Microcirculation-driven mitochondrion dysfunction during the progression of experimental sepsis.

Sepsis is accompanied by a less-known mismatch between hemodynamics and mitochondrial respiration. We aimed to characterize the relationship and time dependency of microcirculatory and mitochondrial functions in a rodent model of intraabdominal sepsis. Fecal peritonitis was induced in rats, and multi-organ failure (MOF) was evaluated 12, 16, 20, 24 or 28 h later (n = 8/group, each) using rat-specific organ failure assessment (ROFA) scores. Ileal microcirculation (proportion of perfused microvessels (PPV), microvascular flow index (MFI) and heterogeneity index (HI)) was monitored by intravital video microscopy, and mitochondrial respiration (OxPhos) and outer membrane (mtOM) damage were measured with high-resolution respirometry. MOF progression was evidenced by increased ROFA scores; microcirculatory parameters followed a parallel time course from the 16th to 28th h. Mitochondrial dysfunction commenced with a 4-h time lag with signs of mtOM damage, which correlated significantly with PPV, while no correlation was found between HI and OxPhos. High diagnostic value was demonstrated for PPV, mtOM damage and lactate levels for predicting MOF. Our findings indicate insufficient splanchnic microcirculation to be a possible predictor for MOF that develops before the start of mitochondrial dysfunction. The adequate subcellular compensatory capacity suggests the presence of mitochondrial subpopulations with differing sensitivity to septic insults.

A Porcine Sepsis Model With Numerical Scoring for Early Prediction of Severity.

Introduction: Sepsis can lead to organ dysfunctions with disturbed oxygen dynamics and life-threatening consequences. Since the results of organ-protective treatments cannot always be transferred from laboratory models into human therapies, increasing the translational potential of preclinical settings is an important goal. Our aim was to develop a standardized research protocol, where the progression of sepsis-related events can be characterized reproducibly in model experiments within clinically-relevant time frames. Methods: Peritonitis was induced in anesthetized minipigs injected intraperitoneally with autofeces inoculum (n = 27) or with saline (sham operation; n = 9). The microbial colony-forming units (CFUs) in the inoculum were retrospectively determined. After awakening, clinically relevant supportive therapies were conducted. Nineteen inoculated animals developed sepsis without a fulminant reaction. Sixteen hours later, these animals were re-anesthetized for invasive monitoring. Blood samples were taken to detect plasma TNF-α, IL-10, big endothelin (bET), high mobility group box protein1 (HMGB1) levels and blood gases, and sublingual microcirculatory measurements were conducted. Hemodynamic, respiratory, coagulation, liver and kidney dysfunctions were detected to characterize the septic status with a pig-specific Sequential Organ Failure Assessment (pSOFA) score and its simplified version (respiratory, cardiovascular and renal failure) between 16 and 24 h of the experiments. Results: Despite the standardized sepsis induction, the animals could be clustered into two distinct levels of severity: a sepsis (n = 10; median pSOFA score = 2) and a septic shock (n = 9; median pSOFA score = 8) subgroup at 18 h of the experiments, when the decreased systemic vascular resistance, increased DO2 and VO2, and markedly increased ExO2 demonstrated a compensated hyperdynamic state. Septic animals showed severity-dependent scores for organ failure with reduced microcirculation despite the adequate oxygen dynamics. Sepsis severity characterized later with pSOFA scores was in correlation with the germ count in the induction inoculum (r = 0.664) and CFUs in hemocultures (r = 0.876). Early changes in plasma levels of TNF-α, bET and HMGB1 were all related to the late-onset organ dysfunctions characterized by pSOFA scores. Conclusions: This microbiologically-monitored, large animal model of intraabdominal sepsis is suitable for clinically-relevant investigations. The methodology combines the advantages of conscious and anesthetized studies, and mimics human sepsis and septic shock closely with the possibility of numerical quantification of host responses.

The microbial composition of the initial insult can predict the prognosis of experimental sepsis.

We hypothesized that the composition of sepsis-inducing bacterial flora influences the course of fecal peritonitis in rodents. Saline or fecal suspensions with a standardized dose range of bacterial colony-forming units (CFUs) were injected intraperitoneally into Sprague-Dawley rats. The qualitative composition of the initial inoculum and the ascites was analyzed separately by MALDI-TOF mass spectrometry. Invasive monitoring was conducted in separate anesthetized groups (n = 12-13/group) after 12, 24, 48 and 72 h to determine rat-specific organ failure assessment (ROFA) scores. Death and ROFA scores peaked at 24 h. At this time, 20% mortality occurred in animals receiving a monomicrobial E. coli suspension, and ROFA scores were significantly higher in the monomicrobial subgroup than in the polymicrobial one (median 6.5; 5.0-7.0 and 5.0; 4.75-5.0, respectively). ROFA scores dropped after 48 h, accompanied by a steady decrease in ascites CFUs and a shift towards intra-abdominal monomicrobial E. coli cultures. Furthermore, we found a relationship between ascites CFUs and the evolving change in ROFA scores throughout the study. Hence, quantitatively identical bacterial loads with mono- or polymicrobial dominance lead to a different degree of sepsis severity and divergent outcomes. Initial and intraperitoneal microbiological testing should be used to improve translational research success.

Kynurenic Acid and Its Synthetic Derivatives Protect Against Sepsis-Associated Neutrophil Activation and Brain Mitochondrial Dysfunction in Rats.

Background and Aims: The systemic host response in sepsis is frequently accompanied by central nervous system (CNS) dysfunction. Evidence suggests that excessive formation of neutrophil extracellular traps (NETs) can increase the permeability of the blood-brain barrier (BBB) and that the evolving mitochondrial damage may contribute to the pathogenesis of sepsis-associated encephalopathy. Kynurenic acid (KYNA), a metabolite of tryptophan catabolism, exerts pleiotropic cell-protective effects under pro-inflammatory conditions. Our aim was to investigate whether exogenous KYNA or its synthetic analogues SZR-72 and SZR-104 affect BBB permeability secondary to NET formation and influence cerebral mitochondrial disturbances in a clinically relevant rodent model of intraabdominal sepsis. Methods: Sprague-Dawley rats were subjected to fecal peritonitis (0.6 g kg-1 ip) or a sham operation. Septic animals were treated with saline or KYNA, SZR-72 or SZR-104 (160 µmol kg-1 each ip) 16h and 22h after induction. Invasive monitoring was performed on anesthetized animals to evaluate respiratory, cardiovascular, renal, hepatic and metabolic parameters to calculate rat organ failure assessment (ROFA) scores. NET components (citrullinated histone H3 (CitH3); myeloperoxidase (MPO)) and the NET inducer IL-1ß, as well as IL-6 and a brain injury marker (S100B) were detected from plasma samples. After 24h, leukocyte infiltration (tissue MPO) and mitochondrial complex I- and II-linked (CI-CII) oxidative phosphorylation (OXPHOS) were evaluated. In a separate series, Evans Blue extravasation and the edema index were used to assess BBB permeability in the same regions. Results: Sepsis was characterized by significantly elevated ROFA scores, while the increased BBB permeability and plasma S100B levels demonstrated brain damage. Plasma levels of CitH3, MPO and IL-1ß were elevated in sepsis but were ameliorated by KYNA and its synthetic analogues. The sepsis-induced deterioration in tissue CI-CII-linked OXPHOS and BBB parameters as well as the increase in tissue MPO content were positively affected by KYNA/KYNA analogues. Conclusion: This study is the first to report that KYNA and KYNA analogues are potential neuroprotective agents in experimental sepsis. The proposed mechanistic steps involve reduced peripheral NET formation, lowered BBB permeability changes and alleviation of mitochondrial dysfunction in the CNS.

Mitochondrial Consequences of Organ Preservation Techniques during Liver Transplantation.

Allograft ischemia during liver transplantation (LT) adversely affects the function of mitochondria, resulting in impairment of oxidative phosphorylation and compromised post-transplant recovery of the affected organ. Several preservation methods have been developed to improve donor organ quality; however, their effects on mitochondrial functions have not yet been compared. This study aimed to summarize the available data on mitochondrial effects of graft preservation methods in preclinical models of LT. Furthermore, a network meta-analysis was conducted to determine if any of these treatments provide a superior benefit, suggesting that they might be used on humans. A systematic search was conducted using electronic databases (EMBASE, MEDLINE (via PubMed), the Cochrane Central Register of Controlled Trials (CENTRAL) and Web of Science) for controlled animal studies using preservation methods for LT. The ATP content of the graft was the primary outcome, as this is an indicator overall mitochondrial function. Secondary outcomes were the respiratory activity of mitochondrial complexes, cytochrome c and aspartate aminotransferase (ALT) release. Both a random-effects model and the SYRCLE risk of bias analysis for animal studies were used. After a comprehensive search of the databases, 25 studies were enrolled in the analysis. Treatments that had the most significant protective effect on ATP content included hypothermic and subnormothermic machine perfusion (HMP and SNMP) (MD = -1.0, 95% CI: (-2.3, 0.3) and MD = -1.1, 95% CI: (-3.2, 1.02)), while the effects of warm ischemia (WI) without cold storage (WI) and normothermic machine perfusion (NMP) were less pronounced (MD = -1.8, 95% CI: (-2.9, -0.7) and MD = -2.1 MD; CI: (-4.6; 0.4)). The subgroup of static cold storage (SCS) with shorter preservation time (< 12 h) yielded better results than SCS ≥ 12 h, NMP and WI, in terms of ATP preservation and the respiratory capacity of complexes. HMP and SNMP stand out in terms of mitochondrial protection when compared to other treatments for LT in animals. The shorter storage time at lower temperatures, together with the dynamic preservation, provided superior protection for the grafts in terms of mitochondrial function. Additional clinical studies on human patients including marginal donors and longer ischemia times are needed to confirm any superiority of preservation methods with respect to mitochondrial function.

Divergent Effects of the N-Methyl-D-Aspartate Receptor Antagonist Kynurenic Acid and the Synthetic Analog SZR-72 on Microcirculatory and Mitochondrial Dysfunction in Experimental Sepsis.

Introduction: Sepsis is a dysregulated host response to infection with macro- and microhemodynamic deterioration. Kynurenic acid (KYNA) is a metabolite of the kynurenine pathway of tryptophan catabolism with pleiotropic cell-protective effects under pro-inflammatory conditions. Our aim was to investigate whether exogenously administered KYNA or the synthetic analog SZR-72 affects the microcirculation and mitochondrial function in a clinically relevant rodent model of intraabdominal sepsis. Methods: Male Sprague-Dawley rats (n = 8/group) were subjected to fecal peritonitis (0.6 g kg-1 feces ip) or a sham operation. Septic animals were treated with sterile saline or received ip KYNA or SZR-72 (160 µmol kg-1 each) 16 and 22 h after induction. Invasive monitoring was performed on anesthetized animals to evaluate respiratory, cardiovascular, renal, hepatic and metabolic dysfunctions (PaO2/FiO2 ratio, mean arterial pressure, urea, AST/ALT ratio and lactate levels, respectively) based on the Rat Organ Failure Assessment (ROFA) score. The ratio of perfused vessels (PPV) of the ileal serosa was quantified with the intravital imaging technique. Complex I- and II-linked (CI; CII) oxidative phosphorylation capacities (OXPHOS) and mitochondrial membrane potential (ΔΨmt) were evaluated by High-Resolution FluoRespirometry (O2k, Oroboros, Austria) in liver biopsies. Plasma endothelin-1 (ET-1), IL-6, intestinal nitrotyrosine (NT) and xanthine oxidoreductase (XOR) activities were measured as inflammatory markers. Results: Sepsis was characterized by an increased ROFA score (5.3 ± 1.3 vs. 1.3 ± 0.7), increased ET-1, IL-6, NT and XOR levels, and decreased serosal PPV (65 ± 12% vs. 87 ± 7%), ΔΨmt and CI-CII-linked OXPHOS (73 ± 16 vs. 158 ± 14, and 189 ± 67 vs. 328 ± 81, respectively) as compared to controls. Both KYNA and SZR-72 reduced systemic inflammatory activation; KYNA treatment decreased serosal perfusion heterogeneity, restored PPV (85 ± 11%) and complex II-linked OXPHOS (307 ± 38), whereas SZR-72 improved both CI- and CII-linked OXPHOS (CI: 117 ± 18; CII: 445 ± 107) without effects on PPV 24 h after sepsis induction. Conclusion: Treatment with SZR-72 directly modulates mitochondrial respiration, leading to improved conversion of ADP to ATP, while administration of KYNA restores microcirculatory dysfunction. The results suggest that microcirculatory and mitochondrial resuscitation with KYNA or the synthetic analog SZR-72 might be an appropriate supportive tool in sepsis therapy.

Endothelin A and B Receptors: Potential Targets for Microcirculatory-Mitochondrial Therapy in Experimental Sepsis.

The hypoxia-sensitive endothelin (ET) system plays an important role in circulatory regulation through vasoconstrictor ETA and ETB2 and vasodilator ETB1 receptors. Sepsis progression is associated with microcirculatory and mitochondrial disturbances along with tissue hypoxia. Our aim was to investigate the consequences of treatments with the ETA receptor (ETA-R) antagonist, ETB1 receptor (ETB1-R) agonist, or their combination on oxygen dynamics, mesenteric microcirculation, and mitochondrial respiration in a rodent model of sepsis. Sprague Dawley rats were subjected to fecal peritonitis (0.6 g kg i.p.) or a sham operation. Septic animals were treated with saline or the ETA-R antagonist ETR-p1/fl peptide (100 nmol kg i.v.), the ETB1-R agonist IRL-1620 (0.55 nmol kg i.v.), or a combination therapy 22 h after induction. Invasive hemodynamic monitoring and blood gas analysis were performed during a 90-min observation, plasma ET-1 levels were determined, and intestinal capillary perfusion (CPR) was detected by intravital videomicroscopy. Mitochondrial Complex I (CI)- and CII-linked oxidative phosphorylation (OXPHOS) was evaluated by high-resolution respirometry in liver biopsies. Septic animals were hypotensive with elevated plasma ET-1. The ileal CPR, oxygen extraction (ExO2), and CI-CII-linked OXPHOS capacities decreased. ETR-p1/fl treatment increased ExO2 (by >45%), CPR, and CII-linked OXPHOS capacity. The administration of IRL-1620 countervailed the sepsis-induced hypotension (by >30%), normalized ExO2, and increased CPR. The combined ETA-R antagonist-ETB1-R agonist therapy reduced the plasma ET-1 level, significantly improved the intestinal microcirculation (by >41%), and reversed mitochondrial dysfunction. The additive effects of a combined ETA-R-ETB1-R-targeted therapy may offer a tool for a novel microcirculatory and mitochondrial resuscitation strategy in experimental sepsis.

Mucosectomy disrupting the enteric nervous system causes contraction and shrinkage of gastrointestinal flaps: potential implications for augmentation cystoplasty.

INTRODUCTION: Augmenting the bladder with a seromuscular gastrointestinal flap is a promising alternative approach aiming for a mucus-free bladder augmentation; however, the contraction (shrinkage) of the flaps remains a major concern. Enteric nervous system (ENS) abnormalities cause a failure of relaxation of the intestinal muscle layers in motility disorders such as Hirschsprung's disease and intestinal neuronal dysplasia. In mammals, the submucosal enteric nervous plexus contains nitrergic inhibitory motor neurons responsible for muscle relaxation. The authors hypothesize that mucosectomy disconnects the submucosal nervous plexus from the myenteric plexus resulting in flap shrinkage. STUDY DESIGN: After ethical approval, mucosectomy was performed on vascularized flaps from the ileum, colon, and stomach in five anesthetized pigs. In Group (I), only the mucosa was scraped off with forceps, creating a sero-musculo-submucosal flap, while in Group (II), the mucosa and submucosa were peeled off as one layer, leaving a seromuscular flap. Isolated and detubularized segments served as control. The width of each flap was measured before and after the mucosectomy. The ENS was assessed by neurofilament immunohistochemistry in conventional sections and by acetylcholinesterase and NADPH-diaphorase enzyme histochemistry in whole-mount preparations. RESULTS: The stomach contracted to a lesser extent of its original width, 92.82 ± 7.86% in Group (I) and 82.24 ± 6.96% in Group (II). The ileum contracted to 81.68 ± 4.25% in Group (I) and to 72.675 ± 5.36% in Group (II). The shrinkage was most noticeable in the colon: 83.89 ± 15.73% in Group (I) and to 57.13 ± 11.51% in Group (II). One-way equal variance test showed significant difference (P < 0,05) between Group (I) and (II), comparing stomach with ileum and ileum with colon. The histochemistry revealed that the submucosal nervous plexus containing nitrergic inhibitory neurons was disconnected from the myenteric plexus in Group (II) of all specimens. CONCLUSION: Mucosectomy resulted in significant immediate shrinkage of the flaps. This was more expressed when also the submucosa was peeled off, thus fully disrupting the ENS. The shrinkage affected the stomach the least and the colon the greatest. This phenomenon should be taken into consideration when planning mucus-free bladder augmentation.

Methane and Inflammation - A Review (Fight Fire with Fire).

Mammalian methanogenesis is regarded as an indicator of carbohydrate fermentation by anaerobic gastrointestinal flora. Once generated by microbes or released by a non-bacterial process, methane is generally considered to be biologically inactive. However, recent studies have provided evidence for methane bioactivity in various in vivo settings. The administration of methane either in gas form or solutions has been shown to have anti-inflammatory and neuroprotective effects in an array of experimental conditions, such as ischemia/reperfusion, endotoxemia and sepsis. It has also been demonstrated that exogenous methane influences the key regulatory mechanisms and cellular signalling pathways involved in oxidative and nitrosative stress responses. This review offers an insight into the latest findings on the multi-faceted organ protective activity of exogenous methane treatments with special emphasis on its versatile effects demonstrated in sepsis models.

Reduction of nitrosative stress by methane: Neuroprotection through xanthine oxidoreductase inhibition in a rat model of mesenteric ischemia-reperfusion.

Our aim was to characterize the main components of the nitrosative response with quantitative changes of the nitrergic myenteric neurons in adjacent intestinal segments after transient superior mesenteric artery occlusion. We also tested the hypothesis that exogenous methane may modulate the evolution of nitroxidation by influencing xanthine oxidoreductase (XOR) activity. The microcirculatory consequences of a 50 min ischemia or ischemia-reperfusion were investigated in anesthetized rats (n = 124) inhaling normoxic air with or without 2.2% methane. XOR activities, nitrogen monoxide (NO), nitrite/nitrate (NOx), and nitrotyrosine levels were measured, together with relative nitrergic neuron ratios from duodenum, ileum and colon samples. The effects of methane on XOR were also examined in vitro. The intramural flow stopped only in the ileum during ischemia. The highest baseline XOR activity was found in the duodenum, which increased further during ischemia. NO and nitrotyrosine levels rose, and the nNOS-immunopositive neuron ratio and NOx level both dropped. Reperfusion uniformly elevated XOR activity and nitrotyrosine formation, with the highest level attained in the duodenum, where the nitrergic neuron ratio remained depressed. These alterations were eliminated in methane-treated animals, XOR activity and nitrotyrosine formation decreased in all sites, and the duodenal nitrergic neuron ratio was re-established. The inhibitory effect of methane on XOR-linked nitrate reductase activity was also demonstrated in vitro. With segment-specific microcirculatory alterations, the risk for nitrosative stress is highest in transiently hypoxic tissues with high endogenous XOR activities. The XOR-inhibitory effect of methane can reduce nitroxidation and protects the nitrergic neuron population in such conditions.

Inhalation of methane preserves the epithelial barrier during ischemia and reperfusion in the rat small intestine.

BACKGROUND: Methane is part of the gaseous environment of the intestinal lumen. The purpose of this study was to elucidate the bioactivity of exogenous methane on the intestinal barrier function in an antigen-independent model of acute inflammation. METHODS: Anesthetized rats underwent sham operation or 45-min occlusion of the superior mesenteric artery. A normoxic methane (2.2%)-air mixture was inhaled for 15 min at the end of ischemia and at the beginning of a 60-min or 180-min reperfusion. The integrity of the epithelial barrier of the ileum was assessed by determining the lumen-to-blood clearance of fluorescent dextran, while microvascular permeability changes were detected by the Evans blue technique. Tissue levels of superoxide, nitrotyrosine, myeloperoxidase, and endothelin-1 were measured, the superficial mucosal damage was visualized and quantified, and the serosal microcirculation and mesenteric flow was recorded. Erythrocyte deformability and aggregation were tested in vitro. RESULTS: Reperfusion significantly increased epithelial permeability, worsened macro- and microcirculation, increased the production of proinflammatory mediators, and resulted in a rapid loss of the epithelium. Exogenous normoxic methane inhalation maintained the superficial mucosal structure, decreased epithelial permeability, and improved local microcirculation, with a decrease in reactive oxygen and nitrogen species generation. Both the deformability and aggregation of erythrocytes improved with incubation of methane. CONCLUSION: Normoxic methane decreases the signs of oxidative and nitrosative stress, improves tissue microcirculation, and thus appears to modulate the ischemia-reperfusion-induced epithelial permeability changes. These findings suggest that the administration of exogenous methane may be a useful strategy for maintaining the integrity of the mucosa sustaining an oxido-reductive attack.

Alleviated mucosal and neuronal damage in a rat model of Crohn's disease.

AIM: To establish a rat model suitable to investigate the repetitive relapsing inflammations (RRI) characteristic to Crohn's disease. METHODS: Colitis was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS). RRI were mimicked by repeating administrations of TNBS. Tissue samples were taken from control, once, twice and three times treated rats from the inflamed and adjacent non-inflamed colonic segments at different timepoints during the acute intestinal inflammation. The means of the ulcerated area were measured to evaluate the macroscopic mucosal damage. The density of myenteric neurons was determined on whole mounts by HuC/HuD immunohistochemistry. Heme oxygenase-1 (HO-1) expression was evaluated by molecular biological techniques. RESULTS: TNBS-treated rats displayed severe colitis, but the mortality was negligible, and an increase of body weight was characteristic throughout the experimental period. The widespread loss of myenteric neurons, and marked but transient HO-1 up-regulation were demonstrated after the first TNBS administration. After repeated doses the length of the recovery time and extent of the ulcerous colonic segments were markedly decreased, and the neuronal loss was on a smaller scale and was limited to the inflamed area. HO-1 mRNA level was notably greater than after a single dose and overexpression was sustained throughout the timepoints examined. Nevertheless, the HO-1 protein up-regulation after the second TNBS treatment proved to be transient. Following the third treatment HO-1 protein expression could not be detected. CONCLUSION: Experimentally provoked RRI may exert a protective preconditioning effect against the mucosal and neuronal damage. The persistent up-regulation of HO-1 mRNA expression may correlate with this.

Gut region-specific rearrangement of the cellular and subcellular compartments of nitric oxide synthase isoforms after chronic ethanol consumption in rats.

We recently provided evidence of cell-type-specific differences in the subcellular distributions of the three nitric oxide synthase (NOS) isoforms in the myenteric neurons, enteric smooth muscle cells and the capillary endothelium of the rat duodenum. We hypothesized that the presence of three NOS isoforms in the same type of cells with differences in subcellular compartmentalization might reflect a functional plasticity. Therefore, investigation of the possible rearrangement of cellular and subcellular NOS compartments in different gut segments following chronic ethanol treatment was the aim of this study. Rats were randomly divided into two groups and received water or 20% ethanol solution, preceded by short periods of adaptation with 10% and 15% ethanol. After 8 weeks, segments of duodenum, ileum and colon of the control and the alcohol-treated rats were processed for post-embedding immunohistochemistry and RT-PCR. The quantitative differences in the numbers of gold particles indicative of the different NOSs and their relative mRNA levels between the two experimental groups varied greatly, depending on the gut segment, and also on the cellular and subcellular compartments investigated. The chronic ethanol administration had the opposite effect on the quantitative distribution of the neuronal and endothelial NOS labelling gold particles in the different cellular compartments and resulted in subcellular rearrangement of NOS labels along the gastrointestinal tract. The intestinal region-specific rearrangement of the cellular and subcellular NOS compartments may possibly result in functional plasticity and help to maintain the optimum NO level under pathological conditions.

Spatiotemporal expression pattern of DsRedT3/CCK gene construct during postnatal development of myenteric plexus in transgenic mice.

Cholecystokinin (CCK) is an early marker of both neuronal and endocrine cell lineages in the developing gastrointestinal tract. To determine the quantitative properties and the spatial distribution of the CCK-expressing myenteric neurones in early postnatal life, a transgenic mouse strain with a CCK promoter-driven red fluorescent protein (DsRedT3/CCK) was established. The cell-specific expression of DsRedT3/CCK was validated by in situ hybridization with a CCK antisense riboprobe and by in situ hybridization coupled with immunohistochemistry involving a monoclonal antibody to CCK. A gradual increase in the DsRedT3/CCK-expressing enteric neurones with clear regional differences was documented from birth until the suckling to weaning transition, in parallel with the period of rapid intestinal growth and functional maturation. To evaluate the proportion of myenteric neurones in which DsRedT3/CCK transgene expression was colocalized with the enteric neuronal marker peripherin, immunofluorescence techniques were applied. All DsRedT3/CCK neurones were peripherin-immunoreactive and the proportion of DsRedT3/CCK-expressing myenteric neurones in the duodenum was the highest after the third week of life, when the number of peripherin-immunoreactive myenteric neurones in this region had decreased. Nearly all of the DsRedT3/CCK-expressing neurones also expressed 5-hydroxytryptophan (5-HT). Thus, by utilizing a new transgenic mouse strain, we have demonstrated a small number of CCK-expressing myenteric neurones with a developmentally regulated spatiotemporal distribution. The coexistence of CCK and 5-HT in the majority of these neurones suggests their possible regulatory role in feeding at the suckling to weaning transition.

Gut region-specific diabetic damage to the capillary endothelium adjacent to the myenteric plexus.

OBJECTIVE: Damage in the capillaries supplying the MP has been proposed as a critical factor in the development of diabetic enteric neuropathy. We therefore investigated connections between STZ-induced diabetes and the BM morphology, the size of caveolar compartments, the width of TJs, the transport of albumin, and the quantitative features of Cav-1 and eNOS expression in these microvessels. METHODS: Gut segments from diabetic rats were compared with those from insulin-treated diabetics and those from controls. The effects of diabetes on the BM, the caveolar compartments, and the TJs were evaluated morphometrically. The quantitative features of the albumin transport were investigated by postembedding immunohistochemistry. The diabetes-related changes in Cav-1 and eNOS expression were assessed by postembedding immunohistochemistry and molecular method. RESULTS: Thickening of the BM, enlargement of the caveolar compartments, opening of the junctions, enhanced transport of albumin, and overexpression of Cav-1 and eNOS were documented in diabetic animals. Insulin replacement in certain gut segments prevented the development of these alterations. CONCLUSIONS: These data provide morphological, functional, and molecular evidence that the endothelial cells in capillaries adjacent to the MP is a target of diabetic damage in a regional manner.