell kinetics (cell proliferation and cell death) and the expression of progesterone receptor (PgR) during growth and regression of pregnancy-dependent mammary tumors (PDMT) of female GRS/A mice were investigated on the histologic level. Cell proliferation was determined using monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (PC10) and cell death was detected using the TUNEL method. PgR expression was determined using monoclonal anti-PgR antibody (10A9). In growing PDMT (type P tumor) obtained during days 16-20 of pregnancy, numerous PCNA labeling was observed in both the epithelial and mesenchymal cells, whereas PgR was found only in epithelial cells and no TUNEL signal was detected. In regressing PDMT obtained during days 0-5 of lactation, the level of PCNA labeling was low and the PgR-positive cells were preferentially labeled by the TUNEL staining, which led to microcyst formation (cystic degeneration). Pale cell carcinoma was shown to be pregnancy-dependent, since the tumor cells were universally PgR-positive, and TUNEL-positive signal with low PCNA-labeling was detected after the delivery.