Effects of diazinon on the ovarian tissue of rats: a histochemical and ultrastructural study.

Despite the negative environmental and biologic effects, organophosphates have currently been widely used. We aimed to examine the possible negative effects of diazinon, a type of organophosphate, on rat ovarian tissue. Wistar Albino rats were divided into four groups. No treatment was given to control, olive oil was applied to sham group. Experimental groups were injected intraperitoneally with 30 and 60 mg/kg/day diazinon, respectively. 24 h later, ovarian tissues were extracted, preparated, examined via light and electron microscope. In the experimental groups granulosa and corpus luteum showed degenerative changes. Dilatation of endoplasmic reticulum cisterns and morphological alterations of mitochondria in granulosa cells were detected utrastructurally. Also, accumulation of lipid droplets and autophagic vacuoles was observed in cells of corpus luteum. A statistically significant dose-dependent decrease in superoxide dismutase and catalase reactivity and a statistically significant increase in caspase-3 expression in cells of atretic follicles and corpus luteum were observed. Results show that exposure to a single dose of diazinon may disrupt antioxidant system, trigger atresia in follicles and negatively effect corpus luteum functions. It was concluded that studies applying possible antioxidant treatments should be carried out to reduce and prevent the negative effects of diazinon on the reproductive system.

Histological and Ultrastructural Changes in Lungs One Year Post Asymptomatic or Mild SARS-CoV-2 Infection: Evaluating the Potential for Active Virus Presence / Cambios Histológicos y Ultraestructurales en los Pulmones un Año Después de una Infección Asintomática o Leve por SARS-CoV-2: Evaluación del Potencial de Presencia Activa del Virus

SUMMARY: Prior research on post-COVID-19 or long COVID primarily focused on the presence of SARS-CoV-2 mostly in symptomatic patients. This study aimed to investigate the persistence of SARS-CoV-2 after 1 year of asymptomatic or mild COVID-19. SARS-CoV-2 infected and control K18-hACE2 transgenic mice (n=25) were studied. Moderate and severe symptomatic subjects were sacrificed after eight days, while mild or asymptomatic mice were kept in BSL-III for twelve months. Analyses included general condition, histochemistry, immunohistochemistry, transmission electron microscopy, and qRT-PCR. Lungs from the twelve-month group showed thickening of alveolar walls, with some lungs exhibiting the recruitment of inflammatory cells, the presence of SARS- CoV-2 mRNA, immunopositivity for the SARS-CoV-2 spike protein, and TEM showed viruses (60-125 nm) within vesicles, indicating continued replication. Certain lung samples showed persistent SARS-CoV-2 presence in Club cells, endothelial cells, and macrophages. The eight-day group exhibited viral interstitial pneumonitis, SARS-CoV-2 immunopositivity, and mRNA. The eight-day hearts displayed viral mRNA, while the twelve-month hearts tested negative. Some asymptomatic twelve-month subjects presented reduced surfactant, basal membrane thickening, fibrosis, and mild autonomic nerve degeneration. In this study conducted on mice, findings indicate the potential for chronic persistence of SARS-CoV-2 in the lungs one year post initial mild or asymptomatic infection, which could suggest the possibility of recurrent episodes in similar human conditions. The observed thickening of alveolar walls and potential fibrotic areas in these mice may imply an increased risk of post-COVID fibrosis in humans. Furthermore, the presence of SARS-CoV-2-positive inflammatory cells in some asymptomatic murine cases could herald a progression toward ongoing inflammation and chronic lung disease in humans. Therefore, the necessity for further studies in human subjects and vigilant monitoring of high-risk human populations is underscored.

Investigaciones anteriores sobre COVID-19 o COVID prolongado se centraron principalmente en la presencia de SARS-CoV-2 principalmente en pacientes sintomáticos. Este estudio tuvo como objetivo investigar la persistencia del SARS-CoV-2 después de 1 año de COVID-19 asintomático o leve. Se estudiaron ratones transgénicos K18-hACE2 infectados con SARS-CoV-2 y de control (n=25). Los animales con síntomas moderados y graves se sacrificaron después de ocho días, mientras que los ratones con síntomas leves o asintomáticos se mantuvieron en BSL-III durante doce meses. Los análisis incluyeron estado general, histoquímica, inmunohistoquímica, microscopía electrónica de transmisión y qRT- PCR. Los pulmones del grupo de doce meses mostraron engrosamiento de las paredes alveolares, y algunos pulmones exhibieron reclutamiento de células inflamatorias, presencia de ARNm del SARS-CoV-2, inmunopositividad para la proteína de la espícula del SARS-CoV-2 y TEM mostró virus (60 -125 nm) dentro de las vesículas, lo que indica una replicación continua. Ciertas muestras de pulmón mostraron una presencia persistente de SARS- CoV-2 en exocrinocitos bronquiolares, células endoteliales y macrófagos. El grupo de ocho días presentó neumonitis intersticial viral, inmunopositividad al SARS-CoV-2 y ARNm. Los corazones de ocho días mostraron ARNm viral, mientras que los corazones de doce meses dieron negativo. Algunos animales asintomáticos de doce meses presentaron disminución del surfactante, engrosamiento de la membrana basal, fibrosis y degeneración leve del nervio autónomo. En este estudio realizado en ratones, los hallazgos indican la posibilidad de persistencia crónica del SARS-CoV-2 en los pulmones un año después de la infección inicial leve o asintomática, lo que podría sugerir la posibilidad de episodios recurrentes en condiciones humanas similares. El engrosamiento observado de las paredes alveolares y las posibles áreas fibróticas en estos ratones puede implicar un mayor riesgo de fibrosis post-COVID en humanos. Además, la presencia de células inflamatorias positivas para SARS- CoV-2 en algunos casos murinos asintomáticos podría presagiar una progresión hacia una inflamación continua y una enfermedad pulmonar crónica en humanos. Por lo tanto, se subraya la necesidad de realizar más estudios en seres humanos y realizar un seguimiento atento de las poblaciones humanas de alto riesgo.

Melamine exposure during the weaning period negatively affects ovarian reserve.

In this study, it was aimed to investigate the effects of melamine exposure since the weaning period on ovarian tissue and ovarian reserve. Melamine is illegally added to milk and formula to provide high false protein positivity. Female rats (the weaning period = 21 days old, n = 18) were divided into 3 groups. 0.1 mL saline was applied to the control group by gavage for 21 days. Fifty mg/kg and seventy-five mg/kg melamine was dissolved in 0.1 mL of saline and applied by gavage for 21 days, respectively. At the end of the experiment, plasma anti-Mullerian hormone (AMH) was measured, follicle count and ovarian diameter measurement were performed in the right ovaries, and flow cytometric analysis for apoptosis was performed in the left ovaries. While a statistically significant decrease was not observed in the number of the follicle and ovarian diameter between the control and melamine-treated groups (p > 0.05), a significant decrease in the corpus luteum and a significant increase in the number of atretic follicles were observed (p < 0.05). Apoptosis (Annexin V) increased in both melamine groups and AMH plasma level decreased significantly in the 75 mg/kg group (p < 0.05). Melamine exposure from the weaning (early postnatal) period may cause a decrease in ovarian reserve in parallel with a dose increase.

Autologously transplanted dermis-derived cells alleviated monobenzone-induced vitiligo in mouse.

Vitiligo is a depigmentation disease which affects skin and hair follicles with a prevalence of 0.5%-1% worldwide. In this study, we aimed to investigate treatmental potential of dermis-derived cells in monobenzone (MBEH)-induced mouse vitiligo model with light and electron microscopy. MBEH (40%) cream was topically applied to C57BL/6 mice until depigmentation occured in vitiligo and experimental groups. In experimental groups, dermis-derived cells obtained from back skin biopsy samples before induction of vitiligo, were injected intradermally to vitiligo mice. On Days 3 and 15 after cell transplantation to experimental groups, skin biopsies were compared with biopsies of control and vitiligo groups. Dermis-derived cells obtained from back skin biopsy samples of experimental groups showed nestin and versican immunoreactivity. Melanin in hair follicles of control group was detected by histochemical stainings (Haematoxylin and eosin and Fontana-Masson) whereas sparse melanin granules were observed in hair follicles of vitiligo group. In experimental groups, there was an increase in the number of hair follicles with melanin compared with vitiligo group. We observed MART-1 immunoreactive cells mostly around the hair follicles in control group and within dermis in vitiligo group. Electron microscopic investigation showed presence of melanosomes in hair follicles of control group and lacking in vitiligo group. In experimental groups, both type of hair follicles were observed with electron microscope. Our data suggest that autologously transplanted dermis-derived cells may be effective in vitiligo treatment by contrubuting to melanin production.

Effects of Monobenzyl ether of hydroquinone on 3T3 mouse fibroblast viability and ultrastructure.

Monobenzyl ether of hydroquinone (MBEH) is a topical depigmentation agent used by vitiligo patients to even the skin tone. We aimed to investigate the effects of MBEH on 3T3 mouse fibroblasts. Fibroblasts were treated with 250 µM, 500 µM, and 750 µM MBEH and vehicle (EtOH:DMSO) for 24 hours. Cell numbers of 250 µM, 500 µM, and 750 µM MBEH treated and vehicle groups decreased significantly compared to control group. TUNEL positive cell rate increased with MBEH concentration. In electron microscopic examination, control and vehicle groups showed active cells features, while mitochondrial swelling and cristae loss were seen in 250 µM MBEH-treated group. In cytoplasm of 500 µM MBEH-treated group, there were many multivesicular bodies and autophagic vacuoles. As an indication of apoptosis, cell membrane blebs and reduction in cell size were observed. In 750 µM MBEH-treated group, cells were completely degenerated. Our findings show that MBEH, which is used as a depigmentation agent to lighten the skin by destroying melanocytes, may also have dose-dependent negative effects on the viability of 3T3 mouse fibroblasts, and these may be mediated through autophagic and apoptotic cell death mechanisms.

Hepatotoxic effects of melamine exposure from the weaning period in rats: a flow cytometric, electron microscopic, and histopathologic study.

This study aims to investigate the effects of melamine exposure from the weaning period (21st postnatal days in rats) on liver tissue. Female Wistar albino rats (n = 18) were divided into three groups. About 0.1-ml saline was applied to the control group by gavage for 21 days from the postnatal 21st day. The second group was taken 50-mg/kg melamine (in 0.1-ml saline) and the third group was taken 75-mg/kg melamine (in 0.1-ml saline) p.o. On the postnatal 45th day, all rats were sacrificed under anesthesia. Then, liver tissues were cut into three parts and two of them placed in neutral formalin for histopathological and flow cytometric analysis, and one of them placed in 2.5% glutaraldehyde. Histopathological analysis was performed with hematoxylin & eosin, Masson trichrome, periodic acid Schiff stained sections, and also with transmission electron microscopy. Apoptosis (Annexin V positivity) was analyzed by flow cytometry. According to histopathological analysis, hepatocyte damage, sinusoidal dilatation, and inflammatory cell infiltration significantly increased in both melamine groups compared with the control group. Apoptosis significantly increased in the 50 and 75-mg melamine groups compared with the control group. In the results of transmission electron microscopy analysis, there was abnormal chromatin distribution in the hepatocyte nuclei, loss in the cristae of the mitochondria, and organelle loss in large areas in the cytoplasm in both melamine exposure groups. As result, melamine exposure from the weaning period causes liver damage with increasing doses.

Assessment of bone healing using (Ti,Mg)N thin film coated plates and screws: Rabbit femur model.

Magnesium (Mg) based implants such as plates and screws are often preferred to treat bone defects because of the positive effects of magnesium in bone growth and healing. Their low corrosion resistance, however, leads to fast degradation and consequently failure before healing was completed. Previously, we developed Mg doped titanium nitrate (TiN) thin film coatings to address these limitations and demonstrated that <10 at% Mg doping led to enhanced mineralization in vitro. In the present study, in vivo performance of (Ti,Mg)N coated Ti6Al4V based plates and screws were studied in the rabbit model. Bone fractures were formed on femurs of 16 rabbits and then fixed with either (Ti,Mg)N coated (n = 8) or standard TiN coated (n = 8) plates and screws. X-ray imaging and µCT analyses showed enhanced bone regeneration on fracture sites fixed with (Ti,Mg)N coated plates in comparison with the Mg free ones. Bone mineral density, bone volume, and callus volume were also found to be 11.4, 23.4, and 42.8% higher, respectively, in accordance with µCT results. Furthermore, while TiN coatings promoted only primary bone regeneration, (Ti,Mg)N led to secondary bone regeneration in 6 weeks. These results indicated that Mg presence in the coatings accelerated bone regeneration in the fracture site. (Ti,Mg)N coating can be used as a practical method to increase the efficiency of existing bone fixation devices of varying geometry.

Autologously transplanted dermal fibroblasts improved diabetic wound in rat model.

Healing of diabetic wounds are delayed due to late initiation and prolongation of the inflammatory phase, and inadequate growth factor synthesis, which may lead to chronic ulcers that may cause limb amputation, besides making the patients vulnerable to infections. In recent years, it has been extensively discussed whether different cell types transplanted to diabetic wound models accelerate wound healing. In this study, the effect of dermis-derived cells on Streptozotocin (STZ) induced experimental diabetic Sprague-Dawley rats were investigated. Animals were divided into 3 groups. First group was control, second group included diabetic animals with wounds. In the third group, firstly, skin specimens were obtained from animal's back, and then primary explant culture was performed. STZ induced experimental diabetes was applied to these animals and then wound was opened. The cells grown in primary culture were transplanted autologously. In all three groups, the samples taken from the wound areas on the 5th and 15th days of the wound were examined at the level of histochemical and immunohistochemical and electron microscopy. In the study, it was observed that the decreasing α-SMA and KGF (FGF-7) expression in the early period especially in the case of experimental diabetes increased as a result of cell transplantation, and in the sections belonging to the experimental diabetic group, a large number of inflammatory cells in the wound area were removed from the environment. In the cell transplanted group, the collagen fiber bundles as if in the control group. As a result, healthy cells of dermis can act as mesenchymal stem cells under certain conditions and have a positive effect on diabetic wound healing.