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Although 17beta-estradiol (estrogen) and estrogen receptor (ER) agonist administration after trauma-hemorrhage improves cardiac function, it remains unknown what the optimal estrogen or ER agonist dosage is to elicit this beneficial effect. To study this, the dose-dependent effects of estrogen, propylpyrazole triol (ER-alpha agonist), and diarylpropionitrile (DPN; ER-beta agonist) on heart performance (+dP/dt) were determined in sham rats and in experimental animals at the time of maximal bleedout (MBO) or at 2 h after trauma-hemorrhage. The results showed that estrogen and DPN induced dose-dependent increases in the maximal rate of left ventricular pressure increase (+dP/dt) in all groups, whereas propylpyrazole triol was ineffective at all doses. The maximal dose and the 50% effective dose of DPN were approximately 100-fold lower than those of estrogen. The half-life of estrogen in plasma was approximately 25 min in sham and MBO groups. A positive correlation between the estrogen-induced increase in +dP/dt and survival in MBO rats were observed. These results collectively suggest that the salutary effects of estrogen on cardiac performance are dose-dependent and mediated via ER-beta.

Although endothelin-1 (ET-1) induces vasoconstriction, it remains unknown whether 17β-estradiol (E2) treatment following trauma-hemorrhage alters these ET-1-induced vasoconstrictive effects. In addition, the role of the specific estrogen receptor (ER) subtypes (ER-α and ER-β) and the endothelium-localized downstream mechanisms of actions of E2remain unclear. We hypothesized that E2attenuates increased ET-1-induced vasoconstriction following trauma-hemorrhage via an ER-β-mediated pathway. To study this, aortic rings were isolated from male Sprague-Dawley rats following trauma-hemorrhage with or without E2treatment, and alterations in tension were determined in vitro. Dose-response curves to ET-1 were determined, and the vasoactive properties of E2, propylpyrazole triol (PPT, ER-α agonist), and diarylpropionitrile (DPN, ER-β agonist) were determined. The results showed that trauma-hemorrhage significantly increased ET-1-induced vasoconstriction; however, administration of E2normalized ET-1-induced vasoconstriction in trauma-hemorrhage vessels to the sham-operated control level. The ER-β agonist DPN counteracted ET-1-induced vasoconstriction, whereas the ER-α agonist PPT was ineffective. Moreover, the vasorelaxing effects of E2were not observed in endothelium-denuded aortic rings or by pretreatment of the rings with a nitric oxide (NO) synthase inhibitor. Cyclooxygenase inhibition with indomethacin had no effect on the action of E2. Thus, E2administration attenuates ET-1-induced vasoconstriction following trauma-hemorrhage via an ER-β-mediated pathway that is dependent on endothelium-derived NO synthesis.

Although gender differences in intestinal perfusion exist following trauma-hemorrhage (T-H), it remains unknown whether endothelin-1 (ET-1) plays any role in these dimorphic responses. To study this, male, proestrus female (female), and 17β-estradiol (E2)-treated male rats underwent midline laparotomy, hemorrhagic shock (blood pressure 40 mmHg, 90 min), and resuscitation (Ringer lactate, 4× shed blood volume, 1 h). Two hours thereafter, intestinal perfusion flow (IPF) was measured using isolated intestinal perfusion. The IPF in sham-operated males was significantly lower than those in other groups and decreased markedly following T-H. In contrast, no significant decrease in IPF was observed in females and E2 males following T-H. The lower IPF in sham-operated males was significantly elevated by ETA receptor antagonist (BQ-123) administration and was similar to that seen in sham-operated females. The decreased IPF in males after T-H was also attenuated by BQ-123 administration. The intestinal ET-1 levels in sham-operated males were significantly higher than in other groups. Although plasma and intestinal ET-1 levels increased significantly after T-H in all groups, they were highest in males. Plasma E2 levels in females and E2 males were significantly higher than in males; however, they were not affected by T-H. There was a negative correlation between plasma ET-1 and E2 following T-H. Thus ET-1 appears to play an important role in intestinal perfusion failure following T-H in males. Because E2 can modulate this vasoconstrictor effect of ET-1, these findings may partially explain the previously observed salutary effect of estrogen in improving intestinal perfusion following T-H in males.

Although gender differences exist in cardiovascular endothelial function, it remains unclear whether such differences are also seen in small intestinal endothelial function. To determine this, untreated male, age-matched proestrus female, castrated male, and 17β-estradiol (E2)-treated noncastrated male rats were studied. Dose response curves to ACh and nitroglycerin (NTG) were determined by measuring changes in perfusion pressure by using an isolated small intestinal perfusion model. Endothelium-derived nitric oxide (NO) production/release was indirectly determined by the ability of intact endothelium to suppress serotonin (10-5M)-induced perfusion pressure changes. Intestinal tissue levels of NO were also measured. Moreover, plasma levels of androgen and E2 were determined and correlated with ACh (10-8M)-induced perfusion pressure reductions. ACh-induced intestinal perfusion pressure reductions in proestrus females, castrated males, and E2-treated noncastrated males were significantly higher than in untreated males. NTG-induced perfusion pressure reductions were not significantly different among groups. Perfusion pressures after administration of serotonin (10-5M) and intestinal tissue levels of NO in proestrus females, castrated males, and E2-treated noncastrated males were also significantly higher than in untreated males. Plasma androgen levels in proestrus females, castrated males, and in E2-treated noncastrated males were significantly lower compared with untreated males. There was a positive correlation between plasma androgen and ACh-reduced perfusion pressure; however, E2 levels did not show a similar relationship. Thus androgens appear to play an inhibitory role in small intestinal endothelial function. These properties in male vessels can be modulated by decreasing the level of circulating androgens or by E2 treatment.

Adrenomedullin (AM), a potent vasodilatory peptide, plays an important role in initiating the hyperdynamic response during the early stage of sepsis. Moreover, the reduced vascular responsiveness to AM appears to be responsible for the transition from the early, hyperdynamic to the late, hypodynamic phase of sepsis. Although the novel specific AM binding protein-1 (AMBP-1) enhances AM-mediated action in a cultured cell line, it remains to be determined whether AMBP-1 plays any role in modulating vascular responsiveness to AM during sepsis. To study this, adult male rats were subjected to sepsis by cecal ligation and puncture (CLP). The thoracic aorta was harvested for determination of AM-induced vascular relaxation. Aortic levels of AMBP-1 were determined by Western blot analysis, and AM receptor gene expression in the aortic tissue was assessed by RT-PCR. The results indicate that AMBP-1 significantly enhanced AM-induced vascular relaxation in aortic rings from sham-operated animals. Although vascular responsiveness to AM decreased at 20 h after CLP (i.e., the late, hypodynamic stage of sepsis), addition of AMBP-1 in vitro restored the vascular relaxation induced by AM. Moreover, the aortic level of AMBP-1 decreased significantly at 20 h after CLP. In contrast, AM receptor gene expression was not altered under such conditions. These results, taken together, suggest that AMBP-1 plays an important role in modulating vascular responsiveness to AM, and the reduced AMBP-1 appears to be responsible for the vascular AM hyporesponsiveness observed during the hypodynamic phase of sepsis.

Although studies have indicated that calcitonin gene-related peptide (CGRP), a potent vasodilatory peptide, is upregulated after endotoxic shock, it remains controversial whether this peptide increases during sepsis and, if so, whether the gut is a significant source of CGRP under such conditions. To study this, polymicrobial sepsis was induced by cecal ligation and puncture (CLP) followed by fluid resuscitation. Plasma levels of CGRP were measured at 2, 5, and 10 h after CLP (i.e., early, hyperdynamic sepsis) and at 20 h after CLP (late, hypodynamic sepsis). The results indicate that plasma CGRP did not increase at 2–5 h but increased by 177% at 10 h after CLP ( P < 0.05). At 20 h after the onset of sepsis, however, the elevated plasma CGRP returned to the sham level. To determine the source of the increased plasma CGRP, the liver, spleen, small intestine, lungs, and heart were harvested, and tissue CGRP was assayed at 10 h after CLP in additional animals. Only the small intestine showed a significant increase in tissue levels of CGRP (by 129%, P < 0.05). Determination of portal vs. systemic levels of CGRP indicates that portal CGRP was 65.7 ± 22.7% higher than the systemic level at 10 h after CLP, whereas portal CGRP in sham-operated rats was only 4.9 ± 2.1% higher. Immunohistochemistry examination revealed that CGRP-positive stainings increased in the intestinal tissue but not in the liver at 10 h after the onset of sepsis. The distribution of CGRP stainings was associated with intestinal nerve fibers. These results, taken together, demonstrate that upregulation of CGRP occurs transiently during the progression of sepsis (at the late phase of the hyperdynamic sepsis), and the gut appears to be a major source of such an increase in circulating levels of this peptide.

Objective Early management of trauma victims includes control of bleeding and rapid restoration of intravascular volume. However, it remains controversial whether infusion of blood products is superior to crystalloids alone. Therefore, it was the aim of the present study to determine whether resuscitation with red blood cells plus lactated Ringer's solution (RL) is more effective than RL alone in improving the cardiovascular and hepatocellular functions after trauma and severe hemorrhage. Design Prospective study. Setting Laboratory. Subjects Sprague-Dawley rats. Interventions and measurements Male adult rats were anesthetized and underwent a laparotomy to induce tissue trauma before hemorrhage. The animals were then bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of the maximal bleed-out (MB) volume was returned in the form of RL, and were then resuscitated with either four times the volume of MB with RL or washed red blood cells (RBC) (-45% the volume of MB) in three times the volume of RL over 60 mins. Various in vivo heart performance variables, cardiac output, and hepatocellular function (ie, the maximum velocity and the overall efficiency of indocyanine green clearance) were determined at 4 hrs after resuscitation. Hemoglobin, systemic oxygen delivery, circulating blood volume, and plasma levels of interleukin-6 were also measured. Main results At 4 hrs after RL resuscitation, heart performance, cardiac output and hepatocellular function were significantly depressed and plasma levels of interleukin-6 were significantly increased. Although infusion of RBC significantly increased mean arterial pressure, hemoglobin, and oxygen delivery compared with animals resuscitated with RL only, infusion of RBC did not further improve the depressed cardiovascular and hepatocellular functions under such conditions. Conclusion Because infusion of RBC and RL resuscitation do not improve organ functions compared with RL resuscitation without RBC, it appears that pharmacologic agents in addition to fluid resuscitation are needed to restore cardiovascular and hepatocellular functions after trauma and hemorrhage.

Although trauma and hemorrhage are associated with tissue hypoperfusion and hypoxemia, changes in oxygen delivery (DO2), oxygen consumption VO2), and oxygen extraction at the organ level in a small animal (such as the rat) model of trauma and hemorrhage have not been examined. Therefore, the objectives of this study were to determine whether blood flow, DO2, VO2, and oxygen extraction ratio in various organs are differentially altered after trauma-hemorrhagic shock and acute resuscitation in the rat.Prospective, randomized animal study.A university research laboratory.Male Sprague-Dawley rats (n = 6-7 animals/group) weighing 275-325 g.Male rats underwent laparotomy (i.e., soft tissue trauma) and were bled to and maintained at a blood pressure of 40 mm Hg until 40% of shed blood volume was returned in the form of lactated Ringer's solution. They were then resuscitated with four times the volume of shed blood with lactated Ringer's solution for 60 mins. At 1.5 hrs postresuscitation, cardiac output and blood flow were determined by using strontium-85 microspheres. Blood samples (0.15 mL each) were collected from the femoral artery and vein and the hepatic, portal, and renal veins to determine total hemoglobin and oxygen content. Systemic and regional DO2, VO2, and oxygen extraction ratio were then calculated.Both the systemic hemoglobin and systemic arterial oxygen content in hemorrhaged animals at 1.5 hrs postresuscitation were50% lower as compared with sham-operated controls. Cardiac output and blood flow in the liver, small intestine, and kidneys decreased significantly, but blood flow in the brain and heart remained unaltered after hemorrhage and resuscitation. Systemic DO2 and VO2 were 73% and 54% lower, respectively, than controls at 1.5 hrs after resuscitation. Similarly, regional DO2 and VO2 in the liver, small intestine, and kidneys decreased significantly under such conditions. In addition, the liver had the most severe reduction in VO2 (76%) among the tested organs. However, the oxygen extraction ratio in the liver of sham animals was the highest (72%) and remained unchanged after hemorrhage and resuscitation.Because the liver experienced the most severe reduction in VO2 associated with an unchanged oxygen extraction capacity, this organ appears to be more vulnerable to hypoxic insult after hemorrhagic shock.

Objectives Studies have shown that endothelium-dependent relaxation (mediated by endothelium-derived nitric oxide) is depressed during the early, hyperdynamic stage of sepsis. Although it is known that ATP-MgCl2 produces beneficial effects following various adverse circulatory conditions, it remains unknown whether this agent attenuates the depressed endothelium-dependent relaxation during early sepsis. The aim of this study, therefore, was to determine whether or not the administration of ATP-MgCl2 early after the onset of sepsis improves or maintains endothelium-dependent relaxation. Design Prospective, controlled animals study. Setting A university research laboratory. Subjects Adult male Sprague-Dawley rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP), followed by administration of 3 mL/100 g body weight normal saline to these and sham-operated rats. Interventions At 1 hr after CLP, ATP-MgCl2 (50 [micro sign]mol/kg body weight) or an equivalent volume of normal saline was infused intravenously over 90 mins. Measurements and Main Results At 5 hrs or 10 hrs after CLP (i.e., the early, hyperdynamic stage of sepsis), the thoracic aorta was isolated, cut into rings, and placed in organ chambers. Norepinephrine was used to preconstrict vessel rings. Dose responses for an endothelium-dependent vasodilator, acetylcholine (ACh, via endothelium-dependent nitric oxide), and an endothelium-independent vasodilator, nitroglycerin, were determined. These results indicate that endothelium-dependent relaxation induced by ACh was significantly depressed at 5 and 10 hrs after CLP. Administration of ATP-MgCl2 after the onset of sepsis, however, maintained ACh-induced vascular relaxation. In contrast, no significant difference in nitroglycerin-induced vascular relaxation as well as norepinephrine-induced contraction was observed, irrespective of administration of ATP-MgCl2. Conclusion Since administration of ATP-MgCl2 prevents the impaired vascular relaxation to the endothelium-dependent vasodilator ACh, this agent may be a useful adjunct for maintaining endothelial cell function during the hyperdynamic stage of sepsis. (Crit Care Med 1999; 27:959-964)

Although hepatocellular dysfunction occurs following trauma and hemorrhagic shock, whether severe hypotension even in the absence of blood loss depresses hepatocellular function remains unknown. The aim of this study, therefore, was to determine whether chemically induced severe hypotension causes hepatocellular dysfunction and, if so, whether IL-6 and PGE 2 are associated with this dysfunction. To study this, hypotension was induced in adult male rats by intravenous infusion of a high dosage of ATP–MgCl 2 solution (3.2 ± 0.45 μmol/min/kg body wt) for 60 min. Blood pressure decreased from 108 ± 6 mm Hg to an average of 43 mm Hg during the infusion period and returned to normal levels immediately after the completion of ATP–MgCl 2 infusion. At 0 and 4 h after hypotension, hepatocellular function [i.e., maximum velocity of indocyanine green clearance ( V max ) and its efficiency ( K m )] was measured using a fiberoptic catheter and in vivo hemoreflectometer. Cardiac output was determined by dye dilution. Microvascular blood flow was assessed by laser Doppler flowmetry. Plasma levels of PGE 2 and IL-6 were measured by radioimmunoassay and bioassay, respectively. The results indicate that severe hypotension in the absence of any blood loss depresses hepatocellular function (i.e., decreased V max and K m ) despite stable cardiac output and hepatic perfusion at 0 and 4 h after the completion of hypotension. Moreover, severe hypotension resulted in significantly increased plasma levels of PGE 2 (only at 0 h) and IL-6. Thus, chemically induced severe hypotension in the absence of any blood loss, which does not significantly reduce cardiac output and hepatic perfusion, depresses hepatocellular function and upregulates IL-6 and PGE 2 production.

Although a burst of immunoresponsiveness may occur during the early stage of sepsis, late sepsis is characterized by severe immunodepression. In addition, although studies have shown that stimulation of macrophage beta-adrenoceptors results in an increase in cAMP and an associated reduction in macrophage phagocytic activity, it remains unknown whether Kupffer cell beta-adrenoceptor characteristics and cAMP levels are altered during polymicrobial sepsis. To study this, Sprague-Dawley rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 h (i.e., the early stage of sepsis) or 20 h (late sepsis) after CLP or sham operation, the liver was perfused with collagenase solution and Kupffer cells were isolated. beta-Adrenoceptor characteristics of the isolated Kupffer cells were determined using [125I]iodopindolol, and basal levels of cAMP were measured by radioimmunoassay. The results indicate that while maximum binding capacity (Bmax) of Kupffer cell beta-adrenoceptors was not altered at 5 h, it increased significantly at 20 h after CLP. Similarly, basal levels of cAMP in Kupffer cells did not change at 5 h but increased markedly at 20 h after the onset of sepsis. In contrast, the dissociation constant (Kd, 1/affinity) of Kupffer cell beta-adrenoceptors was not significantly affected by sepsis at both 5 h and 20 h after CLP. Thus, upregulation of beta-adrenoceptors and increase in cAMP levels in Kupffer cells occur during the late stage of polymicrobial sepsis, and this may contribute to the depression of macrophage phagocytic function under such conditions.

A large number of studies have been and are being carried out to examine the role of nitric oxide in the hyperdynamic and hypodynamic stages of sepsis. It remains unknown, however, whether adrenomedullin (ADM), a novel potent vasodilatory peptide, is up-regulated during hyperdynamic sepsis and, if so, whether its production is sustained during hypodynamic sepsis. To determine this, rats were subjected to sepsis by cecal ligation and puncture (CLP), followed by administration of 3 mL/100 g body weight normal saline to these and sham-operated animals. Blood samples were taken at 1, 1.5, 2, 5, and 10 h (2-10 h post-CLP represents the hyperdynamic stage of sepsis) or at 20 and 30 h after CLP (i.e., the hypodynamic stage). Plasma levels of ADM were measured by radioimmunoassay. Adrenomedullin gene expression in various tissues was examined at 2, 10, or 20 h after CLP by reverse transcription-polymerase chain reaction (RT-PCR). The results indicated that plasma levels of ADM did not increase at 1 and 1.5 h after CLP but increased significantly at 2 h after the onset of sepsis. Moreover, circulating ADM increased progressively at 5-20 h and remained elevated at 30 h after CLP. The increased levels of plasma ADM during sepsis were correlated with up-regulation of ADM mRNA in the small intestine, left ventricle, and thoracic aorta. In contrast, ADM gene expression in renal and hepatic tissues was not significantly altered following the onset of sepsis. The association between the up-regulated ADM and the occurrence of hyperdynamic circulation during the early stage of sepsis (both occur at 2 h after CLP) may indicate a possible cause and effect relationship between the two events. Since we have previously shown that ADM-induced vascular relaxation decreased at 20 h after CLP, it appears that the down-regulation of ADM receptors may be responsible for the transition from the hyperdynamic stage to the hypodynamic stage of sepsis.

Background. Although studies suggest that the gut may be the “motor” responsible for producing sepsis and multiple organ failure after injury, it is not known whether enterectomy prior to the onset of hemorrhage alters proinflammatory cytokines TNF and IL-6 and, if so, whether hepatocellular dysfunction and damage are prevented or attenuated under such conditions. Materials and methods. Under methoxyflurane anesthesia, an enterectomy in the rat was performed by excision of the duodenum, jejunum, and ileum. The rats were then bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of the maximal shed volume was returned in the form of Ringer's lactate. The animals were then resuscitated with four times the volume of shed blood with Ringer's lactate over 1 h. At 1.5 h after the completion of resuscitation, hepatocellular function [i.e., the maximal velocity ( V max ) and transport efficiency ( K m ) of indocyanine green (ICG) clearance] was assessed by an in vivo ICG clearance technique. Blood samples were taken for the measurement of TNF, IL-6, and liver enzymes (i.e., SGPT and SGOT). Cardiac output and microvascular blood flow were determined by ICG dilution and laser Doppler flowmetry, respectively. Results. The increase in circulating levels of TNF but not IL-6 was prevented by enterectomy prior to hemorrhage. The reduced V max and K m and elevated SGPT and SGOT following hemorrhage and resuscitation, however, were not significantly affected by prior enterectomy. Moreover, enterectomy before hemorrhage further reduced hepatic perfusion. Conclusion. Since enterectomy prior to the onset of hemorrhage does not prevent or attenuate the reduced ICG clearance and elevated liver enzymes despite downregulation of TNF production, it appears that the small intestine does not play a significant role in producing hepatocellular dysfunction and injury following trauma and hemorrhagic shock.

Although studies have indicated that hepatocellular function is depressed early after the onset of sepsis, it remains unknown whether liver endothelial cell function is also compromised under such conditions. To study this, male rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP), followed by administration of 3 ml/100 g body wt normal saline subcutaneously to these and to sham-operated animals. Blood samples (0.2-ml aliquots) were taken from the carotid artery, portal vein, and hepatic vein at 2, 5, 10 (i.e., hyperdynamic sepsis), or 20 hr (hypodynamic sepsis) after CLP, and plasma hyaluronic acid (HA) was determined using a Pharmacia assay kit. In addition, HA clearance was assessed at 5, 10, or 20 hr after CLP by injecting 30 μg/100 g body wt HA intravenously. Plasma HA was determined at 2–40 min after the administration of HA. The results indicate that plasma levels of HA in blood from three different sites did not increase significantly until 10 hr after CLP. Clearance of HA decreased only at 20 hr after CLP, compared to sham-operated animals. These results suggest that the increased plasma levels of HA at 10 hr after the onset of sepsis are solely due to the increased release/production of the polysaccharide. Since circulating HA is cleared exclusively by the liver endothelial cell, the results demonstrate that liver endothelial cell dysfunction (i.e., the increased circulating HA levels and decreased HA clearance) occurs only during the late, hypodynamic stage of polymicrobial sepsis.

Background. Although pentoxifylline produces various beneficial effects after endotoxemia or sepsis occurs, it is not known whether this agent attenuates the depressed endothelial cell function during sepsis. Therefore the aim of this study was to determine whether pentoxifylline maintains vascular endothelial cell function (i.e., improves the release of endothelium-derived nitric oxide) during hyperdynamic and hypodynamic stages of polymicrobial sepsis. Methods. Rats were subjected to sepsis by cecal ligation and puncture (CLP), after which 3 ml/100 gm body wt normal saline solution was injected subcutaneously in these and rats in a sham-operated group. At 1 hour after the onset of sepsis, pentoxifylline (50 mg/kg body wt) or an equal volume of normal saline solution was infused intravenously during a 30 minute period. At 10 and 20 hours after CLP was performed (10-hour CLP, hyperdynamic sepsis; 20-hour CLP, hypodynamic sepsis), the thoracic aorta was isolated, cut into rings, and placed in organ chambers. Norepinephrine (2 x 10 −7 mol/L) was used to achieve near maximal tension. Dose responses for an endothelium-dependent vasodilator, acetylcholine, and an endothelium-independent vasodilator, nitroglycerine, were carried out. The changes in percentage relaxation in the aortic rings by these agonists were then determined. Results. Endothelium-dependent (acetylcholine-induced) vascular relaxation decreased significantly at 10 and 20 hours after CLP. Administration of pentoxifylline, however, maintained acetylcholine-induced vascular relaxation at both time points. In contrast, no significant reduction in nitroglycerine-induced vascular relaxation was seen in rats with sepsis irrespective of pentoxifylline treatment. Conclusions. Because pentoxifylline prevented endothelial cell dysfunction at 10 and 20 hours after CLP occurred, this agent appears to be a useful agent for maintaining vascular endothelial function during the hyperdynamic and hypodynamic stages of polymicrobial sepsis.

The objective of this study was to determine whether endothelium-derived nitric oxide (NO) production is reduced at the macrocirculatory and microcirculatory levels during sepsis. To examine this, rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 h after CLP (i.e., midpoint of hyperdynamic sepsis) or sham operation, the aorta and superior mesenteric artery were isolated. Responses to an endothelium-dependent vasodilator, acetylcholine (ACh), and an endothelium-independent vasodilator, nitroglycerin (NTG), were determined. In additional studies, the small intestine was isolated 5 or 20 h (hypodynamic sepsis) after CLP. Responses to ACh and NTG were determined in the isolated intestine. The results indicate that endothelium-dependent relaxation in both the aorta and superior mesenteric artery was depressed at 5 h after CLP. In contrast, there was no significant difference in the relaxation induced by NTG. Moreover, ACh-induced vascular relaxation in the isolated small intestine decreased at 5 and 20 h post-CLP without any significant alterations in NTG-induced relaxation. Since studies have shown that ACh-induced relaxation in the aorta is reduced at 20 h after CLP, it could be concluded that endothelium-derived NO release is depressed during hyperdynamic and hypodynamic stages of sepsis, not only in large arteries, but also at the microcirculatory level.

Although studies have indicated that the levels of catecholamines increase during sepsis, it remains unknown whether the elevated levels of epinephrine, norepinephrine, and dopamine observed in early sepsis are sustained during late, hypodynamic stages of sepsis. In this study, rats were subjected to sepsis by cecal ligation and puncture (CLP, i.e., polymicrobial sepsis). Immediately after CLP or sham operation, animals received 3 mL/100 g body weight normal saline subcutaneously. At .5, 2, 10 (i.e., early sepsis), or 20 h (late sepsis) after CLP, blood samples were drawn and the plasma was separated. Plasma levels of epinephrine, norepinephrine, and dopamine were determined using a [3H]-radioenzymatic assay. The results indicate that plasma levels of epinephrine, norepinephrine, and dopamine increased significantly as early as .5 h after CLP. The increase in catecholamine levels persisted throughout the study periods. Thus, circulating levels of catecholamines were elevated in both early and late stages of polymicrobial sepsis. These results suggest that the increased catecholamine levels at .5-10 h after CLP may contribute to the hypermetabolic conditions that occur during early, hyperdynamic sepsis. However, there is a lack of an association between the elevated plasma catecholamine levels and hypometabolic/hypodynamic state in late sepsis.

Studies indicate that hepatocellular dysfunction occurs at 2 h after cecal ligation and puncture (CLP, i.e., sepsis model) despite the increased cardiac output (CO) and hepatic perfusion. It, however, remains unknown whether hepatocellular function is depressed earlier than the onset of hyperdynamic circulation in sepsis. To determine this, rats were subjected to sepsis by CLP. At .5, 1, 1.5, or 2 h after CLP, CO was measured by dye dilution. Hepatocellular function (i.e., maximum velocity of indocyanine green clearance and the efficiency of the active transport) was determined using an in vivo indocyanine green clearance technique. Microvascular blood flow was measured by laser Doppler flowmetry. To determine whether there is any association between hemodynamics and prostaglandins (PGs), plasma levels of PGE2 and PGI2 were measured by radioimmunoassay. The results indicate that hepatocellular function decreased significantly as early as 1.5 h after CLP. Cardiac output and microvascular blood flow in the liver and small intestine, however, increased and vascular resistance decreased at 2 h after CLP. Thus, hepatocellular dysfunction occurs earlier than the occurrence of hyperdynamic circulation during sepsis. Although circulating PGE2 levels were not altered, plasma PGI2 increased significantly at 2 h after CLP. The elevated circulating PGI2 levels, therefore, may be partially responsible for the decreased vascular resistance and increased tissue perfusion at 2 h after CLP. Our findings also suggest that cellular dysfunction, observed in the very early stage of sepsis, is not due to any hyperdynamic circulation/hypermetabolism-related events, but may be associated with the release of proinflammatory cytokines.

Although ATP-MgCl2 attenuates the release of inflammatory cytokines and restores the defective macrophage (M phi) antigen presentation function after hemorrhage and resuscitation, it is not known whether administration of this agent after hemorrhage affects M phi adenosine 3',5'-cyclic monophosphate (cAMP) levels and beta-adrenergic receptors. To determine this, rats underwent a midline laparotomy (i.e., induction of trauma) and were then bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of Ringer lactate (RL). Animals were resuscitated with four times the volume of shed blood with RL, during and after which ATP-MgCl2 (50 mumol/kg) or saline was administered over 95 min. At 1.5 h postresuscitation (i.e., 10 min after completion of ATP-MgCl2 infusion), peritoneal M phi and Kupffer cells were isolated, and cAMP levels were measured by radioimmunoassay. beta-Receptor binding characteristics were also determined in isolated Kupffer cells. The results indicate that cAMP levels increased significantly in both peritoneal M phi and Kupffer cells after hemorrhage and resuscitation. Maximum binding capacity (Bmax) of beta-receptors increased in Kupffer cells, suggesting that the elevated cAMP may be due to the increased beta-receptor Bmax under such conditions. ATP-MgCl2 treatment, however, markedly decreased beta-receptor Bmax in Kupffer cells and cAMP in both peritoneal M phi and Kupffer cells, and the values were similar to shams. Thus normalization of M phi cAMP levels and beta-receptor binding capacity by ATP-MgCl2 may contribute to the immunoenhancing effects of this agent observed after trauma-hemorrhage and fluid resuscitation.

Although depressed endothelium-dependent relaxation occurs during early sepsis, the precise mechanism responsible for this remains unknown. Because the elevated levels of plasma tumor necrosis factor (TNF) play a major role in the pathophysiology of sepsis, we investigated whether TNF-alpha administration alters endothelium-dependent relaxation. To study this, recombinant TNF-alpha (1.2 x 10(7) U/mg) was infused intravenously (0.25 mg/kg body wt) for 0.5 h in normal rats, and mean arterial pressure was monitored. At 1 h after the completion of TNF-alpha or vehicle infusion, the aorta and a pulmonary artery were isolated, cut into 2.5-mm rings, and placed in organ chambers. Norepinephrine (2 x 10(-7) M) was applied to achieve near-maximal contraction, and dose responses for an endothelium-dependent vasodilator, acetylcholine, and an endothelium-independent vasodilator, nitroglycerine, were determined. In additional studies, aortic rings from normal animals were incubated with TNF-alpha for 2 h in vitro, and vascular reactivity was determined. The results indicate that TNF-alpha administration significantly reduced acetylcholine-induced vascular relaxation both in vivo and in vitro. Such a reduction was sustained at least 80 min after the completion of 2-h incubation with TNF-alpha. In contrast, TNF did not alter nitroglycerine-induced vascular relaxation. Thus TNF-alpha depresses endothelium-dependent relaxation in vitro as well as in vivo. Because TNF-alpha infusion increases plasma TNF levels without decreasing mean arterial pressure, the depressed endothelium-dependent relaxation observed during early sepsis may be due to the elevated circulating levels of TNF.

Although cyclic nucleotides play an important role in regulating the control of metabolism, it is not known whether there are any differential alterations in cyclic nucleotides in Kupffer cells and hepatocytes after trauma-hemorrhage and resuscitation. To study this, rats underwent laparotomy (i.e., trauma-induced) and were rapidly bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of Ringer's lactate. The animals were then resuscitated with Ringer's lactate, equivalent to four times the volume of shed blood. At the time of maximum bleedout or at 1.5 h postresuscitation, a portion of the liver was removed, and the levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were determined by radioimmunoassay. Moreover, Kupffer cells and hepatocytes were isolated in additional groups of animals and cAMP and cGMP levels were measured. The results indicate that hepatic cAMP decreased, whereas hepatic cGMP increased significantly at the time of maximum bleedout. Although resuscitation normalized hepatic cyclic nucleotide levels, the levels of cAMP and cGMP in Kupffer cells increased significantly at 1.5 h after resuscitation. In contrast, cAMP and cGMP levels in hepatocytes were not significantly different from shams under such conditions. Thus, differential alterations in cyclic nucleotide levels in different liver cell populations occur following trauma-hemorrhage and resuscitation.

Although cardiac output (CO) and other hemodynamic variables are used to assess the adequacy of fluid resuscitation after hemorrhage, it is not known whether there is any correlation between restoration of CO and circulating blood volume (CBV). To determine this, rats underwent a midline laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of Ringer lactate (RL). The animals were then resuscitated with four or five times the volume of maximum bleedout in the form of RL. CO and hepatocellular function were measured using an in vivo hemoreflectometer. CBV was monitored by using in vivo indocyanine green clearance. A good correlation between the values of blood volume obtained by this method and the 125I-albumin method indicates that the indocyanine green method is also a reliable technique for measuring CBV. Results indicate that resuscitation after hemorrhage improved the decreased CBV but did not restore it to control levels despite the fact that CO was restored and central venous pressure was more than doubled. A good correlation between depressed CBV and hepatocellular dysfunction was also observed under such conditions. Thus measurement of CBV appears to be useful for evaluating the adequacy of fluid resuscitation after trauma-hemorrhage and hemodilution.

Although interleukin-6 (IL-6) plays an important role in the pathophysiology of trauma-hemorrhage and resuscitation, the cellular origin of this inflammatory cytokine remains unknown. This study was undertaken to determine whether Kupffer cells (KC) are a major source of IL-6 release following trauma-hemorrhage and resuscitation. KC numbers were significantly (p < .05) reduced in vivo with gadolinium chloride (GdCl3; 10 mg/kg IV). KC-reduced (KC(-)) and KC-normal (saline-treated; KC(+)) rats underwent laparotomy (i.e., trauma-induced), followed by either sham operation or hemorrhage. Hemorrhaged rats were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of the shed blood volume was returned as Ringer's lactate, and then resuscitated with Ringer's lactate (four times shed blood volume over 1 h). Results indicate that KC reduction per se had no effect on any measured parameter at any time. At 0.5 and 2.0 h postresuscitation, mean arterial pressure, heart rate, cardiac output, stroke volume, and hematocrit were reduced to a similar extent in both the KC(+) and KC(-) hemorrhage groups. KC reduction did, however, significantly reduce plasma IL-6 concentration (means +/- S.E.; U/ml) at both 0.5 h (KC(+) = 709 +/- 391 vs. KC(-) = 159 +/- 5) and at 2.0 h (KC(+) = 527 +/- 394 vs. KC(-) = 83 +/- 20) postresuscitation. In conclusion, this study demonstrates that KC are a major source of in vivo IL-6 release following trauma-hemorrhage and resuscitation.

Although plasma levels of tumor necrosis factor (TNF) are elevated and hepatocellular dysfunction occurs even in the early hyperdynamic stage of sepsis, the precise mechanism responsible for this dysfunction remains unknown. Although TNF at high doses produces circulatory failure, it is not known whether the dose of TNF that does not adversely affect hemodynamics alters hepatocellular function. To study this, recombinant murine TNF-alpha was infused intravenously (0.05 or 0.25 mg/kg) over 30 min in normal rats. At 1 and 4 h after infusion of TNF-alpha or an equivalent volume of saline, hepatocellular function [i.e., maximum velocity (Vmax) and Michaelis constant (Km)] was assessed using in vivo indocyanine green clearance without blood sampling. Additional parameters measured were as follows: cardiac output by dye dilution, hepatic microcirculation by laser Doppler flowmetry and colloidal carbon infusion, plasma TNF and interleukin-6 (IL-6) by cytokine-dependent cellular assays, and plasma glucose enzymatically. The results indicate that although infusion of 0.05 mg/kg TNF-alpha did not affect Vmax and Km, its infusion at 0.25 mg/kg produced a significant depression of hepatocellular function and markedly increased the synthesis and/or release of IL-6. TNF-alpha-induced hepatocellular dysfunction was not associated with any significant changes in hepatic microcirculation, plasma glucose, cardiac output, and other measured hemodynamic parameters. Thus hepatocellular dysfunction observed after TNF infusion may be due to the direct effect of this cytokine alone or in combination with IL-6.

Although mice are widely used for the study of immune consequences of hemorrhage, the changes of cardiac output (CO) and blood flow (BF) in response to trauma and hemorrhage in this species have not been well defined. To study this, nonheparinized C3H/HeN mice (n = 6 per group) underwent laparotomy (i.e., trauma induced), were bled to a mean arterial pressure of 35 mmHg, and maintained for 90 min by withdrawing more blood or returning Ringer lactate. The animals were then resuscitated with four times the volume of maximal bleedout in the form of Ringer lactate over 60 min. Sham-operated mice underwent the same procedure but were neither bled nor resuscitated. At the end of hemorrhage, 60 min postresuscitation, or corresponding time after sham operation, CO and BF were determined by radioactive microspheres. Results indicate that CO and BF decreased significantly at the end of hemorrhage. Resuscitation, however, restored CO and BF in various organs except the brain and skeletal muscle. Despite this, 9 of 16 mice died within 6 days postresuscitation, whereas none of sham mice died (n = 16 per group in this additional study). Therefore, we have developed a nonheparinized model of trauma-hemorrhage and resuscitation in mice that is associated with late mortality. Furthermore, the microsphere technique provides a reliable method for assessing CO and BF in mice. Thus it may be possible to study the correlation between cardiovascular and immunologic alterations under such conditions.

Although ATP-MgCl2 administration after hemorrhage and resuscitation restores the decreased hepatic blood flow, it is not known whether this is due to the increase in portal blood flow or hepatic arterial blood flow. To study this, rats underwent a midline laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of maximal shed blood volume was returned in the form of Ringer lactate (RL). The animals were resuscitated with four times the volume of the shed blood with RL, during and after which ATP-MgCl2 (50 mumol/kg body wt) or an equal volume of normal saline was infused intravenously over 95 min. Cardiac output and organ blood flow were determined by 85Sr-labeled microspheres at 90 min after the completion of resuscitation. The results indicate that portal blood flow and total hepatic blood flow decreased significantly after hemorrhage and resuscitation. ATP-MgCl2 treatment, however, restored these parameters to sham values. In contrast, hepatic arterial blood flow did not change significantly after either hemorrhage and resuscitation or ATP-MgCl2 infusion. Moreover, the depressed cardiac output was normalized and coronary blood flow was higher than shams after ATP-MgCl2 treatment. Unlike small intestinal blood flow, blood flows to the stomach, spleen, pancreas, mesentery, and cecum were not markedly affected with ATP-MgCl2 infusion. Thus the restoration of hepatic blood flow with ATP-MgCl2 treatment under such conditions is due to the increased portal blood flow, i.e., solely due to the increased small intestinal blood flow.

Although cellular dysfunction occurs very early in sepsis, it remains controversial whether this is solely due to a decrease in tissue perfusion. Recent studies have indicated that while active hepatocellular function was depressed, hepatic surface microvascular blood flow (MBF) increased in early sepsis but decreased in late sepsis as produced by cecal ligation and puncture (CLP). However, it is not known whether microvascular hyperperfusion in early sepsis and microvascular hypoperfusion in late sepsis are common events in other organs under such conditions. To study this, rats were subjected to sepsis by CLP, after which these and the corresponding shams received 3 ml/100 g body wt normal saline. Microvascular perfusion and MBF in various tissues were assessed by colloidal carbon infusion and laser-Doppler flowmetry, respectively, at 5 h (i.e., early sepsis) or 20 h (late sepsis) after CLP or sham operation. Carbon-perfused areas were quantitated by an Optomax image analyzer. The results indicate that the carbon-perfused areas and MBF in the liver, renal cortex, spleen, and small intestinal serosa (only MBF) increased significantly 5 h after CLP. In late sepsis, however, the carbon-perfused areas and MBF were found to be significantly decreased. A highly linear relationship was observed between the changes of carbon-perfused areas and MBF during sepsis in the tested organs. Thus the microvascular responses in the fluid-resuscitated sepsis model are characterized by hyperperfusion in the early stage and hypoperfusion in the late stage of sepsis in the tested tissues. The cellular dysfunctions observed during the early stage of sepsis are, therefore, not due to any reduction in tissue perfusion.

Although calcium antagonists produce salutary effects after shock and ischemia, it is unknown whether such agents restore the depressed cardiac output (CO) and renal function in a nonheparinized model of trauma-hemorrhage and resuscitation. To study this, rats underwent a midline laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of the maximum bleedout was returned in the form of Ringer lactate (RL). They were then resuscitated with four times the volume of shed blood with RL over 60 min. Diltiazem (400 micrograms/kg body wt) or an equal volume of saline was infused intravenously over 95 min. This infusion was started during the last 15 min of resuscitation. CO was determined by indocyanine green dilution. Glomerular filtration rate (GFR) was assessed with [3H]inulin clearance, and cortical microcirculation was examined by laser Doppler flowmetry. Results indicate that crystalloid resuscitation alone transiently restored but did not maintain CO after hemorrhage. Diltiazem infusion in conjunction with crystalloid resuscitation, however, restored and maintained CO and cortical microcirculation. Although GFR decreased in both groups, the values in diltiazem-treated animals were significantly higher than those in the sham-operated animals. Furthermore, diltiazem markedly decreased tissue water content. Thus diltiazem appears to be a promising adjunct in the treatment of hemorrhagic shock even in the absence of blood resuscitation.

Although ATP-MgCl2 improves hepatocellular function in a nonheparinized model of trauma-hemorrhage and crystalloid resuscitation, it remains unknown whether the beneficial effects of this agent are due to downregulation of the release of the inflammatory cytokines, tumor necrosis factor (TNF), and interleukin-6 (IL-6) under those conditions. To study this, rats underwent a 5-cm laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of maximum bleedout volume was returned in the form of Ringer's lactate (RL). The animals were then resuscitated with four times the volume of shed blood with RL over 60 min. ATP-MgCl2 (50 mumoles/kg body weight each) or an equivalent volume of normal saline was infused intravenously for 95 min. This infusion was started during the last 15 min of RL resuscitation. Plasma levels of TNF and IL-6 were measured at 1.5 hr after the completion of resuscitation by cytokine-dependent cellular assays. Hepatic blood flow was determined by in vivo indocyanine green clearance (corrected by hepatic extraction ratio for indocyanine green), radioactive microspheres, and [3H]-galactose clearance techniques. The results indicate that the levels of circulating TNF and IL-6 increased significantly in the hemorrhaged-resuscitated animals. ATP-MgCl2 treatment, however, markedly decreased the synthesis and/or release of these cytokines to levels similar to the sham group. The markedly decreased hepatic blood flow (as determined by three different methods) and hepatic extraction ratio for indocyanine green were also restored by ATP-MgCl2 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Although hepatocellular dysfunction occurs early in sepsis despite fluid resuscitation, it is unknown if an increased volume of resuscitation protects hepatocellular function. To study this, rats were subjected to sepsis by cecal ligation and puncture (CLP). These and sham-treated rats then received either 3 or 6 mL/100 g BW normal saline subcutaneously. Studies were performed at 5 hours (i.e., early sepsis) or 20 hours (late sepsis) after CLP. Hepatic blood flow was determined by radioactive microspheres, 3H-galactose clearance technique, and laser Doppler flowmetry in both groups. Active hepatocellular function (i.e., Vmax and Km) was assessed by an in vivo indocyanine green clearance technique. The results indicate that: (1) hepatic blood flow increased markedly in early sepsis; (2) Vmax and Km decreased significantly at 5 hours and 20 hours after CLP; and (3) the increased volume of fluid resuscitation did not improve the depressed active hepatocellular function 5 hours following CLP. Cardiac output and hepatic microcirculation, however, were significantly increased in early sepsis. These results confirm the notion that the depression in hepatocellular function in early sepsis is not the result of any reduction of hepatic perfusion. The dissociation of increased hepatic blood flow from depressed hepatocellular function remains despite the larger volume of resuscitation. The hepatocellular dysfunction that occurs even in early sepsis cannot be corrected simply by increasing the volume of crystalloid resuscitation.

Although Ringer lactate (RL) is routinely used for resuscitation, it is not known whether the volume of RL that restores cardiac output after severe hemorrhagic shock also restores the depressed effective hepatic blood flow (EHBF). To study this, a 5-cm midline laparotomy was performed in rats (i.e., trauma induced), and the animals were then bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of RL. Animals were then resuscitated with four or five times the volume of maximum bleedout with RL. EHBF was determined during hemorrhage and at various intervals thereafter by an in vivo indocyanine green (ICG) clearance technique and corrected by the appropriate hepatic extraction ratio for ICG. Cardiac output was determined by ICG dilution, and hepatic microvascular blood flow (HMBF) was measured with laser Doppler flowmetry. In addition, hepatic blood flow was assessed by using radioactive microspheres. Results indicate that resuscitation markedly improved but did not restore the depressed EHBF after trauma and hemorrhagic shock despite the fact that cardiac output was restored. Similar changes in EHBF, HMBF, and hepatic blood flow as determined by microspheres were observed, suggesting that the in vivo ICG clearance is a reliable method to assess effective hepatic perfusion. Thus the lack of restoration of EHBF may be responsible for the subsequent hepatocellular dysfunction after trauma and severe hemorrhage.

Although the cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1), and interleukin-6 (IL-6) are important mediators of hemodynamic, metabolic, and immunologic alterations in the host during sepsis, it is not known whether there is any association between the release of these cytokines and prostanoids during sepsis. Sepsis induced by cecal ligation and puncture in rats led to a persistent elevation (p less than 0.05) of plasma TNF until 10 hours, steadily increasing (p less than 0.05) IL-1 plasma levels, and enhanced (p less than 0.05) IL-6 plasma levels at all time points compared to the sham group. Prostaglandin E2 plasma levels were elevated (p less than 0.05) at 5 hours (153 +/- 29 pg/mL; control: 47 +/- 11 pg/mL) and 10 hours (96 +/- 16 pg/mL; control: 21 +/- 5 pg/mL). Prostaglandin E2 production by splenic macrophages (sM phi) from septic animals was increased (p less than 0.05) at 5 hours (9.1 +/- 2.2 ng/mL) and 10 hours (5.6 +/- 1.5 ng/mL) compared to controls (3.3 +/- 0.3 ng/mL at 5 hours; 1.3 +/- 1.3 ng/mL at 10 hours). Incubation of sM phi from septic animals with ibuprofen enhanced (p less than 0.05) IL-1 and TNF synthesis, while IL-6 production was reduced (p less than 0.05). These results indicate that the alterations in prostanoid release and elevated plasma prostanoids may regulate the release and consequently the circulating levels of cytokines during sepsis.

Studies have shown that active hepatocellular function is depressed early after trauma-hemorrhage and persists despite resuscitation with two or three times (x) the volume of maximum bleedout (MB) with lactated Ringer's solution (LR). However, it is not known if a larger volume of fluid resuscitation corrects this dysfunction. To study this, rats were bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of the MB volume was returned in the form of LR, and then resuscitated with 4x or 5x the volume of MB with LR. Three doses of indocyanine green (ICG) were given intravenously and [ICG] measured in vivo using an in-vivo hemoreflectometer. The initial velocity of the clearance of ICG was calculated. Maximal velocity of the clearance (Vmax: the number of functional ICG receptors) and kinetic constant (Km: the efficiency of the active transport) were determined from the Lineweaver-Burk plot. Vmax decreased during hemorrhage, was restored to control levels at 0-4 hours after resuscitation, but decreased at 4-8 hours after resuscitation despite restoration of cardiac output following resuscitation with 5x LR. This could be the result of increased TNF release. The Km also decreased during hemorrhage, but increased at 0-1.5 hours and remained at control levels even 4-8 hours after resuscitation. Thus the failure of Vmax to remain at control levels following adequate fluid resuscitation may form the basis of cellular dysfunction and multiple organ failure after severe hemorrhagic shock.

Tumor necrosis factor (TNF) and interleukin 6 (IL-6) are purported to be important mediators of inflammatory responses. It is not known whether the plasma levels of these cytokines are altered after trauma and hemorrhage. Our objectives were to determine whether there is any elevation of plasma TNF or IL-6 after trauma and hemorrhage and to what extent these changes are due to tissue trauma vs. simple hemorrhage. Trauma was induced in Sprague-Dawley rats under light ether anesthesia by performing a 5-cm midline laparotomy. On closure, animals were catheterized, awakened, hemorrhaged to a mean blood pressure of 40 mmHg, and maintained at that pressure until 40% of maximum shed blood volume was returned in the form of Ringer lactate (RL). Animals were then resuscitated with RL equivalent to four times shed blood volume. Blood samples (0.5 ml) were taken before inducing hemorrhage, at maximal bleed out (45 min), and at 4 and 6 h posthemorrhage to obtain plasma. IL-6 and TNF levels were measured using cytokine-dependent cellular assays. TNF levels were significantly elevated at 45 min into hemorrhage and remained so up to 4 h after hemorrhage. IL-6 levels were also elevated 45 min into hemorrhage and remained so up to 6 h posthemorrhage. IL-6, unlike TNF, was already significantly increased after midline laparotomy and before initiation of hemorrhage compared with unmanipulated animals. Thus induction of IL-6 by trauma may be partially independent of those mechanisms in hemorrhage that are involved in the release of TNF.

Although endothelin-1 (ET-1)-induced organ hypoperfusion after trauma-hemorrhage is improved by estrogen administration, it remains unclear whether estrogen receptor (ER) subtypes play any role in the attenuation of ET-1-induced vasoconstriction in any specific organ bed. To investigate this, isolated perfusion experiments in the heart, liver, small intestine, kidney, and lung were carried out in sham, at the time of maximum bleedout (MBO; i.e., 5-cm midline incision, with removal of 60% of circulating blood volume over 45 min to maintain a mean blood pressure of 40 mmHg), and 2 h after trauma-hemorrhage and resuscitation (T-H/R). Organ-specific ET-1-induced vasoconstriction was evaluated, and the effects of 17β-estradiol (E2) and ER-specific agonists propylpyrazole triol (PPT; ERα agonist) and diarylpropionitrile (DPN; ERβ agonist) were determined. ET-1 induced the greatest vasoconstriction in sham animals, with the strongest response in the kidneys, followed by the small intestine and liver. ET-1-induced responses were weakest in the heart and lungs. ET-1-induced vasoconstriction was evident at the time of MBO but was significantly decreased at 2 h after T-H/R. ERβ plays an important role in cardiac performance, as evidenced by improved heart performance (+dP/d t) in the presence of DPN. DPN also induced a greater effect than PPT in the reduction of ET-1-induced vasoconstriction in the kidneys and lungs. In contrast, PPT attenuated ET-1-induced vasoconstriction in the liver, whereas both DPN and PPT were equally effective in the small intestine. The increased +dP/d t values induced by E2, DPN, or PPT were evident at the time of MBO but were significantly decreased at 2 h after T-H/R. These data indicate that the effects of ET-1 on vasoconstriction and the role of ER subtypes in estrogen-induced vasorelaxation are organ specific and temporally specific after trauma-hemorrhage.

Recent studies indicate that heparinization before hemorrhage maintains microvascular patency in the liver and kidney during and after severe hemorrhagic shock. However, it is not known whether preheparinization has any protective effects on organ function after hemorrhage and resuscitation. To study this, unanesthetized rats (with or without preheparinization) were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of the maximum shed blood volume was returned in the form of Ringer lactate (RL). They were then resuscitated with four times the volume of the shed blood with RL. Cardiac output (CO), [3H]inulin clearance (CIn; renal function), hepatic microvascular blood flow (HMBF), and hepatocellular function (HF), i.e., maximal velocity of indocyanine green clearance (Vmax), were determined 1.5 h after resuscitation. Although CO decreased in both groups, the values in preheparinized rats were significantly higher than in the nonheparinized rats. The improvement in CIn as well as HMBF followed the same trends. HF (Vmax) was significantly depressed in the nonheparinized rats but was maintained in preheparinized rats. Thus administration of heparin before the onset of hemorrhage improves CO and renal function and restores HF to control after hemorrhage and resuscitation. These protective effects of preheparinization could be due to the maintenance of microvascular patency and prevention of blood sludging during and after hemorrhage.

Recent studies from our laboratory have shown that alcohol and burn injury impair intestinal barrier and immune functions. Although multiple factors can contribute to impaired intestinal barrier function, such an alteration could result from a decrease in intestinal blood flow (BF) and oxygen delivery (Do2). Therefore, in this study, we tested the hypothesis that alcohol ingestion before burn injury reduces splanchnic blood flow and oxygen delivery. Rats (250 g) were gavaged with alcohol to achieve a blood ethanol level in the range of 100 mg/dl before burn or sham injury (25% total body surface area). Day 1 after injury, animals were anesthetized with methoxyflurane. Blood pressure, cardiac output (CO), ±dP/d t, organ BF (in ml·min−1·100 g−1), and Do2 (in mg·ml−1·100 g−1) were determined. CO and organ BF were determined using a radioactive microsphere technique. Our results indicate that blood pressure, CO, and +dP/d t were decreased in rats receiving a combined insult of alcohol and burn injury compared with rats receiving either burn injury or alcohol alone. This is accompanied by a decrease in BF and Do2 to the liver and intestine. No significant change in BF to the coronary arteries (heart), brain, lung, skin, and muscles was observed after alcohol and burn injury. In conclusion, the results presented here suggest that alcohol ingestion before burn injury reduces splanchnic BF and Do2. Such decreases in BF and Do2 may cause hypoxic insult to the intestine and liver. Although a hypoxic insult to the liver would result in a release of proinflammatory mediators, a similar insult to the intestine will likely perturb both intestinal immune cell and barrier functions, as observed in our previous study.

Proestrous female rodents are protected from the deleterious effects of trauma-hemorrhage that are observed in males. We hypothesized that the gender dimorphic outcome after trauma-hemorrhage might be related to gender differences in endothelial function and organ perfusion under such conditions. Male and cycle-matched proestrous female Sprague-Dawley rats underwent a midline laparotomy, hemorrhagic shock (40 mmHg for ∼90 min), and resuscitation (Ringer lactate, 4× shed blood volume over 60 min). Various parameters were measured 2 h after completion of resuscitation. In the first set of animals, the left ventricle was cannulated and heart performance (maximal rate of left ventricular pressure increase) as well as cardiac output and organ perfusion rates were determined with 85Sr microspheres. In the second set of animals, aortic vessel rings were harvested and relaxation in response to acetylcholine and nitroglycerin was measured. In the third set of animals, in situ isolated small intestine was perfused to measure the response of the splanchnic vessel bed to acetylcholine and nitroglycerin. After trauma-hemorrhage and resuscitation, females maintained cardiac output and demonstrated increased splanchnic and cardiac perfusion compared with males. Moreover, female intestines did not manifest the endothelial dysfunction that was observed in male intestines after hemorrhagic shock. We conclude that proestrous females show improved endothelial function and tissue perfusion patterns after hemorrhagic shock and that this gender-specific response might be a potential mechanism contributing to the beneficial effects of the proestrus stage under such conditions.

Hypothesis The salutary effects of the testosterone receptor antagonist flutamide on the depressed immune and cardiovascular functions after hemorrhage and resuscitation are related to improved endothelial cell function, which can subsequently lead to an increase in organ blood flow, oxygen delivery, and tissue oxygen consumption. Design, Interventions, and Main Outcome Measures Male adult rats underwent a 5-cm midline laparotomy (ie, trauma) and were bled to and maintained at a mean systemic arterial pressure of 40 mm Hg until 40% maximal blood-out volume was returned in the form of Ringer lactate). The animals were then resuscitated with 4 times the total volume of shed blood with Ringer lactate for 60 minutes. Flutamide (25 mg/kg) or an equivalent volume of the vehicle propanediol was injected subcutaneously 15 minutes before the end of resuscitation. At 20 hours after resuscitation, aortic rings (approximately 2.5 mm in length) were isolated and mounted in an organ chamber. Dose responses for an endothelium-dependent vasodilator (acetylcholine chloride) and endothelium-independent vasodilator (nitroglycerine) were determined. Organ blood flow was measured using strontium 85–labeled microspheres. Total hemoglobin and oxygen content in the femoral artery and portal, hepatic, and renal veins were determined. Oxygen delivery and consumption in liver, small intestine, and kidneys were calculated. Results Administration of flutamide after trauma-hemorrhage attenuated the depressed endothelial function. Furthermore, flutamide treatment restored the reduced blood flow and oxygen delivery and consumption in all organs tested after trauma-hemorrhage and resuscitation. Conclusion Flutamide appears to be a useful adjunct for improving vascular endothelial function and regional hemodynamics after trauma-hemorrhage and resuscitation.

Background. Although acute fluid replacement after trauma and severe hemorrhage remains the cornerstone in the management of trauma victims, it remains unknown whether continuous resuscitation after trauma-hemorrhage and acute fluid replacement produces salutary effects on cardiovascular function and reduces proinflammatory cytokine release. Methods. Adult male rats underwent laparotomy (ie, soft tissue trauma) and were bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of the shed blood volume was returned in the form of Ringer's lactate (RL). The animals were then resuscitated with 4 times the volume of shed blood with RL for 60 minutes, followed by continuous resuscitation with RL at 5 mL/h/kg for 48 hours after the acute fluid replacement. At 48 hours after hemorrhage, mean arterial pressure, cardiac output, and left ventricular contractility parameters, such as the maximal rates of ventricular pressure increase (+dP/dtmax) and decrease (−dP/dtmax), were determined. Microvascular blood flow in the intestine and kidney was assessed by laser Doppler flowmetry. In addition, plasma levels of TNF-α were assayed by enzyme-linked immunosorbent assay. Results. The mean arterial pressure and cardiac output were decreased by 34% and 18%, respectively, at 48 hours after hemorrhage and acute resuscitation. Continuous resuscitation, however, markedly improved these parameters. Similarly, +dP/dtmax and −dP/dtmax decreased significantly after hemorrhage and acute fluid replacement but was restored to sham values after continuous resuscitation. Microvascular blood flow in the gut and kidneys was decreased after hemorrhage and acute resuscitation by 34% and 35%, respectively. However, intestinal and renal perfusion was maintained at the sham levels at 48 hours after continuous resuscitation. In addition, the upregulated TNF-α after acute resuscitation alone was reduced after continuous resuscitation. Conclusions. Continuous resuscitation after acute fluid replacement appears to be a useful approach for restoring and maintaining cardiovascular function and organ perfusion after trauma and severe hemorrhage.(Surgery 2001;129:559-66.)

OBJECTIVE Although polymicrobial sepsis is characterized by an early hyperdynamic phase (2-10 hrs after cecal ligation and puncture [CLP]), followed by a late hypodynamic phase (20 hrs after CLP), it remains unknown whether prostacyclin or prostaglandin I2 (PGI2) plays a significant role in modulating the hyperdynamic state during early sepsis. The aim of this study was to determine whether inhibition of PGI2 synthesis prevents the occurrence of the hyperdynamic response during early sepsis. DESIGN Prospective, controlled animal study. SETTING A university research laboratory. SUBJECTS Adult male Sprague-Dawley rats were subjected to sepsis by CLP. INTERVENTIONS AND MEASUREMENTS Blood samples were collected at 2, 5, 10, or 20 hrs after CLP, and plasma concentrations of PGI2, in the form of its stable product 6-keto-PGF1alpha, were measured by radioimmunoassay. In additional studies, a PGI2 synthase inhibitor, tranylcypromine, was administered subcutaneously at the time of CLP and again at 3 hrs after CLP. At 5 hrs after the onset of sepsis, the maximal rates of the left ventricular pressure rise (+dP/dtmax) and fall (-dP/dtmax) were determined by an in vivo heart performance analyzer. Microvascular blood flow in the liver, small intestine, and spleen was assessed by laser Doppler flowmetry. MAIN RESULTS Plasma concentrations of 6-keto-PGF1alpha increased significantly at 2-20 hrs after CLP. At 5 hrs after the onset of sepsis, +/-dP/dt(max) and microvascular blood flow in the tested tissues increased significantly. Inhibition of PGI2 synthase activity did not prevent the occurrence of hypercardiovascular responses under such conditions. Moreover, the administration of tranylcypromine significantly reduced circulating concentrations of 6-keto-PGF1alpha at 5 hrs after CLP. CONCLUSIONS Because inhibition of PGI2 production did not prevent the occurrence of the hyperdynamic and hypercardiovascular response during the early stage of sepsis, mediators other than PGI2 appear to play a major role in producing the hyperdynamic response under such conditions.

Background. Although sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase, the mechanism responsible for the transition from the hyperdynamic to the hypodynamic state remains unknown. Since recent studies have shown that adrenomedullin (ADM), a novel potent vasodilatory peptide, is upregulated during sepsis, the aim of this study was to determine whether the reduced vascular responsiveness to ADM is associated with the transition from the hyperdynamic phase to the hypodynamic phase of sepsis. Materials and methods. Adult male Sprague–Dawley rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 and 10 h (i.e., the hyperdynamic phase of sepsis) or 20 h (the hypodynamic phase) after CLP, the thoracic aorta or small intestine was harvested and preconstricted with norepinephrine. Adrenomedullin (10 −7 M) was applied and the percentage of ADM-induced vascular relaxation in the aortic ring and isolated small intestine was determined. Results. The responsiveness to ADM in the thoracic aorta was not altered at 5–10 h, but decreased significantly at 20 h after CLP. Although ADM-induced relaxation in resistance blood vessels of the small intestine did not change at 5 h, it decreased markedly at 10 and 20 h after the onset of sepsis. Conclusions. Since the transition from hyperdynamic to hypodynamic sepsis takes place between 10 and 20 h after CLP, it is likely that reduced vascular responsiveness to ADM may be responsible for such an event during the course of polymicrobial sepsis. In view of this, maintenance of vascular ADM responsiveness by pharmacologic agents appears to be a novel approach for preventing or delaying the occurrence of hypodynamic sepsis and septic shock.

Although hepatocellular function is depressed early after trauma and hemorrhage (which are associated with low flow conditions and tissue hypoxemia), it remains unknown whether hypoxemia without blood loss, produces hepatocellular dysfunction and, if so, whether IL-6 and PGE 2 are associated with this dysfunction. To study this, rats were placed in a plastic box which was flushed with a gas mixture containing 6.3% O 2 :93.7% N 2 or room air for 60 min, followed by their return to room air. At 0 and 4 h after hypoxemia, hepatocellular function (i.e., maximum velocity of indocyanine green clearance ( V max ) and the efficiency of the transport ( K m )) was measured using an in vivo hemoreflectometer. Cardiac output was assessed by dye dilution technique. Tissue microvascular blood flow was determined by laser Doppler flowmetry. Plasma IL-6 and PGE 2 were measured by bioassay and radioimmunoassay, respectively. The results indicate that hypoxemia produced a depression in hepatocellular function (i.e., decreased V max by 44–50% and K m by 55–68%) despite stable cardiac output and hepatic microcirculation at 0 and 4 h after hypoxemia. Moreover, hypoxemia resulted in a significant increase in plasma IL-6 (by 372%–389%) as well as PGE 2 (by 38% at 0 h post-hypoxemia). Thus, hypoxemia observed after trauma and hemorrhagic shock appears to be responsible for producing hepatocellular dysfunction possibly through the up-regulation of IL-6 and PGE 2 . In view of this, long-lasting hypoxemia in trauma victims should be avoided, perhaps by early intubation and ventilation so that the potential additional proinflammatory cytokine and PGE 2 release can be prevented.

Although matrix metalloproteinase inhibitors prevent the increase in soluble tumor necrosis factor-α during endotoxemia, it remains unknown whether a novel matrix metalloproteinase inhibitor, GM6001, improves cardiovascular and hepatocellular function after trauma and hemorrhage. To determine this, rats underwent laparotomy (i.e., trauma-induced), and were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of maximal shed volume was returned in the form of Ringer's lactate. The animals were then resuscitated with 3 times the volume of maximal bleedout with Ringer's lactate over 45 min, followed by 2 times Ringer's lactate over 60 min. GM6001, at a dose of 100 mg/kg or an equal volume of normal saline, was administered subcutaneously 15 min before the completion of resuscitation. At 2 and 4 h after resuscitation, cardiac output was measured by indocyanine green (ICG) dilution. Hepatocellular function (i.e., maximum velocity and the efficiency of ICG clearance) was determined by in vivo ICG clearance. Microvascular blood flow in various organs was assessed by laser Doppler flowmetry. The results indicate that cardiac output, hepatocellular function, and tissue microvascular blood flow decreased significantly at 2 and 4 h after resuscitation. GM6001 treatment, however, significantly improved the depressed cardiovascular and hepatocellular function. Since GM6001 improves cardiovascular and hepatocellular function, this agent may be a useful adjunct to fluid resuscitation after trauma and hemorrhagic shock.

Although hepatocellular function is depressed early following hemorrhage, it remains unknown whether liver endothelial cell function is also compromised under such conditions. The aim of this study, however, was to determine if liver endothelial cell function is depressed during hemorrhage and persists following crystalloid resuscitation. To study this, rats underwent a 5-cm laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of maximal bleedout volume was returned in the form of Ringer's lactate. The animals were then resuscitated with 4 times the volume of maximal bleedout volume with Ringer's lactate. Arterial blood was taken before and during hemorrhage and after resuscitation. Plasma hyaluronic acid (HA) levels were determined using a Pharmacia assay kit. To determine whether the elevated HA is due to a decrease in its removal, HA clearance was assessed at 0 and 24 hr after resuscitation by injecting 30 microgram/100 g body wt HA intravenously. The results indicate that plasma HA levels increased significantly at the time of maximal bleedout, which persisted even 24 hr after the completion of resuscitation. Hyaluronic acid clearance decreased significantly at 0 and 24 hr after resuscitation, suggesting that the decreased HA clearance plays a major role in producing the elevated plasma HA levels. Since circulating HA is cleared exclusively by liver endothelial cells, these results, taken together, indicate that liver endothelial cell dysfunction (i.e., the increased plasma HA levels and decreased HA clearance) occurs early during hemorrhage (i.e., approximately 44 min after the onset of the insult) and persists despite resuscitation. Thus, the depressed liver endothelial cell function may directly or indirectly contribute to hepatocellular dysfunction observed under such conditions.

Background and Objective : Although vascular endothelial cell function (i.e., the release of endothelium-derived nitric oxide) decreases and plasma tumor necrosis factor (TNF) increases during sepsis, it is not known whether the elevated TNF is responsible for the depression of endothelial cell function under such conditions. The aim of this study, therefore, was to determine if inhibition of TNF biologic activity by polyethylene glycol dimerized conjugate of the recombinant human form of the p55 soluble TNF receptor (PEG-(rsTNF-R1) 2 ) maintains endothelial function during sepsis. Design, Materials, and Methods : Rats were subjected to sepsis by cecal ligation and puncture (CLP). Immediately before the onset of sepsis, 600 μg/rat PEG-(rsTNF-R1) 2 or an equal volume of saline was infused intravenously. At 10 hours after CLP (i.e., hyperdynamic sepsis), the thoracic aorta was isolated, cut into rings, and placed in organ chambers. Dose responses for an endothelium-dependent vasodilator, acetylcholine (ACh), and an endothelium-independent vasodilator, nitroglycerine (NTG), were determined. Endothelial cell structure was examined by transmission electron microscopy. Results : Endothelium-dependent vascular relaxation was depressed at 10 hours after the onset of sepsis. Administration of PEG-(rsTNF-R1) 2 before CLP, however, maintained ACh-induced relaxation. In contrast, no significant difference in NTG-induced relaxation was seen, irrespective of administration of PEG-(rsTNF-R1) 2 . Furthermore, the deterioration in endothelial structure during sepsis was prevented by PEG-(rsTNF-R1) 2 pretreatment. Conclusion : Since administration of PEG-(rsTNF-R1) 2 maintains vascular endothelial cell structure and function, it can be concluded that TNF plays a pivotal role in producing endothelial dysfunction during sepsis. Thus, pharmacologic agents that inhibit TNF biologic activity and/or its production may be useful for protecting endothelial cells during sepsis.

Although heparinization of animals before hemorrhage improves cell and organ function, the potent anticoagulant activity of conventional heparin sodium precludes its potential clinical use. To determine whether a novel nonanticoagulant heparin, GM1892, would have any beneficial effects on cardiovascular and hapatocellular functions and would decrease susceptibility to sepsis after hemorrhage, laparotomy was performed on rats (i.e., trauma induced), after which they were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of maximal bleedout volume was returned in the form of Ringer lactate solution (RL). The rats were then resuscitated with three times the volume of shed blood with RL over 45 min, followed by infusion of two times RL plus GM1892 (7 mg/kg body wt; approximately 2% the anticoagulant activity of regular heparin) of saline over 60 min. At 2 and 4 h after the completion of resuscitation, cardiac output, hepatocellular function, and microvascular blood flow were determined. The results indicated that cardiac output, hepatocellular function, and microvascular blood flow in the liver, spleen, and small intestine decreased significantly after hemorrhage and resuscitation. Administration of GM1892, however, restored these parameters. The morphological abnormality observed after hemorrhage in the liver, kidney, and small gut was also attenuated with GM1892 treatment. Moreover, GM1892 normalized the elevated plasma prostaglandin E2 levels. Sepsis was induced in additional rats by cecal ligation and puncture (CLP) 20 h after hemorrhage, and the necrotic cecum was excised 10 h thereafter. GM1892 treatment significantly decreased mortality after CLP and cecal excision. Thus GM1892 appears to be a useful adjunct to fluid resuscitation, since it restores the depressed cardiovascular responses and decreases susceptibility to sepsis after trauma and hemorrhage.