Continuous enteral protease inhibition as a novel treatment for experimental trauma/hemorrhagic shock.

PURPOSE: Trauma and hemorrhagic shock (T/HS) is a major cause of morbidity and mortality. Existing treatment options are largely limited to source control and fluid and blood repletion. Previously, we have shown that enteral protease inhibition improves outcomes in experimental models of T/HS by protecting the gut from malperfusion and ischemia. However, enteral protease inhibition was achieved invasively, by laparotomy and direct injection of tranexamic acid (TXA) into the small intestine. In this study, we tested a minimally invasive method of enteral protease inhibitor infusion in experimental T/HS that can be readily adapted for clinical use. METHODS: Wistar rats were exsanguinated to a mean arterial blood pressure (MABP) of 40 mmHg, with laparotomy to induce trauma. Hypovolemia was maintained for 120 min and was followed by reperfusion of shed blood. Animals were monitored for an additional 120 min. A modified orogastric multi-lumen tube was developed to enable rapid enteral infusion of a protease inhibitor solution while simultaneously mitigating risk of reflux aspiration into the airways. The catheter was used to deliver TXA (T/HS + TXA) or vehicle (T/HS) continuously into the proximal small intestine, starting 20 min into the ischemic period. RESULTS: Rats treated with enteral protease inhibition (T/HS + TXA) displayed improved outcomes compared to control animals (T/HS), including significantly improved MABP (p = 0.022) and lactate (p = 0.044). Mass spectrometry-based analysis of the plasma peptidome after T/HS indicated mitigation of systemic proteolysis in T/HS + TXA. CONCLUSION: Minimally invasive, continuous enteral protease inhibitor delivery improves outcomes in T/HS and is readily translatable to the clinical arena.

Volatile Decay Products in Breath During Peritonitis Shock are Attenuated by Enteral Blockade of Pancreatic Digestive Proteases.

There is a need to develop markers for early detection of organ failure in shock that can be noninvasively measured at point of care. We explore here the use of volatile organic compounds (VOCs) in expired air in a rat peritonitis shock model. Expired breath samples were collected into Tedlar gas bags and analyzed by standardized gas chromatography. The gas chromatograms were digitally analyzed for presence of peak amounts over a range of Kovach indices. Following the induction of peritonitis, selected volatile compounds were detected within about 1 h, which remained at elevated amounts over a 6 h observation period. These VOCs were not present in control animals without peritonitis. Comparisons with know VOCs indicate that they include 1,4-diaminobutane and trimethylamine N-oxide. When pancreatic digestive proteases were blocked with tranexamic acid in the intestine and peritoneum, a procedure that serves to reduce organ failure in shock, the amounts of VOCs in the breath decreased spontaneously to control values without peritonitis. These results indicate that peritonitis shock is accompanied by development of volatile organic compounds that may be generated by digestive enzymes in the small intestine. VOCs may serve as indicators for detection of early forms of autodigestion by digestive proteases.

Peptidomic Analysis of Rat Plasma: Proteolysis in Hemorrhagic Shock.

It has been previously shown that intestinal proteases translocate into the circulation during hemorrhagic shock and contribute to proteolysis in distal organs. However, consequences of this phenomenon have not previously been investigated using high-throughput approaches. Here, a shotgun label-free quantitative proteomic approach was utilized to compare the peptidome of plasma samples from healthy and hemorrhagic shock rats to verify the possible role of uncontrolled proteolytic activity in shock. Plasma was collected from rats after hemorrhagic shock (HS) consisting of 2-h hypovolemia followed by 2-h reperfusion, and from healthy control (CTRL) rats. A new two-step enrichment method was applied to selectively extract peptides and low molecular weight proteins from plasma, and directly analyze these samples by tandem mass spectrometry. One hundred twenty-six circulating peptides were identified in CTRL and 295 in HS animals. Ninety-six peptides were present in both conditions; of these, 57 increased and 30 decreased in shock. In total, 256 peptides were increased or present only in HS confirming a general increase in proteolytic activity in shock. Analysis of the proteases that potentially generated the identified peptides suggests that the larger relative contribution to the proteolytic activity in shock is due to chymotryptic-like proteases. These results provide quantitative confirmation that extensive, system-wide proteolysis is part of the complex pathologic phenomena occurring in hemorrhagic shock.

Pancreatic digestive enzyme blockade in the small intestine prevents insulin resistance in hemorrhagic shock.

Hemorrhagic shock is associated with metabolic defects, including hyperglycemia and insulin resistance, but the mechanisms are unknown. We recently demonstrated that reduction of the extracellular domain of the insulin receptor by degrading proteases may lead to a reduced ability to maintain normal plasma glucose values. In shock, transfer of digestive enzymes from the lumen of the intestine into the systemic circulation after breakdown of the intestinal mucosal barrier causes inflammation and organ dysfunction. Suppression of the digestive enzymes in the lumen of the intestine with protease inhibitors is effective in reducing the level of the inflammatory reactions. To determine the degree to which blockade of digestive enzymes affects insulin resistance in shock, rats were exposed to acute hemorrhagic shock (mean arterial pressure of 30 mmHg for 2 h) at which time all shed blood volume was returned. Digestive proteases in the intestine were blocked with a serine protease inhibitor (tranexamic acid in polyethylene glycol and physiological electrolyte solution), and the density of the insulin receptor was measured with immunohistochemistry in the mesentery microcirculation. The untreated rat without enzyme blockade had significantly attenuated levels of insulin receptor density as compared with control and treated rats. Blockade of the digestive proteases after 60 min of hypotension in the lumen of the small intestine led to a lesser decrease in insulin receptor density compared with controls without protease blockade. Glucose tolerance test indicates a significant increase in plasma glucose levels 2 h after hemorrhagic shock, which are reduced to control values in the presence of protease inhibition in the lumen of the intestine. The transient reduction of the plasma glucose levels after an insulin bolus is significantly attenuated after shock but is restored when digestive enzymes in the lumen of the intestine are blocked. These results suggest that in hemorrhagic shock elevated microvascular extracellular digestive enzyme activity causes insulin receptor dysfunction, hyperglycemia, and reduced ability to regulate blood glucose values.

Pancreatic digestive enzyme blockade in the intestine increases survival after experimental shock.

Shock, sepsis, and multiorgan failure are associated with inflammation, morbidity, and high mortality. The underlying pathophysiological mechanism is unknown, but evidence suggests that pancreatic enzymes in the intestinal lumen autodigest the intestine and generate systemic inflammation. Blocking these enzymes in the intestine reduces inflammation and multiorgan dysfunction. We investigated whether enzymatic blockade also reduces mortality after shock. Three rat shock models were used here: hemorrhagic shock, peritonitis shock induced by placement of cecal material into the peritoneum, and endotoxin shock. One hour after initiation of hemorrhagic, peritonitis, or endotoxin shock, animals were administered one of three different pancreatic enzyme inhibitors--6-amidino-2-naphtyl p-guanidinobenzoate dimethanesulfate, tranexamic acid, or aprotinin--into the lumen of the small intestine. In all forms of shock, blockade of digestive proteases with protease inhibitor attenuated entry of digestive enzymes into the wall of the intestine and subsequent autodigestion and morphological damage to the intestine, lung, and heart. Animals treated with protease inhibitors also survived in larger numbers than untreated controls over a period of 12 weeks. Surviving animals recovered completely and returned to normal weight within 14 days after shock. The results suggest that the active and concentrated digestive enzymes in the lumen of the intestine play a central role in shock and multiorgan failure, which can be treated with protease inhibitors that are currently available for use in the clinic.

Receptor cleavage and P-selectin-dependent reduction of leukocyte adhesion in the spontaneously hypertensive rat.

The SHR, a genetic model for hypertension and the metabolic syndrome, has attenuated leukocyte adhesion to the postcapillary endothelium by an unknown mechanism. Based on recent evidence of elevated levels of MMPs in plasma and on microvascular endothelium of the SHR with cleavage of several receptor types, we hypothesize that the reduced leukocyte-endothelial interaction is a result of enhanced proteolytic cleavage of P-selectin on the postcapillary endothelium and PSGL-1 on leukocytes. The attenuated rolling interactions of SHR leukocytes with the endothelium were restored by chronic treatment with a broad-spectrum MMP inhibitor (CGS) for 24 weeks. The SHR MMP levels, in plasma and mesentery, as well as the systolic blood pressure, decreased significantly with treatment. In the SHR mesentery, labeling of P-selectin in the postcapillary venules by immunohistochemistry demonstrated, on average, a 31% lower extracellular P-selectin density compared with the normotensive WKY. A significantly lower extracellular PSGL-1 density on the membranes of SHR neutrophils compared with the WKY also supported our hypothesis. In vivo stimulation of the mesenteric postcapillary venules with histamine demonstrated that the SHR had an attenuated response, as measured by leukocyte rolling velocity on the endothelium. The reduced P-selectin and PSGL-1 density, on SHR postcapillary endothelium and on SHR leukocytes, respectively, was restored significantly by chronic MMP inhibition. The impaired ability of SHR leukocytes to reduce rolling velocity upon inflammatory stimulation led to fewer firmly adhered leukocytes to the endothelium as a contributor to immune suppression.

An elementary analysis of physiologic shock and multi-organ failure: the autodigestion hypothesis.

Physiological shock and subsequent multi-organ failure is one of the most important medical problems from a mortality point of view. No agreement exists for mechanisms that lead to the relative rapid cell and organ failure during this process and no effective treatment. We postulate that the digestive enzymes synthesized in the pancreas and transported in the lumen of the small intestine as requirement of normal food digestion play a central role in multi-organ failure. These powerful enzymes are usually compartmentalized in the lumen of the intestine by the mucosal barrier, but may escape into the wall of the intestine if the permeability of the mucosal lining increases. Entry of the digestive enzymes into the wall of the intestine precipitates an autodigestion process as well as an escape of pancreatic enzymes and breakdown products generated by them into the system circulation. The consequence of autodigestion is multiorgan failure. We discuss the possibility to block the digestive enzymes in acute forms of shock as a potential therapeutic intervention.

Analysis of exhaled volatile compounds following acute superior mesenteric artery occlusion in a pilot rat study.

BACKGROUND: Prompt diagnosis and treatment of acute mesenteric ischemia (AMI) requires a high index of suspicion for timely management. Poor clinical outcomes and delays in surgical treatment are demonstrated even in modern clinical series. Recognition of exhaled volatile organic compounds (VOCs) specific to AMI may facilitate early detection and diagnosis and improve patient outcomes. METHODS: Adult Wistar rats (n = 5) were intubated and anesthetized, and control tracheostomy breath samples were collected using Tedlar gas sample bags. Intestinal ischemia was induced by placing an occlusive clip across the superior mesenteric artery, and breath samples were collected after 1 hour of intestinal ischemia and after 15 minutes of intestinal reperfusion. Gas chromatography was used to identify and measure levels of VOCs obtained, and measured retention indices were compared with known values in the Kovats retention index database. RESULTS: Multiple retention indices (n = 41) were noted on gas chromatography, representing a variety of VOCs detected. Z,Z-farnesol (C15H26O), an isoprenoid, was the only compound detected that was undetectable during the control phase (median = 0 cts/sec) but which significantly elevated during the ischemic (median = 34 cts/sec, range = 25-37) and reperfusion (median = 148 cts/sec, range = 42-246) phases. Three other isoprenoid compounds (E,E-alpha-farnesene, germacrene A, and Z,Z-4,6,8-megastigmatriene) were also detected in all five animals, but their levels did not differ significantly between control, ischemic, and reperfusion phases. CONCLUSIONS: This pilot study demonstrates the feasibility of analyzing exhaled VOCs using a novel rat model for AMI. These findings may be useful for the development and identification of similar assays for the rapid diagnosis of AMI.

Matrix metalloproteinase activity causes VEGFR-2 cleavage and microvascular rarefaction in rat mesentery.

A complication of the spontaneously hypertensive rat (SHR) is microvascular rarefaction, defined by the loss of microvessels. However, the molecular mechanisms involved in this process remain incompletely identified. Recent work in our laboratory suggests that matrix metalloproteinases (MMPs) may play a role by cleavage of the vascular endothelial growth factor receptor 2 (VEGFR-2). In order to further delineate the role for MMPs in microvascular rarefaction, the objective of the current study was to examine the relationship in the same tissue between MMP activity, VEGFR-2 cleavage and rarefaction. Using an in vivo microzymographic technique, we show significantly enhanced levels of MMP-1, -1/-9, -7, and -8 activities, but not MMP-2 and -3 activities, along mesenteric microvessels of the SHR compared to its normotensive control, Wistar Kyoto rat. Based on immunohistochemical methods, the SHR exhibited a decreased labeling of the extracellular, but not the intracellular, domain of VEGFR-2 along mesenteric microvessels. Chronic MMP inhibition served to attenuate VEGFR-2 cleavage and microvascular network rarefaction in the SHR mesentery. These results spatially link MMP-induced VEGFR-2 cleavage and rarefaction in the mesentery of the SHR and thus support the hypothesis that MMPs serve as regulators of microvascular dysfunction in hypertension.

A New Hypothesis for Insulin Resistance in Hypertension Due to Receptor Cleavage.

BACKGROUND: One of the most important unresolved issues in diabetes is the mechanism for the attenuated response to insulin, i.e. insulin resistance. AIMS AND METHODS: We hypothesize that the mechanism for the insulin resistance is due to uncontrolled protease activity in the plasma, on endothelial cells and in the tissue parenchyma. To examine this hypothesis we use of microzymographic techniques in the microcirculation, plasma zymography, and receptor labeling techniques with antibodies against an extracellular domain of the insulin receptor α. RESULTS: The spontaneously hypertensive rat has an enhanced proteolytic activity and significant cleavage of the receptor with attenuated glucose transport. We present evidence for insulin receptor cleavage in a high fat diet and a transgenic model of diabetes. CONCLUSION: These results suggest that cleavage of the extracellular domain of the insulin receptor, a situation that interferes with the ability for insulin to bind and provide an intracellular signal for glucose transport, may be involved in insulin resistance.

Receptor cleavage reduces the fluid shear response in neutrophils of the spontaneously hypertensive rat.

Physiological fluid shear stress evokes pseudopod retraction in normal leukocytes by a mechanism that involves the formyl peptide receptor (FPR) as mechanosensor. In hypertensives, such as the spontaneously hypertensive rat (SHR), leukocytes lack the normal fluid shear response. The increased activity of matrix metalloproteinases (MMPs, including MMP-9) in SHR plasma is associated with cleavage of several cell membrane receptors. We hypothesize that the attenuated fluid shear response in leukocytes (neutrophils) of the SHR is due to extracellular proteolytic cleavage of the FPR. We show that suspended SHR neutrophils in whole blood sheared in a cone-and-plate device or individual neutrophils adherent to a glass surface and subject to fluid shear exhibited reduced pseudopod retractions compared with neutrophils of control Wistar-Kyoto (WKY) rats. SHR neutrophils and naïve Wistar rat neutrophils exposed to SHR plasma also exhibited impaired fluid shear responses as shown by their inability to project pseudopods with fluid shear. Labeling of extracellular FPR revealed that the FPR density in SHR neutrophils is on average 27% reduced compared with those of the WKY rats. Exposure of Wistar rat neutrophils to the gelatinase MMP-9 (final concentration 5 nM) led to attenuation of fluid shear response and decrease in extracellular FPR density. Chronic treatment of the SHR with a broad-acting MMP inhibitor, doxycycline, significantly improved the fluid shear response and increased the FPR extracellular density of SHR neutrophils. These results suggest that proteolytic cleavage of the FPR may interfere with normal fluid shear-induced pseudopod retractions in SHR neutrophils.

Enhanced matrix metalloproteinase activity in the spontaneously hypertensive rat: VEGFR-2 cleavage, endothelial apoptosis, and capillary rarefaction.

Besides an elevated blood pressure, the spontaneously hypertensive rat (SHR) has multiple microvascular complications including endothelial apoptosis with capillary rarefaction. The SHR also has elevated levels of proteolytic (e.g. matrix metalloproteinase, MMP) activity and apoptosis in microvascular cells compared to its normotensive control, but the specific enzymes involved and the molecular mechanism for apoptosis are unknown. We hypothesize that selected MMPs cleave the extracellular domain of vascular endothelial growth factor receptor-2 (VEGFR-2), which in turn causes endothelial apoptosis and capillary rarefaction. Zymographic analysis shows that gelatinase (MMP-2 and MMP-9) and matrilysin (MMP-7) activities are significantly enhanced in SHR plasma. The SHR has lower levels of the extracellular domains of VEGFR-2 in cardiac microvessels. Furthermore, application of plasma from the SHR, or purified MMP-9 and MMP-7 to naïve cells causes cleavage of the extracellular domain of VEGFR-2. The receptor cleavage was blocked by broad-acting MMP inhibitors (GM6001 1 microM, EDTA 10 mM, or doxycycline 11.3 microM). Chronic MMP inhibition (doxycycline, 5.4 mg/kg/day, 24 weeks) attenuated VEGFR-2 cleavage, endothelial apoptosis, and capillary rarefaction in the SHR. These results suggest elevated plasma MMP activities may cleave VEGFR-2, resulting in endothelial apoptosis and capillary rarefaction in the SHR.

Proteinase activity and receptor cleavage: mechanism for insulin resistance in the spontaneously hypertensive rat.

Arterial hypertension is associated with organ dysfunctions, but the mechanisms are uncertain. We hypothesized that enhanced proteolytic activity in the microcirculation of spontaneously hypertensive rats (SHRs) may be a pathophysiological mechanism causing cell membrane receptor cleavage and examine this for 2 different receptors. Immunohistochemistry of matrix-degrading metalloproteinases (matrix metalloproteinase [MMP]-9) protein shows enhanced levels in SHR microvessels, mast cells, and leukocytes compared with normotensive Wistar-Kyoto rats. In vivo microzymography shows cleavage by MMP-1 and -9 in SHRs that colocalizes with MMP-9 and is blocked by metal chelation. SHR plasma also has enhanced protease activity. We demonstrate with an antibody against the extracellular domain that the insulin receptor-alpha density is reduced in SHRs, in line with elevated blood glucose levels and glycohemoglobin. There is also cleavage of the binding domain of the leukocyte integrin receptor CD18 in line with previously reported reduced leukocyte adhesion. Blockade of MMPs with a broad-acting inhibitor (doxycycline, 5.4 mg/kg per day) reduces protease activity in plasma and microvessels; blocks the proteolytic cleavage of the insulin receptor, the reduced glucose transport; normalizes blood glucose levels and glycohemoglobin levels; and reduces blood pressure and enhanced microvascular oxidative stress of SHRs. The results suggest that elevated MMP activity leads to proteolytic cleavage of membrane receptors in the SHR, eg, cleavage of the insulin receptor-binding domain associated with insulin resistance.

The primary valves in the initial lymphatics during inflammation.

BACKGROUND: The primary valve system in the initial lymphatics prevents fluid transport from the initial lymphatics back into the interstitium. The authors hypothesize that since the primary valves are made up of an extraordinarily thin endothelium, they are readily compromised by mechanical or biochemical inflammatory stimuli. Thus, the opening dimension of the primary valves and their ability to prevent reflux into the interstitium during inflammation were investigated. METHODS AND RESULTS: Acute inflammation was generated in the intact rat spinotrapezius muscle by suffusion of f-Met-Leu-Phe and platelet-activating factor. Once inflamed, the effective opening dimensions of the primary valves and the transport back out of the initial lymphatics were determined by examining the transport of fluorescent tracers from the interstitium to the lymphatics. Quantum dots and fluorescently labeled albumin readily enter initial lymphatics from the interstitium. The maximum diameter of microspheres that enter the initial lymphatics is between 0.5 microm and 0.8 microm in both control and inflamed tissue. While under control conditions no quantum dots escaped from initial lymphatics back into the interstitium, during inflammation there was extensive escape of quantum dots. CONCLUSIONS: These results suggest that, in acute inflammation, the function of the endothelial barriers in the initial lymphatics may be compromised. A failure of the primary lymphatic valves has two consequences. First, fluid clearance from the tissue is less efficient, which causes the level of edema to increase. Second, the leaking initial lymphatics allow inflammatory mediators to accumulate in the tissue, therefore enhancing interstitial and lymphatic inflammatory reactions.

Microvascular display of xanthine oxidase and NADPH oxidase in the spontaneously hypertensive rat.

OBJECTIVE: Oxygen free radical production in hypertension may be associated with elevated arteriolar tone and organ injury. Previous results suggest an enhanced level of oxygen free radical formation in microvascular endothelium and in circulating neutrophils associated with xanthine oxidase activity in the spontaneously hypertensive rats (SHR) compared with their normotensive controls, the Wistar Kyoto rats (WKY). The aim of this study was to gain more detailed understanding of where oxidative enzymes are located in the microcirculation. METHODS: An approach was developed to delineate the cellular distribution of two selected oxidative enzymes, xanthine oxidase and nicotinamide adenine dinucleotide phosphate (NADPH) dependent oxidase (protein 67-kDa fraction). Immunolabeling with peroxidase substrate was utilized, which permits full delineation of the primary antibody in all microvascular structures of the mesentery. RESULTS: Xanthine oxidase is present in the endothelium of all segments of the microcirculation, in mast cells, and in parenchymal cells of the mesentery. NADPH oxidase can be detected in the endothelium, leukocytes, and mast cells and with lower levels in parenchymal cells. The mesentery of WKY and SHR has similar enzyme distributions with enhancements on the arteriolar and venular side of the microcirculation that coincide with the sites of enhanced free radical production recently reported. Immune label measurements under standardized conditions indicate that both enzymes are significantly enhanced in the SHR. Adrenalectomy, which serves to reduce the blood pressure and free radical production of the SHR to normotensive levels, leads to a reduction of NADPH and xanthine oxidase to normotensive levels, while supplementation of adrenalectomized SHR with dexamethasone significantly increases the oxidase expression in several parts of the microcirculation to levels above the WKY rats. CONCLUSION: The results indicate that enhanced expression of NADPH and xanthine oxidase in the SHR depends on an adrenal pathway that is detectable in the arteriolar and venular network at high and low pressure regions of the circulation.

Oxidative stress promotes endothelial cell apoptosis and loss of microvessels in the spontaneously hypertensive rats.

OBJECTIVE: Endothelial cell apoptosis caused by oxidative stress may lead to the loss of microvessels (rarefaction) in hypertension. We examine here the effects of antioxidants on cell apoptosis and rarefaction. METHODS AND RESULTS: The juvenile spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were treated with superoxide scavengers, Tempol or Tiron, during growth. After the treatment, oxidative stress status, endothelial cell apoptosis rate, and microvessel length density in skeletal muscle and mesentery were evaluated in comparison with age-matched controls. Untreated 16-week-old SHR had higher oxidative stress (P<0.01) and cell apoptosis rate (P<0.05) and lower microvessel length density (371+/-17 mm/mm3 [P<0.01]) compared with age-matched WKY rats (435+/-15 mm/mm3). In the SHR, but not in WKY rats, systemically applied antioxidants attenuated oxidative stress and cell apoptosis rate (P<0.05 versus untreated controls) and prevented the loss of microvessels (411+/-15 mm/mm3 for Tempol [P<0.01 versus untreated control] and 399+/-17 mm/mm3 for Tiron [P<0.05]). CONCLUSIONS: Antioxidant treatment with cell-permeable superoxide scavengers inhibits endothelial cell apoptosis and prevents microvessel rarefaction in the SHR during growth.

Improvement in early symptoms of shock by delayed intestinal protease inhibition.

HYPOTHESIS: Recent findings indicate that intraintestinal pancreatic protease inhibition before superior mesentery artery occlusion (SMAO) attenuates inflammation and symptoms of shock. Herein we examine the effectiveness of delayed intestinal protease inhibition during reperfusion after SMAO. SUBJECTS: Three groups of male Wistar rats were studied: a nonshock sham group and 2 groups exposed to SMAO for 100 minutes and treated by delayed intestinal lavage starting 40 minutes after reperfusion with buffer (delayed-lavage group) or with the digestive protease inhibitor gabexate mesilate (FOY) (delayed FOY-lavage group). RESULTS: Arterial pressure during reperfusion was significantly lower in the delayed-lavage animals compared with the sham group. Superior mesentery artery occlusion and reperfusion caused the formation of leukocyte activation factors in intestinal homogenates and in plasma, as well as intestinal injury. The delayed-lavage group had a significant increase in activated leukocytes in venules of cremaster muscle. In contrast, in the delayed FOY-lavage group, lavage 40 minutes after reperfusion led to a significant improvement of blood pressure and decreased formation of intestine-derived leukocyte activation factors and intestinal injury compared with the delayed-lavage group. In addition, the delayed FOY-lavage group exhibited fewer rolling leukocytes in venules and reduced apoptosis in the cremaster muscle microcirculation. Intestinal ischemia-induced endotoxemia was attenuated in the delayed FOY-lavage animals. CONCLUSION: Delayed intestinal protease inhibition may improve experimental SMAO-induced shock by reducing intestinal injury, decreasing the level of cell activation in plasma and in the microcirculation, and restoring the blood pressure.

Enhancement of glucocorticoid and mineralocorticoid receptor density in the microcirculation of the spontaneously hypertensive rat.

OBJECTIVE: Elevated blood pressure and abnormal physiological parameters in the microcirculation of the spontaneously hypertensive rat (SHR) can be normalized by adrenalectomy. Thus glucocorticoids and mineralocorticoids may have major control over blood pressure status and organ injury mechanisms in SHRs. As background, this study was designed to examine the distribution of the glucocorticoid and mineralocorticoid receptors in a microvascular network. METHODS: Mature SHR and their normotensive controls, the Wistar-Kyoto (WKY) rat, were studied. An immunohistochemical method was developed that provides a comprehensive display of the receptors in all segments of the mesentery microcirculation and the surrounding tissue parenchyma. RESULTS: All cells in the mesentery exhibit immunolabeling of the glucocorticoid receptor with predominant expression in the nuclei of parenchymal and endothelial cells. The mineralocorticoid receptor is expressed also in most cells of the microcirculation and adjacent parenchymal tissue. Both receptors exhibit the highest levels of immunolabel in the wall of the arterioles and venules, with lower levels in capillaries. Compared with WKY rats, the SHRs exhibit significantly enhanced density of glucocorticoid and mineralocorticoid receptors in endothelial cells of arterioles and venules as well as in parenchymal cells. CONCLUSIONS: These results suggest that the enhanced sensitivity of the SHR to glucocorticoids and aldosterone may be in part associated with enhanced glucocorticoid and mineralocorticoid receptor densities in the microcirculation.

Pancreatic enzymes sustain systemic inflammation after an initial endotoxin challenge.

BACKGROUND: Sepsis is accompanied by severe inflammation whose mechanism remains uncertain. We recently demonstrated that pancreatic proteases in the ischemic intestine have the ability to generate powerful inflammatory mediators that can be detected in the portal vein and in the general circulation. This study was designed to examine several circulatory and inflammatory indices during experimental endotoxemia and intraintestinal pancreatic protease inhibition. METHODS: Immediately after intravenous endotoxin administration, the small intestine was subjected to intraluminal lavage with and without gabexate mesilate, an inhibitor of pancreatic proteases. Shams and rats without lavage served as controls. Hemodynamics, leukocyte (neutrophil and monocyte), and endothelial cell activation, as well as organ injury in the intestine and the cremaster muscle, were examined. RESULTS: After endotoxin administration, control rats developed hypotension, tachycardia, hyperventilation, and leukopenia. The intestine and plasma contained mediators that activated leukocytes. The leukocyte-endothelial interaction within the cremaster muscle microcirculation was enhanced. Endotoxin administration resulted in elevated interleukin-6 plasma levels. Histologic evidence indicated liver and intestinal injury. In contrast, blockade of pancreatic proteases in the intestinal lumen significantly improved hemodynamic parameters and reduced all indices of inflammation in plasma and cell injury in skeletal muscle microcirculation. CONCLUSIONS: Inflammatory mediators derived from the intestine by pancreatic proteases may be involved in the prolonged inflammatory response and sustain symptoms of sepsis after endotoxin challenge.

Microvascular cell death in spontaneously hypertensive rats during experimental inflammation.

OBJECTIVE: Chronic hypertension is associated with an increased risk for tissue injury that may be mediated in part by endothelium and inflammatory cells. To clarify a possible underlying mechanisms, we examined leukocyte migration in the microcirculation and concomitant parenchymal cell death. METHODS: The mesentery of spontaneously hypertensive rats (SHRs) and their normotensive controls, Wistar Kyoto (WKY) rats, was examined with digital fluorescence microscopy after topical stimulation with an inflammatory mediator (f-met-leu-phe, 10(-8)M). The migratory pathways of individual leukocytes were traced, and at the same time cell death was detected by use of a life-death indicator (propidium iodide) over a period of 3 hours. RESULTS: Both WKY and SHR had a progressively increasing number of leukocytes migrating across the endothelium in postcapillary venules into the tissue parenchyma. But parenchymal cell death was detected in a random pattern in the mesentery tissue, without correlation to the migratory positions of the leukocytes. Although mature SHR rats (about 17 weeks) exhibited the same level of cell death as age-matched WKY rats, older WKY rats (about 30 weeks) had significantly lower levels of cell death, whereas the SHR rats maintained the same number of parenchymal cell death as mature animals. CONCLUSIONS: These results suggest that in the presence of an inflammatory mediator, the SHR may exhibit a stronger response to an inflammatory mediator than normotensive WKY rats in a fashion that is age, but not blood pressure, dependent. Parenchymal cell death does not correlate with migration of activated leukocytes at the microvascular level.