Since genetic models for retinal degeneration (RD) in animals larger than rodents have not been firmly established to date, we sought in the present study to develop a new rabbit model of drug-induced RD. First, intravitreal injection of N-methyl-N-nitrosourea (MNU) without vitrectomy in rabbits was performed with different doses. One month after injection, morphological changes in the retinas were identified with ultra-wide-field color fundus photography (FP) and fundus autofluorescence (AF) imaging as well as spectral-domain optical coherence tomography (OCT). Notably, the degree of RD was not consistently correlated with MNU dose. Then, to check the effects of vitrectomy on MNU-induced RD, the intravitreal injection of MNU after vitrectomy in rabbits was also performed with different doses. In OCT, while there were no significant changes in the retinas for injections up to 0.1 mg (i.e., sham, 0.05 mg, and 0.1 mg), outer retinal atrophy and retinal atrophy of the whole layer were observed with MNU injections of 0.3 mg and 0.5 mg, respectively. With this outcome, 0.2 mg MNU was chosen to be injected into rabbit eyes (n=10) at two weeks after vitrectomy for further study. Six weeks after injection, morphological identification with FP, AF, OCT, and histology clearly showed localized outer RD - clearly bordered non-degenerated and degenerated outer retinal area - in all rabbits. We suggest our post-vitrectomy MNU-induced RD rabbit model could be used as an interim animal model for visual prosthetics before the transition to larger animal models.
Animals ; Atrophy ; Intravitreal Injections ; Methylnitrosourea ; Models, Animal ; Models, Genetic ; Photography ; Rabbits ; Retina ; Retinal Degeneration ; Retinaldehyde ; Rodentia ; Tomography, Optical Coherence ; Vitrectomy

The present study investigated the temporal pattern and cellular localization of nestin in the adult mouse retina with pharmaceutically induced retinal degeneration using N-methyl-N-nitrosourea (MNU). After a single intraperitoneal injection of MNU in 8-week-old C57BL/6 mice, the animals were sacrificed at 1, 3, 5, 7, and 21 days (n = 6, in each stage). The eyes were examined by means of immunohistochemical tests using nestin, ionized calcium-binding adaptor molecule (Iba-1), CD11b, F4/80, and glial fibrillary acidic protein (GFAP). Western blot analysis and manual cell counting were performed for quantification. Nestin expression was increased after MNU administration. Nestin+/Iba-1+ cells were migrated into outer nuclear layer (ONL) and peaked at day 3 post injection (PI). Nestin+/CD11b+ cells were also mainly identified in ONL at day 3 PI and peaked at day 5. Nestin+/F4/80+ cells were shown in the subretinal space and peaked at day 3 PI. Nestin+/GFAP+ cells were distinctly increased at day 1 PI and peaked at day 5 PI. The up-regulation of nestin expression after MNU administration in adult mouse retinal microglia, and monocyte/macrophage suggests that when retinal degeneration progresses, these cells may revert to a more developmentally immature state. Müller cells also showed reactive gliosis and differentiational changes.
Adult* ; Animals ; Blotting, Western ; Cell Count ; Glial Fibrillary Acidic Protein ; Gliosis ; Humans ; Injections, Intraperitoneal ; Methylnitrosourea ; Mice* ; Microglia ; Nestin* ; Retina* ; Retinal Degeneration* ; Retinaldehyde* ; Up-Regulation

BACKGROUND: The aim of this study was to investigate the effect of N-methyl-N-nitrosourea (MNU) treatment followed by chronic Helicobacter pylori SS1 and H. felis colonization on the stomachs of C57BL/6 mice. The role of MNU and Helicobacter species in gastric carcinogenesis was also elucidated. METHODS: A total of 69 C57BL/6 mice at 4 weeks of age were divided into 6 groups according to MNU treatment and H. pylori SS1 or H. felis infection. The mice were sacrificed at 21 and 50 weeks. The degree of inflammation was determined by histopathology. The levels of gastric mucosal myeloperoxidase, TNF-α, and interleukin-1β (IL-1β) were measured by ELISA. RESULTS: In the H. felis groups with or without MNU, the incidence of gastric tumors was 21.1% and 35.0% at 21 and 50 weeks, respectively. No gastric tumors were observed in all control mice. At 50 weeks, 37.5% of gastric adenoma cases were observed in the H. felis alone and MNU + H. felis groups. Furthermore, 12.5% of gastric adenocarcinoma cases were observed in the MNU alone and MNU + H. felis groups. The gastric mucosal IL-1β level was significantly higher in the MNU + H. felis group at 21 weeks and H. felis group at 50 weeks, respectively, than that for control mice (P < 0.05). However, the effect of MNU on H. pylori SS1-induced gastric carcinogenesis was low compared to that on H. felis. CONCLUSIONS: Administration of MNU before H. felis infection provokes severe inflammation through IL-1β, and eventually induces gastric cancer. However, the role of MNU in H. pylori SS1-induced gastric carcinogenesis model is minor.
Adenocarcinoma ; Adenoma ; Animals ; Carcinogenesis* ; Cats ; Colon ; Enzyme-Linked Immunosorbent Assay ; Felis ; Helicobacter ; Helicobacter felis ; Helicobacter pylori ; Incidence ; Inflammation ; Methylnitrosourea* ; Mice* ; Peroxidase ; Stomach ; Stomach Neoplasms

Garlic and mugwort have long been used in traditional medicine to prevent various diseases. Several in vitro studies have reported protective efficacies of garlic and mugwort in cases of gastric cancer. In the present study, we investigated the cancer preventive effects of garlic and mugwort mixture extract (GME) in a Helicobacter (H.) pylori-associated gastric carcinogenesis mouse model. To induce gastric cancer, C57BL/6 mice were treated with N-methyl-N-nitrosourea and H. pylori. Various concentrations of GME (0, 100, 500, and 1,000 ppm) were then fed to the mice for 38 weeks, after which the tumor tissues were examined for histopathology, mucin histochemistry and beta-catenin. The incidence of gastric tumors was significantly lower in the highest dose GME-treated mice (46.7%) than control mice (85.7%) (p < 0.05). The multiplicity and size of tumors were also significantly reduced by GME feeding in a dose-dependent manner (p < 0.01). Furthermore, GME suppressed the H. pylori-associated chronic inflammation measured by histologic grading of H. pylori density, chronic gastritis, glandular atrophy and intestinal metaplasia in non-tumorous gastric mucosae. Our data suggest that GME suppresses gastric tumorigenesis via suppression of H. pylori-associated chronic inflammation.
Animals ; Artemisia* ; Atrophy ; beta Catenin ; Carcinogenesis* ; Garlic* ; Gastric Mucosa ; Gastritis ; Helicobacter pylori ; Helicobacter* ; Incidence ; Inflammation ; Medicine, Traditional ; Metaplasia ; Methylnitrosourea ; Mice* ; Mucins ; Stomach Neoplasms

Garlic and mugwort have long been used in traditional medicine to prevent various diseases. Several in vitro studies have reported protective efficacies of garlic and mugwort in cases of gastric cancer. In the present study, we investigated the cancer preventive effects of garlic and mugwort mixture extract (GME) in a Helicobacter (H.) pylori-associated gastric carcinogenesis mouse model. To induce gastric cancer, C57BL/6 mice were treated with N-methyl-N-nitrosourea and H. pylori. Various concentrations of GME (0, 100, 500, and 1,000 ppm) were then fed to the mice for 38 weeks, after which the tumor tissues were examined for histopathology, mucin histochemistry and beta-catenin. The incidence of gastric tumors was significantly lower in the highest dose GME-treated mice (46.7%) than control mice (85.7%) (p < 0.05). The multiplicity and size of tumors were also significantly reduced by GME feeding in a dose-dependent manner (p < 0.01). Furthermore, GME suppressed the H. pylori-associated chronic inflammation measured by histologic grading of H. pylori density, chronic gastritis, glandular atrophy and intestinal metaplasia in non-tumorous gastric mucosae. Our data suggest that GME suppresses gastric tumorigenesis via suppression of H. pylori-associated chronic inflammation.
Animals ; Artemisia* ; Atrophy ; beta Catenin ; Carcinogenesis* ; Garlic* ; Gastric Mucosa ; Gastritis ; Helicobacter pylori ; Helicobacter* ; Incidence ; Inflammation ; Medicine, Traditional ; Metaplasia ; Methylnitrosourea ; Mice* ; Mucins ; Stomach Neoplasms

PURPOSE: To evaluate the expression patterns of F4/80 and nestin in the ciliary body and the optic nerve following N-methyl-N-nitrosourea (NMU)-induced retinal degeneration in adult mice. METHODS: After intraperitoneal injection of MNU (60 mg/kg) in adult mice, the eyes were enucleated at 2, 4, 7 and 30 days. Hematoxylin and eosin (H&E) stain, terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) stain and immunohistochemical stains of F/80 and nestin were performed. RESULTS: After MNU treatment, the photoreceptors were destroyed by cell apoptosis. According to immunohistochemistry, F4/80 and nestin were not co-expressed in the control group, but F4/80 was expressed within the ciliary body and optic nerve in the MNU-treated group; the expression of nestin also increased. In the outer nuclear layer, F4/80 and nestin co-expressing cells were observed. CONCLUSIONS: In response to retinal damage, the F4/80 and nestin co-expressing cells migrated to the retina from the ciliary body and optic nerve and were activated.
Adult ; Animals ; Apoptosis ; Ciliary Body ; Coloring Agents ; Eosine Yellowish-(YS) ; Eye ; Hematoxylin ; Humans ; Immunohistochemistry ; Injections, Intraperitoneal ; Intermediate Filament Proteins ; Methylnitrosourea ; Mice ; Nerve Tissue Proteins ; Optic Nerve ; Retina ; Retinal Degeneration ; Retinaldehyde

The retinal degeneration (RD) is a general cause of blindness. To study its pathophysiology and evaluate the effects of new therapeutic agents before clinical trials, it is essential to establish reliable and stable animal models. This study evaluated a RD animal model in which blindness was induced by N-methyl-N-nitrosourea (MNU), a potent retinotoxin leading to apoptosis of photoreceptors. MNU was applied to the Sprague-Dawley rats by a single intraperitoneal injection in different doses (40, 50, and 60 mg/kg). The retinal functions were examined at 1 week after MNU injection by electroretinogram (ERG). Afterwards, each retina was examined by hematoxylin and eosin stain and immunohistochemistry with anti-glial fibrillary acidic protein antibody. Upon MNU injection of 40, 50 and 60 mg/kg, the ERG amplitude of a-waves showed significant reductions of 7, 26, and 44%, respectively, when compared to that of normal a-waves. The b-wave amplitudes were about 89, 65, and 58% of normal b-waves in the response to scotopic light stimulus. At 1 week, 2 weeks, and 4 weeks after MNU injection (50 mg/kg), all scotopic ERG components decreased progressively. In addition, degeneration of retinal neurons was observed in a time- and dose-dependent manner after MNU injection. Taken together, functional reduction following RD induced by MNU correlates with morphological changes. Thus, this RD rat model may be a useful model to study its pathophysiology and to evaluate the effects of new therapeutic agents before clinical trials.
Animals ; Apoptosis ; Blindness ; Electroretinography ; Eosine Yellowish-(YS) ; Hematoxylin ; Immunohistochemistry ; Injections, Intraperitoneal ; Light ; Methylnitrosourea ; Models, Animal ; Rats ; Rats, Sprague-Dawley ; Retina ; Retinal Degeneration ; Retinal Neurons ; Retinaldehyde

OBJECTIVE: This study aimed to determine the effects of garlic (Allium sativum) on N-Methyl-N-Nitrosourea induced transitional carcinoma in Wistar rats.

METHODOLOGY: Transitional cell carcinoma was induced in thirty male, age-matched Wistar rats (45-50 days old) through intravesical instillation of 0.1mL of N-Methyl-N-Nitrosourea. They were divided into five treatment groups (0.1 mL of NSS; 0.1 mL of Mitomycin C; 0.1 mL of aqueous garlic extract in 10 mg/kg, 20 mg/kg, and 40 mg/kg given daily for the duration of the study); with one rat sacrificed every week (starting two weeks from tumor induction) until all rats were sacrificed after one month. The urinary bladders of the rats were subjected to histopathologic examination by a single veterinary pathologist. One-way ANOVA was used to compare mitotic index, papillomatous growth and vascularization of the specimens at Day 14 (baseline), 21 and 28. A P-value of less than 0.05 was used to detect significant difference.

RESULTS: Statistical analysis comparing mitotic index, papillomatous growth and vascularization showed no significant difference in the indices between the five treatment groups. It can be seen through that the P-value (0.144) for papillomatous growth was the smallest, which may indicate a trend towards a decrease in tumor growth at Day 28 for Mitomycin C and Garlic 40 mg/kg.

CONCLUSIONS: This preliminary study showed a favorable trend towards decreased papillomatous growth in the MNU induced rat bladder carcinoma treated with aqueous extract of Garlic (Allium sativum) at a higher dose and longer duration of time.

Animal ; Male ; Carcinoma, Transitional Cell ; Neoplasms ; Carcinoma ; Garlic ; Plants ; Urinary Bladder ; Rats, Wistar ; rats ; Plant Extracts ; Methylnitrosourea ; Nitrosourea Compounds

The objective of this study was to elevate the tumorigenic rate of animal model with thymic lymphoma induced by N-methyl-N-nitrosourea (MNU) and to reduce the mortality of this mouse model. The injection regimen and experimental cycle were improved on the basis of previous experiments. The male and female C57BL/6 mice were injected by the intraperitoneal route with MNU solution at different dosages in the first week and the 4th week respectively. Following injection of MNU, the general features of the mice were observed. All mice were sacrificed for autopsy before the 24th experimental week. Complete gross examination was performed for detection of tumor masses. The pathologic and immunohistochemical methods were used to identify the origin and subtype classification of the neoplasm. The results showed that at the 25th week the incidence of thymic lymphoma in mice injected with MNU was 83.3% (55/66), the mortality was 7.6%. In conclusion, improving the program and changing the experimental cycle can increase the tumorigenic rate in the mouse model induced by MNU from 67.5% to 83.3% and reduce the mortality from 10% to 7.6%.
Animals ; Female ; Lymphoma ; chemically induced ; Male ; Methylnitrosourea ; adverse effects ; Mice ; Mice, Inbred C57BL ; Neoplasms, Experimental ; chemically induced ; Thymus Neoplasms ; chemically induced

The objective of study was to establish an animal model with thymic lymphoma in mice induced by intraperitoneal injection of DNA alkylating agent N-methyl-N-nitrosourea (MNU). Male and female mice of the C57BL/6 strain were injected by the intraperitoneal route with MNU solution in a dosage of 50 mg/kg body weight. The injection was repeated at week 8. Following injection of MNU, the general status of mice was observed. All mice were sacrificed for autopsy at the 22nd experimental week. Complete gross examination was performed for detection of tumor masses. The results showed that at the 22nd week, the incidence of thymic lymphoma in MNU-treated animals was 67.5% (27/40). No significant sex difference in the incidence of thymic lymphoma was observed. In conclusions, an animal model with thymic lymphoma in mice can be established by twice intraperitoneal administration of MNU. The biological behavior of the induced tumors resembles to those of human thymic lymphoma derived from thymic T-cells.
Animals ; Female ; Lymphoma ; chemically induced ; Male ; Methylnitrosourea ; adverse effects ; analogs & derivatives ; Mice ; Mice, Inbred C57BL ; Neoplasms, Experimental ; chemically induced ; Thymus Neoplasms ; chemically induced ; Trimethylsilyl Compounds ; adverse effects