Polydeoxyribonucleotide Exerts Therapeutic Effect by Increasing VEGF and Inhibiting Inflammatory Cytokines in Ischemic Colitis Rats.

Ischemic colitis is resulted from an inadequate blood supply to a segment or entire colon. Polydeoxyribonucleotide (PDRN), extracted from salmon sperm, has been reported to exert anti-inflammatory and anti-ischemic effects through the adenosine A2A receptor (A2AR). We investigated whether PDRN possesses therapeutic effectiveness on ischemic colitis rats. Ischemic colitis was induced by selective devascularization. The skin temperature on the ischemic colitis-induced region was determined. To assess the colonic damage score and collagen deposition, colonic tissue sections were stained with hematoxylin and eosin (H&E), and Masson trichrome staining was performed. Western blot analysis for A2AR, vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6, Bax, Bcl-2, and extracellular signal-regulated kinase 1/2 (ERK1/2) was performed. Skin temperature was increased and mucosal damage and collagen deposition were observed in the affected colonic tissues in the ischemic colitis rats. Expressions of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) and inflammatory mediator (COX-2) were upregulated in the ischemic colitis rats. Apoptosis was increased by decreasing the ratio of Bcl-2 to Bax and by suppressing the phosphorylated form of ERK1/2 expression in the ischemic colitis rats. Treatment with PDRN alleviated mucosal damage reduced the expressions of inflammatory cytokines and COX-2 and inhibited apoptosis in the ischemic colitis rats. PDRN treatment more enhanced the expressions of A2AR and VEGF in the ischemic colitis rats. PDRN showed therapeutic effectiveness on ischemic colitis by increasing VEGF expression and inhibiting inflammatory cytokines and COX-2 through enhancing A2AR expression.

18F-FDG Uptake in Ischemic Colitis During Follow-up of a Patient With Lung Cancer.

A 61-year-old man with previous history of lung adenocarcinoma underwent 2 F-FDG PET/CT investigations. The first for assessment of enlarged mediastinal lymph nodes revealed an area of increased FDG uptake in the sigmoid colon (located at the right pelvis). Colonoscopy showed a segmental area of ischemic sigmoid colitis, confirmed on histology postbiopsy. The asymptomatic man had no risk factors, and no specific treatment was administered, apart from supportive measures. One year later, there was no significantly abnormal uptake on PET/CT. Incidental ischemic colitis may be demonstrated with FDG and PET/CT needing no specific therapeutic measures for resolution.

Isquemia mesentérica masiva en paciente puerperal por shock tóxico estreptocócico / Massive mesenteric ischemia in a puerperal woman due to streptococcal toxic shock

El estreptococo B hemolítico del grupo A es una bacteria aerobia Gram +, que puede producir una gran variedad de síndromes infecciosos en el puerperio. Hasta un tercio de las infecciones se complicará con un shock tóxico estafilocócico, caracterizado por shock y disfunción multiorgánica. Cultivos, diagnóstico y tratamiento precoces son vitales para el buen pronóstico. Presentamos el caso de una paciente puerperal con un síndrome de shock tóxico estreptocócico, complicado con una isquemia mesentérica masiva, donde tratamiento antibiótico y cirugía precoz fueron claves para la evolución

B hemolytic group A streptococcus is an aerobic Gram-positive bacteria that can produce a wide variety of infectious syndromes in the puerperium. Up to one-third of infections will be complicated by streptococcal toxic shock, characterized by shock and multiorgan dysfunction. Early cultures, diagnosis and treatment are vital to good prognosis. We present the case of a puerperal patient with streptococcal toxic shock, complicated by massive mesenteric ischemia, in whom antibiotic treatment and early surgery were the key to outcome

Dysplasia-like epithelial atypia in ischemic bowel disease.

Inflammatory and reactive conditions are known to mimic dysplasia or malignancy in the gastrointestinal tract. Epithelial atypia that closely mimics low-grade dysplasia (LGD) or high-grade dysplasia (HGD) can sometimes be seen in ischemic bowel. To study this phenomenon, we evaluated surgical resections for ischemic enteritis (n = 65) and ischemic colitis (n = 99) that included sections of viable epithelium adjacent to necrosis. Viable epithelium was classified as normal, obviously reactive, LGD-like atypia or high-grade dysplasia (HGD)-like atypia. Cases with available paraffin blocks were characterized immunohistochemically with antibodies to p16, p53, and MIB-1. Fourteen dysplastic lesions in chronic ulcerative colitis served as controls. Dysplasia-like atypia was found in 13 small bowel resections (20%) and 15 colectomies (15%), most common near re-epithelializing erosions. Two colectomies had extensive dysplasia-like atypia, whereas the other 26 demonstrated focal or several foci of atypia. Nine cases contained HGD-like atypia, 15 contained LGD-like atypia, and 4 showed both HGD- and LGD-like atypia. Features indicating subacute-to-chronic ischemia were more frequent in LGD-like atypia (13/15, 87%) than HGD-like atypia (2/9, 22%; P = .003). Dysplasia-like atypia showed overexpression of p16 (73%), p53 (50%), and MIB-1 (92%), but these markers did not reliably distinguish dysplasia-like atypia from true dysplasia in chronic ulcerative colitis (P = .45 for p16, P = .51 for p53, P = .08 for MIB-1). These results underscore the frequency of dysplasia-like atypia in ischemic bowel, which can occasionally be an extensive and worrisome finding. Distinction from true dysplasia requires recognizing the context of the epithelial atypia because cell cycle markers were not helpful in classifying individual cases.

Ischaemia-induced mucus barrier loss and bacterial penetration are rapidly counteracted by increased goblet cell secretory activity in human and rat colon.

OBJECTIVE: Colonic ischaemia is frequently observed in clinical practice. This study provides a novel insight into the pathophysiology of colon ischaemia/reperfusion (IR) using a newly developed human and rat experimental model. DESIGN: In 10 patients a small part of colon that had to be removed for surgical reasons was isolated and exposed to 60 min of ischaemia (60I) with/without different periods of reperfusion (30R and 60R). Tissue not exposed to IR served as control. In rats, colon was exposed to 60I, 60I/30R, 60I/120R or 60I/240R (n=7 per group). The tissue was snap-frozen or fixed in glutaraldehyde, formalin or methacarn fixative. Mucins were stained with Periodic Acid Schiff/Alcian Blue (PAS/AB) and MUC2/Dolichos biflorus agglutinin (DBA). Bacteria were studied using electron microscopy (EM) and fluorescent in situ hybridisation (FISH). Neutrophils were studied using myeloperoxidase staining. qPCR was performed for MUC2, interleukin (IL)-6, IL-1ß and tumour necrosis factor α. RESULTS: In rats, PAS/AB and MUC2/DBA staining revealed mucus layer detachment at ischaemia which was accompanied by bacterial penetration (in EM and FISH). Human and rat studies showed that, simultaneously, goblet cell secretory activity increased. This was associated with expulsion of bacteria from the crypts and restoration of the mucus layer at 240 min of reperfusion. Inflammation was limited to minor influx of neutrophils and increased expression of proinflammatory cytokines during reperfusion. CONCLUSIONS: Colonic ischaemia leads to disruption of the mucus layer facilitating bacterial penetration. This is rapidly counteracted by increased secretory activity of goblet cells, leading to expulsion of bacteria from the crypts as well as restoration of the mucus barrier.

Expression of intestinal MUC17 membrane-bound mucin in inflammatory and neoplastic diseases of the colon.

AIM: To determine the cellular location and expression of MUC17 mucin in specimens of normal, inflamed and neoplastic colon. METHODS: Immunohistochemical analysis of human surgical resection specimens (n=106) was performed with a specific antibody to the MUC17 apomucin protein. A semi-quantitative scoring system was used to measure MUC17 expression. In various colon cancer cell lines, the MUC17 expression was examined by immunoblot analysis and normal RT-PCR. RESULTS: MUC17 was highly expressed on the surface epithelium and crypts of colonic mucosa. In contrast, the expression of MUC17 was significantly decreased in colonic mucosa of chronic ulcerative colitis (p<0.0001) and ischaemic colitis (p=0.003). Similarly, MUC17 expression was decreased in hyperplastic polyps (p=0.0003), tubular and tubulovillous adenomas (p<0.0001) and colon cancers (p<0.0001). Furthermore, of eight different colon cancer cell lines, MUC17 expression was only detected in LS174T and LS180 cells. CONCLUSION: Results indicate that the potential protective effects of this membrane-bound mucin are primarily or secondarily diminished in inflammatory and neoplastic conditions. Further research is needed to determine the specific role of MUC17 in the pathogenesis of these conditions.

The effect of sildenafil on an animal model for ischemic colitis.

INTRODUCTION: Sildenafil both enhances vasodilatation by relaxing the smooth muscle in the vessels and inhibits platelet aggregation. We have therefore examined the potential benefits of sildenafil on an animal model for ischemic colitis (IC). METHODS: Twenty-eight female Wistar albino rats weighing 250-300 g were randomized into three experimental groups as follows: in Group 1, animals were sham operated (n = 8) and received tap water; in Groups 2 and 3, the rats underwent a standardized surgical procedure to induce IC (n = 10 in each group). Group 2 animals served as the controls, receiving only tap water, while Group 3 animals received 10 mg/kg sildenafil per day as a single dose for a 3-day period. All animals were sacrificed 72 h after devascularization. To determine the severity of the ischemia, we scored the macroscopically visible damage, measured the ischemic area and scored the histopathology. Tissue malondialdehyde levels were also evaluated. RESULTS: The mean area of ischemic changes were 116.80 +/- 189.93 and 0.55 +/- 1.01 mm2 in Group 2 and 3 animals, respectively (p = 0.0001), while the macroscopically mean visible damage score decreased to 0.66 +/- 0.70 (p = 0.0001) for Group 3 animals. The Chiu scores were 0.00, 3.80 +/- 0.91 and 2.66 +/- 1.00 in Group 1, 2 and 3 animals, respectively. There was a statistically significant difference between Group 2 and 3 animals (p = 0.017). CONCLUSIONS: Our findings support the view that sildenafil leads to a improvement in IC due to its well-known effects on the vascular smooth muscle and on the microcirculatory hemodynamics.

Human colonic myocytes are involved in postischemic inflammation through ADAM17-dependent TNFalpha production.

The aim of this study was to identify human colonic resident cells able to initiate an inflammatory response in postischemic injury. Postischemic colonic injury, a condition relevant to various clinical settings, involves an inflammatory cascade in intestinal tissues through the recruitment of circulating inflammatory cells. However, there is no information on the nature of resident cells of the different intestinal layers able to initiate a postischemic inflammatory response. It is however an important issue in the context of a pharmacological approach of the early phase of intestinal ischemia. We reasoned that maintaining the different colonic layers as explant cultures in an oxygenated medium immediately after colonic resection, that is, after an ischemic period, would allow one to identify the resident cells able to initiate an inflammatory cascade, without interference of recruited inflammatory/immune cells. To this end, we designed an explant culture system that operationally defines three compartments in surgical specimens of the human colon, based on the microdissected layers, that is, mucosa, submucosa (containing muscularis mucosae) and muscularis propria. To validate the results obtained in explant cultures in the clinical setting of ischemic colitis, eight cases of sigmoid volvulus were examined. Only the myocytes-containing explants produced tumor necrosis factor alpha (TNFalpha), via an ADAM17 (a disintegrin and metalloproteinase-17)-dependent pathway, as shown by the abrogation of TNFalpha production by the inhibitor Tapi-2. Immunofluorescence studies identified nonvascular and vascular myocytes as resident cells coexpressing TNFalpha and ADAM17, both in our postischemic explant system and in surgical specimens from ischemic colitis patients. Finally, time-course experiments on explanted tissues showed that TNFalpha production by myocytes was an early event triggered by a postischemic oxidative stress involving nuclear factor kappa B (NF-kappaB). In conclusion, this study identifies human intestinal myocytes as resident cells able to initiate an inflammatory reaction through TNFalpha production in postischemic conditions, and delineates two points of control in TNFalpha production, NF-kappaB and ADAM17, which can be targeted by pharmacological manipulation.

Hypoxia-inducible factor 1 alpha and vascular endothelial growth factor overexpression in ischemic colitis.

AIM: To examine the etiology and pathophysiology in human ischemic colitis from the viewpoint of ischemic factors such as hypoxia-inducible factor 1 alpha (HIF-1 alpha and vascular endothelial growth factor (VEGF). METHODS: Thirteen patients with ischemic colitis and 21 normal controls underwent colonoscopy. The follow-up colonoscopy was performed in 8 patients at 7 to 10 d after the occurrence of ischemic colitis. Biopsy samples were subjected to real-time RT-PCR and immunohistochemistry to detect the expression of HIF-1 alpha and VEGF. RESULTS: HIF-1 alpha and VEGF expression were found in the normal colon tissues by RT-PCR and immunohistochemistry. HIF-1 alpha and VEGF were overexpressed in the lesions of ischemic colitis. Overexpressed HIF-1 alpha and VEGF RNA quickly decreased to the normal level in the scar regions at 7 to 10 d after the occurrence of ischemic colitis. CONCLUSION: Constant expression of HIF-1 alpha and VEGF in normal human colon tissue suggested that HIF-1 alpha and VEGF play an important role in maintaining tissue integrity. We confirmed the ischemic crisis in ischemic colitis at the molecular level, demonstrating overexpression of HIF-1 alpha and VEGF in ischemic lesions. These ischemic factors may play an important role in the pathophysiology of ischemic colitis.

Membrane permeability and antipyrine absorption in a rat model of ischemic colitis.

The aim of this study was to determine whether the duration of ischemia affects antipyrine absorption in the large intestine. This was carried out in a rat model of ischemic colitis in which ischemia and associated inflammation was induced by marginal vessel ligation. Blood flow was disrupted by positioning an o-ring around the distal rectum and ligating the marginal vessel at two locations in the hind-gut ligament artery region. Ligation was performed for 1, 2, 3, and 5h. We assessed large intestine damage by measuring key indicators of inflammation, myeloperoxidase (MPO) activity and thiobarbituric acid reactant substrates (TBARS) in the mucosa and by histological staining with hematoxylin-eosin stain. Antipyrine membrane permeability was assessed in Ussing-type diffusion chambers, and related pharmacokinetics were calculated from antipyrine plasma concentration measurements following colon administration of the drug. Vessel ligation caused some sloughing of epithelial cells and elevated the MPO and TBARS levels. Prolonged ligation failed to affect the apparent permeability coefficient (P(app)) of antipyrine. Prolonged ligation, however, gradually increased plasma antipyrine concentrations to near control levels. This increase was paralleled by increases in the absorption rate constant AUC and antipyrine bioavailability. Taken together, these results suggest that the absorption kinetics of antipyrine may depend on blood flow changes in the large intestine that occur with inflammation.

Endothelin-1 induces mucosal mast cell degranulation and tissue injury via ETA receptors.

The effects of endothelin-1 (ET-1) on mucosal mast cells are of special interest, since they may be an important component of the tissue response that occurs during ischaemic preconditioning or ischaemia/re-oxygenation injuries. Increasing doses of ET-1 were administered intravenously to anaesthetized rats. In a second series of experiments, animals were pretreated with the ET(A) receptor antagonists BQ-610 or ETR-P1/fl peptide, or with the ET(B) receptor antagonist IRL-1038. Intestinal perfusion changes were recorded, and the proportion of degranulated mast cells and the degree of mucosal damage were determined in ileal biopsies. ET-1 induced dose-dependent alterations in the haemodynamic and morphological parameters, and caused significant mast cell degranulation. These changes were inhibited significantly by pretreatment with the ET(A) receptor antagonists, but not with the ET(B) receptor antagonist. We conclude that a cross-talk exists between endothelial cell-derived humoral mediators and the intestinal mast cell system.

Oxidative stress and antioxidants in intestinal disease.

Oxidative injury caused by free radicals is an important cause of tissue injury now recognized to occur in inflammation, ischemia and by the action of xenobiotics. It is also recognized to induce gene mutation and promote carcinogenesis. In this review the general concept of nett free radical injury counterbalanced by antioxidants is discussed as oxidative stress. The role of oxidative stress in intestinal ischemia, radiation enteritis, inflammatory bowel disease and the promotion of gastric and colorectal cancer is discussed. The data for the role of oxidative stress in the pathogenesis of ischemic, inflammatory and radiation induced disease are strong, but interventional studies with antioxidants have shown only weak beneficial effects in the above diseases. Therefore the role of antioxidants in the therapy of gastrointestinal diseases remains controversial and should be the subject of controlled trials.

Human intestinal fatty acid binding protein: report of an assay with studies in normal volunteers and intestinal ischemia.

BACKGROUND: Human intestinal fatty acid binding protein (hIFABP) is a cytoplasmic protein of mature small intestinal epithelium. Work with the rat demonstrated that serum levels of IFABP correlated with early phases of intestinal mucosal injury. The aim of this study was to develop an assay for hIFABP and assess its usefulness as a marker for intestinal mucosal injury in human beings. METHODS: Recombinant hIFABP (r-hIFABP) was used to produce rabbit anti-hIFABP. Specificity and avidity of binding were tested with immunoprecipitation and Scatchard analysis. r-hIFABP was labeled with 125I, and a competitive assay was developed. Urine and serum from normal volunteers and from patients with necrotizing enterocolitis (NEC), acute thromboembolic related intestinal ischemia, and systemic inflammatory response syndrome were tested for hIFABP. RESULTS: Molecular weight was 10(-12) kd, limit of detection was 1.87 ng/ml, and no cross-reactivity occurred when tested against rat IFABP or human heart FABP. Mean levels of hIFABP (ng/ml) were controls (serum less than 1.87, urine less than 1.87), NEC (serum 14.7 ng/ml), intestinal ischemia (serum 50 ng/ml, urine 52.3 ng/ml), systemic inflammatory response syndrome (serum 5.3 ng/ml, urine 13.2 ng/ml). CONCLUSIONS: This assay is quantitative for hIFABP in serum and urine. Results from both normal persons and those with various causes of intestinal ischemia parallel our previous findings in the rat. Preliminary findings suggest that hIFABP may serve as a diagnostic marker for early intestinal mucosal compromise and, in addition, that it should prove useful as a tool in developing rationale therapeutic regimens to treat these complex clinical problems.

Methods for detecting local intestinal ischemic anaerobic metabolic acidosis by PCO2.

Gut ischemia is often assessed by computing an imaginary tissue interstitial Ph from arterial plasma HCO3- and the PCO2 in a saline-filled balloon tonometer after equilibration with tissue PCO2 and (PtiCO2). PtiCO2 may alternatively be assumed equal to venous PCO2 (PVCO2) in that region of gut. The idea is that as blood flow decreases, gut PtiCO2 and PVCO2 will increase to the maximum aerobic value, i.e., maximum respiratory PVCO2 (PVCO2rmax). Above a "critical" anaerobic threshold, lactate (La-) generation, by titration of tissue HCO3-, should raise PtiCO2 above PVCO2rmax. During progressive selective whole intestinal flow reduction in six pentobarbital-anesthetized pigs, we used PCO2 electrodes to test the hypotheses that critical PtiCO2 is achieved earlier in mucosa than in serosa and that PVCO2rmax, computed using an in vitro model, predicts critical PtiCO2. We defined critical PtiCO2 as the inflection of PtiCO2-PVCo2 vs. O2 delivery (QO2) plots. Critical QO2 for O2 uptake was 12.55 +/- 2 ml.kg-1.min-1. Critical PtiCO2 for mucosa and serosa was achieved at similar whole intestine QO2 (13.90 +/- 5 and 13.36 +/- 5 ml.kg-1.min-1, P = NS). Critical PtiCO2 (129 +/- 24 and 96 +/- 21 Torr) exceeded PVCO2rmax (62 +/- 3 Torr). During ischemia, La- excretion into portal venous blood was matched by K+ excretion, causing PVCO2 to increase only slightly, despite PtiCO2 rising to 380 +/- 46 (mucosa) and 280 +/- 38 (serosa) Torr. These results suggest that mucosa and serosa become dysoxic simultaneously, that ischemic dysoxic gut is essentially perfused, and that in vitro predicted PVCO2rmax underestimates critical PtiCO2.

Intestinal pseudo-obstruction and ischemia secondary to both beta 2-microglobulin and serum A amyloid deposition.

We present the case of a 61-year-old man on long-term hemodialysis in whom early colonic ischemia and pseudo- obstruction were caused by amyloid deposition of both the beta 2-microglobulin and AA type. The light microscopic diagnosis of amyloidosis was confirmed by Congo red birefringence, immunohistochemistry, and electron microscopy. The two types of amyloid had separate distributions within the colon. While both types affected submucosal and serosal blood vessels, the beta 2- microglobulin amyloid also formed large masses that displaced smooth muscle cells and was associated with Class II major histocompatibility-positive multinucleated giant cells. The pathogenesis of this uncommon occurrence of two amyloids and the role of chronic inflammation in amyloidogenesis are discussed.

Beneficial effects of L-arginine on intestinal epithelial restitution after ischemic damage in rats.

The polyamines are involved in repair processes after intestinal ischemia. Arginine and ornithine, both precursors of polyamines were therefore expected to exert beneficial effects on mucosal barrier dysfunction. Arginine may also generate NO and there is support for the view that NO may be beneficial after an ischemic insult. Male Wistar rats were given, by gavage, isonitrogenous solutions of L-arginine (0.5 g/kg) or L-ornithine (0.7 g/kg) 17 and 2 h before ischemia. Controls received an isonitrogenous solution of casein hydrolysate (1 g/kg). Transient intestinal ischemia was produced in anesthetized rats by occluding the superior mesenteric artery for 90 min. Intestinal morphology, hydrolase activities, polyamine and cGMP contents, and cell proliferation rates were determined 4 h after reperfusion. Administration of arginine or ornithine did not prevent ischemic damage but accelerated morphological repair, enhanced cell proliferation, and polyamine content was observed. Arginine was significantly more effective than ornithine. Formation of cGMP was enhanced after arginine administration. NG-nitroarginine methylester, an inhibitor of NO synthase, prevented the arginine effects on mucosal repair. We conclude that arginine-derived NO is an important mediator in the restitution of intestinal mucosa by minimizing cell injury during reperfusion.

[Experimental study on the etiology of ischemic colitis].

A reliable experimental model for ischemic colitis in rat could be obtained by ligating the marginal vessels of the colon 3 days after attaching the ring to induce partial colon obstruction. This model has the advantage of enabling to observe the chronic progress and the organ of the sideration to clarify the cause of ischemic colitis. The decrease of blood flow within the colonic mucosa continued up to 7 days after ligation in rat with partial obstruction, while no apparent abnormal changes were observed, and the previous level for several hours in the rats with out attaching the ring. This fact proved that the RT group is in a state easily developing sideration due to the sustained partial colon obstruction. The reactant reacting with thiobarbituric acid (TBA) was found to increase, in particular, 6 or more hours after ligation of vessels. A superoxide scavenger, liposomal-encapsulated superoxide dismutase (1-SOD) suppressed development of the ischemic lesion. The change in free radicals is therefore, considered to be involved in the occurrence of ischemic colitis.