Joerg Glatzle

Long-Term Quality of Life in Patients with Crohn’s Disease and Perianal Fistulas: Influence of Fecal Diversion

PurposeSymptomatic perianal fistulas impair quality of life in patients with Crohn’s disease. Fecal diversion improves symptoms but may impair quality of life. This study was designed to compare long-term quality of life in patients with Crohn’s disease with symptomatic perianal fistulas who were treated with or without fecal diversion.MethodsFrom 1996 to 2002, perianal fistulas were treated in 116 patients with Crohn’s disease. A questionnaire, including four quality of life instruments, was mailed to each patient (Short-Form General Health Survey, Gastrointestinal Quality of Life Index, Cleveland Global Quality of Life Score, Short Inflammatory Bowel Disease Questionnaire).ResultsQuestionnaires were returned by 77 of 116 patients (66 percent). Thirty-four of these patients had undergone fecal diversion, whereas 43 had not. Median follow-up was 49 (range, 18–97) months in diverted and 44 (range, 14–98) months in undiverted patients (not significant). In the diverted group, 44 percent complained of Crohn’s disease-related symptoms, which was less compared with 79 percent in undiverted patients (P < 0.05). Diverted patients achieved 68 ± 1 percent of the maximum possible score on the Gastrointestinal Quality of Life Index compared with 60 ± 2 percent in undiverted patients (mean ± standard error of the mean; P < 0.001); diverted patients scored better on the subscale “gastrointestinal symptoms” of the Gastrointestinal Quality of Life Index (81 ± 1 percent vs. 67 ± 2 percent; P < 0.001). There was no difference in the Short Inflammatory Bowel Disease Questionnaire between diverted and undiverted patients except for the subscale “bowel function” (91 ± 2 percent vs. 76 ± 2 percent; P < 0.0001). No difference in quality of life was detected by the Short-Form General Health Survey and Cleveland Global Quality of Life Score.ConclusionsIn the investigated population of patients with Crohn’s disease, quality of life seems to be similar or potentially superior in diverted patients suffering from perianal fistulas compared with undiverted patients. A diverting stoma, therefore, may improve quality of life in patients with severe perianal Crohn’s disease.

Circadian Expression of Clock- and Tumor Suppressor Genes in Human Oral Mucosa

Purpose: Circadian rhythms are daily oscillations of multiple biological processes driven by endogenous clocks. Imbalance of these rhythms has been associated with cancerogenesis in humans. To further elucidate the role circadian clocks have in cellular growth control, tumor suppression and cancer treatment, it is revealing to know how clock genes and clock-controlled genes are regulated in healthy humans. Materials and Methods: Therefore comparative microarray analyses were conducted investigating the relative mRNA expression of clock genes throughout a 24-hour period in cell samples obtained from oral mucosa of eight healthy diurnally active male study participants. Differentially expressed selected genes of interest were additionally evaluated using qRT-PCR. Results: Microarray analysis revealed 33 significant differentially regulated clock genes and clock- controlled genes, throughout a one day period (6.00h, 12.00h, 18.00h, 24.00h). Hereof were 16 clock genes and 17 clock- controlled genes including tumor suppressor- and oncogenes. qRT-PCR of selected genes of interest, such as hPER2, hCRY1, hBMAL1, hCCRN4L and hSMAD5 revealed significant circadian regulations. Conclusion: Our study revealed a proper circadian regulation profile of several clock- and tumor suppressor genes at defined points in time in the participants studied. These findings could provide important information regarding genes displaying the same expression profile in the gastrointestinal tract amounting to a physiological expression profile of healthy humans. In the future asynchronous regulations of those genes might be an additional assistant method to detect derivations distinguishing normal from malignant tissue or assessing risk factors for cancer.

Cholecystokinin-58 is more potent in inhibiting food intake than cholecystokinin-8 in rats

Abstract Introduction: Several cholecystokinin (CCK) forms have been detected in plasma, but most studies on food intake investigated the effects of CCK-8 only. Recently, it has been demonstrated that CCK-58 is the only endocrine-active form of CCK in rats. Methods: CCK-58 was synthesized with a peptide synthesizer using FMOC chemistry and CCK-58 effects on food intake were compared to CCK-8 in rats. Results: Both CCK-58 and CCK-8 inhibited food intake in a dose-dependent manner and were equally potent at 30 min. CCK-58 showed a prolonged inhibition of food intake compared to CCK8 at the higher dose tested (7 nmol/kg), inhibiting food intake also at 60 min, and cumulative food intake was inhibited for up to 210 min by CCK-58. Conclusions: CCK-58 has the same potency in inhibiting food intake as CCK-8 in rats, but inhibits food intake longer. This might be due to its tertiary structure resulting in a delayed plasma degradation or a prolonged binding at the CCK receptor. As CCK-58 is the major CCK form in the gut wall and possibly in the circulating blood in humans, the effects of CCK on food intake might have been underestimated in the past.Zinc deficiency has been associated with impaired learning and memory function in animals and human beings. However, the molecular mechanisms remain obscure. In light of evidence that ubiquitin C-terminal hydrolase L1 (Uch-L1) and cAMP-responsive element-binding protein (CREB) are required for synaptic and memory function and the possible regulation of CREB by Uch-L1, this present study was conducted to investigate the effect of zinc depletion on Uch-L1 protein expression and on Uch-L1 and CREB mRNA expression in cultured hippocampal neurons. Cultured hippocampal neurons were exposed to a cell membrane-permeant zinc chelator TPEN (2 microM), and to TPEN plus zinc sulphate (5 microM) for 24 h. Cultures were then processed to detect neuronal injury by lactate dehydrogenase (LDH) assay, Uch-L1 protein levels by Western blot, and Uch-L1 and CREB mRNAs levels by RT-PCR. The LDH release rate in TPEN-incubated neurons was notably increased compared to non-treated controls. Significant down-regulation of Uch-L1 protein level and mRNA levels for Uch-L1 and CREB were observed in TPEN-treated neurons. Co-addition of zinc almost completely reversed TPEN-induced neuronal injury and the alterations in Uch-L1 and CREB expression. The results demonstrated that zinc modulated the expression of Uch-L1 and CREB at the protein and/or transcription levels in hippocampal neurons, which implies that down-regulation of both Uch-L1 and CREB might participate in memory dysfunction induced by zinc deficiency.

Differential Sensitization of Afferent Neuronal Pathways During Postoperative Ileus in the Mouse Jejunum

Background:Postoperative ileus induces reflex inhibition of gastrointestinal motility and an intestinal inflammatory response. We aimed to determine whether afferent sensitivity is increased during postoperative ileus and whether alterations are cyclooxygenase-2 (COX-2)-dependent. Methods:C57BL/6 mice underwent laparotomy followed by standardized small bowel manipulation to induce ileus or sham treatment. After 24 hours, extracellular multiunit mesenteric afferent nerve discharge was recorded in vitro from 2-cm segments of jejunum. Fos immunoreactivity was determined for neuronal activation in the vagal nucleus of the solitary tract (nTS) of the brain stem and leukocyte infiltration in the intestinal muscularis by myeloperoxidase stains. Results:Serosal bradykinin (1 μM) was followed by an increase in afferent discharge to 65 ± 5 imp·s−1 in ileus segments compared with 37 ± 6 imp·s−1 in sham controls (P < 0.05). The response was attenuated to 31 ± 7 imp·s−1 after the selective COX-2 inhibitor 5,5-dimethyl-3-(flurorophenyl)-4-(4-methylsulfonyl) phenyl-2(5H)-furanone (DFU) in ileus segments. Afferent firing during ileus was augmented at luminal distension at 20 mm Hg but not at pressures up to 60 mm Hg. The number of Fos-positive neurons in the nTS was 110 ± 45 during ileus compared with 7 ± 4 in sham controls (−7.32 mm from bregma, P < 0.05) and did not differ after DFU. The intestinal muscularis contained more leukocytes during ileus compared with ileus segments after DFU and controls (both P < 0.05). Conclusion:This study provides direct evidence from afferent nerve recordings that sensitivity to bradykinin, which stimulates predominantly spinal afferents, is augmented during postoperative ileus involving a COX-2 pathway. Vagal afferents were also sensitized because low-threshold mechanosensitivity and neuronal activation in the nTS were increased.

Mediators of neuronal activation in the rat brainstem following intestinal anaphylaxis

Brainstem neurones become activated following intestinal antigen challenge but the signalling mechanisms have not been resolved. Our aim was to determine the extent of brainstem activation after intestinal anaphylaxis induced by chicken egg albumin (EA). An increase in Fos-positive neurones in the nucleus tractus solitarius (nTS) was observed following EA (P<0.05). Fos-expression was decreased following pretreatment with pyrilamine and ondansetron i.p. and to a similar extent when both antagonists were administered together (all P<0.05 vs. control). Indomethacin had no effect on Fos-expression after antigen challenge. 5-HT and histamine but not prostanoids, released following intestinal anaphylaxis, induce nTS activation via histamine H(1)- and 5-HT(3) receptors. Information on the intestinal inflammatory status is relayed centrally and may play a role in reflexes and behavioural responses to activation of the immune system.

Neuronal activation in the nucleus of the solitary tract following jejunal lipopolysaccharide in the rat

INTRODUCTION Inflammation during systemic lipopolysaccharide (LPS) seems to be modulated by the CNS via afferent and efferent vagal pathways. We hypothesized that similar to systemic inflammation, local LPS in the gut lumen may also activate central neurons and aimed to identify potential molecular mechanisms. METHODS Male Wistar rats were equipped with an exteriorized canula in the proximal jejunum. LPS or vehicle were administered into the jejunum (10 mg ml(-1)). For further study of molecular mechanisms, LPS or vehicle were administered systemically (1 mg kg(-1)). Brain stem activation was quantified by Fos-immunohistochemistry in the vagal nucleus of the solitary tract (NTS) and the Area postrema which is exposed to systemic circulation. Serum LPS concentrations were also determined. RESULTS Jejunal LPS exposure entailed 91+/-12 (n=7) Fos-positive neurons in the NTS compared to 39+/-9 in controls (n=6; p<0.01), while serum LPS concentrations and Fos-positive neurons in the Area postrema were not different. Systemic LPS triggered 150+/-25 (n=6) and vehicle 52+/-6 Fos-positive neurons (n=7; p<0.01). The Fos count after systemic LPS was reduced to 99+/-30 following pretreatment with the cyclooxygenase inhibitor Naproxen (10 mg kg(-1); p>0.05 versus vehicle controls) and increased to 242+/-66 following the iNOS-inhibitor Aminoguanidine (15 mg kg(-1); p<0.01). In the Area postrema, 97+/-17 (n=6) neurons were counted in animals pretreated with systemic LPS compared to 14+/-4 in controls (n=7, p<0.001). CONCLUSIONS Central neuronal activation following inflammation after systemic LPS is modulated by cyclooxygenase and NO pathways. Local exposure to bacterial LPS in the gut lumen activates the NTS which may set the stage for efferent vagal modulation of intestinal inflammation.

Extrinsic afferent nerve sensitivity and enteric neurotransmission in murine jejunum in vitro

Enteric and extrinsic sensory neurons respond to similar stimuli. Thus they may be activated in series or in parallel. Because signal transmission via synapses or mediator release would depend on calcium, we investigated its role for extrinsic afferent sensitivity to chemical and mechanical stimulation. Extracellular multiunit afferent recordings were made in vitro from paravascular nerve bundles supplying the mouse jejunum. Intraluminal pressure and afferent nerve responses were recorded under control conditions and under four conditions designed to interfere with enteric neurotransmission. We found that phasic intestinal contractions ceased after switching perfusion to Ca(2+)-free buffer with or without a purinergic P2 receptor antagonist, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) or cadmium (blocking all Ca(2+)-channels) but not following omega-conotoxin GVIA (N-type Ca(2+)-channel blocker). Luminal HCl (pH 2) and 5-HT (500 microM) evoked peak firing of 17 +/- 4 impulses per second (imp/s) (n = 10) and 21 +/- 4 imp/s (n = 13) under control conditions. These responses were reduced to 4 +/- 2 imp/s and 5 +/- 2 imp/s by cadmium (n = 7, P < 0.05), to 7 +/- 2 imp/s and 6 +/- 1 imp/s by Ca(2+)-free perfusion (n = 6, P < 0.05), and to 3 +/- 1 imp/s and 4 +/- 1 imp/s by Ca(2+)-free perfusion with PPADS (n = 6, P < 0.05). Responses were unchanged by omega-conotoxin GVIA. Mechanical ramp distension of the intestinal segment to 60 cmH(2)O was not altered by any of the experimental conditions. We concluded that HCl and 5-HT activate extrinsic afferents via a calcium-dependent mechanism, which is unlikely to involve enteric neurons carrying N-type calcium channels. Extrinsic mechanosensitivity is independent of enteric neurotransmission. It appears that cross talk from the enteric to the extrinsic nervous system does not mediate extrinsic afferent sensitivity.

Stress-induced attenuation of brain stem activation following intestinal anaphylaxis in the rat.

Intestinal anaphylaxis triggers neuronal activation in the nucleus tractus solitarius (nTS) of the rat brain stem. Stress may modulate reflex circuitry in the brain stem and facilitate intestinal inflammatory responses. We hypothesized that stress would modulate central neuronal activation during intestinal anaphylaxis. NTS neurons were activated following intestinal antigen challenge in sensitized Hooded Lister rats but not in negative controls (P < 0.05). The number of Fos-positive neurons following intestinal anaphylaxis decreased in animals exposed to water-avoidance stress (P < 0.05), although serum levels of rat mast cell protease II were not different in stressed and unstressed animals, indicating a similar degree of mast cell degranulation. Stress seems to inhibit neuronal activation in the rat brain stem during intestinal inflammation without modulation of the inflammatory response itself. This may have implications for a potential efferent neuronal modulation of inflammatory responses in the gut.

Aktivierung vagaler Hirnstammkerne nach Gabe des Neurotoxins Capsaicin wahrend des postoperativen Ileus bei der Maus

Introduction: For the condition of postoperative ileus neuronal inhibitory reflex mechanism activation has been shown in the spinal cord to induce postoperative ileus [1]. Capsaicin sensitive visceral afferent neurons are an important component of that reflex mechanism [2]. We aimed to examine central vagal afferent sensitivity by measuring brainstem Fos expression in mice during postoperative ileus after modulation of afferent neronal transmission by using the Capsaicin. Methods: Under enflurane anesthesia, C57BL/6 mice underwent laparotomy followed by small bowel manipulation to induce an ileus or left untouched as a sham-treatment group. Ileus animals were pretreated by Capsaicin (1 µm/kg i. p.) 48 h prior to small bowel manipulation. The animals were killed 24 h later. The brainstem was removed for Fos immunocytochemistry. Fos IR was quantified in the nucleus of the solitary tract (nTS). Leucocyte infiltration into the intestinal muscularis was used an index of post-operative inflammation. Data are mean ± SEM and were compared by one-way ANOVA. Results: The number of leucocytes infiltrating the muscularis was elavated during ileus (39 ± 9 vs. 1.8 ± 1/mm 2 ; P = 0.008, N = 6). Capsaicin increased the number of leucocytes in the muscularis in ileus animals to 72 ± 28/mm 2 , (N = 6). This was associated with an increase in the number of Fos-positive neurons in the nTS following surgery compared to sham controls (Bregma:–7.70 mm, 30 ± 9 vs. 6 ± 2; P = 0.01, –7.32 mm, 107 ± 26 vs. 6 ± 2, P = 0.016, N = 4). A reduction in Fos-expression occurred after Capsaicin treatment in ileus animals (Bregma: –7.70 mm 8 ± 3, n = 5, –7.32 mm 16 ± 8, n = 5, P < 0.05). Conclusion: Small bowel manipulation leads to a markely increase in brainstem Fos expression indicating that vagal afferent pathways are activated during postoperative ileus. Capsaicin does not reduce local intestinal inflammation and neither improve gastrointestinal motility, but brainstem Fos activation in ileus animals. Thus, ablation of intestinal afferent neurons and subsequently impaired afferent reflex mechanisms might be not benificial in the late phase of postoperative ileus. Einleitung

Aktivierung vagaler Hirnstammkerne und Sensibilität extrinsischer afferenter Nervenfasern während des postoperativen lleus bei der Maus

Introduction: Small bowel manipulation leads to a profound ileus and an increase in spinal Fos expression, suggesting prolonged activation of spinal afferent pathways. The extent to which vagal afferents may also be influenced has not been investigated. We aimed to examine intestinal afferent sensitivity and brainstem Fos expression in mice during postoperative ileus. Methods: Under enflurane anesthesia, C57BL/6 mice underwent laparotomy followed by small bowel manipulation to induce an ileus or left untouched as a sham-treatment group. The animals were killed 24 h later. The brainstem was removed for Fos immunocytochemistry. 3 cm segments of jejunum were placed in Krebs buffer. Extracellular multiunit mesenteric afferent nerve discharge was recorded at baseline during distension and following serosal application of bradykinin (BK 1 µM). Whole nerve afferent discharge was quantified as peak increase above baseline. Fos IR was quantified in the nucleus of the solitary tract (nTS). Leukocyte infiltration into the intestinal muscularis was used an index of post-operative inflammation. Data are mean ± SEM and were compared by unpaired Students’ t-test. Results: The number of leukocytes infiltrating the muscularis was elevated during ileus (39 ± 9 vs. 1.8 ± I/mm2 in sham controls; P = 0.008). This was associated with an increase in the number of Fos-positive neurons in the nTS following surgery compared to sham controls (Bregma:- 7.70 mm, 30 ± 9 vs. 6 ± 2; P = 0.01, - 7.32 mm, 107 ± 26 vs. 6 ±2, P = 0.016, N = 4). Spontaneous discharge was higher in ileus animals compared to sham segments (17 ± 1 vs. 12 ± 2 imp/s, P = 0.02, N = 6), but not in the peak response to ramp distension (at 60 mmHg 85 ± 6 vs. 70 ± 4 imp/s respectively, P = 0.07). The response profile was different in ileus with a significantly increased response at low distending pressures (2 -20 mmHg) compared to controls (18 ±2 imp/s vs 9 ±2, P < 0.05). The level of firing after BK adminstration was not significantly different during ileus (58 db 8 imp/s vs 39 db 9 imp/s, N = 8, n.s.). Conclusion: Small bowel manipulation leads to a markely increase in brainstem Fos expression indicating that vagal afferent pathways are activated during postoperative ileus. Mechanical manipulation leads to an augmented afferent sensitivity to low distending pressures. The increased sensitivity to low threshold distension might be due to local inflammatory processes. This activation is not reflected by changes in afferent sensitivity due to chemical stimulation, suggesting there may be compensatory changes to limit inflammation-induced hypersensitivity.