Naval Medical Research Institute Research Articles

Insulin Ameliorates Neurodegeneration in an Experimental Animal Model of Brain Aging by a Decrease of GFAP Expression and Apoptosis Inhibition

Background: Aging is one of the factors that will cause gradual changes in brain tissue and its destruction. One of the protective factors against aging is insulin. Objectives: Our study investigated insulin's neuroprotective effect by preventing oxidative stress-mediated neuronal damage. Methods: A total of 48 adult male NMRI (the Naval Medical Research Institute) mice were divided into 2 groups: control and insulin. Insulin was administered intraperitoneally (IP) for 8 weeks to the control group and for 12 weeks to the insulin group. In this study, animals were treated with insulin, and insulin's role in the aging process was evaluated. Then, brain tissue was extracted for evaluation of oxidative stress by assessment of glutathione disulfide (GSH) and reactive oxygen species (ROS) levels, the expression of the glial fibrillary acidic protein (GFAP) as a biomarker for astrogliosis, stereological study, and the real-time polymerase chain reaction (PCR) technique evaluated the level of apoptosis biomarker. Results: The results of stereological parameters show that the volumes of the brain cortex and the number of neurons improved in the insulin group by aging as compared to the control group. The results of real-time PCR showed that the expression level of apoptotic markers decreased in the insulin group compared to the control group. In the insulin group, GSH levels were higher than in the control group, and the control group produced more ROS than the insulin group. As compared with insulin, the control group expressed more GFAP protein and increased the number of glial cells by aging. Conclusions: The results showed that insulin could have neuroprotective effects against aging changes and reduce apoptosis and cell death.

Anethole via increase in the gene expression of PI3K/AKT/mTOR mitigates the autistic-like behaviors induced by maternal separation stress in mice

Autism spectrum disorder (ASD) is a neurodegenerative disease with increasing incidence in the world. The maternal separation (MS) stress at early life with its own neuroendocrine and neurostructural changes can provide the basis for development of ASD. Previously it has been reported neuroprotective characteristics for anethole. The PI3K/AKT/mTOR signaling pathway has pivotal role in the function of central nervous system (CNS). This study aimed to evaluate the possible effects of anethole on the autistic-like behaviors in the maternally separated (MS) mice focusing on the potential role of the PI3K/AKT/mTOR pathway. Forty male Naval Medical Research Institute (NMRI) mice were assigned to five groups (n = 8) comprising a control group (treated with normal saline) and four groups subjected to MS and treated with normal saline and or anethole at doses of 31.25, 62.5 and 125 mg/kg, respectively. All gents were administrated via intraperitoneal (i.p.) route for 14 constant days. Behavioral tests were conducted, including the three-chamber test, shuttle box and resident-intruder test. The gene expression of the PI3K, AKT and mTOR assessed in the hippocampus by qRT-PCR. Findings indicated that MS is associated with autistic-like behaviors. Anethole increased the sociability and social preference indexes in the three-chamber test, increased duration of secondary latency in the shuttle box test and decreased aggressive behaviors in the resident-intruder test. Also, anethole increased the gene expression of PI3K, AKT and mTOR in the hippocampus of MS mice. We concluded that anethole through increase in the gene expression of PI3K/ AKT/mTOR mitigated autistic-like behaviors induced by MS in mice.

Possible role of NO/NMDA pathway in the autistic-like behaviors induced by maternal separation stress in mice.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder. Maternal separation (MS) stress is an established model of early-life stress associated with autistic-like behaviors. Altered glutamatergic and nitrergic neurotransmissions may contribute to the pathophysiology of ASD. However, the specific mechanisms underlying these alterations and their relationship to MS-induced autistic-like behaviors remain unclear. Addressing this knowledge gap, this study aims to elucidate the involvement of the nitric oxide (NO)/ N-methyl-D-aspartate (NMDA) pathway in MS-induced autistic-like behaviors in mice. This knowledge has the potential to guide future research, potentially leading to the development of targeted interventions or treatments aimed at modulating the NO/NMDA pathway to ameliorate ASD symptoms. Ninety male Naval Medical Research Institute (NMRI) mice were assigned to six groups (n = 15) comprising a control group (treated with saline) and five groups subjected to MS and treated with saline, ketamine, NMDA, L-NAME, and L-arginine. Behavioral tests were conducted, including the three-chamber test, shuttle box, elevated plus-maze, and marble burying test. Gene expression of iNOS, nNOS, and NMDA-R subunits (NR2A and NR2B), along with nitrite levels, was evaluated in the hippocampus. The findings demonstrated that MS induced autistic-like behaviors, accompanied by increased gene expression of iNOS, nNOS, NR2B, NR2A, and elevated nitrite levels in the hippocampus. Modulation of the NO/NMDA pathway with activators and inhibitors altered the effects of MS. These results suggest that the NO/NMDA pathway plays a role in mediating the negative effects of MS and potentially contributes to the development of autistic-like behaviors in maternally separated mice.

Comparison of the effects of mycophenolate mofetil alone and with testosterone on sperm, sex hormones and antioxidant enzymes in male rats

Background: Mycophenolate mofetil (MMF) is an immunosuppressant drug which is used to inhibit organ transplant rejection and to treat autoimmune diseases. Theoretically, such medications have spermatotoxic and genotoxic effects. However, evidence on the effects of MMF on fertility is limited and controversial.This study evaluated the effects of MMF and testosterone on sperm, sex hormones and antioxidant enzymes in male rats. Materials and Methods: In this experimental study 24 adult male Naval Medical Research Institute (NMRI) mice randomly divided into 3 groups: 1- saline serum (control), 2- MMF and 3- MMF along with testosterone by subcutaneous injection, for 65 days. Change in body weight, sperm quality (concentration, motility and morphology), serum level of gonadotropin hormones, and testosterone, superoxide dismutase (SOD), glutathione (GSH), catalase, total antioxidant capacity (TAC) in serum and epididymis were evaluated. Findings: Both MMF treatment groups compared to control showed a decrease in body weight, number and motility of sperm, level of testosterone, antioxidant enzymes (SOD, GSH, and catalase) and TAC (P<0.05), and increase of gonadotropin hormones and sperm morphology abnormality (P<0.05). The adverse effects on sperm count and epididymis testosterone levels in the Mycophenolatemofetil +testosterone group was less than the Mycophenolate mofetil group (P<0.05). Conclusion: This study showed that Mycophenolatemofetil has adverse effects on sperm count, gonadotropins , testosterone and antioxidant pathway enzymes, and the use of testosterone cannot reduce the adverse effects of MMF. The adverse effects of Mycophenolate mofetil may be due to a decrease in the level of antioxidants.

A novel experimental model to investigate fungal involvement shows expression of Dectin-1 in periapical lesion pathogenesis.

Candida albicans is linked to persistent endodontic lesions. However, the recognition receptor that identifies it is not explored previously. The aim of this study was to (1) establish a zymosan-induced model of apical periodontitis in mouse, (2) observe the expression of Dectin-1 and its possible relationship with toll-like receptor (TLR) 2 and (3) observe relationship between Osteopontin (OPN) and inflammatory cytokines. A total of 138 Naval Medical Research Institute (NMRI) mice were randomly divided into; Experimental Group n = 69 and Zymosan Group n = 69. Periapical periodontitis was developed in right maxillary molar. The animals were sacrificed at 7, 21 and 42 days. Bone blocks containing the mesial root (n = 15 for qRT-PCR, n = 45 for enzyme-linked immune sorbent assay (ELISA)) were collected for mRNA expression and ELISA. While whole maxilla (n = 3 from each time interval) were used for histology and immunohistochemical analysis. One way analysis of variance (ANOVA) and Tuckey's posthoc was used for statistical analysis at p ≤ .05. TLR-2, Dectin-1 and TLR4-positive cells was detected at all time intervals in both groups. A strong positive correlation was observed between TLR-2 and Dectin-1 in both lesions (regular r = .680, p = .015, zymosan (r = .861, p < .001)). A significant correlation was found between OPN and tumour necrosis factor-alpha (TNF-α) in zymosan lesion (r = .827, p = .001). Immune cells of inflamed periapical tissue expressed Dectin-1 receptor in response to the microbial challenge from infected root canals and showed positive correlation with TLR-2 and OPN suggesting a possible receptor collaboration mediated by OPN. The expression of OPN and TNF-α showed positive correlation in response to fungal antigen, indicating a possible relationship.

The Effects of Vitamin A and Magnesium Sulfate on the Expression of Nox4 Following Irradiation in Bone Marrow Cells

Purpose: Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase is one of the probable sources of Reactive Oxygen Species generated by ionizing radiation. This study aimed to determine the expressions of Nox4 and radioprotective effects of magnesium sulfate and vitamin A following whole-body 2 Gy X-ray irradiation.&#x0D; Materials and Methods: In this research, 6-7 weeks old Naval Medical Research Institute (NMRI) male mice were kept in a standard cage with an appropriate temperature and 12 on 12 off light cycle. Three concentrations of vitamin A and magnesium sulfate were intraperitoneally injected into mice 2 hours before irradiation. The dose rate was 50 cGy/min at Source Skin Distance (SSD) = 100 cm and a field size of 10 × 10cm2. The mice were anesthetized and sacrificed by cervical dislocation 24 hours after irradiation. Then, the expression of Nox4 was assessed by Real-Time Polymerase Chain Reaction (PCR).&#x0D; Results: There were significant differences between the mean of gene expression in groups treated with vitamin A and magnesium sulfate compared to only radiation group (P &lt; 0.05).&#x0D; Conclusion: Based on the results of this study, it is likely that vitamin A and magnesium sulfate neutralize the harmful effects of free radicals due to their antioxidant properties.

Effects of Dioxin on DNA Damage in the Testes of Adult Male Mice

Background: Given the importance of environmental factors and the impact of environmental pollutants, such as dioxins, on organ systems, especially the reproductive system, it is necessary to study these issues and the mechanisms of their effects. Objectives: This study aimed to investigate the effects of different doses of dioxins on DNA damage in the testes of adult male mice. Methods: In this experimental study, 32 male Naval Medical Research Institute (NMRI) rats were randomly divided into four groups: The control group received normal saline, and the dioxin groups were treated with different doses (0.1, 0.5, and 1 µg/kg) for two weeks. Apoptosis in the testes was then examined using a TUNEL assay kit. Results: The mean number of TUNEL-positive spermatogonia cells was 5.91±5.28 in the dioxin group 1, 7.20±10.03 in the dioxin group 2, and 8.73±4.63 in the dioxin group 3, which was higher than that in the control group (0.16±0.40; P=0.073, P=0.034, and P=0.007, respectively). The mean number of TUNEL-positive spermatocyte cells was 5.16±1.99 in the dioxin group 1, 2.50±4.62 in the dioxin group 2, and 3.33±2.94 in the dioxin group 3, which was higher than that in the control group (P=0.034, P=0.14, and P=0.037, respectively). The mean number of TUNEL-positive spermatocyte cells in the dioxin group 1 was significantly higher than that in the dioxin group 2 (2.50±4.62, P=0.047). The average number of TUNEL-positive spermatid cells was 11.58±6.90 in the dioxin group 1, 11.10±12.19 in the dioxin group 2, and 10.20±7.32 in the 3-dioxin group, which was higher than that in the control group (0.16±0.40; P=0.008, P=0.014, and P=0.015, respectively). Conclusion: The results of the present study showed that dioxin caused dose-dependent apoptosis in the testes.

The Effect of Vitamin D3 Supplemented with PhytoSolve on Genes Involved in Implantation in an NMRI Mice Model of Polycystic Ovary Syndrome: An Experimental Study.

Vitamin D3 has been shown to be effective in the treatment of PCOS. However, due to its poor solvability and bioavailability, effective time is delayed and dosage requirements are increased. In our previous study, we demonstrated that PhytoSolve containing VD3 is more effective than vitamin D3 alone in the treatment of PCOS. In this study, we aimed to investigate the effect of this vitamin D3 formulation on gene expression involved in implantation in patients with PCOS. To create PhytoSolve, Lipid S75, glycerol, and MCT oil were combined using a sonicator probe. Six groups, each consisting of 36 female Naval Medical Research Institute (NMRI) mice, were included in the following groups: control; sham; PCOS; PhytoSolve; PhytoSolve containing VD3; and vitamin D3. The mice were given DHEA injections to induce PCOS. After administering PhytoSolve containing VD3 and vitamin D3 by gavage for one week from the 13th day of model creation, the female mice were mated and endometrial tissue was collected for analysis of LIF, β-integrin, and HOXA10 proteins and genes. Compared to the group receiving vitamin D3 alone, the group receiving PhytoSolve containing vitamin D3 showed a significant increase in the expression of LIF, β-integrin, and HOXA10 genes (p<0.05). Although there was an increase in the expression of β-integrin and HOXA10 proteins in the group given PhytoSolve containing vitamin D3 compared to the group given vitamin D3, this increase was not significant. However, the increase in LIF protein expression in the group given PhytoSolve containing vitamin D3 was significant when compared to the group given vitamin D3 (p<0.05). The use of PhytoSolve containing vitamin D3 was more effective than vitamin D3 alone. The PhytoSolve formulation might be a useful solution for medications with limited solubility and bioavailability.

Experimental Cardiac Toxicity Induced by the Injection of Uracoan Rattlesnake (Crotalus vegrandis) and the Black Rattlesnake (Crotalus pifanorum) Venoms

Background Snakebite is a collective health problem that afflicts areas with poor healthcare coverage. Venezuela has an important population of snakes, including the endemic species Crotalus vegrandis and Crotalus pifanorum, whose venom has not been fully characterized, especially of those aspects related to cardiac electrophysiology. Aims In this sense, this work aims to characterize the electrocardiographic and histopathological effect of crude venom of C. vegrandis and C. pifanorum on albino Naval Medical Research Institute (NMRI) mice. Results For this, mice were gathered in C. pifanorum and C. vegrandis experimental groups, including normal controls and envenomed mice injected with commercial antivenom. C. vegrandis venom showed a significant T and S wave flattening and pulmonic (pulmonary) regurgitation (PR) enlargement, in addition to atrial ectopic activity, notched R wave, triggered activity, and T wave inversion. C. pifanorum was the only group that registered triggered activity. Antivenom was able to revert conduction disorders showing a statistical increase in arrhythmogenic compared by χ2 . The multidimensional comparison confirmed the statistical differences between C. vegrandis and C. pifanorum venoms and between antivenom vs non-antivenom groups, detecting variables associated with cardiac conduction, as the most important variables. Conclusion In conclusion, this work demonstrated, as far as we know, for the first time the cardiotoxic effects associated with C. vegrandis and C. pifanorum venom injection, subsequently suggesting the duty of including an electrocardiogram in the consultation of any accident caused by these species.

Chronic stress but not acute stress decreases the seizure threshold in PTZ-induced seizure in mice: role of inflammatory response and oxidative stress.

Seizure is paroxysmal abnormal electrical discharges in the cerebral cortex. Inflammatory pathways and oxidative stress are involved in the pathophysiology of seizures. Stress can induce an oxidative stress state and increase the production of inflammatory mediators in the brain. We investigated the effects of acute and chronic stresses on the seizure threshold in pentylenetetrazol (PTZ)-induced seizures in mice, considering oxidative stress and inflammatory mediators in the prefrontal cortex. In this study, 30 male Naval Medical Research Institute (NMRI) mice were divided into 3 groups, including acute stress, chronic stress, and control groups. PTZ was used for the induction of seizures. The gene expression of inflammatory markers (IL-1β, TNF-α, NLRP3, and iNOS), malondialdehyde (MDA) level, nitrite level, and total antioxidant capacity (TAC) were assessed in the prefrontal cortex and serum. Our results showed that stress could increase the expression of inflammatory cytokines genes and oxidative stress in the prefrontal cortex of the brain and serum following PTZ-induced seizures, which is associated with increased seizure sensitivity and decreased the seizure threshold. The effects of chronic stress were much more significant than acute stress. We concluded that the effects of chronic stress on seizure sensitivity and enhancement of neuroinflammation and oxidative stress are much greater than acute stress.

Differences in lipid metabolism in acquired versus preexisting glucose intolerance during gestation: role of free fatty acids and sphingosine-1-phosphate

BackgroundThe prevalence of gestational diabetes mellitus (GDM) is increasing worldwide. There is increasing evidence that GDM is a heterogeneous disease with different subtypes. An important question in this context is whether impaired glucose tolerance (IGT), which is a typical feature of the disease, may already be present before pregnancy and manifestation of the disease. The latter type resembles in its clinical manifestation prediabetes that has not yet manifested as type 2 diabetes (T2DM). Altered lipid metabolism plays a crucial role in the disorder's pathophysiology. The aim was to investigate the role of lipids which are relevant in diabetes-like phenotypes in these both models with different time of initial onset of IGT.MethodsTwo rodent models reflecting different characteristics of human GDM were used to characterize changes in lipid metabolism occurring during gestation. Since the New Zealand obese (NZO)-mice already exhibit IGT before and during gestation, they served as a subtype model for GDM with preexisting IGT (preIGT) and were compared with C57BL/6 N mice with transient IGT acquired during gestation (aqIGT). While the latter model does not develop manifest diabetes even under metabolic stress conditions, the NZO mouse is prone to severe disease progression later in life. Metabolically healthy Naval Medical Research Institute (NMRI) mice served as controls.ResultsIn contrast to the aqIGT model, preIGT mice showed hyperlipidemia during gestation with elevated free fatty acids (FFA), triglycerides (TG), and increased atherogenic index. Interestingly, sphingomyelin (SM) concentrations in the liver decreased during gestation concomitantly with an increase in the sphingosine-1-phosphate (S1P) concentration in plasma. Further, preIGT mice showed impaired hepatic weight adjustment and alterations in hepatic FFA metabolism during gestation. This was accompanied by decreased expression of peroxisome proliferator-activated receptor alpha (PPARα) and lack of translocation of fatty acid translocase (FAT/CD36) to the hepatocellular plasma membrane.ConclusionThe preIGT model showed impaired lipid metabolism both in plasma and liver, as well as features of insulin resistance consistent with increased S1P concentrations, and in these characteristics, the preIGT model differs from the common GDM subtype with aqIGT. Thus, concomitantly elevated plasma FFA and S1P concentrations, in addition to general shifts in sphingolipid fractions, could be an interesting signal that the metabolic disorder existed before gestation and that future pregnancies require more intensive monitoring to avoid complications.Graphical This graphical abstract was created with BioRender.com.

Expression of Toll-like receptor 2, Dectin-1, and Osteopontin in murine model of pulpitis.

This in vivo animal study aimed to develop a murine model of pulpitis induced by pulp exposure with or without application of zymosan in Naval Medical Research Institute (NMRI) mice and observe expressions of Toll-like receptor (TLR)-2, TLR-4, Dectin-1, Osteopontin (OPN), tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-1ß. A total of 168 NMRI mice were divided into two groups, i.e., group A (n = 84) (pulpitis induced by pulp exposure only) and group B (n = 84) (pulpitis induced by pulp exposure and zymosan application). Right maxillary molar pulps were exposed with ¼ round bur, and animals were sacrificed at 0, 6, 9, 12, 24, 48, and 72h. The exposed teeth were obtained for real-time polymerase chain reaction (qRT-PCR) analysis and histological and immunohistochemistry (IHC) analysis. Histological evaluation revealed a time-dependent steady increase in inflammation. Similar time-dependent increase in the expression of inflammatory cytokines was noted. Group A exhibited an increase in TLR-4, Dectin-1, and OPN at 6h, while TLR-2 was expressed at 24h. Group B expressed TLR-2, Dectin-1, and OPN at 9, 48, and 72h, respectively (p ≤ 0.05). Expression of OPN and TNF-α exhibited a similar pattern in both groups. IHC also detected expression of TLR-2, Dectin-1, TLR4, and CD68 in some cells at 6 and 9h. NMRI mice provided for a stable pulp inflammation model. Zymosan may be used to develop pulp inflammation model and study inflammatory response towards fungal antigens. Dental pulp expressed Dectin-1 receptor. OPN and TNF-α exhibited a similar expression pattern. Innate immunity of dental pulp is capable of detecting fungal pathogens.

EF24, a schistosomicidal curcumin analog: Insights from its synthesis and phenotypic, biochemical and cytotoxic activities

Praziquantel (PZQ) is the only drug available for community-based control programs which aim to reduce the prevalence and morbidity associated with schistosomiasis. Here, we synthesized and evaluated the schistosomicidal, biochemical and cytotoxic activities of EF24, a synthetic curcumin analog, against different isolates of Schistosoma mansoni. EF24 elicited marked phenotypic alterations at 10 μM against schistosomula and 42-day-old adult worms of the Naval Medical Research Institute (NMRI) isolate. EF24 had 50% effective concentration (EC50) values of <10 μM against the Luis Evangelista (LE), Sergipe (SE), Belo Horizonte (BH) and Belo Horizonte less sensitive to PZQ (BH < PZQ) isolates of adult S. mansoni; however, the respective sensitivities of these isolates differed. Changes in the parasite included, vacuolization of the tegument and focal lysis of the interstitial tissue and muscle layers. Against 28-day-old juvenile worms (LE isolate), EF24 was about three times more potent than PZQ. After 6 h at 12.5 μM, EF24 increased reactive oxygen species (ROS) and the activity of the antioxidant enzyme, glutathione-S-transferase (GST), by 32 and 19% in female and male adult worms, respectively. By contrast, after 6 h at 12.5 μM glutathione reductase (GR) activity decreased by 43 and 30%, and glutathione peroxidase (GPx) activity decreased by 67 and 44% in females and males, respectively. EF24 was less cytotoxic to mammalian host cells than to S. mansoni, with selectivity indexes (SIs) of 1.8–3.4 and 2.7–7.5 for juvenile and adult worms, respectively. Given the current evidence for the in vitro schistosomicidal effect of EF24, the structure-activity relationship of additional analogs to identify new candidates for schistosomiasis treatment is warranted.

Effect of Eight Weeks of Endurance Training in Light and Dark Phases of Circadian Rhythm on the Oxidative Stress Index in Pancreas of Diabetic Mice

Introduction: Chronic hyperglycemia is associated with an increase in cellular damage due to oxidative stress in pancreatic tissue. The effect of exercise in different phases of the circadian cycle on protecting pancreatic tissue from oxidative stress in diabetic patients is unknown. The aim of this study was to investigate the effect of eight weeks of endurance training in light and dark phases of circadian rhythm on the oxidative stress index in pancreas of diabetic mice.&#x0D; Methods: In this study, 18 mice from the Naval Medical Research Institute  (26.3±3.22 gr) were selected and after inducing diabetes through high-fat food and Streptozotocin injection (20 mg/kg), randomly divided into 6 groups: light phase healthy control, dark phase healthy control, light phase diabetic control, dark phase diabetic control, light phase diabetic training and dark phase diabetic training. The endurance training protocol (50-60 %Vmax) was performed 5 d/w for 8 weeks. After anesthesia, blood samples and pancreatic tissues were removed. Insulin resistance markers, oxidative stress index and expression of Brain and Muscle ARNT-Like1 protein expression were measured in pancreas of diabetic rats. Data were analyzed by one way analysis of variance at the significance level of p &lt;0.05.&#x0D; Results: The eight weeks of endurance training significantly decreased insulin resistance markers (p= 0.005), oxidative stress index (p ˂0.05) and significantly increased the Bmal1 protein expression (p = 0.009). The mean values of all variables showed significant differences between light and dark phases.&#x0D; Conclusion: Endurance training may improve insulin sensitivity and oxidant damage in diabetic conditions by increasing the function of the antioxidant system and the expression of Circadian regulation proteins. Activity in the dark phase causes further increases the metabolism of cells. As a result, performing these types of exercises in the dark phase is recommended to these patients as a new treatment strategy.

Development of a murine model of oral carcinogenesis: an accelerated tool for biomarker and anti-tumour drug discovery.

Oral squamous cell carcinoma (OSCC) is the most common cancer in Pakistani men and the second most common cancer in women. The objective of our study was to devise a novel accelerated murine model of oral carcinogenesis that can be exploited as a tool to investigate the cancer circuitry involved in OSCC and to identify molecules of diagnostic, therapeutic and prognostic significance. A total of 40 healthy male, 6–8 weeks old, 22 ± 2 gram, Naval Medical Research Institute (NMRI) outbred strain mice were recruited in the experiment. NMRI mice are commonly used for animal experiments in various fields of biology and for drug toxicity. Of these, 25 mice underwent the oral carcinogenesis regimen via topical application of 0.5% 9,10-dimethyl-1,2-benzanthracene (DMBA) on the lower left lip for a maximum of 20 weeks and 15 mice were used as controls (without the carcinogenic regimen). Exophytic tissue masses were harvested, fixed in 10% formalin and stained with haematoxylin and eosin (H&E) for microscopic diagnosis. Additionally, the expression levels of CK 5/6, p53 and Ki-67 were investigated using immunohistochemistry. Of the 25 mice which underwent the carcinogenic regimen, 21 developed moderately differentiated squamous cell carcinoma and 1 showed dysplastic features with foci of invasion. Three mice were found dead with lesion(s). CK 5/6 showed strong positivity (100%) and p53 and Ki-67 showed patchy (<30%) strong positivity in OSCC, suggesting the similarity of our model to human OSCC. We present an accelerated, close-to-human carcinogenesis, model of oral carcinogenesis using DMBA in NMRI mice that can be exploited to study the pathogenesis of oral squamous cell carcinoma and subsequently devise immunotherapy or targeted therapy.

Effects of N-Acetyl-L-Cystein Antioxidant on Ex Vivo Culture of Vitrified Premature Mouse Ovarian Tissue.

Optimization of practical ways to obtain mature follicles from cryopreserved ovarian tissues, especially in patients suffering from ovarian dysfunction, is very important. In vitro ovarian tissue culture allows faster screening of follicle development and reduces follicle isolation damage. During ovarian tissue culture, controlling oxidative stress is critical to support better follicular development and less damage. Immature Naval Medical Research Institute (NMRI) mouse ovaries (8-days-old) were randomly distributed into four cultured groups; non-vitrified, vitrified, non-vitrified N-acetyl-L-cysteine (NAC)+, and vitrified NAC+. Ovaries of vitrified groups along with non-vitrified ovaries were cultured on agar gel in the presence or absence of NAC for 5 days. Afterward, morphological evaluations, mRNA expressions of Gdf9, Bmp6, Lif, Amh, Bax, and Bcl2 genes, malondialdehyde, and total antioxidant capacities were compared between four groups at the first and last day of culture. Good preservation of tissue integrity and an increase of follicular development were observed in all groups. In addition, the expression of Gdf9, Lif, Bax, and Bcl2 genes were increased and Amh was decreased in groups cultured in the presence of NAC compared to groups cultured without NAC. Although total antioxidant capacity was not significantly different between the experimental groups, the lipid peroxidation and apoptotic index were significantly reduced in the presence of NAC. Thus, it appears that NAC antioxidant acts as a contributory factor for the ex vivo culture of ovarian tissue and reduces oxidative stress, apoptotic index, and improves follicular development, especially in non-vitrified groups.

The monitoring of Francisella tularensis in surface water of East Azerbaijan province, Iran

BackgroundFrancisella tularensis could be disseminated through arthropod bites and exposure to infected animals, water, and aerosols. Water sources that are contaminated with rodent excrement could be a source of contamination; therefore, an analysis of water samples is an appropriate method to investigate the routes of dissemination. Since an outbreak occurred in one of the villages in East Azerbaijan. The current study aimed to investigate the Francisella isolation in the different water samples from East Azerbaijan, Iran. Sampling was carried out in East Azerbaijan province. Forty-six specimens of surface water were collected. Filtration, culture, and inoculation of the water sample into NMRI (Naval Medical Research Institute) inbreed mice were performed. DNA was extracted from filtered water samples, different organs of inoculated mice, and bacterial isolates and was tested by TaqMan real-time PCR by targeting ISFtu2 and fopA genes. Despite the unsuccessfulness in isolation of F. tularensis, molecular test results indicate the presence of bacteria in surface water. The highest rate of F. tularensis (ten from 46 water samples, 21.7%) was detected from injected mice based on molecular methods. Despite the high efforts of researchers to isolate Francisella spp. in Iran, in recent years, and also the evidence that shows the presence of this bacterium in different parts of the country, the culture was not successful again in this study and the molecular method still is recommended to identify the possible sources of Francisella spp.

Pharmacological evidence for the possible involvement of the NMDA receptor pathway in the anticonvulsant effect of tramadol in mice.

Previous studies have shown controversial results regarding the pro- or anticonvulsant effects of tramadol. Additionally, the underlying mechanism of seizure induction or alleviation by tramadol has not been fully understood. In the current study, the effects of tramadol on pentylenetetrazole (PTZ)-induced seizure and the possible involvement of the N-methyl-D-aspartate (NMDA) pathway were assessed in mice. Male Naval Medical Research Institute (NMRI) mice were treated with intravenous infusion of PTZ in order to induce clonic seizures and determine seizure threshold. Tramadol was injected intraperitoneally (0.1-150 mg/kg) 30 minutes prior to elicitation of seizures. The possible effects of intraperitoneal injections of NMDA receptor antagonists, ketamine (0.5 mg/kg) and MK-801 (0.5 mg/kg) on the anticonvulsant property of tramadol were investigated subsequently. Tramadol (1-100 mg/kg) increased PTZ-induced seizure threshold in a dose-dependent, time-independent manner, with optimal anticonvulsant effect at a dose of 100 mg/kg. Acute administration of either ketamine (0.5 mg/kg) or MK-801 (0.5 mg/kg) potentiated the anticonvulsant effect of a subeffective dose of tramadol (0.3 mg/kg). These results suggest a possible role of the NMDA pathway in the anticonvulsant effect of tramadol.

The effect of curcumin on embryonic in vitro development in experimental polycystic ovary syndrome: An experimental study.

BackgroundPolycystic ovary syndrome (PCOS) is a common disease in women. Some plant compounds which have antioxidant properties, such as curcumin, may be useful for these patients when delivered orally or in vitro.Objective The aim of this study was to evaluate the impact of PCOS on oocyte quality and the effect of curcumin on in vitro fertilization of oocytes.Materials and Methods In this experimental study, Naval Medical Research Institute mice aged six to eight wk were used. Mice were divided into five experimental groups (control, experimental PCOS, curcumin 6, 12 and 24 μM). To induce experimental PCOS, estradiol valerate (100 mg/kg, IP) was injected. The total antioxidant capacity and production of malondialdehyde in ovarian tissue and blood serum were evaluated in all groups. Finally, 6, 12 and 24 μM of curcumin were added to the culture medium of the PCOS group oocytes and development in the different groups was evaluated.ResultsA high percentage of oocytes for fertilization were not in good condition in terms of number and quality in the group of PCOS. The addition of curcumin to the embryo culture medium was associated with a higher percentage of fertilized oocytes, two-cells and blastocysts. This increase was significant at a concentration of 24 μM (p 0.01).ConclusionGiven that adding curcumin seemed to improve fetal growth and prevent the harmful effects of oxygen free radicals on the culture medium, it is recommended to add a certain concentration of curcumin under normal conditions without oxidative stress.

The reaggregation of normal granulosa-cumulus cells and mouse oocytes with polycystic ovarian syndrome in vitro: An experimental study.

BackgroundThe dialogue between oocytes and their surrounding cells plays a major role in the progress of oocyte meiosis and their developmental potential.ObjectiveThis study aimed to evaluate the effect of co-culture of normal granulosa-cumulus cells (GCCs) with oocytes from polycystic ovarian syndrome (PCOS) mice.Materials and MethodsNormal GCCs were collected from 10 virgin adult Naval Medical Research Institute female mice (30-35 gr, 7-8 wk old), and were cultured in an alpha-minimum essential medium supplemented with 5% fetal calf serum for 24-48 hr (110 cells/well). Then, germinal-vesicle oocytes from PCOS mice were cultured in the presence of cultured normal GCCs (experimental group) and without GCCs (control group). The maturation rate and quality of the PCOS oocytes were examined by evaluating TFAM and Cx43 gene expression (real-time PCR) and the connection among PCOS oocytes and normal GCCs after 24 hr of culture.ResultsThe co-culture of normal GCCs and PCOS oocytes in the experimental group led to the formation of a complex called a PCOS oocyte-normal GCCs complex. The maturation rate of these complexes was significantly increased compared to that of the control group (p 0.001). A significant difference was also found in the expression of Cx43 (p 0.001) and TFAM (p 0.05) genes in the experimental group compared with the control group. The connection between PCOS oocytes and normal GCCs was observed in the scanning electron microscope images.ConclusionCo-culture with normal GCCs improves the capacity of PCOS oocytes to enter meiosis, which may result in the promotion of assisted reproduction techniques.