High activity of the endogenous opioid system and acute but not chronic stress influence experimental colitis development in mice.

Exposition to environmental factors is one of the major underlying causes in inflammatory bowel diseases (IBD), with several endogenous systems involved. Our aim was to characterize the impact of stress on the colitis development in relation to the endogenous opioid system (EOS) activity in mice. A unique mouse model of high and low activity of EOS (namely high (HA)/low (LA) stress-induced analgesia) was employed. Mice were bred using bidirectional selection and classified as HA or LA line based on the measurement of analgesia. Colitis was induced by instillation of trinitrobenzenesulfonic acid in 30% EtOH/0.9% NaCl. After 4 days, the macroscopic score was assessed and samples for molecular and histological studies were collected. To evaluate the influence of stress on colitis development, chronic mild stress (exposure to stress stimuli for 2 and 5 weeks) and acute stress (short restraint over 3 days) were applied before colitis induction. We observed a difference in the colitis development between non-stressed HA and LA mice, as indicated by macroscopic and ulcer scores. Acute stress improved colitis in HA mice but did not change the inflammation score in LA line as compared to respective non-stressed mice. Chronic mild stress had no influence on colitis in either of mouse lines. Our study supports the hypothesis that the activity of EOS may be crucial in IBD development. We also evidence that acute, but not chronic stress influenced IBD exacerbation, depending on EOS function.

Prosecretory effect of loperamide in ileal and colonic mucosae of mice displaying high or low swim stress-induced analgesia associated with high and low endogenous opioid system activity.

BACKGROUND: Irritable bowel syndrome (IBS) is characterized by abdominal pain, bloating, and changes in bowel habit. The aim of this study was to characterize the effect of loperamide hydrochloride (LOP) and naloxone hydrochloride (NLX), an opioid agonist and antagonist, respectively, on electrolyte equilibrium in ileal and colonic mucosae and to estimate the possible influence of divergent activity of the endogenous opioid system (EOS) on IBS therapy. METHODS: Two mouse lines bidirectionally selected for high (HA) and low (LA) swim stress-induced analgesia associated with high and low EOS activity were used in this study. To assess the effect of LOP and NLX on HA/LA lines in vivo, we used the castor oil-induced diarrhea model. Changes in electrolyte equilibrium were determined on the basis of short-circuit current (ΔIsc ) in isolated mouse ileum and colon exposed to LOP and NLX and stimulated by forskolin (FSK), veratridine (VER), and bethanechol (BET). KEY RESULTS: In vivo, we found that LOP significantly prolonged time to appearance of diarrhea in HA and LA lines. In vitro, LOP and NLX increased ΔIsc in FSK- and VER-stimulated colonic tissue, respectively, in HA line. In the ileum, LOP increased ΔIsc in FSK- and VER-stimulated tissue and decreased ΔIsc in BET-stimulated tissues in HA line. CONCLUSIONS & INFERENCES: Individual differences in EOS activity may play a crucial role in the response to the IBS-D therapy, thus some patients may be at an increased risk of side effects such as constipation or diarrhea.

Modulation of the endocannabinoid system by the fatty acid amide hydrolase, monoacylglycerol and diacylglycerol lipase inhibitors as an attractive target for secretory diarrhoea therapy.

Secretory diarrhoea is a leading cause of mortality and morbidity worldwide. Our aim was to characterize the effect of inhibition of selected enzymes involved in the synthesis or degradation of endocannabinoids on electrolyte equilibrium in the mouse colonic tissue. The aim of this study was to evaluate the effects of PF-3845, JZL-184 and RHC-80267, as inhibitors of fatty acid amide hydrolase (FAAH), monoacylglycerol (MAGL) and diacylglycerol lipase (DAGL), respectively on epithelial ion transport in isolated mouse colon stimulated by forskolin (FSK), veratridine (VER) and bethanechol (BET). Next, colonic tissue was co-incubated with selected inhibitors and cannabinoid receptor antagonists: AM 251 and AM 630 (CB1 and CB2 antagonists, respectively). We found that PF-3845 induced antisecretory effect in FSK-stimulated colonic tissue (P < 0.01), which was significantly reversed by AM 251 (P < 0.001) and AM 630 (P < 0.01). JZL-184 significantly reduced ΔIsc (P < 0.05) in FSK-stimulated conditions and co-incubation with AM 630, but not AM 251 reversed this effect when compared to JZL-184 alone (P < 0.05). After addition of PF-3845 and JZL-184 to colon tissue stimulated by VER, we did not observe any significant effect on ΔIsc. PF-3845, JZL-184 or RHC-80267 were without any statistically significant effect on BET-evoked ion transport when compared to control. Our findings showed that indirect modulation of the endocannabinoid system could be an attractive target for novel effective treatment of secretory diarrhoea, which is devoid of side effects on the central nervous system caused by direct administration of cannabinoid receptor agonists.

The effect of opioid agonists and antagonists on gastrointestinal motility in mice selected for high and low swim stress-induced analgesia.

BACKGROUND: The opioid system in the gastrointestinal (GI) tract plays an important physiological role, but is also responsible for the side effect of opioid drugs, including troublesome constipation in chronic pain treatment. The aim of this study was to characterize and validate a new mouse model to study the effects of opioid agonists and antagonists in the GI tract. METHODS: Six-week-old male Swiss-Webster mice, divergently bred for high (HA) and low (LA) swim stress-induced analgesia (SSIA), were used in the study. To assess the influence of opioid agonists (morphine and loperamide) and antagonists (naloxone hydrochloride, NLX and naloxone methiodide, NLXM) on GI motility, whole GI transit (whole GIT) and upper GIT assays were conducted. To evaluate the expression of opioid receptors in the ileum and colon of HA and LA mice, immune staining was performed. KEY RESULTS: The effect of morphine was more pronounced in HA line, whereas loperamide exerted a stronger effect in LA mice. Furthermore, NLX and NLXM differentially abolished the inhibitory action of the central and peripheral opioid system on whole and upper GIT in HA and LA mice. The differences in GI motility between HA and LA mice coexisted with parallel changes in the expression of opioid receptors in the ileum and colon. CONCLUSIONS & INFERENCES: Differences in the activity of the endogenous opioid system are responsible for the vulnerability to changes in GI motility during treatment with opioids. Our findings validate the HA/LA model for further studies on opioids in the GI tract.

Salvinorin A analogues PR-37 and PR-38 attenuate compound 48/80-induced itch responses in mice.

BACKGROUND AND PURPOSE: The opioid system plays a crucial role in several physiological processes in the CNS and in the periphery. It has also been shown that selective opioid receptor agonists exert potent inhibitory action on pruritus and pain. In this study we examined whether two analogues of Salvinorin A, PR-37 and PR-38, exhibit antipruritic properties in mice. EXPERIMENTAL APPROACH: To examine the antiscratch effect of PR-37 and PR-38 we used a mouse model of compound 48/80-induced pruritus. In order to elucidate the mechanism of action of tested compounds, specific antagonists of opioid and cannabinoid receptors were used. The effect of PR-37 on the CNS was assessed by measuring motor parameters and exploratory behaviours in mice. KEY RESULTS: PR-37 and PR-38, jnjected s.c., significantly reduced the number of compound 48/80-induced scratching behaviours in mice in a dose- and time-dependent manner. PR-38 was also active when orally administered. The antiscratch activity of PR-37 was blocked by the selective κ opioid receptor antagonist, nor-binaltorphimine, and that of PR-38 by the selective µ opioid receptor antagonist, ß-funaltrexamine. CONCLUSION AND IMPLICATIONS: In conclusion, a novel framework for the development of new antipruritic drugs derived from salvinorin A has been validated.

78,XX testicular DSD syndrome in a mongrel dog.

Because of an apparent sexual ambiguity (enlarged clitoris), a 1-year-old mongrel dog was presented to the clinic. A positive result on a GnRH stimulation test revealed the existence of functional testicular tissue. A midline laparotomy was performed, and gonads resembling testes were resected along with the adherent parts of the uterine horns. Microscopic examination confirmed that the sampled gonads were testes. Cross-sections of the head and tail of the epididymis revealed their typical structures. All layers of the uterine wall were well-developed. The lumen was stellar, covered by columnar cylindrical epithelium, although locally some epithelial cells had changed in height from columnar to flat. The uterine glands were distributed in functional layer of endometrium in a non-uniform way. Cytogenetic analysis based on the evaluation of metaphase plates of blood lymphocytes showed a female karyotype, 78,XX. PCR amplification of the SRY gene was negative in the studied mongrel dog. This canine disorder may be genetically heterogeneous, potentially with a different mutation in different breeds. An autosomal recessive inheritance for the XX male is suggested in such cases. The present case of sex reversal syndrome concerns a non-purebred dog. In mongrels, it is definitely less likely for the defect to be inherited because of a recessive disorder. According to the recently proposed nomenclature, the described case should be classified as 78,XX testicular DSD syndrome.

Genetic characteristics of the ostrich population using molecular methods.

A genetic analysis was performed on Polish ostriches from the 3 principal ostrich breeds: red-, blue-, and black-necks. The analysis was based on 2 molecular methods: DNA fingerprinting and microsatellites. The DNA fingerprinting patterns were obtained using the restriction enzyme HinfI and Jeffrey's 33.15 probe. The second method consisted of a PCR procedure, for which 5 VIAS-OS primers specific to the ostrich were used. The PCR products were separated on polyacrylamide gel using ALFexpress (Authomated Laser Fluorescent DNA Sequencer). The study aimed at assessing the genetic variability within and among the 3 ostrich breeds as well as evaluating the genetic distance between them, and represents the first report on the genetic characteristics of the ostrich breeds. The results obtained by both methods showed considerable compatibility, especially with regard to the relationship among the breeds analyzed. The diversity within breeds, obtained on the basis of the DNA fingerprinting analysis, proved to be low. Among the ostrich populations analyzed, the highest variability potential was observed for black-necked ostriches (the mean diversity of patterns amounted to 29.04%, whereas the mean heterozygosity was 0.30) and the lowest was observed for the red-necks. The largest genetic similarity was recorded between red- and blue-necked ostriches, but the greatest genetic distance was between the red- and black-necks. This means that the use of birds of those breeds in crosses should result in the highest heterotic effect. Both of these methods measured the genetic distance between the analyzed ostrich breeds that was expected from the geographic origin of these birds. The results obtained in the present study showed that both analytic methods used can be successfully applied when elaborating on the genetic characteristics of the ostrich.

Gene expression profiling of hereditary exencephaly in chickens.

In this preliminary study, differentially expressed genes were investigated in cranial tissues from chickens with hereditary exencephaly using cDNA microarrays containing 1,152 genes and expressed sequence tags (ESTs). Genes showing twofold or greater differences at P < 0.05 between affected and normal cranial cells were considered to be candidates for hereditary exencephaly in chicken. Eighteen ESTs (11 known genes/homologues) were upregulated and 108 ESTs (51 known genes/homologues) were downregulated. The EST AL584231 (ROS006C9), orthologous to human MTHFD1, a known candidate gene for human neural tube defects (NTDs), was expressed at the same level both in normal and affected chicken cranial tissues. ESTs AL584253 (ROS006F7, thioredoxin reductase 1) and AL585511 (ROS024H9, thioredoxin), both involved in NTD pathogenic pathways in mice, were downregulated and had mean ratios of 0.41 and 0.04 for expression in affected vs. normal cells respectively. Expression differences of these two ESTs were confirmed by quantitative real-time polymerase chain reaction. These data indicate that ESTs AL584253 and AL585511 are candidates for hereditary exencephaly in chickens.