Macroscopic Scores Research Articles

Effect of methylene blue on experimental postoperative adhesion: A systematic review and meta-analysis

Adhesion is a primary challenge following surgery, and the anti-adhesive effect of methylene blue (MB) has been investigated. This systematic review and meta-analysis aimed to evaluate the effect of MB on postoperative adhesions in experimental studies. We initially searched OVID-MEDLINE, EMBASE, and Google Scholar in February 2021, and then in May 2021. The anti-adhesive efficacy of MB was compared with that of the control (either placebo or nothing) after the surgical procedure. The primary and secondary outcomes were the macroscopic and microscopic adhesion scores, respectively. Traditional meta-analysis, meta-regression, and trial sequential analysis (TSA) were performed to analyze the retrieved outcomes. We included 13 experimental studies of 367 rats (200 rats received MB and 167 rats received placebo or nothing). The macroscopic adhesion scores were significantly lower in the MB-administered group than in the control group (standardized mean difference, 2.313; 95% confidence interval, 1.104 to3.523; I2 = 94.0%, Tau = 2.059). Meta-regression analysis showed that macroscopic adhesion tended to decrease with an increase in MB dose. TSA demonstrated that the cumulative Z curve crossed both the conventional test and trial sequential monitoring boundary for the macroscopic adhesion score. MB had a beneficial effect on intraperitoneal adhesion following laparotomy, and adhesions decreased with increase in dose.

Morus macroura Miq. Fruit extract protects against acetic acid-induced ulcerative colitis in rats: Novel mechanistic insights on its impact on miRNA-223 and on the TNFα/NFκB/NLRP3 inflammatory axis

Nod-like receptor pyrin domain-1 containing 3 (NLRP3) inflammasome/tumor necrosis factor alpha (TNFα)/nuclear factor kappa B (NFκB) inflammatory pathway is known to be involved in the pathogenesis of ulcerative colitis (UC). Inversely, miRNA-223 can exert counter-regulatory effect on NLRP3 expression. The mulberry tree (Morus macroura) fruit is attaining increased importance for its antioxidant and anti-inflammatory activity in addition to its high safety profile. Accordingly, we attempted to explore the possible protective effect of mulberry fruit extract (MFE) in acetic acid (AA)-induced UC rat model. Phytochemical constituents of MFE were characterized using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). In the in vivo study, three doses of MFE were orally given for seven days before intra-rectal induction of UC by AA on day eight. Screening study revealed that MFE (300 mg/kg) significantly reduced macroscopic and microscopic UC scores. Biochemically, MFE ameliorated oxidative stress, levels of TNFR1, NLRP3, p-NFκB p65, TNFα, IL-1β, and IL-18, caspase-1 activity, but enhanced miRNA-223 expression. In conclusion, our study provided a novel protective impact for MFE against UC, in which miRNA-223 and TNFα/NFκB/NLRP3 pathway are involved. These results provide a promising step that might encourage further investigations of MFE as a protective agent in UC patients.

Endometriosis Induces Colonic Inflammation and Reactive Gliosis in an Animal Model

IntroductionEndometriosis is a complex gynecological disorder characterized by the ectopic growth of endometrial tissue. Symptoms of endometriosis are known to impair the quality of life of patients, among these symptoms are dysmenorrhea, chronic pelvic pain, and gastrointestinal (GI) issues. These GI issues are one of the common reasons for misdiagnosis with irritable bowel syndrome (IBS), a pain syndrome characterized by abdominal pain, painful bowel movements, diarrhea, and constipation, all of which are often present in endometriosis and persist even after treatment. Enteric glial cells (EGC) are known to play a role in pain associated with IBS, and reactive gliosis has been reported in patients with IBS, but the role of EGC in endometriosis has yet to be elucidated.HypothesisWe hypothesize that endometriosis will induce reactive gliosis, with increased expression of the glial fibrillary acidic protein (GFAP) and S100B, in the myenteric plexus of colonic sections in an animal model of endometriosis.MethodsWe used 15 female Sprague Dawley rats and divided them into three groups (n=5 per group): Naïve, Sham, and Endo. Endometriosis was induced in the Endo group by suturing 4 pieces of autologous uterine tissue in the intestinal mesentery, while the Sham group received only sutures without tissue. Von Frey and Hot Plate tests were done the day before surgery, two weeks after surgery, and before sacrifice to assess pain behavior. 21 days after surgery, animals were sacrificed, and endometriosis development and colonic macroscopic inflammation were assessed. Colonic tissue was collected to assess GFAP and S100B immunoreactivity, microscopic damage, and mast cell whole‐tissue cell count.ResultsWe found Endo animals showed a decrease in allodynia threshold, but no hyperalgesia. All Endo animals developed endometriosis with an average area per vesicle of 13.89±1.43mm2, and we found Endo animals had a significant increase in the colonic macroscopic score (3.01±0.22;p<0.001) compared to Sham (0.35±0.12) and Naïve (0.47±0.20). Paraffin‐embedded colon sections stained with H&E didn’t show statistical differences in microscopic damage between groups, yet toluidine blue staining for mast cells showed a 36% increase in the mean number of cells in Endo compared to Naïve but didn’t reach statistical difference. GFAP whole‐mount immunostaining showed significantly increased integrated density in Endo (25655.00±2395.00) when compared to Sham (13558.00±719.80;p<0.001) or Naïve (9035.00±1362.00;p<0.001), while no statistical difference was found in S100B integrated density between groups.ConclusionEndometriosis can alter GI homeostasis by inducing macroscopic inflammation, reactive gliosis, and some mast cell infiltration. Taken together these suggest endometriosis can mimic IBS histopathology beyond the symptomatology, reinforcing this disease’s complexity and the need to treat it beyond the gynecological setting.

Lycopodium Mitigates Oxidative Stress and Inflammation in the Colonic Mucosa of Acetic Acid-Induced Colitis in Rats.

Inflammatory bowel diseases (IBDs) such as ulcerative colitis (UC) and Crohn’s disease (CD) are diseases of the gastrointestinal system involving genetic and environmental factors attributed to oxidative stress and inflammation. Targeting oxidative stress and inflammation by novel dietary compounds of natural origin convincingly appears to be one of the important therapeutic strategies to keep the disease in remission. As there is no permanent cure for IBD except for chronic long-term treatment or surgery, it is therefore imperative to investigate plant-based agents that are receiving attention for their therapeutic benefits to overcome the debilitating clinical conditions of IBD. Lycopodium (LYCO), a plant of tropical and subtropical origin and known by numerous names such as ground pine, club moss, or devil’s claw, has been popularly used for centuries in traditional medicine including Chinese and Indian medicines. In the present study, the effect of LYCO has been investigated in an acetic acid (AA)-induced colitis model in Wistar rats. LYCO was orally administered at the dose of 50 mg/kg/day either 3 days before or 30 min after the induction of IBD and continued for 7 days by intrarectal administration of AA. The changes in body weight and macroscopic and microscopic analysis of the colon of rats of different experimental groups were observed on days 0, 2, 4, and 7. The levels of myeloperoxidase (MPO), reduced glutathione (GSH), and malondialdehyde (MDA) were measured. AA caused a significant reduction in body weight and increased macroscopic and microscopic ulcer scores along with a significant decline in antioxidant enzymes, superoxide dismutase (SOD), and catalase and antioxidant substrate, glutathione (GSH). There was a concomitant increased formation of malondialdehyde (MDA), a marker of lipid peroxidation, and raised myeloperoxidase (MPO) activity, a marker of neutrophil activation. Treatment with LYCO significantly improved IBD-induced reduction in body weight, improved histology, inhibited MDA formation, and restored antioxidants along with reduced MPO activity. AA also caused the release of proinflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-23 (IL-23). Furthermore, AA also increased the levels of calprotectin, a protein released by neutrophils under inflammatory conditions of the gastrointestinal tract. LYCO treatment significantly reduced the release of calprotectin and proinflammatory cytokines. The results demonstrate that LYCO treatment has the potential to improve disease activity by inhibiting oxidative stress, lipid peroxidation, and inflammation along with histological preservation of colonic tissues.

Fingolimod ameliorates acetic acid-induced ulcerative colitis: An insight into its modulatory impact on pro/anti-inflammatory cytokines and AKT/mTOR signalling.

Because of the approved immunomodulatory activities of fingolimod, the current study aimed at studying the curative potential of fingolimod against experimentally induced ulcerative colitis (UC) via modulating pro/anti-inflammatory cytokines release and AKT/mTOR signalling. UC was induced in rats by intracolonic instillation of acetic acid. Fingo (0.5mg/kg/day, p.o.) was given for 8 consecutive days that started 48 h after UC induction. Fingolimod increased body weight growth rate and colon body/weight and colon weight/length indices compared to the UC group. Fingolimod significantly decreased clinical evaluation score and macroscopic score compared to the UC group. The curative potential of fingolimod was further confirmed by histopathological examination revealing marked attenuation of mucosal injury and inflammatory cells infiltration. Fingolimod significantly decreased colon malondialdehyde content and increased colon glutathione contents compared to the UC group. Fingolimod also significantly decreased the expressions of pro-inflammatory cytokines interleukin-9 and T-helper 17 along with increasing the expression of anti-inflammatory interleukin-10 and transforming growth factor-β compared to the UC group. In addition, fingolimod decreased the expressions of AKT and mTOR compared to the UC group. Fingolimod attenuated acetic acid-induced UC through its immunomodulatory effect by shifting the balance to favour anti-inflammatory cytokine production rather than pro-inflammatory cytokines and modulating the AKT/mTOR signalling.

The protective impact of adapted trimebutine maleate-loaded nanostructured lipid carriers for alleviating the severity of acute colitis

Nanoparticles for colon-drug delivery were designed and evaluated to solve many discrepancy issues such as high adverse effects of released drugs, insufficient drug amount at diseased areas, and unintentionally premature drug release to noninflamed GIT regions. Herein, the goal of this work was to convert trimebutine maleate (TMB) into nanostructured lipid carriers (NLC) in order to improve its protective effects in ulcerative colitis. NLC of TMB was prepared by the hot homogenization followed by ultra-sonication method. A full 42-factorial design was used to estimate the produced TMB-NLC. The study design included the exploration of the impact of two independent variables namely lipid mix amount and ratio (glyceryl mono stearate and capryol 90), surfactant concentration (0.5, 1, 1.5, and 2%), on the particle size, polydispersity index, and the entrapment efficiency (EE%). The protective activity of F9 was examined through macroscopical scores, histopathological changes, immunohistochemical localization of tumor necrosis factor-α (TNF-α) and examination of oxidative stress such as reduced glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) against acetic acid-induced colitis in rats. Consistent with our expectations, the orally administered optimized formula (F9) alleviated the severity of colitis in acetic acid-induced rat model of colitis likely owing to the controlled release compared to free TMB. We aimed to develop TMB-loaded NLC for the treatment of acute colitis with the goal of providing a superior drug safety profile over long-term remission and maintenance therapy.

Intra-articular ozone slows down the process of degeneration of articular cartilage in the knees of rats with osteoarthritis

BackgroundOsteoarthritis (OA) is a joint disease of multifactorial etiology, affecting mainly the knees. We aimed to evaluate the effects of two different doses of gaseous ozone intra-articularly on the knee cartilage morphology of rats with osteoarthritis (OA). MethodsThe articular lesion was induced by sodium monoiodoacetate (MIA). 40 Wistar rats were divided into 4 groups: G1 control (without lesion and without treatment), G2 articular lesion (AL) (only lesion MIA-induced), G3 AL + treatment with 5 μg/mL of ozone intra-articular, and G4 AL + treatment with 10 μg/mL of ozone intra-articular. The experiment was carried out for 60 days. ResultsBoth doses of ozone intra-articular demonstrated less reduction in joint space (G3 and G4) compared to the G2, formation of osteophytes, but without subchondral sclerosis. Ozone decreased the volumetric density of the articular lesion (VV(AL)) of tibial. The treatments recovered VV(AL) of the femur similar to G1. Ozone lower dose (G3) showed lower tibia and femur macroscopic scores. ConclusionIntra-articular gaseous ozone can delay the degeneration of articular cartilage and can represents an integrative therapy in the OA treatment of knee after 60 days of treatment. For the first time the role of ozone in articular cartilage degeneration was evaluated helping to understand this therapy.

Non-Inferiority Field Study Comparing the Administrations by Conventional Needle-Syringe and Needle-Free Injectors of a Trivalent Vaccine Containing Porcine Circovirus Types 2a/2b and Mycoplasma hyopneumoniae.

The objective of this study was to assess the clinical, immunological, microbiological, and pathological evaluation of trivalent vaccine containing porcine circovirus types 2a/b (PCV2a/b) and Mycoplasma hyopneumoniae given by two different needle-free injection devices compared with conventional needle-syringe injection in a herd with subclinical PCV2d infection and enzootic pneumonia. A total of 240 21-day-old pigs, which weighed between 5 to 6 kg, were randomly divided into four groups (60 pigs per group, 30 = male and 30 = female per group). Injection site reactions in the pigs were minimal for the two needle-free injection devices and needle-syringe injection. Trivalent vaccination of pigs with two needle-free injection devices was not inferior to conventional needle-syringe injection for growth performance. Trivalent vaccination of pigs with two different needle-free injection devices reduced levels of PCV2d loads in serum and M. hyopneumoniae loads in the larynx equally compared to the conventional needle-syringe injection. The amount of PCV2d load in serum from the needle-free Pulse FX injection device at 49 days post vaccination showed non-inferiority to conventional needle-syringe injection. The immune response against PCV2 and M. hyopneumoniae to trivalent vaccine given with the needle-free Pulse FX injection device was non-inferior to conventional needle-syringe injection. The pigs from the two needle-free injection device and conventional needle-syringe injection had significantly (p < 0.05) lower macroscopic and microscopic lung lesion scores, and microscopic lymphoid lesions than from unvaccinated. The results of this study demonstrated that vaccination of trivalent vaccine by the two needle-free Pulse injection devices used in the study was non-inferior to that by conventional needle-syringe injection for growth performance, immune response against PCV2 and M. hyopneumoniae, and reduction of PCV2 viremia.

A182 THE LACK OF CHROMOGRANIN A IMPACTS COLONIC EPITHELIAL CELLS MARKERS IN AN EXPERIMENTAL MODEL OF ULCERATIVE COLITIS

BackgroundUlcerative colitis (UC) is a chronic and acute inflammatory disorder of the colon linked to dysregulated gut mucosal immune response and compromised colonic epithelial barrier function and integrity. Chromogranin-A (CHGA), a pro-peptide secreted by enteroendocrine cells, is highly expressed in colonic tissues of patients with UC. Elevated CHGA has been shown to correlate with UC disease activity and severity. Moreover, complete deletion of CHGA was shown to result in a diminution of pro-inflammatory markers known to disrupt the colonic epithelial barrier function and gut mucosal healing process. However, little is known about the effect of the absence of CHGA on colonic epithelial barrier function and gut mucosal integrity.AimsHere, we characterized the impact of the lack of CHGA on the colonic mucosa and epithelial barrier structure and function using CHGA knockout (CHGA-/-) mice treated with dextran sulfate sodium (DSS) to induce colitis.Methods13–17 weeks old male C57BL/6 wild-type (CHGA+/+) and C57BL/6 CHGA-/- mice were treated for 5 days with 5% DSS to induced acute colitis, control mice received regular water. CHGA mRNA expression, disease activity index (DAI), and macroscopic score (MS) were analyzed. Distal colonic tissues were isolated, and mRNA expression of markers associated with regenerative stem cells (fast-cycling stem cells [Lgr5+] and reserve stem cells [HOPX+] and [LY6a+], Goblet cells functions mucus barrier mucin 2 [MUC2], resistin-like molecule β [RELMβ], WAP 4-disulphide core domain 2 [WFDC2]) and trefoil factor 3 (TFF3) was evaluated by qRT-PCR.ResultsWe validated a beneficial effect of the Lack of CHGA on colitis severity, associated with significantly lower DAI and MS. In colitic CHGA+/+ and CHGA-/- mice, Lgr5+ and HOPX+ were both highly down-regulated, although, compared to CHGA+/+, CHGA-/- mice presented a 10.5fold higher expression of HOPX+. Compared to non-colitic states, Ly6a+ expression was significantly elevated in both colitic CHGA+/+ and CHGA-/- mice, however, no differences in Lgr5+ and Ly6a+ expression were noted between CHGA+/+ and CHGA-/- mice in all conditions. In CHGA+/+ mice, inflammatory conditions led to higher MUC2 and RELMB expression, although, compared to CHGA+/+, these markers were significantly lower in CHGA-/- mice. In colitic conditions, compared to CHGA+/+, CHGA-/- had a significant increase of WFDC2. In non-colitic conditions, mRNA expressions of all markers evaluated between CHGA+/+ and CHGA-/- in this study were unaltered. Finally, no differences were observed in TFF3 gene expression.ConclusionsThese results indicate in the absence of CHGA, the colonic epithelial barrier integrity and function are maintained through the modulation of goblet cells functions and elevated gut mucosa regenerative potential, thus enhancing the mucosal protection to colitis damage.Funding AgenciesCIHR

Dioscin promotes autophagy by regulating the AMPK-mTOR pathway in ulcerative colitis

Background Dioscin is reported to alleviate the dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) in mice. Autophagy plays an anti-inflammatory role in UC. We herein aimed to explore the biological functions of dioscin in autophagy in UC. Methods To explore the effects of dioscin on UC progression, a DSS-induced mouse model of UC was established. Body weight, disease activity index and macroscopic damage index scores were recorded for seven days. Hematoxylin & Eosin (HE) staining was used to stain colon sections and an BX53 microscope was prepared to observe pathological changes. The activities of glutathione, superoxidative dismutase, and malondialdehyde were determined by commercially available kits. Western blotting was performed to measure the protein levels of p-AMPK/AMPK, p-mTOR/mTOR and autophagy-related genes. Results The DSS-induced colitis and oxidative stress in mice were ameliorated after dioscin treatment. Dioscin promoted the phosphorylation of AMPK to inhibit mTOR activation and facilitated autophagy in DSS-induced mice model of UC. Conclusion Dioscin promotes autophagy by promoting the phosphorylation of AMPK to inhibit mTOR activation in ulcerative colitis.

The Anti-Inflammatory Effect of Acidic Mammalian Chitinase Inhibitor OAT-177 in DSS-Induced Mouse Model of Colitis.

Inflammatory bowel diseases (IBD) are chronic and relapsing gastrointestinal disorders, where a significant proportion of patients are unresponsive or lose response to traditional and currently used therapies. In the current study, we propose a new concept for anti-inflammatory treatment based on a selective acidic mammalian chitinase (AMCase) inhibitor. The functions of chitinases remain unclear, but they have been shown to be implicated in the pathology of various inflammatory disorders regarding the lung (asthma, idiopathic pulmonary fibrosis) and gastrointestinal tract (IBD and colon cancer). The aim of the study is to investigate the impact of AMCase inhibitor (OAT-177) on the dextran sulfate sodium (DSS)-induced models of colitis. In the short-term therapeutic protocol, OAT-177 given intragastrically in a 30 mg/kg dose, twice daily, produced a significant (p < 0.001) anti-inflammatory effect, as shown by the macroscopic score. Additionally, OAT-177 significantly decreased TNF-α mRNA levels and MPO activity compared to DSS-only treated mice. Intraperitoneal administration of OAT-177 at a dose of 50 mg/kg caused statistically relevant reduction of the colon length. In the long-term therapeutic protocol, OAT-177 given intragastrically in a dose of 30 mg/kg, twice daily, significantly improved colon length and body weight compared to DSS-induced colitis. This is the first study proving that AMCase inhibitors may have therapeutic potential in the treatment of IBD.

Miconazole Mitigates Acetic Acid-Induced Experimental Colitis in Rats: Insight into Inflammation, Oxidative Stress and Keap1/Nrf-2 Signaling Crosstalk.

Simple SummaryThe protective effect of miconazole, sulfasalazine (as a reference drug) and their combination on acetic acid (AA)-induced ulcerative colitis (UC) in a rat model was investigated. Pretreatment with miconazole significantly reduced wet colon weight and macroscopic scores, accompanied by a significant amelioration of the colonic architecture disorder. Miconazole and/or sulfasalazine revealed protective effects on AA-induced ulcerative colitis via activation of the Nrf2 pathway that improved the antioxidant defense system against oxidative stress and inflammation in UC, as demonstrated by the alleviation of colonic immunopathology, suppression of malondialdehyde (MDA) and elevation in GSH, SOD and HO-1, as well as downregulation of the levels of TNF-α, IL-6 and CRP and upregulation of the IL-10 level. Therefore, miconazole alone—particularly the high dose—or in combination treatment with sulfasalazine was the most efficient candidate compared with sulfasalazine alone and may be an alternative strategy for the treatment of UC.Ulcerative colitis (UC) is the most common type of inflammatory bowel disease, characterized by oxidative stress and elevated pro-inflammatory cytokines. Miconazole is an azole antifungal that stimulates the expression of antioxidant enzymes via Nrf2 activation, which consequently inhibits ROS formation and NF-κB activation. Hence, the present study aimed to investigate the protective effect of miconazole, sulfasalazine (as a reference drug) and their combination on acetic acid (AA)-induced UC in a rat model which was induced by intra-rectal administration of 4% AA. Rats were pretreated with miconazole (20 and 40 mg/kg, orally) or sulfasalazine (100 mg/kg, orally), or their combination (20 mg/kg miconazole and 50 mg/Kg of sulfasalazine, orally). Pretreatment with miconazole significantly reduced wet colon weight and macroscopic scores, accompanied by a significant amelioration of the colonic architecture disorder. Moreover, the treatment also significantly decreased the malondialdehyde (MDA) level and prevented the depletion of superoxide dismutase (SOD) activity and GSH content in inflamed colons. Additionally, the treatment showed suppressive activities on pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and C-reactive protein (CRP), and upregulated the anti-inflammatory cytokine interleukin-10 (IL-10). Moreover, the treatment upregulated the protein levels of Nrf-2 and heme oxygenase-1 (HO-1) in the colon tissue. Taken together, miconazole is effective in alleviating AA-induced colitis in rats, and the mechanism of its action is associated with the activation of Nrf2-regulated cytoprotective protein expression.

A flavonoid rich standardized extract of Glycyrrhiza glabra protects intestinal epithelial barrier function and regulates the tight-junction proteins expression

BackgroundIntestinal epithelial barrier dysfunction predisposes to many gastrointestinal, metabolic, and psychological disorders. A flavonoid rich extract of Glycyrrhiza glabra (FREG) has previously been reported to possess anti-inflammatory, antioxidant, and antiulcer properties.AimTo investigate the effect of FREG (GutGard®) on restoring intestinal barrier function in tumor necrosis factor-alpha (TNF-α) stimulated human colonic adenocarcinoma cell monolayer (Caco-2) and 2,4,6-Trinitrobenzenesulfonic acid (TNBS) induced ulcerative colitis in rats.MethodsIn in vitro, human intestinal Caco-2 cell monolayers were treated with TNF-α in the presence or absence of FREG and the paracellular permeability to FITC-conjugated 4-kD dextran (FD4) was measured to evaluate protection against the barrier dysfunction. In in vivo, intestinal barrier dysfunction was induced in male albino Wistar rats via intrarectal instillation of TNBS. Subsequently, the rats were treated orally with either FREG at 6.25, 12.5, and 25 mg/kg body weight, or Mesacol (250 mg/kg) for 5 days. On day 5, intestinal epithelial permeability was assessed with FD4 leakage into the serum. Also, colonic inflammation, colon morphology, histology and macroscopic score, weight to length ratio were evaluated. The activity of myeloperoxidase (MPO), TNF- α, secretory IgA levels and tight junction proteins expression were evaluated in rat’s colon.ResultsFREG protected the intestinal epithelial barrier integrity in human intestinal Caco-2 cells in vitro. FREG administration significantly improved the intestinal epithelial barrier function as evident from significant reduction in FD4 leakage. The colon morphology, histology score, macroscopic score, colon weight to length ratio also indicates beneficial effects of FREG on barrier function. In addition, FREG regulated the tight junction proteins, and markedly decreased TNF-α, MPO levels and significantly increased the secretory IgA levels in TNBS induced colitis rats.ConclusionThe study findings support the protective action of FREG on intestinal epithelial barrier integrity indicating its potential in protecting from implications of leaky gut.

Mesenchymal Stem Cell Exosomes Promote Functional Osteochondral Repair in a Clinically Relevant Porcine Model

Background: Previous studies have reported the efficacy of human mesenchymal stem cell (MSC) exosomes for the repair of osteochondral defects in rats and rabbits. However, the safety and efficacy of MSC exosomes remain to be validated in a clinically relevant large animal model. Purpose: To validate the safety and efficacy of human MSC exosomes for osteochondral repair in a clinically relevant micropig model. Study Design: Controlled laboratory study. Methods: Bilateral osteochondral defects (6-mm diameter and 1-mm depth) were surgically created in the medial femoral condyles in knees of 12 micropigs. The pigs then received 2-mL intra-articular injections of MSC exosomes and hyaluronic acid (HA) (Exosome+HA) or HA alone after surgery and thereafter at 8 and 15 days. Osteochondral repair was assessed by magnetic resonance imaging (MRI) at 15 days and at 2 and 4 months after surgery as well as by macroscopic, histological, biomechanical, and micro—computed tomography (micro-CT) analyses at 4 months after surgery. Results: Exosome+HA–treated defects demonstrated significantly better MRI scores than HA-treated defects at 15 days and at 2 and 4 months. Additionally, Exosome+HA–treated defects demonstrated functional cartilage and subchondral bone repair, with significantly better macroscopic and histological scores and biomechanical properties (Young modulus and stiffness) than HA-treated defects at 4 months. Micro-CT further showed significantly higher bone volume and trabecular thickness in the subchondral bone of Exosome+HA–treated defects than that of HA-treated defects. Importantly, no adverse response or major systemic alteration was observed in any of the animals. Conclusion: This study shows that the combination of MSC exosomes and HA administered at a clinically acceptable frequency of 3 weekly intra-articular injections can promote functional cartilage and subchondral bone repair, with significantly improved morphological, histological, and biomechanical outcomes in a clinically relevant porcine model. Clinical Relevance: Our findings provide a robust scientific rationale to support a phase 1/2 clinical trial to test MSC exosomes in patients with osteochondral lesions.

Characteristics of broiler chicken breast myopathies (spaghetti meat, woody breast, white striping) in Ontario, Canada

Spaghetti meat (SM), woody breast (WB), and white striping (WS) are myopathies affecting breast muscle of broiler chickens, and are characterized by a loss of myofibers and an increase in fibrous tissue. The conditions develop in intensive broiler chicken production systems, and cause poor meat process-ability and negative customer perception leading to monetary losses. The objectives of the present study were to describe the physical and histological characteristics of breast myopathies from commercial broiler chicken flocks in Ontario, Canada, and to assess the associations between the severity of myopathies with the physical and histological characteristics of the affected breast muscle fillets. Chicken breast fillets (n = 179) were collected over 3 visits from a processing plant and scored macroscopically to assess the severity of myopathies, following an established scoring scheme. For each fillet, the surface area, length, width, thickness, weight, and hardness (compression force) were measured. A subset of 60 fillets was evaluated microscopically. Multinomial logistic regression models were built to evaluate associations between physical parameters and macroscopic scores. The odds of SM co-occurring with severe WB (SM1WB2) were significantly associated with increased fillet thickness (OR = 1.59, 95% CI 1.31–1.94) and weight (OR = 1.06, 95% CI 1.03–1.09). Histologically, myopathies had overlapping lesions consisting of polyphasic myodegeneration, perivascular inflammatory cuffing and accumulation of fibrous tissue and fat. The pairwise correlation between macroscopic and microscopic scores was moderate (rho 0.45, P < 0.001). This is the first study to characterize breast myopathies in Canadian broiler flocks. Results show that the morphologic and microscopic changes of fillets from this cohort are similar to data from other countries, and provide database to benchmark these parameters in future studies. Our standardized categorization can be applied to broiler breast fillets in other regions of the world.

TOFACITINIB TREATMENT PREVENTS POST-OPERATIVE RECURRENCE OF CROHN’S DISEASE MODELED BY ILEOCECAL RESECTION IN HLA-B27 TRANSGENIC RATS

Abstract BACKGROUND Post-operative recurrence (POR) after ileocecal resection (ICR) affects most of Crohn’s disease (CD) patients within 3-5 years after surgery. Current immunomodulatory treatments have not proven efficiency in preventing POR, except for anti-TNFα biologic agents. AIM To evaluate the efficacy of the Janus Kinase inhibitor Tofacitinib to prevent POR after ICR in HLA-B27 transgenic rat model (TgB27) operated on an ICR. METHODS Thirty-three rats (strain F344, 29 TgB27, 4 control non Tg (nTg) rats) were operated on an ICR at the 12th week of life and were sacrificed at the 18th week of life. Tofacitinib (15 mg/kg, n=17) or placebo (n=16) were daily administered by oral gavage two weeks before surgery (10th week of life) until sacrifice. Anastomotic recurrence was blindly analyzed using macroscopic (from 0 to 4) and histologic (from 0 to 6) scores. Luminal microbiome was analyzed by shotgun metagenomic sequencing (Hiseq Illumina) in 9/33 rats before (week 9) and after Tofacitinib treatment or placebo (week 12), as well as 6 weeks following surgery (week 18). STAT3 phosphorylation was quantified in ileal samples by western blotting. Data were expressed by median with IQR. Comparisons were performed using not parametric two tailed Mann-Whitney analysis for unmatched data and Wilcoxon matched-pairs test for matched data. Two tailed χ2 test was used for contingency analysis. Spearman rank correlation was used for microbiota analysis. RESULTS 8/14 TgB27 rats receiving placebo (57%) showed signs of POR (macroscopic score ≥ 2) which were significantly prevented in 2/15 TgB27 rats (13%) treated with Tofacitinib (p= 0.013). nTg animals did not show macroscopic signs of POR. Macroscopic lesions were located 2 cm around the anastomosis in the TgB27 placebo group and consisted in ulcerations with a score of 2 (1-2.25). 11/15 TgB27 rats (73%) treated with Tofacitinib did not show macroscopic lesions and their score were significantly improved (0 (0-1), p= 0,0006). Histologic scores were significantly higher in TgB27 placebo group vs TgB27 Tofacitinib-treated rats (3 (2-4) vs 2 (1-3), p=0.036). Tofacitinib did not impact on alpha diversity and the relative abundance of bacterial taxa either before and after surgery. In contrast, surgery led to a significant 47% decrease of alpha diversity (p=0.0008) and loss of specific taxa in both TgB27 and nTg animals. No correlation was found between individual lesion scores and alpha diversity indexes. Ileal pSTAT3/STAT3 ratios were decreased by 80% in TgB27 rats treated with Tofacitinib (n=4) compared to placebo group (n=4) (0.3 ± 0,16 vs 1.58 ± 0,3, p=0,028). CONCLUSION We develop a reliable model of ICR in TgB27 rats mimicking the POR of CD. Tofacitinib prevents the macroscopic and histologic POR after ICR, without major modifications of the microbioma. ACKNOWLEDGMENTS Charity Foundation DigestScience and Pfizer SAS.

P602 Tofacitinib treatment prevents post-operative recurrence of Crohn’s Disease modeled by ileocecal resection in HLA-B27 transgenic rats

Abstract Background Post-operative recurrence (POR) after ileocecal resection (ICR) affects most of Crohn’s disease (CD) patients. Immunomodulatory treatments have not proven efficiency in preventing POR except for anti-TNFα biologic agents. We evaluate the efficacy of the JAK inhibitor Tofacitinib to prevent POR after ICR in HLA-B27 transgenic rat model (TgB27). Methods Thirty-three rats (strain F344, 29 TgB27, 4 control non Tg (nTg) rats) were operated on an ICR at the 12th week (W12) of life and were sacrificed at W18. Tofacitinib (15 mg/kg, n=17) or placebo (n=16) were daily administered by oral gavage at W10 until W18. Anastomotic recurrence were blindly analyzed using macroscopic (from 0 to 4) and histologic (from 0 to 6) scores. Luminal microbiome was analyzed by shotgun metagenomic sequencing (Hiseq Illumina) in 9/33 rats before (W9) and after Tofacitinib treatment or placebo (W12 and W18). STAT3 phosphorylation was quantified in ileal samples by western blotting. Data were expressed by median with IQR. Comparisons were performed using not parametric two tailed Mann-Whitney analysis for unmatched data and Wilcoxon matched-pairs test for matched data. Two tailed χ2 test was used for contingency analysis. Spearman rank correlation was used for microbiota analysis. Results 8/14 TgB27 rats receiving placebo (57%) showed signs of POR (macroscopic score ≥ 2) which were significantly prevented in 2/15 TgB27 rats (13%) treated with Tofacitinib (p= 0.013). nTg animals did not show macroscopic signs of POR. Macroscopic lesions were located 2 cm around the anastomosis in the TgB27 placebo group and consisted in ulcerations with a score of 2 (1–2.25). 11/15 TgB27 rats (73%) treated with Tofacitinib did not show macroscopic lesions and their score were significantly improved (0 (0–1), p= 0,0006). Histologic scores were significantly higher in TgB27 placebo group vs TgB27 Tofacitinib-treated rats (3 (2–4) vs 2 (1–3), p=0.036). Tofacitinib did not impact on alpha diversity and the relative abundance of bacterial taxa either before and after surgery. In contrast, surgery led to a significant 47% decrease of alpha diversity (p=0.0008) and loss of specific taxa in both TgB27 and nTg animals. No correlation was found between individual lesion scores and alpha diversity indexes. Ileal pSTAT3/STAT3 ratios were decreased by 80% in TgB27 rats treated with Tofacitinib (n=4) compared to placebo group (n=4) (0.3 ± 0,16 vs 1.58 ± 0,3, p=0,028). Conclusion We develop a reliable model of ICR in TgB27 rats mimicking the POR of CD. Tofacitinib prevents the macroscopic and histologic POR after ICR, without major modifications of the microbioma. Acknowledgments: Charity Foundation DigestScience and Pfizer SAS

P069 Design and delivery of anti-oxidant inorganic complexes mimicking the superoxide dismutase for inflammatory bowel diseases treatment

Abstract Background Oxidative stress (OS), defined by an increased flux of reactive oxygen species (ROS), plays an important role in the pathogenesis, the perpetuation and the amplification of inflammatory bowel diseases. In normal state, enzymatic defenses regulate the concentration of ROS but in IBD, some of them, including the superoxide dismutase (SOD), are weakened. The use of molecules mimicking the SOD activity may compensate for this deficiency and reduce the OS. 4 molecules were characterized for their SOD activity, their stability and their inertness. Their anti-inflammatory activity were assessed in a cellular model of epithelial inflammation and in a murine model of colitis and correlated to their physico-chemical properties. Methods The studied SOD mimics display similar structures differing by the presence of a propyl and a cyclohexyle group aiming to improve their kinetic inertness. To generate an inflammation associated to ROS production, intestinal epithelial (HT29) cells were stably transfected to over-express MD2, a co-receptor for LPS binding to TLR-4 receptor. The cells were stimulated by LPS (0.1µg/mL) and incubated with the SOD mimics (6 hours, 0,1-100µM). The secretion of IL-8 was measured by ELISA and the MnSOD expression by Western Blot. The SOD mimics were administrated to a murine model of colitis chemically-induced by DNBS. To protect them from the gastric acidity, the mimics were encapsulated in delivery vector. The monitoring of weight and the determination of intestinal permeability and macroscopic scores were performed. Results The mimics containing a cyclohexyle group display kinetic rates for metal exchanges that are up to 30-fold lower meaning that they are more inert. At 100µM, the four SOD mimics induced a significative decrease in IL-8 secretion (&amp;lt;35%/positive control (PT), p&amp;lt;0,02) and in MnSOD expression (&amp;lt;20%/PT, p&amp;lt;0,033). At 10µM, only the mimics containing the cyclohexyle group are still able to significatively counteract the LPS-induced secretion in IL-8 (&amp;lt;30/PT, p&amp;lt;0,001) and over-expression of MnSOD (20%/PT, p&amp;lt;0,02). Their bioactivity at lower doses can be correlated to their higher inertness. Concerning in vivo activity, preliminary assays show a faster recovery of the mice, that have received the SOD mimics with the cyclohexyle group. 3 days following DNBS injection, the mice of this group recovered in average 89% of their starting weight while the control mice recovered only 83%. Conclusion The SOD mimics display anti-inflammatory and antioxidant activity on HT29-MD2 cells, which is dependent on their inertness. Assays on a murine model of colitis seems to follow the same trend. The use of antioxidant treatments based on SOD mimics may reduce the inflammation rising from oxidative stress in IBD.

P710 Evaluation of helminth extracellular vesicles as a novel therapeutic for inflammatory bowel disease in murine models of colitis

Abstract Background With the increasing complexity of inflammatory bowel disease (IBD) new therapies are clearly required. Recent evidence is coming to light demonstrating beneficial effects of helminth proteins. We aim to assess the prophylactic and therapeutic effects of extracellular vesicles (EVs) from the nematode N. brasiliensis, which serves as a model organism for human hookworm infections, in an acute and chronic model of colitis. Methods Prophylactic and therapeutic effects of EVs were assessed in an acute model of colitis by the administration of 20µg intraperitoneally (i.p) of Nb EVs or grape EVs. Mice were sacrificed after 5 days. The therapeutic effects of Nb EVs were also assessed in a chronic model of colitis. Here, Rag1 knockout mice were treated twice weekly for three weeks with 20µg of either Nb EV, grape EV or aIL-12. Outcomes assessed for both experiments included weight loss, clinical disease, as well as macroscopic and histologic disease scores. Groups were statistically compared for significant differences. Results The prophylactic administration of Nb EVs significantly reduced weight loss, clinical disease, and histologic pathology compared to grape EV-treated negative controls. Therapeutic administration of Nb EVs significantly reduced weight loss and histologic pathology in the colon. Conclusion The current study provides important evidence that the anti-inflammatory effects of helminth EVs can be harnessed to prevent and reverse inflammation in an acute and chronic mouse model of colitis. With further characterization of helminth EVs, these compounds have the potential to generate much needed new anti-inflammatory therapies for IBD.

P073 Eosinophil depletion partially protects from colonic inflammation, but increases colonic collagen deposition in a DSS colitis model

Abstract Background The role of eosinophils in intestinal inflammation and fibrosis in inflammatory bowel disease (IBD) is largely unknown. Therefore, we assessed the functional role of eosinophils in a chronic murine model of colitis and associated fibrosis via anti-CCR3 mediated eosinophil depletion. Methods 6-8-week-old C57BL/6 RAG-/- mice received three cycles of dextran sodium sulphate (DSS) (1.75% - 2.25% - 2.25%) each interspersed with 14 days of recovery. Twice weekly, anti-CCR3 antibody (n=8), isotype (n=8) or saline injections (n=8) were given intraperitoneally. At the same timepoints, the disease activity index (DAI; mouse weight, stool consistency and presence of blood) was determined. At sacrifice, colonic damage was scored macroscopically (presence of hyperaemia, adhesions and length and degree of colon affected by inflammation). Colonic single cells were isolated and stained for flow cytometry, where eosinophils were characterized as CD45+ CD11b+ Siglec-F+ CD117- cells. Intestinal fibrosis was scored via colon weight/length, collagen deposition (using a colorimetric hydroxyproline assay) and COL1A1 expression by PCR. Results Anti-CCR3 mediated eosinophil depletion resulted in decreased disease activity compared to the other DSS treated groups injected with saline or isotype, determined by the area under the curve of the DAI (74.6±18.4 vs. 127.5±42.9 and 136.9±33.6, p=0.01 and p=0.0008 respectively) (figure 1). The macroscopic damage score also suggested eosinophil depleted mice to be partially protected from colonic inflammation compared to the saline and isotype injected mice that received DSS (1.1±1.0 vs. 2.1±1.2 and 3.0±0.7, p=0.09 and p=0.001 respectively) (figure 1). Colon weight/length and hydroxyproline assay showed a trend towards increased fibrosis in the anti-CCR3 injected group compared to saline (p=0.03 and 0.07, respectively) but not isotype (p=0.3 and 0.1, respectively) injected groups (figure 2). However, COL1A1 expression levels were significantly increased in the eosinophil depleted mice compared to the saline and isotype injected mice receiving DSS (43.2±11.4 vs. 23.3±8.7 and 30.1±11.0, p=0.002 and 0.04 respectively) (figure 2), indicating increased collagen deposition. Conclusion Eosinophil depletion via intraperitoneal anti-CCR3 injections resulted in partial protection against colonic inflammation, but was associated with increased collagen production. Caution is therefore needed when designing therapeutic interventions targeting eosinophils.