Granulocyte Marker Protein Research Articles

Does a single exposure to social defeat render rats more vulnerable to chemically induced colitis than brief inescapable foot-shocks?

All mammals are to different degrees exposed to stressors being physical or social, which may affect health and well-being. Stressful and traumatic situations have direct effects on immune responses that may alter susceptibility to developing somatic illnesses. In animal research, different types of stressors have been investigated in studying the effect on bowel disorders, some stressors being more or less of environmental origin. We aimed, therefore, to explore whether a more natural stressor would differ from a stressor of more unnatural characteristics on dextran sulphate sodium (DSS) induced colitis in adult rats. Specifically, whether social stress within a single social defeat (SD) paradigm would be a more potent stressor than brief inescapable foot-shocks (IFS) in causing elevated faecal granulocyte marker protein (GMP), and crypt- and inflammation scores in colonic tissue. Three groups of male Wistar rats were used; socially defeated rats; inescapable foot-shock rats; and comparison rats. Main findings showed no difference between the groups on GMP levels. However, there was a significant difference on inflammation and crypt scores for the distal part of colon, detected through histology, where socially defeated rats were more susceptible. A single SD seems to be more adverse than inescapable foot-shock on DSS induced colitis, but further studies are recommended to validate a broader range of different outcomes comparing two such different rodent stress models.

Neonatal maternal separation in male rats increases intestinal permeability and affects behavior after chronic social stress

Prolonged maternal separation in rats has several effects on health and behavior. Here we investigated how maternal separation might interact with social stress in adulthood on behavior and gastrointenstinal permeability. The effects of either daily 180min long term pup-dam separation (LMS) during the stress hyporesponsive period or daily 10min brief maternal separation (BMS) on behavior, corticosterone and intestinal permeability were investigated, compared to a non-handling (NH) condition in male offspring. The animals from each separation condition were then randomly assigned to adult stress and control conditions, where the stress condition was exposure to 14days of social instability (CSI). Sucrose preference, elevated plus maze behavior and corticosterone were measured. Colitis was experimentally induced by dextran sulfate sodium for 7days, followed by measurement of intestinal permeability using the 51CrEDTA method. Granulocyte marker protein was measured in feces and colons were examined histologically for inflammation. Prior to the social stress, the LMS offspring showed elevated corticosterone levels, lower elevated plus maze activity and less fluid consumption. After social stress, corticosterone levels were suppressed in LMS animals and again they showed less fluid consumption. LMS animals had significantly higher intestinal permeability, but only when also exposed to the social stress in adulthood. The current results support a two-hit model, whereby early life events interact with adult life events in altering animals' vulnerability.

The effects of AZD3582 [4-(nitroxy)butyl-(2S)-2-(6-methoxy-2-naphthyl) propanoate], and naproxen on key pathogenic steps in NSAID-enteropathy in the rat

The pathogenesis of NSAID-induced enteropathy may involve dual inhibition of the cyclooxygenase (1 and 2) and a topical effect with sequential increased intestinal permeability, development of inflammation and ulcers. It has been suggested that nitric-oxide donating drugs cause significantly less gastrointestinal injury by counteracting for NSAID-induced reductions in blood flow. To compare the effects of AZD3582 [4-(nitroxy)butyl-(2S)-2-(6-methoxy-2-naphthyl) propanoate], and naproxen on key pathogenic steps in NSAID-enteropathy in the rat. Single doses of AZD3582, naproxen (dose range 10-300 micromol/kg) or vehicle were given to male Sprague Dawley rats. Intestinal permeability (51CrEDTA) and intestinal inflammation (granulocyte marker protein) was quantitated and ulcer counts made. Intestinal permeability (all doses) and inflammation (highest dose of the drugs) increased significantly from control levels following naproxen and AZD3582 and there was no significant difference between the drugs. Median ulcer counts were, however, significantly (p < 0.01) lower with AZD3582 (4 +/- 2) than with naproxen (17 +/- 4). Naproxen and AZD3582 are equally associated with increased small intestinal permeability and inflammation, which is the consequence of their topical effect. The reduced small bowel ulcer counts with AZD3582 accords with the suggestion that vascular factors are the main driving force for NSAID-induced ulcer formation.

No Protection against DSS-induced Colitis by Short-term Pretreatment with Seal or Fish Oils in Rats

Background Omega-3 (n-3) polyunsaturated fatty acids (PUFAs) have modulating effects in several chronic inflammatory conditions. The aim of the present study was to test whether prior short-term dietary supplementation with n-3 (fish or seal oil) or n-6 (soy oil) PUFA rich oils would protect the development of dextran sulfate sodium (DSS)-induced colitis in rats. Methods Forty-eight male Wistar rats were divided into 6 groups: no intervention, sham, DSS, seal oil + DSS, fish oil + DSS and soy oil + DSS. Following 7 days of acclimatisation, 1 mL oil (seal, fish or soy) or distilled water (sham) was administered by gavage day 8 to 14. Colitis was induced by 5% DSS in drinking water from day 15 to 21. Rats were sacrificed on day 23. Histological colitis (crypt and inflammation) scores, faecal granulocyte marker protein (GMP) and quantitative fatty acid composition in red blood cells were measured. Results Pretreatment with fish or seal oils did not significantly influence DSS induced inflammation. In fact, all the oils tended to exacerbate the inflammation. Soy oil increased the mean crypt score ( P &lt; 0.04), but not the inflammation score or GMP. The ratio of n-6 to n-3 fatty acids (FAs) was 11 to 1 and 10 to 1 in standard diet and in red blood cells of control rats, respectively. Following administration of DSS, the ratio fell in all treatment groups ( P &lt; 0.001). The lowest ratios were seen in the groups receiving DSS + fish or seal oils (around 6 to 1). Conclusion Short-term pretreatment with fish or seal oils did not protect against subsequent induction of colitis by DSS in this rat model. Whether the high ratio of n-6 to n-3 FAs in the standard diet concealed effects of n-3 FA supplementation should be further investigated.

Low-Dose Oral Ferrous Fumarate Aggravated Intestinal Inflammation in Rats With Dss-Induced Colitis

Oral ferrous iron therapy may reinforce intestinal inflammation. One possible mechanism is by catalyzing the production of reactive oxygen species. We studied the effects of low-dose oral ferrous fumarate on intestinal inflammation and plasma redox status in dextran sulfate sodium (DSS)-induced colitis in rats. Forty male Wistar rats were divided into 5 groups: no intervention, sham gavage (distilled water), ferrous fumarate, DSS, and ferrous fumarate + DSS. Ferrous fumarate was dissolved in distilled water (0.60 mg Fe/kg per day) and administered by gavage on days 1 to 14. All rats were fed a standard diet. Colitis was induced by 5% DSS in drinking water on days 8 to 14. Rats were killed on day 16. Histologic colitis scores, fecal granulocyte marker protein, plasma malondialdehyde, plasma antioxidant vitamins, and plasma aminothiols were measured. DSS significantly increased histologic colitis scores (P < 0.001) and fecal granulocyte marker protein (P < 0.01). Ferrous fumarate further increased histologic colitis scores (P < 0.01) in DSS-induced colitis. DSS + ferrous fumarate decreased plasma vitamin A compared with controls (P < 0.01). Otherwise, no changes were seen in plasma malondialdehyde, plasma antioxidant vitamins, or plasma aminothiols. Low-dose oral ferrous iron enhanced intestinal inflammation in DSS-induced colitis in rats.

Polyethylene glycol reduces inflammation and aberrant crypt foci in carcinogen-initiated rats

Polyethylene glycol 8000 inhibits the formation of tumors and of aberrant crypt foci (ACF) in carcinogen-initiated rats. We asked: is the inhibition associated with a reduction of colonic inflammation and an increase in colonic cell permeability? Twenty-eight, male F 344 rats were divided into two groups, 10 control animals and 18 animals initiated with azoxymethane. Nine of the rats in the carcinogen-initiated group were given a diet with 5% PEG 8000 in an AIN-93 based, high fat diet. The other nine, and the control group received the diet without the addition of PEG. Nine weeks later, the rats receiving the diet containing PEG had a 43% reduction in ACF ( P<0.001) compared with the carcinogen-initiated rats on the control diet, a result confirming earlier observations that PEG inhibits colon carcinogenesis. The animals receiving the diet containing PEG also had a 10-fold reduction in fecal granulocyte marker protein (GMP) ( P<0.001) compared with both the carcinogen-treated and the control animals. PEG reduced inflammation below the levels of carcinogen-treated and of untreated animals. Fecal water from the rats receiving PEG did not reduce transepithelial resistance of, or manitol flux through, human Caco-cells grown as monolayers in vitro. PEG may reduce colon carcinogenesis through a mechanism involving colonic inflammation.

Proactive Sensitizing Effects of Acute Stress on Acoustic Startle Responses and Experimentally Induced Colitis in Rats: Relationship to Corticosterone

In humans, some individuals develop a syndrome after trauma (post-traumatic stress disorder, PTSD) characterized by increased startle responses and lower than normal cortisol secretion. We explored a rat model using the acoustic startle response (ASR) as a behavioral indicator of the effect of a short series of shocks. Because gastrointestinal disorders have been associated with prior stress, we also studied the rats' vulnerability to a chemically-induced colitis. After initial blood sampling, 12 rats were exposed to ten 1 mA 5 s foot-shocks while 12 rats served as controls. Nineteen days later the rats were tested for ASR. Thirty trials (10 trials at each of 95, 105, and 115 dB, pseudo-randomized) were given. After exposure for 6 days to dextran sulphate sodium in their drinking water, the rats were killed and the colons examined for erosions. Shocked rats showed greater startle responses and more colonic erosion than unshocked rats, but the shock effects were significant only for animals with low initial plasma corticosterone levels. Shocked rats also showed higher levels of granulocyte marker protein (GMP) in their faeces. These results suggest that low corticosterone secretion may represent a marker for vulnerability to long term effects of shocks as indicated by increased startle responses and colonic pathology.

COX-1 and 2, intestinal integrity, and pathogenesis of nonsteroidal anti-inflammatory drug enteropathy in mice

Background & Aims: The pathogenesis of nonsteroidal anti-inflammatory drug–induced enteropathy is controversial, but it is thought that cyclooxygenase-1 (COX-1) inhibition is of pivotal importance. We compared small intestinal function and morphology in untreated wild-type, COX-1– and COX-2–deficient mice and the effect of indomethacin, selective COX-1 (SC-560), and COX-2 (celecoxib) inhibition. Methods: Intestinal permeability (51CrEDTA), inflammation (fecal granulocyte marker protein), prostaglandin E2 (PGE2) levels, and macroscopic and microscopic appearances were assessed at baseline and after the drugs. Results: COX-1−/− animals were normal except for a 97% decrease in intestinal PGE2 levels. COX-1+/+ and COX-1−/− animals reacted in a similar way to indomethacin. However, celecoxib, having caused no damage in COX-1+/+ animals, caused small bowel ulcers in COX-1−/− animals. Selective inhibition of COX-1 decreased intestinal PGE2 levels in COX-2+/+ and COX-2−/− animals by 95%–97%, but caused only small bowel ulcers in the latter group. Dual inhibition of COX-1 and COX-2 in wild-type animals resulted in similar small bowel damage. Between 40% and 50% of untreated COX-2−/− animals had increased intestinal permeability and inflammation. Some had ileal ulcers that were distinctively different from indomethacin-induced ulcers. Furthermore, long-term celecoxib administration in wild-type animals was associated with similar damage as in the COX-2−/− mice. Conclusions: COX-1 deficiency or inhibition and short-term COX-2 inhibition are compatible with normal small intestinal integrity. Dual inhibition of the COX enzymes leads to damage similar to that seen with indomethacin. Long-term COX-2 deficiency or inhibition is associated with significant intestinal pathology despite normal intestinal PGE2 levels, suggesting a role for COX-2 in the maintenance of small intestinal integrity in the mouse.GASTROENTEROLOGY 2002;122:1913-1923

Comparison of Faecal and Intestinal Concentrations of Granulocyte Marker Protein and Localization of Gastrointestinal Tumours in Rats

Background: Increased faecal concentrations of the granulocyte marker protein (GMP) have been found in rats with azoxymethane (AOM) induced carcinoma of the colon, but the origin of this GMP is unknown. The aims were to investigate the concentrations of GMP in different parts of the gastrointestinal (GI) tract in rats with or without AOM-induced carcinoma and to correlate the GMP concentrations to localization of the carcinomas. Methods: Nineteen rats were given intramuscular injections of AOM, 15 mg/kg, once weekly for 6 weeks and were killed after 22 weeks. Five rats that were not given AOM injections served as controls. Results: All rats given AOM developed tumours; 18 developed a total of 33 adenocarcinomas in the GI tract and one developed an adenoma in the colon. Nine animals had carcinoma in the small bowel, seven of which also had carcinoma of the colon, and nine animals had carcinomas in the large bowel only. No other tumours were found. All except one of the animals that had carcinoma of the colon had elevated faecal GMP concentrations, and from week 11 there was a significant difference in the GMP values between the control group and the group that developed colon carcinoma. In all rats that developed carcinoma in the small bowel, the tumour was localized in the proximal part. In the rats that had been given AOM, the luminal GMP concentrations were significantly higher in the proximal part of the small bowel than in the distal part, but there were no significant differences in the GMP concentrations between animals with and without carcinoma in the small bowel. Sixteen rats developed a total of 24 carcinomas in the colon, and one rat developed an adenoma. Luminal GMP concentration in the distal part of the colon was elevated in all animals with carcinomas in the colon, and the GMP concentrations were significantly higher in the distal part than in the proximal part. Rats with one carcinoma in the colon had significantly lower GMP values in the distal part, compared to rats that had two carcinomas in the colon. Conclusions: The animal model described is suitable for further studies on many aspects of tumour development in the colon. Furthermore, it is likely that increased faecal GMP concentration in rats with colon carcinoma is a result of an inflammatory process in or around tumours.

Granulocyte Marker Protein Is Increased in Stools from Rats with Azoxymethane-Induced Colon Cancer

Background: We wanted to investigate the relationship between the fecal levels of granulocyte marker protein (GMP) and the presence of aberrant crypt foci (ACF) and colorectal cancer in rats given injections of azoxymethane (AOM) and fed either of two different diets, a basal diet plus 20% corn oil or 20% beef suet, respectively. Methods: The rats received intraperitoneal injections of AOM, 15 mg/kg, once weekly for 6 weeks and were killed after 22 weeks. Results: In the group fed beef suet 17 of 19 rats developed colon cancer, whereas in the group fed corn oil 4 of 14 rats developed cancer. None of the 20 control rats fed either the beef suet or corn oil diets developed cancer or aberrant crypts, and GMP remained unchanged. Surprisingly, the numbers of ACF were significantly higher (467 versus 295; P = 0.004) in the group fed corn oil than in the group fed beef suet. On the other hand, the size (crypts/focus) of the ACF was significantly higher (P = 0.03) in the beef suet group. Furthermore, fecal GMP was significantly higher in the beef suet group than in the corn oil group after 18 weeks, and this difference increased further toward the end of the study. GMP was greatly increased in all rats with colorectal cancer. Conclusions: Fecal GMP may have provided us with a valuable tool for further studies of the induction and progression of neoplasia in rats and, possibly, in mice, since the anti-GMP antibody cross-reacts with feces extracts from mice.

Correlation between Faecal Excretion of Indium-111-Labelled Granulocytes and Calprotectin, a Granulocyte Marker Protein, in Patients with Inflammatory Bowel Disease

Background: Several studies have suggested that clinical indices of disease activity in inflammatory bowel disease (IBD) do not adequately reflect the degree of inflammation in most such patients. Faecal excretion of indium-111-labelled neutrophilic granulocytes has been suggested as the gold standard of disease activity, but its complexity and high cost and the exposure of patients to ionizing irradiation have limited the use of this technique. The aim of this study was to investigate the correlation between the faecal excretion of the granulocyte marker protein calprotectin and that of 111In-labelled granulocytes. Methods: Calprotectin in stool extracts from 19 patients with Crohn's disease (CD), 10 with ulcerative colitis (UC), and 9 presumably healthy controls was assessed with a simple enzyme-linked immunosorbent assay. Simultaneously, the faecal excretion of autologous 111In-labelled granulocytes was measured. Results: There was a strong correlation between the average daily excretion of calprotectin and that of the total 3-day excretion of 111In-labelled granulocytes (r = 0.87, P < 0.0001). Furthermore, the concentration of calprotectin, assessed in a small stool sample on day 1, also correlated well with the excretion 111In-labelled granulocytes (r = 0.80, P < 0.0001). Conclusion: The results suggest that faecal calprotectin reflects the granulocyte migration through the gut wall in patients with IBD and hence might serve as a simple, inexpensive alternative to the indium-111 technique.

Association of granulocyte transmigration with structural and cellular parameters of injury in experimental radiation enteropathy.

Inflammatory cells are involved in the pathogenesis of tissue injury through release of cytokines and biologically active compounds. This study used a novel, noninvasive method to assess the association between granulocyte transmigration and structural and molecular changes in radiation enteropathy. A 4 cm loop of rat small intestine was exposed to 0, 2.8, 12, or 23 Gy localized irradiation. Feces was collected in metabolic cages before and 3, 7, 14, 28, and 42 days after irradiation. Granulocyte marker protein (GMP) was measured in buffer extracts of feces by enzyme-linked immunosorbent assay (ELISA). Irradiated and shielded intestine were procured at 2 and 26 weeks and assessed for histopathologic injury [radiation injury score (RIS)], ED-2 positive macrophages, and interleukin-1 alpha (IL-1 alpha) positive cells. Irradiated intestine exhibited characteristic histopathologic alterations and increased numbers of macrophages and IL-1 alpha positive cells. There was a highly significant dose-dependent increase in post-radiation GMP (P < 0.0001). Maximal GMP excretion occurred 3-7 days after irradiation. Six weeks after irradiation, GMP excretion had returned to normal in the 2.8 and 12 Gy groups, but was still 3.5 times higher in the 23 Gy group than in controls. The associations between early GMP excretion and RIS and fibrosis at 26 weeks were highly significant (P < 0.001 and P < 0.0001, respectively). Post-radiation granulocyte transmigration is dose-dependent and correlates with structural and molecular changes, as well as with subsequent chronic injury. The GMP assay is a sensitive, non-invasive indicator of acute intestinal radiation injury and a promising biological predictor of chronic toxicity. Our data underscore the importance of consequential mechanisms in radiation enteropathy.

Association of granulocyte transmigration with structural and cellular parameters of injury in experimental radiation enteropathy

Inflammatory cells are involved in the pathogenesis of tissue injury through release of cytokines and biologically active compounds. This study used a novel, non-invasive method to assess the association between granulocyte transmigration and structural and molecular changes in radiation enteropathy. A 4 cm loop of rat small intestine was exposed to 0, 2.8, 12, or 23 Gy localized irradiation. Feces was collected in metabolic cages before and 3, 7, 14, 28, and 42 days after irradiation. Granulocyte marker protein (GMP) was measured in buffer extracts of feces by enzyme-linked immunosorbent assay (ELISA). Irradiated and shielded intestine were procured at 2 and 26 weeks and assessed for histopathologic injury [radiation injury score (RIS)], ED-2 positive macrophages, and interleukin-1α (IL-1α) positive cells. Irradiated intestine exhibited characteristic histopathologic alterations and increased numbers of macrophages and IL-1α positive cells. There was a highly significant dose-dependent increase in post-radiation GMP (P < 0.0001). Maximal GMP excretion occurred 3–7 days after irradiation. Six weeks after irradiation, GMP excretion had returned to normal in the 2.8 and 12 Gy groups, but was still 3.5 times higher in the 23 Gy group than in controls. The associations between early GMP excretion and RIS and fibrosis at 26 weeks were highly significant (P < 0.001 and P < 0.0001, respectively). Post-radiation granulocyte transmigration is dose-dependent and correlates with structural and molecular changes, as well as with subsequent chronic injury. The GMP assay is a sensitive, non-invasive indicator of acute intestinal radiation injury and a promising biological predictor of chronic toxicity. Our data underscore the importance of consequential mechanisms in radiation enteropathy. Radiat. Oncol. Invest. 5:275–282, 1997. © 1997 Wiley-Liss, Inc.

Assessment of disease activity in ulcerative colitis by faecal calprotectin, a novel granulocyte marker protein.

This study comprised 62 outpatients with ulcerative colitis who underwent 64 colonoscopies. The disease activity was evaluated according to endoscopic and histological criteria. The results revealed a significant correlation between both the endoscopic as well as the histological gradings of disease activity and faecal calprotectin. The median faecal calprotectin levels in the control group (6 mg/l) and in the patients with no or low disease activity (11.5 mg/l) were significantly different (p < 0.0001). The median calprotectin level among patients with active disease was 68 mg/l which was significantly different from the latter group (p < 0.0001). Furthermore, we suggest that the degree of inflammation rather than the extent of the disease determined the faecal calprotectin levels. In conclusion, assessment of faecal calprotectin seems to be a marker of disease activity in patients with ulcerative colitis.