Bilateral Nerve Alterations in a Unilateral Experimental Neurotrophic Keratopathy Model: A Lateral Conjunctival Approach for Trigeminal Axotomy

To study bilateral nerve changes in a newly developed novel mouse model for neurotrophic keratopathy by approaching the trigeminal nerve from the lateral fornix. Surgical axotomy of the ciliary nerve of the trigeminal nerve was performed in adult BALB/c mice at the posterior sclera. Axotomized, contralateral, and sham-treated corneas were excised on post-operative days 1, 3, 5, 7 and 14 and immunofluorescence histochemistry was performed with anti-β-tubulin antibody to evaluate corneal nerve density. Blink reflex was evaluated using a nylon thread. The survival rate was 100% with minimal bleeding during axotomy and a surgical time of 8±0.5 minutes. The blink reflex was diminished at day 1 after axotomy, but remained intact in the contralateral eyes in all mice. The central and peripheral subbasal nerves were not detectable in the axotomized cornea at day 1 (p<0.001), compared to normal eyes (101.3±14.8 and 69.7±12.0 mm/mm2 centrally and peripherally). Interestingly, the subbasal nerve density in the contralateral non-surgical eyes also decreased significantly to 62.4±2.8 mm/mm2 in the center from day 1 (p<0.001), but did not change in the periphery (77.3±11.7 mm/mm2, P = 0.819). Our novel trigeminal axotomy mouse model is highly effective, less invasive, rapid, and has a high survival rate, demonstrating immediate loss of subbasal nerves in axotomized eyes and decreased subbasal nerves in contralateral eyes after unilateral axotomy. This model will allow investigating the effects of corneal nerve damage and serves as a new model for neurotrophic keratopathy

Hypoxia-inducible factor (HIF1alpha) inhibition modulates cumulus cell function and affects bovine oocyte maturation in vitro

Various metabolic and hormonal factors expressed in cumulus cells are positively correlated with the in vitro maturation (IVM) of oocytes. However, the role of hypoxia sensing both during maturation of cumulus-oocyte complexes (COCs) as well as during the resumption of meiosis remains uncertain. HIF1alpha plays major roles in cellular responses to hypoxia, and here we investigated its role during bovine COC maturation by assessing the expression of related genes in cumulus cells. COCs were divided into the following groups: immature (control), in vitro matured (IVM/control), or matured in the presence of a blocker of HIF1alpha activity (echinomycin, IVM/E). We found an inhibition of cumulus cell expansion in IVM/E, compared with the IVM/control. Transcript levels of several factors (n = 13) were assessed in cumulus cells. Decreased expression of HAS2, TNFAIP6, TMSB4, TMSB10, GATM, GLUT1, CX43, COX2, PTGES and STAR was found in IVM/E (P &amp;lt; 0.05). Additionally, decreased protein levels were detected for STAR, HAS2 and PCNA (P &amp;lt; 0.05), while activated-Caspase 3 remained unaffected in IVM/E. Progesterone output decreased in IVM/E. The application of PX-478, another blocker of HIF1alpha expression, yielded identical results. Negative effects of HIF1alpha suppression were further observed in the significantly decreased oocyte maturation and blastocyst rates from COCs matured with echinomycin (P &amp;lt; 0.05) or PX-478 (P &amp;lt; 0.05). These results support the importance of HIF1alpha for COC maturation and subsequent embryo development. HIF1alpha is a multidirectional factor controlling intercellular communication within COCs, steroidogenic activity, and oocyte development rates, and exerting effects on blastocyst rates

Changes of sFas and sFasL, oxidative stress markers in serum and follicular fluid of patients undergoing IVF

The Fas-Fas Ligand interaction is one of the essential events for the induction of apoptosis whereas the exact role of their soluble forms in the reproductive system is still not fully understood. Also oxidative stress in the pathogenesis of infertility causing diseases in women and has been suggested as one of the important factors that negatively affect IVF outcome. In this study, our aim was to evaluate serum and follicular fluid levels of soluble Fas soluble Fas Ligand, malondialdehyde, superoxide dismutase and total antioxidant capacity in patients undergoing IVF and compared with controls. This study included 109 patients. Patients were classified as unexplained infertility (N = 31), PCOS (N = 19), tubal factor (N = 9) and endometriosis (N = 10) and compared with male factor infertility (N = 40) that was the control group. sFas and sFasL levels were measured by immunoassay method. MDA, SOD and TAC levels were measured by colorimetric method. Patients with unexplained infertility, PCOS and tubal factor had significantly lower sFas levels compared with their controls (respectively, p < 0.01, p < 0.05, p < 0.05). However, SOD activity in unexplained infertility, PCOS and endometriosisgroupswere significantly higher than control group (p < 0.01).Decreased follicular fluid TAC levels were found in all patient groups compared with controls (respectively, p < 0.01, p < 0.05, p < 0.01, p < 0.01).Patients with tubal factor had significantly higher serum sFasL (p < 0.05), but lower follicular fluid sFasL levels (p < 0.05) compared with unexplained infertility. Tubal factor and endometriosis groups had lowerfollicular fluid TAC levels compared to unexplained infertility and PCOSgroups (p < 0.01). In this study, serum and follicular fluid sFas levels were decreased and antioxidant activity was impaired in infertility, possibly implying increased apoptosis. Especially in unexplained infertility group changes in this parametres more remarkable

Effect of intraluminal pillars on particle motion in bifurcated microchannels.

A central feature of intussusceptive angiogenesis is the development of an intravascular pillar that bridges the opposing sides of the microvessel lumen. In this report, we created polydimethyl siloxane (PDMS) microchannels with geometric proportions based on corrosion casts of the colon microcirculation. The structure of the PDMS microchannels was a bifurcated channel with an intraluminal pillar in the geometric center of the bifurcation. The effect of the intraluminal pillar on particle flow paths was investigated using an in vitro perfusion system. The microchannels were perfused with fluorescent particles, and the particle movements were recorded using fluorescence videomicroscopy. We found that the presence of an intravascular pillar significantly decreased particle velocity in the bifurcation system (p \u3c 0.05). In addition, the pillar altered the trajectory of particles in the center line of the flow stream. The particle trajectory resulted in prolonged pillar contact as well as increased residence time within the bifurcation system (p \u3c 0.001). Our results suggest that the intravascular pillar not only provides a mechanism of increasing resistance to blood flow but may also participate in spatial redistribution of cells within the flow stream

Spatial calibration of structured illumination fluorescence microscopy using capillary tissue phantoms.

Quantitative assessment of microvascular structure is relevant to the investigations of ischemic injury, reparative angiogenesis and tumor revascularization. In light microscopy applications, thick tissue specimens are necessary to characterize microvascular networks; however, thick tissue leads to image distortions due to out-of-focus light. Structured illumination confocal microscopy is an optical sectioning technique that improves contrast and resolution by using a grid pattern to identify the plane-of-focus within the specimen. Because structured illumination can be applied to wide-field (nonscanning) microscopes, the microcirculation can be studied by sequential intravital and confocal microscopy. To assess the application of structured illumination confocal microscopy to microvessel imaging, we studied cell-sized microspheres and fused silica microcapillary tissue phantoms. As expected, structured illumination produced highly accurate images in the lateral (X-Y) plane, but demonstrated a loss of resolution in the Z-Y plane. Because the magnitude of Z-axis distortion was variable in complex tissues, the silica microcapillaries were used as spatial calibration standards. Morphometric parameters, such as shape factor, were used to empirically optimize Z-axis software compression. We conclude that the silica microcapillaries provide a useful tissue phantom for in vitro studies as well as spatial calibration standard for in vivo morphometry of the microcirculation

Nerve alteration in the center after axotomy and the contralateral eyes.

<p>Representative histological photographs of corneal nerves stained with anti-βIII tubulin FITC-conjugated antibody. Normal subbasal (A) and stromal (E) nerve plexus. Subbasal nerve plexus completely disappeared (B; day 1, C; day 7, D; day 14) and stromal nerve definitely decreased from day1 (F; day 1, G; day 7, H; day 14) and after trigeminal axotomy. The nerve of the contralateral eye decreased from day 1 (J; day 1, K; day 7, L; day 14).</p

Potential associations between alterations in gut microbiome and obesity-related traits after the bariatric surgery

Background This study aimed to examine the effects of both obesity and bariatric surgery on gut microbiome, dietary intake, as well as metabolic and inflammatory parameters. Methods All participants (15 with morbid obesity who had bariatric surgery, 8 with morbid obesity and 11 non-obese) were followed up for a 6-month period with interviews at baseline (M0), at the end of 3 (M3) and 6 months (M6). Dietary assessment was done, and blood and faecal samples were collected. Results Dietary energy and nutrient intakes as well as serum glucose levels, total cholesterol, low-density lipoprotein (LDL)-cholesterol and high sensitivity C-reactive protein (hs-CRP) levels decreased after surgery (p 0.05) after the surgery, leading to a significant decrease in Firmicutes/Bacteroidetes ratio (p = 0.01). At sub-phylum level, the abundances of Lactobacillus and Bifidobacterium decreased, whereas those of Akkermansia increased after the surgery (p 0.05) at M3 and M6. Similarly, alpha-diversity indices were lower in subjects with morbid obesity at M0, but became similar to levels in non-obese controls at M6. Conclusion This study confirmed that bariatric surgery has substantial impacts on gut microbiome's composition and diversity, as well as anthropometrical measurements and biochemical parameters, which were associated with the alterations in dietary intake patterns