Comparative Analysis of the Healing Effects of the Transplanted Cell Sheets to the Experimentally Injured Maxillary Sinuses.

BACKGROUND: In our study, the authors aimed to obtain a live and functional sinus epithelium with mesenchymal stem cells and nasal mucosa epithelial cells from rabbits which are cultured in temperature-responsive culture plates to get a single-layer. METHODOLOGY/PRINCIPAL: Twenty-two female New Zealand rabbits were included in the study. Two of them were used to obtain mesenchymal stem cells. A total of 40 maxillary sinuses were randomly divided into 5 groups: 1) control group which is used to investigate normal rabbit maxillary mucosa, 2) secondary healing group, 3) mesenchymal stem cell graft group, 4) differentiated mesenchymal stem cell group, and 5) nasal mucosal graft group. The animals were sacrificed at the 28th day after the surgery.Scanning electron microscopy, transmission electron microscopy, and immunohistochemical investigations were performed. RESULTS: With these investigations, it was shown that; all graft groups were histologically better than secondary healing group and when the authors compared the graft groups, differentiated mesenchymal stem cell group were the best. CONCLUSION: Our study results showed that endoscopic sinus surgery and treatment with cell sheets, which were generated in temperature-responsive culture dishes, had more functional respiratory epithelium.

Is the effect of melatonin on vascular endothelial growth factor receptor-2 associated with angiogenesis in the rat ovary?

OBJECTIVES: Vascular endothelial growth factor (VEGF) and its receptors play important roles in angiogenesis. Melatonin plays an important role in gonadal development; thus, its effect on the reproductive system is evident. We investigated the influence of melatonin on the expression of VEGF, vascular endothelial growth factor receptor-1 (VEGFR1) and vascular endothelial growth factor receptor-2 (VEGFR2), as well as on changes in oxidative stress markers and follicle numbers in rat ovaries. METHODS: For this purpose, 45 Wistar rats were separated into the following groups: Group 1, control; Group 2, vehicle; and Group 3, melatonin. Rats in Group 3 were treated with melatonin at 50 mg/kg/day for 30 days. The effects of melatonin on the expression of VEGF, VEGFR1 and VEGFR2 were established by immunohistochemistry analysis. The effects of melatonin on antioxidant enzyme activities were demonstrated by spectrophotometric analysis. RESULTS: Based on immunohistochemistry analysis, VEGFR2 was predominantly localized to theca cells in the ovary. Our data indicate that melatonin treatment can significantly increase VEGF and VEGFR1 expression in the ovary ( p <0.05). Additionally, the number of degenerated follicles significantly decreased with melatonin treatment ( p <0.05). Melatonin administration also led to significant increases in antioxidant enzyme levels in the ovary. CONCLUSION: Melatonin treatment exerts protective effects on follicles against increased lipid peroxidation through modulating tissue antioxidant enzyme levels. These effects may be related to angiogenesis and antioxidant activities.

Decellularization of Trachea With Combined Techniques for Tissue-Engineered Trachea Transplantation.

OBJECTIVES: The purpose of this study is to shorten the decellularization time of trachea by using combination of physical, chemical, and enzymatic techniques. METHODS: Approximately 3.5-cm-long tracheal segments from 42 New Zealand rabbits (3.5±0.5 kg) were separated into seven groups according to decellularization protocols. After decellularization, cellular regions, matrix and strength and endurance of the scaffold were followed up. RESULTS: DNA content in all groups was measured under 50 ng/mg and there was no significant difference for the glycosaminoglycan content between group 3 (lyophilization+deoxycholic acid+de-oxyribonuclease method) and control group (P=0.46). None of the decellularized groups was different than the normal trachea in tensile stress values (P>0.05). Glucose consumption and lactic acid levels measured from supernatants of all decellularized groups were close to group with cells only (76 mg/dL and 53 mg/L). CONCLUSION: Using combination methods may reduce exposure to chemicals, prevent the excessive influence of the matrix, and shorten the decellularization time.

Is the effect of melatonin on vascular endothelial growth factor receptor-2 associated with angiogenesis in the rat ovary?

OBJECTIVES Vascular endothelial growth factor (VEGF) and its receptors play important roles in angiogenesis. Melatonin plays an important role in gonadal development; thus, its effect on the reproductive system is evident. We investigated the influence of melatonin on the expression of VEGF, vascular endothelial growth factor receptor-1 (VEGFR1) and vascular endothelial growth factor receptor-2 (VEGFR2), as well as on changes in oxidative stress markers and follicle numbers in rat ovaries. METHODS For this purpose, 45 Wistar rats were separated into the following groups: Group 1, control; Group 2, vehicle; and Group 3, melatonin. Rats in Group 3 were treated with melatonin at 50 mg/kg/day for 30 days. The effects of melatonin on the expression of VEGF, VEGFR1 and VEGFR2 were established by immunohistochemistry analysis. The effects of melatonin on antioxidant enzyme activities were demonstrated by spectrophotometric analysis. RESULTS Based on immunohistochemistry analysis, VEGFR2 was predominantly localized to theca cells in the ovary. Our data indicate that melatonin treatment can significantly increase VEGF and VEGFR1 expression in the ovary ( p <0.05). Additionally, the number of degenerated follicles significantly decreased with melatonin treatment ( p <0.05). Melatonin administration also led to significant increases in antioxidant enzyme levels in the ovary. CONCLUSION Melatonin treatment exerts protective effects on follicles against increased lipid peroxidation through modulating tissue antioxidant enzyme levels. These effects may be related to angiogenesis and antioxidant activities.

Effect of Melatonin on the Expression of VEGF-A and on the Degeneration of Follicle Reserve in Rat Ovary.

OBJECTIVE: To analyze the effects of melatonin on vascular endothelial growth factor A (VEGF-A) expression and follicle reserve in rat ovary. MATERIALS AND METHODS: A total of 45 female Wistar rats were used in the present study. Rats were divided into three groups: group 1 (control), group 2 (vehicle), and group 3 (melatonin). Rats in the melatonin group were treated with an intraperitoneal injection of melatonin at a dose of 50 mg/kg/day for 56 days. We investigated VEGF-A expression in rat ovary in all the groups using Western blot and reverse transcription-polymerase chain reaction. Histopathological parameters were evaluated using light microscopy. RESULTS: The number of atretic follicles was significantly lower in the melatonin treatment rats than in the control rats (p<0.05); however, the number of antral follicles was significantly higher in the former (p<0.05). Additionally, we observed a weak immunoblot stain in the melatonin group for VEGF-A protein. Interestingly, melatonin treatment induced a significant decrease in VEGF-A expression in the ovary of group 3 rats (p<0.05), whereas no such difference was observed between group 1 and group 2 rats (p>0.05). CONCLUSION: The present study demonstrates that the protective effect of melatonin on the degeneration of follicles in rat ovary is reduced by decreasing the VEGF-A expression. These results suggest that melatonin is effective against follicular atresia and preserves antral follicles, thus, offering a therapeutic advantage in clinical use.

Live birth after Laser Assisted Viability Assessment (LAVA) to detect pentoxifylline resistant ejaculated immotile spermatozoa during ICSI in a couple with male Kartagener's syndrome.

Primary ciliary dyskinesia (PCD) is a rare, autosomal recessive disease with abnormalities in the structure of cilia, causing impairment of muco-ciliary clearance with respiratory tract infections, heterotaxia and abnormal sperm motility with male infertility. Here, with a comprehensive literature review, we report a couple with an infertility history of 9 years and three unsuccessful IVF treatments, where male partner has Kartagener's Syndrome, a subtype of PCD, displaying recurrent respiratory infections, dextrocardia and total asthenozoospermia. His diagnosis was verified with transmission electron microscopy and genetic mutation screening, revealing total absence of dynein arms in sperm tails and homozygous mutation in the ZMYND10, heterozygous mutations in the ARMC4 and DNAH5 genes. Laser assisted viability assay (LAVA) was performed by shooting the sperm tails during sperm retrieval for microinjection, following detection of pentoxifylline resistant immotile sperm. Live births of healthy triplets, one boy and two monozygotic girls, was achieved after double blastocyst transfer.

Neutral sphingomyelinase inhibition alleviates apoptosis, but not ER stress, in liver ischemia-reperfusion injury.

Previous studies have revealed the activation of neutral sphingomyelinase (N-SMase)/ceramide pathway in hepatic tissue following warm liver ischemia reperfusion (IR) injury. Excessive ceramide accumulation is known to potentiate apoptotic stimuli and a link between apoptosis and endoplasmic reticulum (ER) stress has been established in hepatic IR injury. Thus, this study determined the role of selective N-SMase inhibition on ER stress and apoptotic markers in a rat model of liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reactions monitoring (MRM) method using ultrafast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared with controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. A significant increase was observed in ER stress markers C/EBP-homologous protein (CHOP) and 78 kDa glucose-regulated protein (GRP78) in IR injury, which was not significantly altered by N-SMase inhibition. Inhibition of N-SMase caused a significant reduction in phospho-NF-kB levels, hepatic TUNEL staining, cytosolic cytochrome c, and caspase-3, -8, and -9 activities which were significantly increased in IR injury. Data herein confirm the role of ceramide in increased apoptotic cell death and highlight the protective effect of N-SMase inhibition in down-regulation of apoptotic stimuli responses occurring in hepatic IR injury.

Pentoxifylline Alleviates Early Brain Injury in a Rat Model of Subarachnoid Hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a severe cerebrovascular disease frequently caused by ruptured aneurysms. Early brain injury (EBI) is the primary cause of morbidity and mortality in patients diagnosed with SAH and is associated with increased intracranial pressure, decreased cerebral blood flow and cerebral ischemia. Pentoxifylline (PTX) is a methylxanthine derivative clinically proven to improve perfusion in the peripheral microcirculation and has been shown to have neuroprotective effects in brain trauma and global cerebral ischemia in experimental animal models. This study aimed to determine the effect of PTX in experimental SAH, which has not been investigated yet. METHODS: An experimental SAH model was induced in male Wistar rats by autologous blood injection into the prechiasmatic cistern, and PTX was injected intraperitoneally immediately after SAH. The effects of PTX were evaluated 24 h after SAH via assessing the cerebral ultrastructure via transmission electron microscopy (TEM). Brain edema, blood-brain barrier (BBB) permeability, red blood cell deformability, tumor necrosis factor-alpha (TNF-alpha), nitrite-nitrate levels and apoptotic neuron death were also determined 24 h after SAH. The BBB permeability was measured by Evans blue (EB) extravasation, erythrocyte deformability was determined by filtration technique, and TNF-alpha and reactive nitrogen metobolites were analyzed in brain tissue by ELISA and spectral analysis, respectively. Apoptotic neurons were determined in brain sections by cleaved caspase-3 immunohistochemical analysis, and expression intensity was quantified using image J software. RESULTS: Cerebral ultrastructure in SAH group animals revealed intense perivascular edema and distortion in the astrocyte foot processes. PTX treatment attenuated structural deterioration due to SAH. Brain water content, BBB permeability, TNF-alpha, nitrite-nitrate levels and apoptotic neuronal death were significantly increased 24 h after SAH and were significantly alleviated by PTX treatment. There was no significant change in red cell deformability after SAH. CONCLUSIONS: Our results show that PTX reduces brain edema, BBB permeability, TNF-alpha expression, reactive nitrogen metobolites and apopotosis in experimental SAH. Based on our findings we suggest that PTX exerts neuroprotection against SAH-induced EBI, which might be associated with the inhibition of inflammation and apoptotic neuronal cell death.

The Healing Effects of Autologous Mucosal Grafts in Experimentally Injured Rabbit Maxillary Sinuses.

OBJECTIVES: Healing processes of the nose and paranasal sinuses are quite complex, and poorly understood. In this study, we aimed to compare the effect of mucosal autologous grafts on the degenerated rabbit maxillary sinus mucosa with spontaneous wound healing. It is hypothesized that mucosal grafts will enhance ciliogenesis and improve the morphology of regenerated cilia. METHODS: Ten female New Zealand rabbits were included in the study. They underwent external maxillary sinus surgery through a transcutaneous approach. A total of 20 maxillary sinuses were randomly divided into 2 groups: 'spontaneous healing group' and 'autologous graft group.' The animals were sacrificed at the 14th day after the surgery. Scanning electron microscope (SEM), and light microscope were used for the evaluation. RESULTS: Cellular composition of the graft group is better than the spontaneous healing group. The graft group had larger areas covered with ciliary epithelium than the spontaneous healing group, and the mean length of the cilias were also longer. Additionally, there were wider cilia with abnormal morphology areas in the spontaneous healing group. CONCLUSION: In our opinion, covering of the denuded areas with a graft improves re-epithelization, and may prevent the early complications after sinus surgeries.

Neutral sphingomyelinase inhibition decreases ER stress-mediated apoptosis and inducible nitric oxide synthase in retinal pigment epithelial cells.

Endoplasmic reticulum (ER) stress and excessive nitric oxide production via the induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of ocular diseases characterized by retinal degeneration. Previous studies have revealed the sphingomyelinase/ceramide pathway in the regulation of NOS2 induction. Thus, the objective of this study was to determine the activity of the sphingomyelinase/ceramide pathway, assess nitric oxide production, and examine apoptosis in human retinal pigment epithelial (RPE) cells undergoing ER stress. Sphingomyelinase (SMase) activity; nuclear factor κB (NF-κB) activation; NOS2, nitrite/nitrate, and nitrotyrosine levels; and apoptosis were determined in cultured human RPE cell lines subjected to ER stress via exposure to tunicamycin. Induction of ER stress was confirmed by increased intracellular levels of ER stress markers including phosphorylated PKR-like ER kinase, C/EBP-homologous protein, and 78-kDa glucose-regulated protein. ER stress increased nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, and nitrotyrosine formation and caused apoptosis in RPE cell lines. Inhibition of neutral SMase (N-SMase) activity via GW 4869 treatment caused a significant reduction in nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, nitrotyrosine formation, and apoptosis in ER-stressed RPE cells. In conclusion, N-SMase inhibition reduced nitrative stress and apoptosis in RPE cells undergoing ER stress. Obtained data suggest that NOS2 can be regulated by N-SMase in RPE cells experiencing ER stress.

Measurement of intracellular biomolecular oxidation in liver ischemia-reperfusion injury via immuno-spin trapping.

Hepatic ischemia-reperfusion (I/R) can lead to liver failure in association with remote organ damage, both of which have significant rates of morbidity and mortality. In this study, novel spin trapping and histopathological techniques have been used to investigate in vivo free radical formation in a rat model of warm liver I/R injury. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was administered to rats via intraperitoneal injection at a single dose of 1.5g of pure DMPO/kg body wt 2h before the initiation of liver ischemia. Blood vessels supplying the median and left lateral hepatic lobes were occluded with an arterial clamp for 60min, followed by 60min reperfusion. The effects of DMPO on I/R injury were evaluated by assessing the hepatic ultrastructure via transmission electron microscopy and by histopathological scoring. Immunoelectron microscopy was performed to determine the cellular localization of DMPO nitrone adducts. Levels of nitrone adducts were also measured to determine in situ scavenging of protein and DNA radicals. Total histopathological scoring of cellular damage was significantly decreased in hepatic I/R injury after DMPO treatment. DMPO treatment significantly decreased the hepatic conversion of xanthine oxidase and 4-hydroxynonenal formation in I/R injury compared to the untreated I/R group. The distribution of gold-nanoparticle-labeled DMPO nitrone adducts was observed in mitochondria, cytoplasm, and nucleus of hepatocytes. The formation of protein- and DNA-nitrone adducts was increased in DMPO-treated I/R livers compared to DMPO controls, indicating increased in situ protein and DNA radical formation and scavenging by DMPO. These results suggest that DMPO reduces I/R damage via protection against oxidative injury.