Alejandra G. Garrido

Experimental Models Of Sepsis And Their Clinical Relevance

Sepsis remains a major cause of morbidity and mortality mainly because of sepsis-induced multiple organ dysfunction. In contrast to preclinical studies, most clinical trials of promising new treatment strategies for sepsis have failed to demonstrate efficacy. Although many reasons could account for this discrepancy, the misinterpretation of preclinical data obtained from experimental studies and especially the use of animal models that do not adequately mimic human sepsis may have been contributing factors. In this review, the potentials and limitations of various animal models of sepsis are discussed to clarify to which extent these findings are relevant to human sepsis. Such models include intravascular infusion of endotoxin or live bacteria, bacterial peritonitis, cecal ligation and perforation, soft tissue infection, pneumonia or meningitis models using different animal species including rats, mice, rabbits, dogs, pigs, sheep, and nonhuman primates. Despite several limitations, animal models remain essential in the development of all new therapies for sepsis and septic shock because they provide fundamental information about the pharmacokinetics, toxicity, and mechanism of drug action that cannot be replaced by other methods. New therapeutic agents should be studied in infection models, even after the initiation of the septic process. Furthermore, debility conditions need to be reproduced to avoid the exclusive use of healthy animals, which often do not represent the human septic patient.

Experimental models of sepsis and septic shock: an overview

Sepsis remains a major cause of morbidity and mortality in surgical patients and trauma victims, mainly due to sepsis-induced multiple organ dysfunction. In contrast to preclinical studies, most clinical trials of promising new treatment strategies for sepsis have fails to demonstrate efficacy. Although many reasons could account for this discrepancy, the misinterpretation of preclinical data obtained from experimental studies, and especially the use of animal models that do not adequately mimic human sepsis may have been contributing factors. In this review, the benefits and limitations of various animal models of sepsis are discussed to clarify the extend to which findings are relevant to human sepsis, particularly with respect to the subsequent design and execution of clinical trials. Such models include intravascular infusion of endotoxin or live bacteria, bacterial peritonitis, cecal ligation and perforation, soft tissue infection, pneumonia or meningitis models, using different animal species including rats, mice, rabbits, dogs, pigs, sheep and nonhuman primates. Despite several limitations, animal models remain essential in the development of all new therapies for sepsis and septic shock, because they provide fundamental information about the pharmacokinetics, toxicity, and mechanism of drug action that cannot be duplicated by other methods. New therapeutic agents should be studies in infection models, even after the initiation of the septic process. Furthermore, debility conditions need to be reproduced to avoid the exclusive use of healthy animals, which often do not represent the human septic patient.

Small volume of hypertonic saline as the initial fluid replacement in experimental hypodynamic sepsis

IntroductionWe conducted the present study to examine the effects of hypertonic saline solution (7.5%) on cardiovascular function and splanchnic perfusion in experimental sepsis.MethodsAnesthetized and mechanically ventilated mongrel dogs received an intravenous infusion of live Escherichia coli over 30 minutes. After 30 minutes, they were randomized to receive lactated Ringers solution 32 ml/kg (LR; n = 7) over 30 minutes or 7.5% hypertonic saline solution 4 ml/kg (HS; n = 8) over 5 minutes. They were observed without additional interventions for 120 minutes. Cardiac output (CO), mean arterial pressure (MAP), portal and renal blood flow (PBF and RBF, respectively), gastric partial pressure of CO2 (pCO2; gas tonometry), blood gases and lactate levels were assessed.ResultsE. coli infusion promoted significant reductions in CO, MAP, PBF and RBF (approximately 45%, 12%, 45% and 25%, respectively) accompanied by an increase in lactate levels and systemic and mesenteric oxygen extraction (sO2ER and mO2ER). Widening of venous-arterial (approximately 15 mmHg), portal-arterial (approximately 18 mmHg) and gastric mucosal-arterial (approximately 55 mmHg) pCO2 gradients were also observed. LR and HS infusion transiently improved systemic and regional blood flow. However, HS infusion was associated with a significant and sustained reduction of systemic (18 ± 2.6 versus 38 ± 5.9%) and mesenteric oxygen extraction (18.5 ± 1.9 versus 36.5 ± 5.4%), without worsening other perfusional markers.ConclusionA large volume of LR or a small volume of HS promoted similar transient hemodynamic benefits in this sepsis model. However, a single bolus of HS did promote sustained reduction of systemic and mesenteric oxygen extraction, suggesting that hypertonic saline solution could be used as a salutary intervention during fluid resuscitation in septic patients.

Short-lasting systemic and regional benefits of early crystalloid infusion after intravenous inoculation of dogs with live Escherichia coli

We investigated the systemic and regional hemodynamic effects of early crystalloid infusion in an experimental model of septic shock induced by intravenous inoculation with live Escherichia coli. Anesthetized dogs received an intravenous infusion of 1.2 x 10(10) cfu/kg live E. coli in 30 min. After 30 min of observation, they were randomized to controls (no fluids; N = 7), or fluid resuscitation with lactated Ringers solution, 16 ml/kg (N = 7) or 32 ml/kg (N = 7) over 30 min and followed for 120 min. Cardiac index, portal blood flow, mean arterial pressure, systemic and regional oxygen-derived variables, blood lactate, and gastric PCO2 were assessed. Rapid and progressive cardiovascular deterioration with reduction in cardiac output, mean arterial pressure and portal blood flow (approximately 50, approximately 25 and approximately 70%, respectively) was induced by the live bacteria challenge. Systemic and regional territories showed significant increases in oxygen extraction and in lactate levels. Significant increases in venous-arterial (approximately 9.6 mmHg), portal-arterial (approximately 12.1 mmHg) and gastric mucosal-arterial (approximately 18.4 mmHg) PCO2 gradients were also observed. Early fluid replacement, especially with 32 ml/kg volumes of crystalloids, promoted only partial and transient benefits such as increases of approximately 76% in cardiac index, of approximately 50% in portal vein blood flow and decreases in venous-arterial, portal-arterial, gastric mucosal-arterial PCO2 gradients (7.2 +/- 1.0, 7.2 +/- 1.3 and 9.7 +/- 2.5 mmHg, respectively). The fluid infusion promoted only modest and transient benefits, unable to restore the systemic and regional perfusional and metabolic changes in this hypodynamic septic shock model.

Fluid replacement with hypertonic or isotonic solutions guided by mixed venous oxygen saturation in experimental hypodynamic sepsis.

BACKGROUND Splanchnic perfusion is prone to early injury and persists despite normalization of global hemodynamic variables in sepsis. Volume replacement guided by oxygen derived variables has been recommended in the management of septic patients. Our hypothesis was that a hypertonic isoncotic solution would improve the benefits of crystalloids replacement guided by mixed venous oxygen saturation. METHODS Seventeen anesthetized and mechanically ventilated mongrel dogs received an intravenous infusion of live E. coli in 30 minutes. They were then randomized into three groups: control group (n = 3) bacterial infusion without treatment; normal saline (n = 7), initial fluid replacement with 32 mL/kg of normal saline during 20 minutes; hypertonic solution (n = 7), initial fluid replacement with 4 mL/kg of hypertonic solution during 5 minutes. After 30 and 60 minutes, additional boluses of normal saline were administered when mixed venous oxygen saturation remained below 70%. Mean arterial pressure, cardiac output; regional blood flows, systemic and regional oxygen-derived variables, and lactate levels were assessed. Animals were observed for 90 minutes and then killed. Hystopathological analysis including apoptosis detection using terminal deoxynucleotidil transferase mediated dUTP-biotin nick end labeling was performed. RESULTS A hypodynamic septic shock was observed after bacterial infusion. Both the fluid-treated groups presented similar transient benefits in systemic and regional variables. A greater degree of gut epithelial cells apoptosis was observed in normal saline-treated animals. CONCLUSIONS Although normalization of mixed venous oxygen saturation was not associated with restoration of markers of splanchnic or other systemic perfusion variables, the initial fluid savings with hypertonic saline and its latter effect on gut apoptosis may be of interest in sepsis management.

Regional Blood Flow Distribution and Oxygen Metabolism During Mesenteric Ischemia and Congestion

BACKGROUND Acute mesenteric ischemia is a potentially fatal vascular emergency with mortality rates ranging between 60% and 80%. Several studies have extensively examined the hemodynamic and metabolic effects of superior mesenteric artery occlusion. On the other hand, the cardiocirculatory derangement and the tissue damage induced by intestinal outflow obstruction have not been investigated systematically. For these reasons we decided to assess the initial impact of venous mesenteric occlusion on intestinal blood flow distribution, and correlate these findings with other systemic and regional perfusion markers. METHODS Fourteen mongrel dogs were subjected to 45 min of superior mesenteric artery (SMAO) or vein occlusion (SMVO), and observed for 120 min after reperfusion. Systemic hemodynamics were evaluated using Swan-Ganz and arterial catheters. Regional blood flow (ultrasonic flow probes), intestinal O(2)-derived variables, and mesenteric-arterial and tonometric-arterial pCO(2) gradients (D(mv-a)pCO(2) and D(t-a)pCO(2)) were also calculated. RESULTS SMVO was associated with hypotension and low cardiac output. A significant increase in the regional pCO(2) gradients was also observed in both groups during the ischemic period. After reperfusion, a progressive reduction in D(mv-a)pCO(2) occurred in the SMVO group; however, no improvement in D(t-a)pCO(2) was observed. The histopathologic injury scores were 2.7 +/- 0.5 and 4.8 +/- 0.2 for SMAO and SMVO, respectively. CONCLUSIONS SMV occlusion promoted early and significant hemodynamic and metabolic derangement at systemic and regional levels. Additionally, systemic pCO(2) gradient is not a reliable parameter to evaluate the local intestinal oxygenation. Finally, the D(t-a)pCO(2) correlates with histologic changes during intestinal congestion or ischemia. However, minor histologic changes cannot be detected using this methodology.

Systemic and regional hemodynamic effects of enalaprilat infusion in experimental normotensive sepsis

Angiotensin-converting enzyme inhibitors have been shown to improve splanchnic perfusion in distinct shock states. We hypothesized that enalaprilat potentiates the benefits of early fluid resuscitation in severe experimental sepsis, particularly in the splanchnic region. Anesthetized and mechanically ventilated mongrel dogs received an intravenous infusion of live Escherichia coli over a period of 30 min. Thereafter, two interventions were performed: fluid infusion (normal saline, 32 mL/kg over 30 min) and enalaprilat infusion (0.02 mg kg(-1) min(-1) for 60 min) in randomized groups. The following groups were studied: controls (fluid infusion, N = 4), E1 (enalaprilat infusion followed by fluid infusion, N = 5) and E2 (fluid infusion followed by enalaprilat infusion, N = 5). All animals were observed for a 120 min after bacterial infusion. Mean arterial pressure, cardiac output (CO), portal vein blood flow (PVBF), systemic and regional oxygen-derived variables, and lactate levels were measured. Rapid and progressive reductions in CO and PVBF were induced by the infusion of live bacteria, while minor changes were observed in mean arterial pressure. Systemic and regional territories showed a significant increase in oxygen extraction and lactate levels. Widening venous-arterial and portal-arterial pCO2 gradients were also detected. Fluid replacement promoted transient benefits in CO and PVBF. Enalaprilat after fluid resuscitation did not affect systemic or regional hemodynamic variables. We conclude that in this model of normotensive sepsis inhibition of angiotensin-converting enzyme did not interfere with the course of systemic or regional hemodynamic and oxygen-derived variables.

Efeitos iniciais da reposição volêmica com solução salina hipertônica a 7,5% na perfusão e oxigenação esplâncnica após choque hemorrágico

OBJETIVO: Avaliar os efeitos hemodinâmicos sistemicos e esplâncnico da expansao volemica inicial com SSH em modelo de choque hemorragico controlado. METODOS: Dez caes foram submetidos a sangramento controlado (20 ml/min) ate uma pressao arterial media de 40±5 mmHg (PAM). Apos 30 minutos de choque, receberam 4 ml/Kg de SSH em 5 minutos e posteriormente observados sem intervencoes adicionais durante 60 minutos. As variaveis hemodinâmicas sistemicas foram obtidas de um cateter arterial e de um cateter de Swan-Ganz, enquanto as regionais atraves da cateterizacao da veia porta, fluxometro ultrassonico na veia porta e um tonometro na cavidade. A oferta, taxa de extracao e consumo esplâncnico de oxigenio, pH intramucoso e os gradientes veno-arterial, porta-arterial e mucosa-arterial da pCO2 (Dap-apCO2, Dvp-apCO2 e Dt-apCO2, respectivamente), foram calculados. RESULTADOS: A hemorragia (29,8±2,4 ml/Kg) reduziu a pressao arterial media (125±6 para 42±1 mmHg), o DC (1,9±0,2 para 0,6±0,1 L/min) e o fluxo porta (504±73 para 126±12 ml/min), enquanto elevou o Dap-apCO2 (5,3±0,8 para 19,9±1,6 mmHg), Dvp-apCO2 (5,4±1,4 para 22,6±2,1 mmHg) e o Dt-apCO2 (6,1±1,1 para 43,8±7,5 mmHg). A infusao de SSH resultou em recuperacao parcial dos fluxos sistemico e porta. Atenuou os gradientes de CO2 com menor impacto sobre o Dt-apCO2. CONCLUSAO: A SSH promoveu beneficios parciais na perfusao sistemica e esplâncnica, os quais foram especialmente limitados na microcirculacao regional, como demonstrado pelo Dt-apCO2. Alem disso, as variaveis sistemicas e regionais dependentes de oxigenio, nao refletem a adequacao da perfusao da mucosa gastrica, enfatizando a importância da monitorizacao deste territorio - pela tonometria - durante os estados de choque.

Short-term effects of hydroxyethylstarch resuscitation on systemic and regional hemodynamics and metabolism in a brain-dead canine model.

BACKGROUND Hydroxyethylstarch (HES) is a synthetic polymer of glucose that has been suggested for therapeutic use in long-term plasma expansion. The aim of this study was to test the hypothesis that the infusion of a small volume of HES may provide benefits in systemic and regional hemodynamics and metabolism in a brain-dead canine model compared with large volume crystalloid resuscitation. METHODS Fourteen mongrel dogs were subjected to a brain-death protocol by consecutive insufflations of a balloon catheter in the epidural space. One hour after induction of brain-death, the animals were randomly assigned to two groups: NS (0.9% NaCl, 33 mL/kg), and HES (6%HES 450/0.7, 17 mL/Kg). Systemic and regional hemodynamics were evaluated using Swan-Ganz, ultrasonic flowprobes, and arterial catheters. Serial blood samples were collected for blood gas, electrolyte, and serum chemistry analysis. Systemic, hepatic, and splanchnic O(2)-derived variables were also calculated. RESULTS Epidural balloon insufflations induced a significant increase in mean arterial pressure, cardiac output (MAP and CO, respectively), regional blood flow, and systemic vascular resistance. Following the hyperdynamic phase, severe hypotension with normalization of systemic and regional blood flow was observed. Fluid resuscitation induced a prompt increase in MAP, CO, and portal vein blood flow, and a significant reduction in systemic and pulmonary vascular resistance. There were no differences between groups in metabolic indices, liver function tests (LFTs), or renal function tests. HES was more effective than NS in restoring cardiac performance in the first 2h after fluid resuscitation (P < 0.05). Both tested solutions partially and temporarily restored systemic and regional oxygen delivery. CONCLUSION Small volumes of 6% HES 450/0.7 improved cardiovascular performance and provided the same regional hemodynamic and metabolic benefits of large volumes of isotonic crystalloid solutions.

Early hemodynamics and metabolic changes after total abdominal evisceration for experimental multivisceral transplantation

PURPOSE Multivisceral transplantation (MVTx) has been accepted as standard therapeutic modality for patients with short-bowel syndrome associated with irreversible liver failure. Even nowadays, experimental models of MVTx grounds high incidence of intraoperative or early recipient mortality. Despite the known deleterious effects of hepatosplanchnic exenteration the impact of this procedure on systemic hemodynamics and metabolism remains to be determined. METHODS Nine dogs (20.1+/-0.5 kg) were subjected to an en bloc resection of all abdominal organs including, stomach, duodenum, pancreas, liver, spleen, small bowel, and colon. A woven double velour vascular graft was interposed between the suprahepatic and infrahepatic vena cava. Systemic hemodynamic were evaluated through a Swan-Ganz catheter, ultrasonic flowprobes, and arterial lines. Systemic O2-derived variables, glucose, and lactate metabolism were analyzed throughout the experiment. RESULTS Complete abdominal exenteration was associated with significant reduction in cardiac output, and mean arterial pressure (57% and 14%, respectively). Two hours after reperfusion a significant reduction in arterial pH and glucose were also observed. Oxygen consumption remained unaltered during the first two hours of the experiment, with a significant increase of lactate levels (1.4+/-0.3 vs. 7.6+/-0.4, p<0.05). Three animals died before the 3 hours of reperfusion were completed. Total abdominal exenteration for MVTx in dogs is associated with early major hemodynamics, and metabolic changes. CONCLUSION The deleterious hemodynamic alterations observed are probably related with the association of severe acidosis, hyperlactemia, hypoglycemia, and reduction of total circulating blood volume. Close hemodynamic and metabolic monitoring should be provided during experimental MVTx in order to promote an increase in successful rates of this complex and challenging procedure.