Macroscopic Scores Research Articles

Heterotopic autotransplantation of equine ovarian tissue using intramuscular versus subvulvar grafting sites: Preliminary results

Ovarian tissue transplantation (OTT) is a technique well established and successfully applied in humans using mainly orthotopic or heterotopic transplantation sites. In livestock, OTT is still in its infancy and, therefore, different aspects of the technique, including the efficiency of different heterotopic OTT sites as well as the potential effect of age (i.e., young vs. old mares) in the ovarian graft quality, need to be investigated. The present study investigated the efficacy of the intramuscular (IM) or the novel subvulvar mucosa (SV) heterotopic autotransplantation sites to maintain the survivability of the grafts for 3 and 7 days post-OTT. Ovarian biopsy fragments were obtained in vivo and distributed to the following treatments: Fresh control group (ovarian fragments immediately fixed), SV-3, IM-3, SV-7, and IM-7. During and after graft harvesting, the macroscopic characteristics of the grafts (i.e., adherence, morphology, and bleeding) were scored, and the percentages of morphologically normal and developing preantral follicles as well as the follicular and stromal cell densities of the grafts were evaluated. The results were that similar (P > 0.05) macroscopic scores were observed between both transplantation sites 7 days post-OTT, with positive correlations (P < 0.01) found among adherence, morphology, and bleeding of the grafts. A lower (P < 0.05) percentage of morphologically normal follicles was found 7 days post-OTT in the SV site (82%) compared with the Fresh control group (99%) and IM site (95%); however, the percentages of developing follicles were similar (P > 0.05) between both transplantation sites 7 days post-OTT (30–43%). Although similar (P > 0.05) follicular densities were found in both transplantation sites in young and old mares at 3 and 7 days post-OTT, large individual variation in the follicular depletion rate was observed regardless of transplantation site. The Fresh control group and SV-7 treatments had higher (P < 0.05) stromal cell densities in young and old mares compared with both IM-7 treatments. When comparing transplant sites between young and old mares, the follicular density in old mares and the stromal cell density in young mares were greater (P < 0.05) in the SV than in the IM site. In conclusion, even though the transplantation sites differentially affected some end points, overall comparable findings of the OTT technique using both heterotopic autotransplantation sites (i.e., IM and SV) for equine ovarian tissue were observed.

AdipoRon, an Orally Active, Synthetic Agonist of AdipoR1 and AdipoR2 Receptors Has Gastroprotective Effect in Experimentally Induced Gastric Ulcers in Mice

Introduction: Adiponectin is a hormone secreted by adipocytes, which exhibits insulin-sensitizing and anti-inflammatory properties and acts through adiponectin receptors: AdipoR1 and AdipoR2. The aim of the study was to evaluate whether activation of adiponectin receptors AdipoR1 and AdipoR2 with an orally active agonist AdipoRon has gastroprotective effect and to investigate the possible underlying mechanism. Methods: We used two well-established mouse models of gastric ulcer (GU) induced by oral administration of EtOH (80% solution in water) or diclofenac (30 mg/kg, p.o.). Gastroprotective effect of AdipoRon (dose 5 and 50 mg/kg p.o.) was compared to omeprazole (20 mg/kg p.o.) or 5% DMSO solution (control). Clinical parameters of gastroprotection were assessed using macroscopic (gastric lesion area) and microscopic (evaluation of the gastric mucosa damage) scoring. To establish the molecular mechanism, we measured: myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities; glutathione (GSH) level; and IL-1β, adenosine monophosphate-activated protein kinase (AMPK), and phosphorylated AMPK expression in gastric tissue. Results: AdipoRon produced a gastroprotective effect in both GU mouse models as evidenced by significantly lower macroscopic and microscopic damage scores. AdipoRon exhibited anti-inflammatory effect by reduction in MPO activity and IL-1β expression in the gastric tissue. Moreover, AdipoRon induced antioxidative action, as demonstrated with higher GSH levels, and increased SOD and GPX activity. Conclusions: Activation of AdipoR1 and AdipoR2 using AdipoRon reduced gastric lesions and enhanced cell response to oxidative stress. Our data suggest that AdipoR1 and AdipoR2 activation may be an attractive therapeutic strategy to inhibit development of gastric ulcers.

Autologous Platelet-Rich Fibrin Membrane to Augment Healing of Microfracture Has Better Macroscopic and Histologic Grades Compared With Microfracture Alone on Chondral Defects in a Rabbit Model

To determine the invivo effectiveness of a single-stage surgical procedure that combines microfracture and an autologous platelet-rich fibrin (PRF) membrane for cartilage repair in a rabbit model. Cartilage defects were created in the trochlear groove of the knees of adult white rabbits. Defects were divided into 2 treatment groups: microfracture only (control group) and microfracture covered by a PRF membrane (PRF group). To evaluate the repair cartilage, assessments were performed at 4, 12, and 24 weeks postoperatively using the International Cartilage Repair Society (ICRS) macroscopic scoring system and modified Wakitani histologic grading system. The mean ICRS macroscopic scores in the control and PRF groups were 4.1 and 5.8, respectively, at 4 weeks (P= .0623); 6.3 and 9.8, respectively, at 12 weeks (P= .006); and 6.5 and 10.3, respectively, at 24 weeks (P= .010). The mean modified Wakitani scores in the control and PRF groups were 4.0 and 3.9, respectively, at 4 weeks (P > .999); 5.3 and 10.4, respectively, at 12 weeks (P= .006); and 2.6 and 7.4, respectively, at 24 weeks (P= .012). The ICRS macroscopic scores and modified Wakitani scores showed that a single-stage surgical procedure combining microfracture and a PRF membrane was more effective than surgery with only microfracture for promoting cartilage repair. A single-stage surgical procedure combining microfracture and an autologous PRF membrane is a potentially beneficial treatment method for cartilage defects that does not require using any xenocollagen membrane.

Anti‐inflammatory Effect of Betulin in Acetic Acid Induced Ulcerative Colitis: Effect on TLR4/NF‐kB

Background Ulcerative colitis (UC) is a common inflammatory bowel disease. The importance of experimental models for investigating the pathogenic mechanisms and assessment of potential therapeutic agents for UC is well established. Betulin is a natural triterpene compound that has been reported to exert wide spectrum of pharmacological and biological activities. Aim of the work The present study aims to evaluate the potential therapeutic effect of betulin against acetic acid (AA)-induced UC in rats. Material and methods Thirty adult Sprague Dawley rats were randomly assigned into three groups: normal control, UC and UC+betulin (8 mg/kg). UC was induced by intracolonic instillation of AA (3% v/v) in 0.9% saline in all study groups except for the normal group. Four days post instillation of AA, UC+betulin group was treated by intraperitoneal administration of betulin once daily for 14 consecutive days. Results Results showed that betulin attenuated AA-induced UC as evident by reduced macroscopic scores, decreased serum C reactive protein (CRP) titer and lactate dehydrogenase (LDH) activity and attenuated histopathological hallmarks including mucosal necrosis, haemorrhage, congestion and inflammatory cells infiltration. Moreover, betulin treatment dampened colonic inflammatory status via downregulation of toll like receptor-4 (TLR4)/Nuclear factor-κB NF-kB axis accompanied by reduction of colonic inflammatory cytokines tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) as well as CD68 immunostaining. Additionally, betulin administration suppressed signs of apoptosis as shown by reduced caspase-3 and caspase-8 immunostaining compared with UC group. Conclusion Our findings demonstrate a novel therapeutic effect of betulin against UC via suppression of TLR4/NF-κB signalling.

Voluntary Exercise Modulates Neuroimmune Response in Colonic Inflammation Induced by Endometriosis

Endometriosis is a complex gynecological disease characterized by the growth of endometrial tissue outside of the uterine cavity. Some symptoms that are prevalent in women with endometriosis are dysmenorrhea, infertility, pelvic pain, and gastrointestinal symptoms (GI). Among the GI symptoms, women with endometriosis experience painful bowel movements, diarrhea, constipation, and bloating similar to irritable bowel syndrome (IBS). Enteric glial cells (EGC), when reactive, are known to play a role in the inflammation of IBS. Reactive EGC are also involved in the polarization of macrophages to a proinflammatory phenotype; while exercise is known to promote activation of PPARγ, a nuclear receptor known for its anti-inflammatory activity, which is expressed in anti-inflammatory macrophages. We hypothesize that exercise can reduce endometriosis-induced expression of the glial fibrillary acidic protein (GFAP) and S100B, markers of reactive enteric gliosis; and promote expression of PPARγ in colonic macrophages. Methods Three groups of female Sprague Dawley rats were used: Sham Control (n=4), Endo Control (n=5), and Endo Exercise (n=5). Endometriosis was induced by suturing 4 pieces of autologous uterine tissue from the right uterine horn on the intestinal mesentery, while the Sham group only received the sutures without implants. After surgery Endo Exercise group was exposed to voluntary wheel running for 60 days, while control groups were individually housed in standard cages. On Day 60 we sacrificed the animals, examined them for endometriosis development, assessed colonic macroscopic score, and collected a piece of distal colon. We performed double immunofluorescence staining using GFAP/S100B and CD68/PPARγ antibodies, respectively, on slides containing formalin-fixed paraffin-embedded colonic tissue. Results After normalizing the mean fluorescent intensity (MFI) to DAPI, we found that the Endo Control group has a significant increase in GFAP MFI (0.57±0.06; p<0.01) compared to Sham Control (0.32±0.03), which was reduced significantly by exercise (0.38±0.01; p<0.05). Similarly, exercise significantly decreased S100B MFI (0.67±0.06; p<0.05) compared to Endo Control (1.34±0.16), although Endo Control was not significantly higher than Sham Control (0.94±0.20). For CD68/PPARγ, we found that Endo Exercise increased significantly the percentage of macrophages expressing PPARγ (88.43±3.45%; p<0.05) compared to Endo Control (51.14±10.44%). Endo Control had significantly higher macroscopic colonic damage (2.04±0.41; p<0.01) compared to Sham Control (0.26±0.14) and Endo Exercise (0.38±0.12). Conclusions These results show that endometriosis induces colonic inflammation and enteric reactive gliosis, while exercise intervention restores gut homeostasis, possibly through the increase of anti-inflammatory macrophages expressing PPARγ. Altogether, these results suggest exercise may prove beneficial to improve endometriosis-induced GI symptoms.

Resilience to height loss of articular cartilage of osteoarthritic stifle joints of old pigs, compared with healthy cartilage from young pigs in a tribological pin-on-plate exposure, revealing similar friction forces.

IntroductionWe saw a lack of data on the biomechanical behavior of degenerated articular cartilage (OA) compared with that of healthy cartilage, even though the susceptibility to wear and tear of articular cartilage plays a key role in the progression of osteoarthritis (OA). Therefore, we performed a comparison between naturally occurring OA and healthy cartilage from pigs, before and after tribological stress.AimThe aim of the study was to compare OA-cartilage with healthy cartilage and to analyze the resilience to tribological shear stress, which will be measured as height loss (HL), and to friction forces of the cartilage layers. The findings will be substantiated in macro- and microscopical evaluations before and after tribological exposure.MethodsWe assessed stifle joints of fifteen old and sixteen young pigs from the local abattoir radiologically, macroscopically and histologically to determine possible OA alterations. We put pins from the femoral part of the joints and plates from the corresponding tibial plateaus in a pin-on-plate tribometer under stress for about two hours with about 1108 reciprocating cycles under a pressure of approximately 1 MPa. As a surrogate criterion of wear and tear, the HL was recorded in the tribometer. The heights of the cartilage layers measured before and after the tribological exposure were compared histologically. The condition of the cartilage before and after the tribological exposure was analyzed both macroscopically with an adapted ICRS score and microscopically according to Little et al. (2010). We assessed the friction forces acting between the surfaces of the cartilage pair–specimens.ResultsArticular cartilage taken from old pigs showed significant degenerative changes compared to that taken from the young animals. The macroscopic and microscopic scores showed strong alterations of the cartilage after the tribological exposure. There was a noticeable HL of the cartilage specimens after the first 100 to 300 cycles. The HL after tribological exposure was lower in the group of the old animals with 0.52 mm ± 0.23 mm than in the group of the young animals with 0.86 mm ± 0.26 mm (p < 0.0001). The data for the HL was validated by the histological height measurements with 0.50 mm ± 0.82 mm for the old and 0.79 mm ±0.53 mm for the young animals (p = 0.133). The friction forces measured at the cartilage of the old animals were 2.25 N ± 1.15 N and 1.89 N ± 1.45 N of the young animals (p = 0.3225).ConclusionUnlike articular cartilage from young pigs, articular cartilage from old pigs showed OA alterations. Tribological shear stress exposure revealed that OA cartilage showed less HL than healthy articular cartilage. Tribological stress exposure in a pin–on–plate tribometer seemed to be an appropriate way to analyze the mechanical stability of articular cartilage, and the applied protocol could reveal weaknesses of the assessed cartilage tissue. Friction and HL seemed to be independent parameters when degenerated and healthy articular cartilage were assessed under tribological exposure in a pin–on- plate tribometer.

Salvia miltiorrhiza hydroalcoholic extract inhibits postoperative peritoneal adhesions in rats

BackgroundOne of the most prevalent postoperative complications is believed to be intra-abdominal peritoneal adhesions, which is followed by several complications. Several adhesion prevention products have been examined, yet none of them were found to be completely effective. The current research is conducted to evaluate the beneficial effects of Salvia miltiorrhiza hydroalcoholic extract in inhibiting postoperative peritoneal adhesions in rats.MethodsForty rats were randomly classified into five equal groups (n = 8): 1) the normal group did not undergo surgical operations, 2) the control group in which the adhesion was induced, and which did not receive any treatment, 3) distilled water group that received distilled water, and 4,5) treatment groups treated with 1 and 5% of Salvia miltiorrhiza hydroalcoholic extract. The rats were euthanized 14 days following the surgery and the macroscopic score, the microscopic score of granulomatous inflammation and granulation tissue formation, IHC markers (vimentin, CD31, IL-1β, COX-2, and iNOS), and oxidative stress biomarkers (MDA, GPx, CAT, and TAC) were assessed in the experimental groups of the study.ResultsThe difference between the control group and other groups for the adhesions macroscopic score, microscopic score, IHC markers, and oxidative stress biomarkers was significant (p < 0.05). Distilled water had no protective effect on the formation of peritoneal adhesions. Salvia miltiorrhiza treatment in two different doses significantly reduced macroscopic and microscopic scores, MDA concentration, Vimentin, IL-1β, COX-2, and iNOS compared to the control group (p < 0.05). The levels of GPx, CAT, and TAC in the treatment groups increased significantly compared with the control group (p < 0.05). Our findings revealed that a higher dose of Salvia miltiorrhiza was more effective in reducing peritoneal adhesions, proinflammatory and mesenchymal cell markers, and oxidative stress.ConclusionsSalvia miltiorrhiza extract, owing to its strong antioxidant and anti-inflammatory properties, could effectively reduce peritoneal adhesions. Therefore, Salvia miltiorrhiza is recommended to be used as an effective anti-peritoneal post-operative adhesive agent.

Shaoyao Decoction Inhibits Inflammation and Improves Intestinal Barrier Function in Mice With Dextran Sulfate Sodium-Induced Colitis.

Shaoyao decoction (SYD), a classical traditional Chinese medicine formula, is effective for the treatment of inflammatory bowel disease (IBD). This study was designed to investigate the therapeutic effects of SYD on IBD and possible mechanisms. Dextran sulfate sodium (DSS, 3.5%) was used to induce colitis in C57BL/6 mice. Disease phenotypes were investigated based on disease activity index (DAI), colon length, and microscopic and macroscopic scores. Additionally, the presence of proinflammatory cytokines, immune cell infiltrates, intestinal cell proliferation, apoptosis, epithelial permeability, signal transducer and activator of transcription 3 (STAT3), and nuclear factor-κB (NF-κB) signaling, as well as the intestinal mucosal barrier function, were investigated. The administration of SYD significantly ameliorated the clinical signs, suppressed the levels of proinflammatory cytokines, and reduced immune cell infiltrates into colonic tissues of DSS-induced colitis model mice. SYD also significantly reduced the DSS-induced activation of STAT3 and NF-κB signaling. Furthermore, SYD promoted epithelial integrity by regulating epithelial cell apoptosis and epithelial permeability. Finally, we demonstrated that SYD protected the intestinal barrier function by significantly regulating the mucus layer genes Muc1, Muc2, Muc4, and Tff3, as well as the epithelial barrier genes Z O -1 and Occludin. Our results indicate that SYD has a protective effect on DSS-induced colitis, which is attributable to its anti-inflammatory activity and intestinal barrier function-enhancing effects. These results provide valuable insights into the pharmacological actions of SYD for the treatment of IBD.

Ascorbic Acid and Iron Supplement Treatment Improves Stem Cell–Mediated Cartilage Regeneration in a Minipig Model

Background: The transplantation of mesenchymal stem cells (MSCs) into cartilage defects has led to variable cartilage repair outcomes. Previous in vitro studies have shown that ascorbic acid and reduced iron independently can improve the chondrogenic differentiation of MSCs. However, the combined effect of ascorbic acid and iron supplementation on MSC differentiation has not been investigated. Purpose: To investigate the combined in vivo effects of ascorbic acid and a US Food and Drug Administration (FDA)–approved iron supplement on MSC-mediated cartilage repair in mature Göttingen minipigs. Study Design: Controlled laboratory study. Methods: We pretreated bone marrow–derived MSCs with ascorbic acid and the FDA-approved iron supplement ferumoxytol and then transplanted the MSCs into full-thickness cartilage defects in the distal femurs of Göttingen minipigs. Untreated cartilage defects served as negative controls. We evaluated the cartilage repair site with magnetic resonance imaging at 4 and 12 weeks after MSC implantation, followed by histological examination and immunofluorescence staining at 12 weeks. Results: Ascorbic acid plus iron–pretreated MSCs demonstrated a significantly better MOCART (magnetic resonance observation of cartilage repair tissue) score (73.8 ± 15.5), better macroscopic cartilage regeneration score according to the International Cartilage Repair Society (8.6 ± 2.0), better Pineda score (2.9 ± 0.8), and larger amount of collagen type II (28,469 ± 21,313) compared with untreated controls (41.3 ± 2.5, 1.8 ± 2.9, 12.8 ± 1.9, and 905 ± 1326, respectively). The obtained scores were also better than scores previously reported in the same animal model for MSC implants without ascorbic acid. Conclusion: Pretreatment of MSCs with ascorbic acid and an FDA-approved iron supplement improved the chondrogenesis of MSCs and led to hyaline-like cartilage regeneration in the knee joints of minipigs. Clinical Relevance: Ascorbic acid and iron supplements are immediately clinically applicable. Thus, these results, in principle, could be translated into clinical applications.

Effects of red ginseng on gut, microbiota, and brain in a mouse model of post-infectious irritable bowel syndrome

BackgroundIrritable bowel syndrome (IBS), the most common functional gastrointestinal disorder, is characterized by chronic abdominal pain and bowel habit changes. Although diverse complicated etiologies are involved in its pathogenesis, a dysregulated gut–brain axis may be an important factor. Red ginseng (RG), a traditional herbal medicine, is proven to have anti-inflammatory effects and improve brain function; however, these effects have not been investigated in IBS. MethodsThree-day intracolonic zymosan injections were used to induce post-infectious human IBS-like symptoms in mice. The animals were randomized to receive either phosphate-buffered saline (CG) or RG (30/100/300 mg/kg) for 10 days. Amitriptyline and sulfasalazine were used as positive controls. Macroscopic scoring was performed on day 4. Visceral pain and anxiety-like behaviors were assessed by colorectal distension and elevated plus maze and open field tests, respectively, on day 10. Next-generation sequencing of gut microbiota was performed, and biomarkers involved in gut–brain axis responses were analyzed. ResultsCompared to CG, RG significantly decreased the macroscopic score, frequency of visceral pain, and anxiety-like behavior in the IBS mice. These effects were comparable to those after sulfasalazine and amitriptyline treatments. Moreover, RG significantly increased the proliferation of beneficial microbes, including Lactobacillus johnsonii, Lactobacillus reuteri, and Parabacteroides goldsteinii. RG significantly suppressed expression of IL-1β and c-fos in the gut and prefrontal cortex, respectively. Further, it restored the plasma levels of corticosterone to within the normal range, accompanied by an increase in adrenocorticotropic hormone. ConclusionRG may be a potential therapeutic option for the management of human IBS.

Early failure of a polyvinyl alcohol hydrogel implant with foreign body reaction and osteolysis in an ovine model of cartilage repair

Purpose: Hemiarthroplasty with polyvinyl alcohol hydrogel (PVA) cartilage implants in the treatment of hallux rigidus showed initial promise with a 90% success rate. However, recent studies suggest that the implant may shrink and subside, leading to higher than anticipated failure rates. The purpose of this study was to characterize the healing response to polyvinyl implantation by using an ovine model. Methods: An osteochondral defect was created unilaterally in the medial femoral condyle of six sheep and filled with a PVA implant (Cartiva, Wright Medical) (n=4) or left empty (n=2). Sheep were followed for six months. Post-mortem quantitative CT (qCT) evaluations were performed to measure bone density using 5mm-wide ROI delineated in 3D reconstruction around the defect/implant area. Joints were then dissected and evaluated using a modified International Cartilage Repair Society (ICRS) macroscopic score (I=best, IV=worst). Osteochondral sections were assessed using a modified Osteoarthritis Research Society International (OARSI) histopathology net score (0=best, 20=worst). Synovial membrane histology was scored using the aseptic lymphocyte-dominated vasculitis-associated lesions (ALVAL) scoring system for soft tissue reaction (0=best, 19=worst). Both the synovial membrane and osteochondral sections underwent immunohistochemistry with anti-ionized calcium-binding adapter molecule 1 antibody (Iba1) to identify macrophages. Results: Two of four sheep with the PVA implant showed unrelenting pain and lameness necessitating early euthanasia. These were considered failed implants. qCT scans revealed 2-fold more osteolysis around failed implants than the other two (successful) implants or empty defects (Figure 1A). Grossly, failed implant sheep had ICRS scores of III and IV/V. The two successful implant sheep had ICRS scores of II/V and both empty defect sheep had scores of III/V. Osteochondral histology of failed implants had a mean OARSI score of 13/20 with extensive GAG loss and a stromal inflammatory reaction surrounding the implant that was up to 5-fold wider than successful implants (Figure 1B). This reaction contained abundant fibrovascular tissue with aggregates of lymphocytes and plasma cells. Failed implants revealed strong and diffuse Iba1 staining of macrophages along the implant/bone interface with cells densely packed in multiple rows (Figure 1C). Multinucleated giant cells and macrophages were identified surrounding presumptive implant debris in one failed implant (Figure 1C, red arrow and X). Successful implants and empty defects had few Iba1 positive cells randomly distributed (Figure 1C). Mean synovial membrane ALVAL score for failed implants was 11/19 with presence of an adverse local tissue reaction with lymphocytes and macrophages, vascular reactivity, and synovial hyperplasia (Figure 1D, E). In contrast, synovial membrane in successful implants and empty defects were normal (ALVAL 0/19). Conclusions: Polyvinyl alcohol hydrogel implants had a 50% early failure rate with uncontrollable pain, severe osteolysis, inflammation and an adverse local tissue reaction. Furthermore, the recruitment of macrophages and multinucleated giant cells are evidence of a foreign body reaction typically associated with implant wear debris. Non-degradable biomaterials such as PVA will result in continuous infiltration by macrophages and secretion of proinflammatory cytokines, leading to periprosthetic osteolysis and ultimately implant failure. These Results suggest that the use of this PVA implant should not be continued in human patients without further in vivo safety studies.

Protective Effect of a Formulation Containing Pistacia atlantica Oleo-Gum-Resin and Honey on Experimental Model of Acetic Acid-Induced Colitis in Rats

Background and objectives: Inflammatory bowel disease (IBD) is a recurrent chronic inflammatory disease of the gastrointestinal tract. In Iranian traditional medicine, the oleo-gum-resin of the genus Pistacia is recommended for treatment of various diseases including gastrointestinal disorders. The present study investigated the therapeutic action of a combination of Pistacia atlantica subspecies kurdica oleo-gum-resin and honey in acetic acid-induced colitis in rats. Methods: Pistacia atlantica oleo-gum-resin was mixed with honey. The mixture was suspended in distilled water. Following induction of colitis with 4% acetic acid in all animals, except in sham group,themixture was orally administered for two consecutive days at the concentrations of 100, 200, 400 mg/kg. Other groups included the control, sham and a standard group (dexamethasone). Microscopic and histopathologic examinations were conducted in inflamed colonic tissue. The inflammatory biomarkers of colitis including interleukin 6 (IL-6), tumor necrosis factor (TNF-α), and myeloperoxidase (MPO) and the gene expression level of toll like receptor-4 (TLR-4) were assessed. Results: Pistacia atlantica oleo-gum-resin+ honey induced significant progress in macroscopic and microscopic scores. Colonic levels of MPO, IL-6, and TNF-α significantly declined in rats treated with the mixture; while significant decrease in mucosal gene expression of TLR-4 and significant improvement of colitis were observed. Pistacia atlantica oleo-gum-resin (400 mg/kg) + honey (400 mg/kg)reduced inflammation of the bowel and colonic ulcer severity shown by downregulation of inflammation cytokines, reduction of neutrophil infiltration, and suppression of TLR-4 expression. Conclusion: The combination might be a promising supplement for treatment of inflammatory disorders.

Protective Efficacy of Different Live Attenuated Infectious Bronchitis Virus Vaccine Combination against Challenge with GI11 (BR-Type) Strain

The infectious bronchitis virus (IBV) is an avian coronavirus that causes a highly contagious disease that results in substantial economic losses to the poultry industry worldwide. The prevention is mostly based on biosecurity measures and vaccination. The vaccination programs are defined by epidemiological status and cross-immunity provided against different serotypes. Programs using vaccine combinations can be designed to increase protection. We conducted an experimental study to compare two vaccine programs on protection against a virulent strain belonging to the GI-11 genotype (BR-type) isolated in Brazil using two different Mass-type vaccines combined with a BR-type live vaccine. Two groups of commercial chicks were vaccinated at day-1 using two commercially available Mass-type vaccines + BR-type vaccine. They were challenged intranasally at 28-day using 104 EID50/0.1 microliter/chick of wild-type G11 IBV (IBV/24W). Ciliostasis, macroscopic and microscopic lesion scores were evaluated at 4 and 11-days post vaccination (dpv) and 5 days post infection (dpi) and not showed differences between vaccinated groups. MLV+BR-Type group presented highest amount of BR strain vaccine and lowest amount of Mass strain vaccine in tracheal and cloacal swabs at 4 and 11 dpv. In the other hand, the Ma5+BR-type group showed fewer BR-type positive in both samples tested. After challenge, MLV+BR group showed higher titer of antibody measured by ELISA. These results suggest that the selection of a Mass-type vaccine strain can impact directly in the BR vaccine replication, when used associated, and consequently the induction of humoral immune response. Despite that, both protocols provided protection against the challenge with G11 strain.

New Class of Anti-Inflammatory Therapeutics Based on Gold (III) Complexes in Intestinal Inflammation-Proof of Concept Based on In Vitro and In Vivo Studies.

Inflammatory bowel diseases (IBD) are at the top of the worldwide rankings for gastrointestinal diseases as regards occurrence, yet efficient and side-effect-free treatments are currently unavailable. In the current study, we proposed a new concept for anti-inflammatory treatment based on gold (III) complexes. A new gold (III) complex TGS 121 was designed and screened in the in vitro studies using a mouse macrophage cell line, RAW264.7, and in vivo, in the dextran sulphate sodium (DSS)-induced mouse model of colitis. Physicochemical studies showed that TGS 121 was highly water-soluble; it was stable in water, blood, and lymph, and impervious to sunlight. In lipopolysaccharide (LPS)-stimulated RAW264.7 cells, the complex showed a potent anti-inflammatory profile, as evidenced in neutral red uptake and Griess tests. In the DSS-induced mouse model of colitis, the complex administered in two doses (1.68 μg/kg, intragastrically, and 16.8 μg/kg, intragastrically, once daily) produced a significant (* p < 0.05) anti-inflammatory effect, as shown by macroscopic score. The mechanism of action of TGS 121 was related to the enzymatic and non-enzymatic antioxidant system; moreover, TGS 121 induced changes in the tight junction complexes expression in the intestinal wall. This is the first study proving that gold (III) complexes may have therapeutic potential in the treatment of IBD.

Toll-like receptor 4-mediated necroptosis in the development of necrotizing enterocolitis

BackgroundDramatic intestinal epithelial cell death leading to barrier dysfunction is one of the mechanism of neonatal necrotizing enterocolitis (NEC), in which Toll-like receptor 4 (TLR4) plays a pivotal role. This study explored the role of necroptosis, a drastic way of cell death in NEC.MethodsThe expression of necroptotic proteins was tested in NEC intestinal tissue and compared with controls. NEC was induced in neonatal wild-type mice and a necroptosis inhibitor was given to investigate whether NEC could be relieved. The general condition, macroscopic scoring, and histological evaluations were performed. The expression of tight junction proteins, inflammatory cytokines, and necroptosis-related proteins was measured, and barrier function was examined. Then, NEC was induced in TLR4-knockout pups to confirm the role of TLR4 in necroptosis.ResultsNecroptotic proteins were significantly upregulated in both NEC patient and animal models, together with the expression of TLR4. NEC could be relieved and inflammatory infiltration was decreased by necrostatin-1s. TLR4-knockout mice showed milder tissue degradation and less necroptosis after NEC induction.ConclusionsNecroptosis is an essential pathological process of NEC. TLR4 may be one stimulator of necroptosis in NEC. Inhibiting the intestinal cell necroptosis might be a useful strategy in the treatment of NEC.ImpactNecroptosis is a key pathological process in NEC, which appears to involve TLR4.Anti-necroptosis treatment is a promising strategy that could significantly relieve the symptoms of NEC.

Beta-Chitosane as a Treatment for Ulcerative Colitis: Therapeutic Effectiveness and Possible Mechanisms of Action

Purpose: Colitis is a widespread inflammatory bowel disease with heterogeneous etiology (genetic and immunological). It is treated with drugs such as steroidal and non-steroidal anti-inflammatory that, in the long term, can cause side effects. For this reason, the exploitation of natural resources to combat this type of disease is the concern of researchers. The purpose of our study was to evaluate the anti-colitis (anti-inflammatory) effect of β-Chitosane induced in albino mice by acetic acid (5%). Methods: Mices were separated into six groups: the witness (untreated and not ulcerated), negative control group (ulcerated and untreated), the positive control ulcerated and treated with the Dexaméthasone® (1 mg/kg), and test groups ulcerated and treated with different doses of β-Chitosane (0.5 g/ kg; 0.75 g/ kg and 1 g/ kg) for the entire treatment estimated to six-days. β-Chitosane efficacy was evaluated by macroscopic and microscopic scores. Results: The clinical scores showed that β-Chitosane with a dose of 1 g/kg for the entire treatment significantly reduced the damage caused by acetic acid with a score of (3.41 ± 1.45) compared to those of the positive control which reduced less the inflammation (6.26 ± 1.23). The histological study of the colons was able to validate the effect of β-Chitosane by decreasing neutrophil infiltration and ulceration in the colon as well as by structural recovery of the mucosa. Conclusion: These results provide evidence that β-Chitosane has a protective effect against ulcerative colitis that may be due to its antioxidant, anti infectious, anti-inflammatory and healing activities.

A230 ISOMALTODEXTRIN DOSE-DEPENDENTLY REDUCES COLITIS DEVELOPMENT IN HLA-B27 RAT COLITIS MODEL WITH ASSOCIATED CHANGES TO GUT MICROBIOME COMPOSITION AND SHORT CHAIN FATTY ACID PRODUCTION

Abstract Background Inflammatory bowel disease (IBD) is characterized by altered intestinal microbiome (i.e. dysbiosis), described by reduced strict butyrate-producing anaerobes versus increased facultative anaerobes. Data from preclinical studies and clinical trials show prebiotic inulin-type fibers can prevent and reduce colitis. However, other dietary fibers (e.g. resistant starches, RS) are understudied and it is unknown if RS-induced microbial shifts are protective in experimental colitis. Aims Assess efficacy of isomaltodextrin (IMD), a novel RS, to reduce intestinal inflammation in HLA-B27 transgenic (TG) rat colitis model and identify protective mechanisms associated with gut microbial composition and function. Methods 4 week old HLA-B27 TG rats were fed standard chow supplemented with: 7.5% IMD (low dose, LD), 15% IMD (high dose, HD), negative control (15% cellulose, NC), or positive control (15% fructooligosaccharides, PC) for 12 weeks. Body weight and food intake were measured. Cecal and colonic inflammation assessed by weight/length ratio, macroscopic scoring and mucosal IL-1β secretion. Changes in microbial energy metabolism evaluated by measuring short chain fatty acid (SCFA) production in stool and cecal contents. Endpoint fecal and cecal microbiota composition differences assessed by 16S rRNA gene sequencing (Illumina MiSeq platform). Results IMD showed dose-dependent effect on cecal inflammation, measured by macroscopic tissue scoring, weight/length ratio and IL-1β secretion. HD rats had significantly lower cecum IL-1β concentration compared to NC (q=0.01), while LD showed only a trend (q=0.09). HD had significantly higher cecal amounts of Bacteroidaceae and Allobaculum spp. and lower amounts of Peptostreptococcaceae, Eubacterium and Barnesiella spp. versus the LD and NC. HD was associated with significantly higher total SCFA compared to NC (q&amp;lt;0.01) and showed a trend of higher total SCFA than LD (q=0.06). Analysis of SCFAs revealed propionate, isobutyrate and valerate ratios were significantly lower in HD than LD and NC. HD showed a trend of higher ratio of butyrate + acetate compared to NC. This suggests increased carbohydrate fermentation by acetate-producing and -converting microbial groups. Correlation analysis confirmed IL-1β concentrations were positively associated with isobutyrate (r=0.52, q&amp;lt;0.01), valerate (r=0.54, q&amp;lt;0.01), and propionate (r=0.48, q&amp;lt;0.01), suggesting their use as chronic inflammation markers in HLA-B27 models. Conclusions IMD was dose-dependently effective in reducing chronic cecal inflammation in experimental colitis. Benefits were associated with specific shifts in gut microbiome composition and SCFA production. Results from this preclinical study warrant future microbiota-altering intervention trials using IMD in clinical IBD. Funding Agencies Hayashibara Co., Ltd.

Local Administration of Low-Dose Nerve Growth Factor Antibody Reduced Pain in a Rat Osteoarthritis Model.

Systemic injection of a nerve growth factor (NGF) antibody has been proven to have a significant relevance in relieving osteoarthritis (OA) pain, while its adverse effects remain a safety concern for patients. A local low-dose injection is thought to minimize adverse effects. In this study, OA was induced in an 8-week-old male Sprague–Dawley (SD) rat joint by monoiodoacetate (MIA) injection for 2 weeks, and the effect of weekly injections of low-dose (1, 10, and 100 µg) NGF antibody or saline (control) was evaluated. Behavioral tests were performed, and at the end of week 6, all rats were sacrificed and their knee joints were collected for macroscopic and histological evaluations. Results showed that 100 µg NGF antibody injection relieved pain in OA rats, as evidenced from improved weight-bearing performance but not allodynia. In contrast, no significant differences were observed in macroscopic and histological scores between rats from different groups, demonstrating that intra-articular treatment does not worsen OA progression. These results suggest that local administration yielded a low effective NGF antibody dose that may serve as an alternative approach to systemic injection for the treatment of patients with OA.

Linagliptin mitigates experimental inflammatory bowel disease in rats by targeting inflammatory and redox signaling

AimsDipeptidyl peptidase-4 (DPP-4) has been involved in the pathogenesis of inflammatory bowel diseases (IBD), yet the underlying mechanisms remain inconclusive. The present study aimed to investigate the potential of linagliptin, a potent/selective DPP-4 inhibitor with marked anti-inflammatory actions, to attenuate trinitrobenzene sulfonic acid (TNBS)-evoked colitis in rats; an experimental model of IBD, and the implicated molecular mechanisms. This may add to the clinical utility of linagliptin for the management of patients with coexisting IBD and diabetes mellitus. Notably, no former studies have linked JAK2/STAT3, HMGB1/NF-κB, and Nrf2/HO-1 signaling in TNBS-evoked colitis. Materials and methodsWestern blotting and ELISA were used to determine the levels of target signals. Key findingsAdministration of linagliptin (1.5 mg/kg; p.o.) mitigated the colitis severity via diminishing the disease activity index, colon weight/length ratio, and macroscopic scores. Linagliptin also lowered the colonic histologic scores and leukocyte invasion. Notably, linagliptin inhibited the colonic DPP-4 activity and upregulated the expression of intestinotrophic GLP-2 without incurring hypoglycemia in animals. Linagliptin curbed inflammation through the suppression of colonic IL-6, TNF-α, and myeloperoxidase and upregulation of IL-10. It also inhibited the IL-6/JAK2/STAT3 pathway via downregulating p-JAK2/JAK2 and p-STAT3/STAT3 protein expression and HMGB1/RAGE/NF-κB cascade through lowering HMGB1, RAGE, and p-NF-κB p65/NF-κB p65 protein expression. In the context of mucosal oxidative stress, linagliptin diminished lipid peroxides and augmented GSH, GPx, and total antioxidant capacity. It also activated Nrf2/HO-1 pathway via upregulating Nrf2 and HO-1 protein expression. SignificanceLinagliptin shows a promise for the management of IBD via targeting IL-6/JAK2/STAT3, HMGB1/RAGE/NF-κB, and Nrf2/HO-1 pathways.

Valorization of Algerian Grape Pomace Seeds: Extraction of Bioactive Compounds, Prevention and Treatment of Experimental Inflammatory Bowel Diseases

The global production of agroindustry wastes generate high amounts of wastes that represent an economical and environmental issues. Hence, the objective of this study was to characterize the phenolic composition profile recovered from Vitis vinifera waste and its related in vitro antioxidant and anti-inflammatory activities. In this study, we investigated the effects of different amounts of V. vinifera grape pomace seeds polyphenols (GPEs) on an experimental ulcerative colitis mouse model. We induced colitis using acetic acid and estimated disease activity index (DAI), which included weight loss, stool consistency and bleeding. The colonic mucosal injury was assessed by clinical, macroscopic, biochemical and histopathological examinations. Microscopic damage score and changes in weight and length of colon and digestive system segment were analyzed. Macroscopic activity of acetic acid-induced colitis was observed, the colon of acetic acid-treated mice showed severe inflammation characterized by marked ulceration, edema and erosions. The severity of acetic acid-induced colitis was assessed by macroscopic score analysis. Treatment with phenolic extract (0.15 and 0.1 mg) significantly (P < 0.001) reduced the disease index and significantly improved the macroscopic score. Histological analysis shows the therapeutic effect of GPEs (0.15 and 0.1 mg), with a decrease in the intensity and distribution of lesions in the duration of treatment. In conclusion, grape pomace seeds polyphenols may serve as a natural antioxidant and anti-inflammatory treatment for ulcerative colitis.