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Enhanced gastric mucosal damage of desensitization of capsaicin-sensitive afferent neurons after chronic treatment with sodium taurocholate in rats
A176
AGA ABSTRACTS
EFFECT OF OMEPRAZOLETHERAPY ON HELICOBACTER' PYLORI, UREASE ACTIVITY AND ANTRAL GASTRIC HISTOLOGY IN PATIENTS WITH DUODENAL ULCER. I M Nakshabendi, Q B Zhang, M Mokhashi, C Gemmell, F D Lee*, R I Russell• Department of Gastroenterology & Department of Pathology*, Royal Infirmary, Glasgow, Scotland. Helicobacter pylori (HP) is a s s o c i a t e d with chronic active gastritis and peptic ulceration (PU). O m e p r a z o l e is a proton pump inhi~bitor which is v e r y e f f e c t i v e fn healing PU and gastritis• It was found that omeprazole has some activity agai.nst HP both in vitro a n d in vivo a s w e l l a s inhibiting urease activity in v i t r o . A I M S : ~ t o evaluate the effect of omeprazole on urease activity (in vivo), HP colonization of the gastric mucosa, associated gastritis and CLO-test in patients with duodenal ulcer '(DU). PATIENTS AND METHODS: 12 p a t i e n t s (17 m e n a n d 5 w o m e n , age 22-68 yr ) with DU > 5 mm in diameter and a positive CLO test were studied. Omeprazole 20 mg b.d was given to each patient and re-scoped 8 weeks later to check for healing of DU and repeat biopsies from the gastric antrum for histology, CLO test culture and urease activity. RESULTS: The results of this study showed that all patients had complete healing of the DU, significant reduction in the urease activity ( p < 0.00!, 95% ci of the difference between means is 1 4 . 1 t o 3 2 . 7 ), H P d e n s i t y ( p < 0.001, 95% CI of the difference between means iff 590.5 to 1486.1) and gastritis activity l:The number of HP microorganisms were reduced on the histological sections of the gastric antrum after omeprazole therapy and disappeared in three cases which corresponded to negative CLO-tests results after 24 hours reading. CONCLUSION: Omeprazole at a dose of 20 mg b.d is capable of reducing HP density, urease activity ( i n vivo) a n d a n t r a l gastritis activity i n patients w i t h DU.
• GASTRIC JUICE POLYMERASE CHAIN REACTION (PCR) FOR HELICOBACTER PYLORI (Hp) DIAGNOSIS: AN ALTERNATIVE TO HISTOLOGY? . Navaglia, F. DI Marlo*, D. Basso, M. Scrlgner, A. Toma¢ • Rugge-, M. ~ Pl-ebani. Departments of Laboratory Medicine, *Gastroenterology and ~ Pathology, University of Padua, Italy. Sp infection is involved In the pathogenesis of several gastr0duodenal diseases, as type n chronlc gastritis, duodenal ulcer and gastric cancer. The diagnosis of this infection as well as the assessment of its eradication after therapy need therefore to be extremely accurate and precise. The recent availability of molecular techniques, specifically the PCR, allows u s to detect very low amounts of thls bacterium in biological samples. Aims Of the present study were to verify the clinical usefulness :of Hp PCR identification in different blologloal samples and to compare the results with those obtained with histology. We studied 50 consecutive patients (30 males, 20 females, age range 19-79). From each subject s before endoscopy, a saliva sample was obtained. During endoscopy, gastric Juice (immediately neutralized with NaOH i mol/L), 4 antral a n d 4 body biopsies were taken. Hp was histologically evaluated in 2 antral and 2 body biopsies (Giemsa and/or warthln Starry staining}. Sallvaf gastric Juice, 2 antral and 2 body biopsies were stored at -20 C untlll DNA extractlont which was performed using a standard protelnase K digestion a n d phenol-chloroform method. DNA was then subjected to 35 amplification cloles using the primers HPU1 (5.GCCAATGGTAAATTAGTT3') and HPU2 (5,CTCCTTAATTGTTTTTAC3') Which amplified a 411~bp product from the uraase gene A. PCR products were identified after agarose gel electrophoresis. Results: at • histology 26 patients were Hp positive; of them 25 (96%) had also PCR positive findings in gastric Juice and in antra1 (1case) or body (2 cases) biopsies or both (20 cases). 8/26 (31~) Hp positive patients had also positive PCR saliva findings. Among the 24 Sp negative patients at histology, 14 (58%) had PCR positive results. The table reports PCR findings i n histologically np negative patients. Histology for Hp~ negative (24 cases) PCR positive in: - antral biopsies (AS) 2 cases - body biopsies (BB) 0 cases - gastric Juice (GJ) 6 cases - AB+BB 0 cases - AN+GJ 3 cases - BB+GJ 2 Cases - AB+BB+GJ i case PCR negative in: i0 cases 8 of the 14 PCR positive, histology negative patients had also positive saliva PCR findings, while all the r e m a i n l n g l 0 had saliva negative findings. Conoluslons: i. PCR Hp detection in saliva samples supports the hypothesis of a fecal oral transmission of this infection; 2. PCR Hp detection in. gastrlc Juice is a sensitive method to diagnose this infection; 3. positive PCR findings In patients otherwise negative is probably due to a higher sensitivity of PCR than histology. We suggest Hp PCR detection in gastric Juice In addition or even In alternative to histology for diagnosing and monitoring this infection.
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GASTROENTEROLOGY, Vol. 108, NO. 4
ENHANCED GASTRIC MUCOSAL DAMAGE BY DESENSITIZATION OF CAPSAICIN-SENSITIVE AFFERENT NEURONS AFTER CHRONIC TREATMENT WITH SODIUM TAUROCHOLATE IN RATS. M. Narita, S. Nakamura, S. Okabe Department of Applied Pharmacology, Kyoto Pharmaceutical University, Misasagl, Yamashina, Kyotu 607, Japan Chronic treatment with sodium taurecholate (TC) induces atrophic gastritis in rats, most probably through the broken mucozal barrier. However. the underlying mechanism remains unclear. Capsaleinsensitive afferent neurons (CS neurons) have bean found to be involved in the regulation of some functions such as gastric manozal blood flow (GMBF). We thus examined whether or not chronic treatment with TC may cause any deleterious effect on CS neurons and then enhance the development of gastritis and/or gastric damage. [Method] Male Douryu rats (250-300 g) were given 10 mM TC or tap water (as control) for 4 wk. Thereafter, the stomachs of these animals anesthetized with urethane were mounted on lucite chamber and perfused with 100 mM HC1. The stomachs were exposed for 30 rain to 10 mM TC dissolved in 100 mM HC1 (acidified TC), and transmueozal potential difference (PD), luminal pH and GMBF were determined simultaneously. When the experiments were finished, the stomachs were removed, and examined for mucosal damage .under a dissecting microscope. The amount of CGRF release from the oxyntic macoca was measured by ehemiluminesoent enzyme immunoaszay. [Resultsl In TC treated group, the basal PD was significantly reduced and luminal pH tended to increase. GMBF in these animals was profoundly suppressed in response to TC or capsaiein. The basal amount of CGRP released from the oxyntie muceza was not affected by TC treatment. However, the amount of CGRP released in response to capsaiein (10-5M) wa~ significantly reduced after TC treatment (1.74_+ 0.28 vs. 2.23 + 0.34 pg/mg tissue wt). 'PC caused a sizable amount of H ~ back-dlffusion and a more pronounced and persistent rise in GMBF in the stomach perfnsed with 100 mM HCI, yet only minimal damage was observed in the control animals. Under these conditions, TC treatment inhibited such GMBF responses, leading to hemorrhagic damage without affecting the degree of H ÷ back-diffusian. [Conclusion] These results indicate that chronic treatment with TC causes desensitization of CS neurons, reanlting in a weakened mueozal defensive mechanism (mainiy GMBF) leading to the enhanced development of gastric injury by acidified TC.
SUPEROXIDE ANION GENERATION PLAYS AN IMPORTANT ROLE IN ESOPHAGITIS INDUCED BY ACID AND PEPSIN IN RABBITS. MJ.Naya, A.Lanas: O. Alda. S. of Gastroenterology and Physiology. H . Clinico and University of Zaragoza. Zaragoza. Spain. Reactive oxygen metabolites have been associated with gastric and intestinal injury and play an important role as mediators of inflammation. Aims and Methods:To investigate whether mucosal reactive oxygen metabolite production is important in the pathophysiology of peptic esoplmgitis, we have studied the effects of several free radical scavengers (dimetylsulfoxide -DMSO lmg/ml; catalase 9;000U/ks; and superoxide dismutase-SOD 6 mg/kg) on an "in vivo" esophagitis model in rabbits induced by a reeimulating 50 ml solution o f acidified pepsin (saline at pH 2 + 2000 U of pepsirdml for 80 min (Gut 1990;39:11). The extent of both macroscopic and microsoopic macosal damage was graded from 0= Normal to 3=Confluent hemorrhage and/or erosions. Also, mucosal damage was evaluated by measuring flux rates of H+0tEq/h), K~(gEq/h) and total hemoglobin (Hb) content (mg). In a separate set of experiments, rabbit esophageal mucosal cells were isolated following collagenase digestion, mechanical disruption, filtration and eentrifugation (Gastroenterology 1990; 99:909). Generation of reactive oxygen metabolites were studied in these cells exposed at pH 7, 5, and 2 with pepsin (1 mg/ml) for 15 min. Oxygen metabolites and cell vitality were analyzed by flow cyt0met~ with the fluorochromes hydrotidine(O2") , dihydrorhodamine (H202) and propidium iodide (cell v i t . ) . Each experimental group contained 6-8 animals and results (x-~SE) were analyzed using one way ANOVA and unpaired Student's t test. Results: Of the radical sca'Jengers tested, SOD, which reacts with superoxide anion, significantly decreased esophagitis induced by acid and pepsin.* p<0.05; **p <0.01 Exneriment Mac. Damane H+ K+ Fib Control 2.25a-0.25 -607±92 1007±164 98±23 DMSO 2.09±0.25 -500±106 900~218 113±31 Catalase 1.33±0.21" -429±103 608±144 71±25 SOD 0.33±0.21"* -309±46* 314±79"* 12A:5 ** Isolated cells from the esophageal mucosa generated increased amounts of 02" metabolites when exposed to acid and pepsin (pH7 = 26±13 U. vs pH5 = 174a:67 U. vs pH2 = 5874-217 U *). Cell death was negligible at pH 7 and 5 and lower' than <20% at pH 2 in these experimental conditions. Conclusions: Reactiv~ oxygen metabolites especially superoxide anion seem to play a significant role in~ the pathophysiology of esopahgitis induced by acid and pepsin in rabbits.
AGA ABSTRACTS
EFFECT OF OMEPRAZOLETHERAPY ON HELICOBACTER' PYLORI, UREASE ACTIVITY AND ANTRAL GASTRIC HISTOLOGY IN PATIENTS WITH DUODENAL ULCER. I M Nakshabendi, Q B Zhang, M Mokhashi, C Gemmell, F D Lee*, R I Russell• Department of Gastroenterology & Department of Pathology*, Royal Infirmary, Glasgow, Scotland. Helicobacter pylori (HP) is a s s o c i a t e d with chronic active gastritis and peptic ulceration (PU). O m e p r a z o l e is a proton pump inhi~bitor which is v e r y e f f e c t i v e fn healing PU and gastritis• It was found that omeprazole has some activity agai.nst HP both in vitro a n d in vivo a s w e l l a s inhibiting urease activity in v i t r o . A I M S : ~ t o evaluate the effect of omeprazole on urease activity (in vivo), HP colonization of the gastric mucosa, associated gastritis and CLO-test in patients with duodenal ulcer '(DU). PATIENTS AND METHODS: 12 p a t i e n t s (17 m e n a n d 5 w o m e n , age 22-68 yr ) with DU > 5 mm in diameter and a positive CLO test were studied. Omeprazole 20 mg b.d was given to each patient and re-scoped 8 weeks later to check for healing of DU and repeat biopsies from the gastric antrum for histology, CLO test culture and urease activity. RESULTS: The results of this study showed that all patients had complete healing of the DU, significant reduction in the urease activity ( p < 0.00!, 95% ci of the difference between means is 1 4 . 1 t o 3 2 . 7 ), H P d e n s i t y ( p < 0.001, 95% CI of the difference between means iff 590.5 to 1486.1) and gastritis activity l:The number of HP microorganisms were reduced on the histological sections of the gastric antrum after omeprazole therapy and disappeared in three cases which corresponded to negative CLO-tests results after 24 hours reading. CONCLUSION: Omeprazole at a dose of 20 mg b.d is capable of reducing HP density, urease activity ( i n vivo) a n d a n t r a l gastritis activity i n patients w i t h DU.
• GASTRIC JUICE POLYMERASE CHAIN REACTION (PCR) FOR HELICOBACTER PYLORI (Hp) DIAGNOSIS: AN ALTERNATIVE TO HISTOLOGY? . Navaglia, F. DI Marlo*, D. Basso, M. Scrlgner, A. Toma¢ • Rugge-, M. ~ Pl-ebani. Departments of Laboratory Medicine, *Gastroenterology and ~ Pathology, University of Padua, Italy. Sp infection is involved In the pathogenesis of several gastr0duodenal diseases, as type n chronlc gastritis, duodenal ulcer and gastric cancer. The diagnosis of this infection as well as the assessment of its eradication after therapy need therefore to be extremely accurate and precise. The recent availability of molecular techniques, specifically the PCR, allows u s to detect very low amounts of thls bacterium in biological samples. Aims Of the present study were to verify the clinical usefulness :of Hp PCR identification in different blologloal samples and to compare the results with those obtained with histology. We studied 50 consecutive patients (30 males, 20 females, age range 19-79). From each subject s before endoscopy, a saliva sample was obtained. During endoscopy, gastric Juice (immediately neutralized with NaOH i mol/L), 4 antral a n d 4 body biopsies were taken. Hp was histologically evaluated in 2 antral and 2 body biopsies (Giemsa and/or warthln Starry staining}. Sallvaf gastric Juice, 2 antral and 2 body biopsies were stored at -20 C untlll DNA extractlont which was performed using a standard protelnase K digestion a n d phenol-chloroform method. DNA was then subjected to 35 amplification cloles using the primers HPU1 (5.GCCAATGGTAAATTAGTT3') and HPU2 (5,CTCCTTAATTGTTTTTAC3') Which amplified a 411~bp product from the uraase gene A. PCR products were identified after agarose gel electrophoresis. Results: at • histology 26 patients were Hp positive; of them 25 (96%) had also PCR positive findings in gastric Juice and in antra1 (1case) or body (2 cases) biopsies or both (20 cases). 8/26 (31~) Hp positive patients had also positive PCR saliva findings. Among the 24 Sp negative patients at histology, 14 (58%) had PCR positive results. The table reports PCR findings i n histologically np negative patients. Histology for Hp~ negative (24 cases) PCR positive in: - antral biopsies (AS) 2 cases - body biopsies (BB) 0 cases - gastric Juice (GJ) 6 cases - AB+BB 0 cases - AN+GJ 3 cases - BB+GJ 2 Cases - AB+BB+GJ i case PCR negative in: i0 cases 8 of the 14 PCR positive, histology negative patients had also positive saliva PCR findings, while all the r e m a i n l n g l 0 had saliva negative findings. Conoluslons: i. PCR Hp detection in saliva samples supports the hypothesis of a fecal oral transmission of this infection; 2. PCR Hp detection in. gastrlc Juice is a sensitive method to diagnose this infection; 3. positive PCR findings In patients otherwise negative is probably due to a higher sensitivity of PCR than histology. We suggest Hp PCR detection in gastric Juice In addition or even In alternative to histology for diagnosing and monitoring this infection.
~
GASTROENTEROLOGY, Vol. 108, NO. 4
ENHANCED GASTRIC MUCOSAL DAMAGE BY DESENSITIZATION OF CAPSAICIN-SENSITIVE AFFERENT NEURONS AFTER CHRONIC TREATMENT WITH SODIUM TAUROCHOLATE IN RATS. M. Narita, S. Nakamura, S. Okabe Department of Applied Pharmacology, Kyoto Pharmaceutical University, Misasagl, Yamashina, Kyotu 607, Japan Chronic treatment with sodium taurecholate (TC) induces atrophic gastritis in rats, most probably through the broken mucozal barrier. However. the underlying mechanism remains unclear. Capsaleinsensitive afferent neurons (CS neurons) have bean found to be involved in the regulation of some functions such as gastric manozal blood flow (GMBF). We thus examined whether or not chronic treatment with TC may cause any deleterious effect on CS neurons and then enhance the development of gastritis and/or gastric damage. [Method] Male Douryu rats (250-300 g) were given 10 mM TC or tap water (as control) for 4 wk. Thereafter, the stomachs of these animals anesthetized with urethane were mounted on lucite chamber and perfused with 100 mM HC1. The stomachs were exposed for 30 rain to 10 mM TC dissolved in 100 mM HC1 (acidified TC), and transmueozal potential difference (PD), luminal pH and GMBF were determined simultaneously. When the experiments were finished, the stomachs were removed, and examined for mucosal damage .under a dissecting microscope. The amount of CGRF release from the oxyntic macoca was measured by ehemiluminesoent enzyme immunoaszay. [Resultsl In TC treated group, the basal PD was significantly reduced and luminal pH tended to increase. GMBF in these animals was profoundly suppressed in response to TC or capsaiein. The basal amount of CGRP released from the oxyntie muceza was not affected by TC treatment. However, the amount of CGRP released in response to capsaiein (10-5M) wa~ significantly reduced after TC treatment (1.74_+ 0.28 vs. 2.23 + 0.34 pg/mg tissue wt). 'PC caused a sizable amount of H ~ back-dlffusion and a more pronounced and persistent rise in GMBF in the stomach perfnsed with 100 mM HCI, yet only minimal damage was observed in the control animals. Under these conditions, TC treatment inhibited such GMBF responses, leading to hemorrhagic damage without affecting the degree of H ÷ back-diffusian. [Conclusion] These results indicate that chronic treatment with TC causes desensitization of CS neurons, reanlting in a weakened mueozal defensive mechanism (mainiy GMBF) leading to the enhanced development of gastric injury by acidified TC.
SUPEROXIDE ANION GENERATION PLAYS AN IMPORTANT ROLE IN ESOPHAGITIS INDUCED BY ACID AND PEPSIN IN RABBITS. MJ.Naya, A.Lanas: O. Alda. S. of Gastroenterology and Physiology. H . Clinico and University of Zaragoza. Zaragoza. Spain. Reactive oxygen metabolites have been associated with gastric and intestinal injury and play an important role as mediators of inflammation. Aims and Methods:To investigate whether mucosal reactive oxygen metabolite production is important in the pathophysiology of peptic esoplmgitis, we have studied the effects of several free radical scavengers (dimetylsulfoxide -DMSO lmg/ml; catalase 9;000U/ks; and superoxide dismutase-SOD 6 mg/kg) on an "in vivo" esophagitis model in rabbits induced by a reeimulating 50 ml solution o f acidified pepsin (saline at pH 2 + 2000 U of pepsirdml for 80 min (Gut 1990;39:11). The extent of both macroscopic and microsoopic macosal damage was graded from 0= Normal to 3=Confluent hemorrhage and/or erosions. Also, mucosal damage was evaluated by measuring flux rates of H+0tEq/h), K~(gEq/h) and total hemoglobin (Hb) content (mg). In a separate set of experiments, rabbit esophageal mucosal cells were isolated following collagenase digestion, mechanical disruption, filtration and eentrifugation (Gastroenterology 1990; 99:909). Generation of reactive oxygen metabolites were studied in these cells exposed at pH 7, 5, and 2 with pepsin (1 mg/ml) for 15 min. Oxygen metabolites and cell vitality were analyzed by flow cyt0met~ with the fluorochromes hydrotidine(O2") , dihydrorhodamine (H202) and propidium iodide (cell v i t . ) . Each experimental group contained 6-8 animals and results (x-~SE) were analyzed using one way ANOVA and unpaired Student's t test. Results: Of the radical sca'Jengers tested, SOD, which reacts with superoxide anion, significantly decreased esophagitis induced by acid and pepsin.* p<0.05; **p <0.01 Exneriment Mac. Damane H+ K+ Fib Control 2.25a-0.25 -607±92 1007±164 98±23 DMSO 2.09±0.25 -500±106 900~218 113±31 Catalase 1.33±0.21" -429±103 608±144 71±25 SOD 0.33±0.21"* -309±46* 314±79"* 12A:5 ** Isolated cells from the esophageal mucosa generated increased amounts of 02" metabolites when exposed to acid and pepsin (pH7 = 26±13 U. vs pH5 = 174a:67 U. vs pH2 = 5874-217 U *). Cell death was negligible at pH 7 and 5 and lower' than <20% at pH 2 in these experimental conditions. Conclusions: Reactiv~ oxygen metabolites especially superoxide anion seem to play a significant role in~ the pathophysiology of esopahgitis induced by acid and pepsin in rabbits.