Local carbachol application induces oral microvascular recruitment and improves gastric tissue oxygenation during hemorrhagic shock in dogs.

Introduction: Hemorrhagic shock is characterized by derangements of the gastrointestinal microcirculation. Topical therapy with nitroglycerine or iloprost improves gastric tissue oxygenation but not regional perfusion, probably due to precapillary adrenergic innervation. Therefore, this study was designed to investigate the local effect of the parasympathomimetic carbachol alone and in combination with either nitroglycerine or iloprost on gastric and oral microcirculation during hemorrhagic shock. Methods: In a cross-over design five female foxhounds were repeatedly randomized into six experimental groups. Carbachol, or carbachol in combination with either nitroglycerine or iloprost were applied topically to the oral and gastric mucosa. Saline, nitroglycerine, or iloprost application alone served as control groups. Then, a fixed-volume hemorrhage was induced by arterial blood withdrawal followed by blood retransfusion after 1h of shock. Gastric and oral microcirculation was determined using reflectance spectrophotometry and laser Doppler flowmetry. Oral microcirculation was visualized with videomicroscopy. Statistics: 2-way-ANOVA for repeated measurements and Bonferroni post-hoc analysis (mean ± SEM; p < 0.05). Results: The induction of hemorrhage led to a decrease of gastric and oral tissue oxygenation, that was ameliorated by local carbachol and nitroglycerine application at the gastric mucosa. The sole use of local iloprost did not improve gastric tissue oxygenation but could be supplemented by local carbachol treatment. Adding carbachol to nitroglycerine did not further increase gastric tissue oxygenation. Gastric microvascular blood flow remained unchanged in all experimental groups. Oral microvascular blood flow, microvascular flow index and total vessel density decreased during shock. Local carbachol supply improved oral vessel density during shock and oral microvascular flow index in the late course of hemorrhage. Conclusion: The specific effect of shifting the autonomous balance by local carbachol treatment on microcirculatory variables varies between parts of the gastrointestinal tract. Contrary to our expectations, the improvement of gastric tissue oxygenation by local carbachol or nitroglycerine application was not related to increased microvascular perfusion. When carbachol is used in combination with local vasodilators, the additional effect on gastric tissue oxygenation depends on the specific drug combination. Therefore, modulation of tissue oxygen consumption, mitochondrial function or alterations in regional blood flow distribution should be investigated.

Pravastatin Improves Colonic and Hepatic Microcirculatory Oxygenation during Sepsis without Affecting Mitochondrial Function and ROS Production in Rats.

Microcirculatory and mitochondrial dysfunction are considered the main mechanisms of septic shock. Studies suggest that statins modulate inflammatory response, microcirculation, and mitochondrial function, possibly through their action on peroxisome proliferator-activated receptor alpha (PPAR-α). The aim of this study was to examine the effects of pravastatin on microcirculation and mitochondrial function in the liver and colon and the role of PPAR-α under septic conditions. This study was performed with the approval of the local animal care and use committee. Forty Wistar rats were randomly divided into 4 groups: sepsis (colon ascendens stent peritonitis, CASP) without treatment as control, sepsis + pravastatin, sepsis + PPAR-α-blocker GW6471, and sepsis + pravastatin + GW6471. Pravastatin (200 µg/kg s.c.) and GW6471 (1 mg/kg) were applied 18 h before CASP-operation. 24 h after initial surgery, a relaparotomy was performed, followed by a 90 min observation period for assessment of microcirculatory oxygenation (µHbO2) of the liver and colon. At the end of the experiments, animals were euthanized, and the colon and liver were harvested. Mitochondrial function was measured in tissue homogenates using oximetry. The ADP/O ratio and respiratory control index (RCI) for complexes I and II were calculated. Reactive oxygen species (ROS) production was assessed using the malondialdehyde (MDA)-Assay. Statistics: two-way analysis of variance (ANOVA) + Tukey's/Dunnett's post hoc test for microcirculatory data, Kruskal-Wallis test + Dunn's post hoc test for all other data. In control septic animals µHbO2 in liver and colon deteriorated over time (µHbO2: -9.8 ± 7.5%* and -7.6 ± 3.3%* vs. baseline, respectively), whereas after pravastatin and pravastatin + GW6471 treatment µHbO2 remained constant (liver: µHbO2 pravastatin: -4.21 ± 11.7%, pravastatin + GW6471: -0.08 ± 10.3%; colon: µHbO2 pravastatin: -0.13 ± 7.6%, pravastatin + GW6471: -3.00 ± 11.24%). In both organs, RCI and ADP/O were similar across all groups. The MDA concentration remained unchanged in all groups. Therefore, we conclude that under septic conditions pravastatin improves microcirculation in the colon and liver, and this seems independent of PPAR-α and without affecting mitochondrial function.

Gemfibrozil Improves Microcirculatory Oxygenation of Colon and Liver without Affecting Mitochondrial Function in a Model of Abdominal Sepsis in Rats.

Recent studies observed, despite an anti-hyperlipidaemic effect, a positive impact of fibrates on septic conditions. This study evaluates the effects of gemfibrozil on microcirculatory variables, mitochondrial function, and lipid peroxidation levels with regard to its potential role as an indicator for oxidative stress in the colon and liver under control and septic conditions and dependencies on PPARα-mediated mechanisms of action. With the approval of the local ethics committee, 120 Wistar rats were randomly divided into 12 groups. Sham and septic animals were treated with a vehicle, gemfibrozil (30 and 100 mg/kg BW), GW 6471 (1 mg/kg BW, PPARα inhibitor), or a combination of both drugs. Sepsis was induced via the colon ascendens stent peritonitis (CASP) model. Then, 24 h post sham or CASP surgery, a re-laparotomy was performed. Measures of vital parameters (heart rate (HR), mean arterial pressure (MAP), and microcirculation (µHbO2)) were recorded for 90 min. Mitochondrial respirometry and assessment of lipid peroxidation via a malondialdehyde (MDA) assay were performed on colon and liver tissues. In the untreated sham animals, microcirculation remained stable, while pre-treatment with gemfibrozil showed significant decreases in the microcirculatory oxygenation of the colon. In the CASP animals, µHbO2 levels in the colon and the liver were significantly decreased 90 min after laparotomy. Pre-treatment with gemfibrozil prevented the microcirculatory aberrations in both organs. Gemfibrozil did not affect mitochondrial function and lipid peroxidation levels in the sham or CASP animals. Gemfibrozil treatment influences microcirculation depending on the underlying condition. Gemfibrozil prevents sepsis-induced microcirculatory aberrances in the colon and liver PPARα-independently. In non-septic animals, gemfibrozil impairs the microcirculatory variables in the colon without affecting those in the liver.

A Toolbox to Investigate the Impact of Impaired Oxygen Delivery in Experimental Disease Models.

Impaired oxygen utilization is the underlying pathophysiological process in different shock states. Clinically most important are septic and hemorrhagic shock, which comprise more than 75% of all clinical cases of shock. Both forms lead to severe dysfunction of the microcirculation and the mitochondria that can cause or further aggravate tissue damage and inflammation. However, the detailed mechanisms of acute and long-term effects of impaired oxygen utilization are still elusive. Importantly, a defective oxygen exploitation can impact multiple organs simultaneously and organ damage can be aggravated due to intense organ cross-talk or the presence of a systemic inflammatory response. Complexity is further increased through a large heterogeneity in the human population, differences in genetics, age and gender, comorbidities or disease history. To gain a deeper understanding of the principles, mechanisms, interconnections and consequences of impaired oxygen delivery and utilization, interdisciplinary preclinical as well as clinical research is required. In this review, we provide a "tool-box" that covers widely used animal disease models for septic and hemorrhagic shock and methods to determine the structure and function of the microcirculation as well as mitochondrial function. Furthermore, we suggest magnetic resonance imaging as a multimodal imaging platform to noninvasively assess the consequences of impaired oxygen delivery on organ function, cell metabolism, alterations in tissue textures or inflammation. Combining structural and functional analyses of oxygen delivery and utilization in animal models with additional data obtained by multiparametric MRI-based techniques can help to unravel mechanisms underlying immediate effects as well as long-term consequences of impaired oxygen delivery on multiple organs and may narrow the gap between experimental preclinical research and the human patient.

Local Mucosal CO2 but Not O2 Insufflation Improves Gastric and Oral Microcirculatory Oxygenation in a Canine Model of Mild Hemorrhagic Shock.

Introduction: Acute hemorrhage results in perfusion deficit and regional hypoxia. Since failure of intestinal integrity seem to be the linking element between hemorrhage, delayed multi organ failure, and mortality, it is crucial to maintain intestinal microcirculation in acute hemorrhage. During critical bleeding physicians increase FiO2 to raise total blood oxygen content. Likewise, a systemic hypercapnia was reported to maintain microvascular oxygenation (µHbO2). Both, O2 and CO2, may have adverse effects when applied systemically that might be prevented by local application. Therefore, we investigated the effects of local hyperoxia and hypercapnia on the gastric and oral microcirculation. Methods: Six female foxhounds were anaesthetized, randomized into eight groups and tested in a cross-over design. The dogs received a local CO2-, O2-, or N2-administration to their oral and gastric mucosa. Hemorrhagic shock was induced through a withdrawal of 20% of estimated blood volume followed by retransfusion 60 min later. In control groups no shock was induced. Reflectance spectrophotometry and laser Doppler were performed at the gastric and oral surface. Oral microcirculation was visualized by incident dark field imaging. Systemic hemodynamic parameters were recorded continuously. Statistics were performed using a two-way-ANOVA for repeated measurements and post hoc analysis was conducted by Bonferroni testing (p < 0.05). Results: The gastric µHbO2 decreased from 76 ± 3% to 38 ± 4% during hemorrhage in normocapnic animals. Local hypercapnia ameliorated the decrease of µHbO2 from 78 ± 4% to 51 ± 8%. Similarly, the oral µHbO2 decreased from 81 ± 1% to 36 ± 4% under hemorrhagic conditions and was diminished by local hypercapnia (54 ± 4%). The oral microvascular flow quality but not the total microvascular blood flow was significantly improved by local hypercapnia. Local O2-application failed to change microvascular oxygenation, perfusion or flow quality. Neither CO2 nor O2 changed microcirculatory parameters and macrocirculatory hemodynamics under physiological conditions. Discussion: Local hypercapnia improved microvascular oxygenation and was associated with a continuous blood flow in hypercapnic individuals undergoing hemorrhagic shock. Local O2 application did not change microvascular oxygenation, perfusion and blood flow profiles in hemorrhage. Local gas application and change of microcirculation has no side effects on macrocirculatory parameters.