Fig 2: EphB2 expression in different segments of the rat GI tract.A–C, EphB2 expression was examined by western blot analysis of full-thickness tissue sections from indicated regions of the rat GI tract. Pgp9.5 was used as a loading control (A). Note that pgp9.5 is expressed at higher levels in the distal colon. Furthermore, we observed the apparition of putative EphB2-derived bands in the colon that might be degradation products. Ponceau S (B) and Coomassie (C) stainings are provided as addition to the traditional loading control. Although equal amount of proteins was loaded for analysis, those stainings show the variability in protein migration profil and relative protein abundance between intestinal segments. Each data point represents the relative adjusted volume of protein band intensity measured by using a rectangle tool of constant volume on each lane of the WB membrane. Data are represented as relative values of protein density in arbitrary units. D, densitometric quantitation of western blot signals revealed that EphB2 is present in all segments of the gut (n = 12 sections per segment from 12 rats; jej., jejunum; prox. colon, proximal colon; dist. colon, distal colon; n = 12 sections per segment from 12 animals, F (3, 44) = 18.91, p = 0.00000005, ANOVA; Tukey's post-hoc test; **p < 0.001, ***p < 0.0001). E, quantitative RT-PCR analysis of EphB2 mRNA levels in full-thickness tissue sections in the GI tract of adult rats. Note that there is no strict proportionality between EphB2 mRNA levels and translation product levels, likely due to posttranscriptional and posttranslational mechanisms. F–H, double immunostaining for EphB2 and a specific marker for neural processes ßIII tubulin (Tuj1) (F), neuronal cell body (Hu) (G), or glial marker (GFAP) (H) in a whole-mount preparation of myenteric plexus from distal colon of adult rat (6-month-old). Images illustrate that mostly neuronal processes and interganglionic fibers display similar pattern as EphB2 stainings. Images show that pattern of GFAP stainings differs from that of EphB2.

Fig 3: Enteric neuronal network activity. (A–K) Analysis of neural network activity of highly pure cultures of enteric neurons by multi-electrode array (MEA) at D28. (A,B) Representative image of enteric neurons in MEA, (A) brightfield and (B) double immunostaining of enteric neurons at D28 with neuronal marker PGP 9.5 (red), MAP2 (green) and the cell nucleus marker DAPI (blue). (C) Representative voltage recording at one electrode, showing spontaneous firing and its inhibition by TTX treatment. (D) Numbers of spikes per minutes before and after TTX, average of 15 electrodes. (E) Detail of a representative enteric neuron spike measured by MEA. (F) Histogram representing an example of Inter-Spike Interval (ISI) measured on one electrode (n°26). (G) Single electrode recordings showing examples representative traces of two consecutive burst before and after TTX treatment. (H–J) Different parameters of burst measured by MEA before and after addition of TTX in the culture, i.e., (H) numbers, (I) duration, and (J) average of inter-burst interval. (K) Detail of a representative burst trace measured by MEA. Results were compared using t-test (*p < 0.05; ***p < 0.001). Data are presented as mean ± SEM of n = 3 chips from 3 independent rat embryos.

Fig 4: Tadalafil mediated activation of mitochondrial mediated apoptosis pathway. Protein extracted from individual groups [Control (Normal saline, 3 ml/kg, p.o.); Toxic control (MNU 47 mg/kg, i.v.); MNU + Tadalafil (47 mg/kg i.v. + 2 mg/kg p.o.) and MNU + Tadalafil (47 mg/kg i.v. + 4 mg/kg p.o.)] were subjected to immunoblotting of proapoptotic (BAD) and anti-apoptotic (Bcl-xl) marker along with COX, NF?Bp65 and UCHL-1. ß-actin was used as internal loading control. Each experiment was performed in triplicate. The data was represented as mean ± SD. The groups were significantly different by one-way ANOVA followed by Bonferroni multiple tests. All groups were compared to the toxic control group (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig 5: (A–C) CD68+ pattern distribution within the non-OA (A) and diseased (B,C) AFUs by immunohistochemistry. (D, E) CD68+ elements characteristics by morphometric analysis: Welsch-corrected t-test of CD68+ particle size (D) and density (E) reveals statistically significant differences between OA and OA-free subjects (****p < 0.0001). (F–H) PGP9.5 immunoreactivity in the healthy (F) and diseased (G, H) AFUs. Scale bars: 100 µm (A,C,F,H); 50 µm (B,G). Scale bars inserts: 25 µm.