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Beta Adrenergic Antagonists in Sepsis: Effects on Survival in A Murine Model
258
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
macrophages are thought to be responsible for the increased susceptibility to sepsis which is seen after severe burn. This supports a two-hit model in which the macrophage inflammatory response is dependent on pre-existing levels of immune cell activation. We, and others, have shown that activation of the parasympathetic nervous system via vagal nerve stimulation (VNS) can alter the inflammatory response to injury. We hypothesized that VNS would decrease peritoneal macrophage responsiveness to an inflammatory insult after severe burn injury. Methods: Male balb/c mice were subjected to a 30% total body surface area steam burn with and without electrical stimulation to the right cervical vagus nerve. Peritoneal macrophages were collected at various time-points after injury. to assess the degree of macrophage hyper-responsiveness, peritoneal macrophages were incubated in LPS or vehicle. Flow cytometry was then used to identify F4/80+ macrophages and to analyze the phosphorylation state of the NFkb pathway mediator, p65 Rel A in this population. Results: There was a significant increase in macrophage NFkb p65 Ser 536 phosphorylation in macrophages exposed burn injury, followed by LPS treatment. Neither burn alone, nor LPS alone was capable of increasing macrophage NFkb p65 Ser 536 phosphorylation. Increased NFkb p65 Ser 536 phosphorylation was seen within 4 hours after burn injury, with phosphorylation returning to baseline by 24 hours post-burn. Treatment with VNS significantly reduced NFkb p65 Ser 536 phosphorylation in burn-primed macrophages that were exposed to LPS. Finally, VNS mediated an unexpected reduction in baseline NFkb p65 Ser 536 phosphorylation levels in sham animals. Conclusions: VNS prevented priming of peritoneal macrophages after severe burn injury, characterized by decreased phosphorylation of NFkb p65 Ser 536. Moreover, VNS limited the baseline inflammatory state of macrophages in uninjured animals. These studies suggest that VNS mediates the inflammatory response in peritoneal macrophages by affecting the set point of LPS-responsiveness. 28.2. Beta Adrenergic Antagonists in Sepsis: Effects on Survival in A Murine Model. R. S. Friese, J. Weller, P. Rhee; University of Arizona: College of Medicine Department of Surgery, Tucson, AZ Introduction: Distinct from their actions on the cardiovascular system, beta-blockers also have profound effects on modulation of the inflammatory response and metabolism. However, sepsis-induced myocardial depression has resulted in clinical avoidance of beta-blockers as treatment in severe sepsis. Potential benefits of beta-blockade in severe sepsis include blunting activation of the sympathetic nervous system resulting in decreased myocardial oxygen demand as well as modulation of the immune response. the purpose of this study was to examine the effect of beta-1 selective blockade on survival after endotoxemia. We hypothesized that beta-1 selective blockade initiated after septic insult would prolong survival in a murine model. Methods: 812 week old C57BL/6J male mice underwent intra-peritoneal injection of 12.5mg/kg of lipopolysaccharide (LPS). Four hours post LPS injection anesthetized animals underwent cannulation of the right external jugular vein with placement of an osmotic pump (Alzet; Cupertino, CA) in the subcutaneous space of the back. Pumps were primed to continuously deliver a beta-1 selective antagonist (esmolol hydrocholide; delivered at 6.7ug/kg/min) or an equal volume of normal saline (control). Primary outcome was survival at 120 hours (5 days) post LPS injection. KaplanMeier survival analysis with log-rank test was utilized to explore for mortality differences between groups. Significance was defined as p<0.05. Results: Mean pre-protocol weight was 25.6 + 2.2 mg. Continuous beta-1 selective antagonist infusion resulted in increased survival at five days post LPS injection. (Figure) Mean survival after LPS injection with beta blockade was 70.4 + 36.4 hours (n¼24). While mean survival after LPS injection without beta blockade was 48.1 + 25.4 hours (n¼23). Hazard ratio for mortality with beta-1 selective blockade is 0.40 (95% CI; 0.20-0.79). Conclusions: Continuous infusion of
a beta-1 selective antagonist initiated after septic insult improves survival at five days in a murine model. Our findings support the use of beta-1 selective blockade in the treatment of sepsis. Further studies exploring mechanisms for this improved survival are warranted.
28.3. Single Nucleotide Polymorphisms and Type of Steroid Impact Functional Response of the Human Glucocorticoid Receptor. A. C. Baker,1,2 V. W. Chew,2 K. Tung,1 D. Lim,2 T. L. Green,2 A. Amini,1,2 K. Cho,2 D. G. Greenhalgh1,2; 1University of California Davis Medical Center, Sacramento, CA; 2Shriners Hospitals for Children Northern California, Sacramento, CA Introduction: Clinical trials evaluating the use of steroids in septic shock have shown variable outcomes, yet little insight has developed as to why some patients improve with steroids and others do not. Studies have implicated polymorphisms of the human glucocorticoid receptor (hGR) coding sequence as a possible cause of altered steroid response. We previously identified a combination of 3 single nucleotide polymorphisms (SNP) of the hGR coding sequence that altered response. in order to further evaluate the role of hGR variations in humans, we further hypothesize that hGR polymorphisms along with type of steroid will influence the stress response and may lead to individualized tailoring of steroid therapy. Methods: Total RNA was isolated from 97 healthy human blood samples and surveyed for the hGR gene. the National Center for Biotechnology Information hGRa sequence was used as a reference for comparison. Two unique SNPs we previously studied, and shown to result in decreased hGRa activity in the absence of steroid (A214G and T962C), were selected for further evaluation with various steroids. Derivative hGRa isoform constructs which isolated the non-synonymous SNPs were created (A214G-hGRa and T962C-hGRa). Luciferase activity was measured to assess functional response of HEK293 cells transfected with these hGR isoforms using a reporter assay in the presence of graded concentrations of 0.9% saline, hydrocortisone(HYD), methylprednisolone(MPS) and dexamethasone(DEX). Significance was determined using ANOVA. Results: Each isoform had a unique dose-response curve to each steroid with a peak activity depending on concentration and type of steroid. the presence of either SNP A214G or T962C within hGRa, resulted in a decreased response when compared to hGRa when stimulated with HYD at a concentration range from 1x106 mM to 1 mM (P<.05). the same decreased response compared to hGRa occurred for the SNPs with DEX stimulation, but at a much lower concentration range than HYD (1x109 mM to 1x106 mM). in the presence of MPS, the presence of SNP A214G resulted in greater activity when compared to hGRa at a concentration range of 1x106 mM to 1x102 mM (P<.05), whereas the presence T962C resulted in activity equivalent to hGRa (Figure). Conclusions: SNPs alter the response of the hGR in both the presence and absence of steroids. in addition, different steroids appear
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
macrophages are thought to be responsible for the increased susceptibility to sepsis which is seen after severe burn. This supports a two-hit model in which the macrophage inflammatory response is dependent on pre-existing levels of immune cell activation. We, and others, have shown that activation of the parasympathetic nervous system via vagal nerve stimulation (VNS) can alter the inflammatory response to injury. We hypothesized that VNS would decrease peritoneal macrophage responsiveness to an inflammatory insult after severe burn injury. Methods: Male balb/c mice were subjected to a 30% total body surface area steam burn with and without electrical stimulation to the right cervical vagus nerve. Peritoneal macrophages were collected at various time-points after injury. to assess the degree of macrophage hyper-responsiveness, peritoneal macrophages were incubated in LPS or vehicle. Flow cytometry was then used to identify F4/80+ macrophages and to analyze the phosphorylation state of the NFkb pathway mediator, p65 Rel A in this population. Results: There was a significant increase in macrophage NFkb p65 Ser 536 phosphorylation in macrophages exposed burn injury, followed by LPS treatment. Neither burn alone, nor LPS alone was capable of increasing macrophage NFkb p65 Ser 536 phosphorylation. Increased NFkb p65 Ser 536 phosphorylation was seen within 4 hours after burn injury, with phosphorylation returning to baseline by 24 hours post-burn. Treatment with VNS significantly reduced NFkb p65 Ser 536 phosphorylation in burn-primed macrophages that were exposed to LPS. Finally, VNS mediated an unexpected reduction in baseline NFkb p65 Ser 536 phosphorylation levels in sham animals. Conclusions: VNS prevented priming of peritoneal macrophages after severe burn injury, characterized by decreased phosphorylation of NFkb p65 Ser 536. Moreover, VNS limited the baseline inflammatory state of macrophages in uninjured animals. These studies suggest that VNS mediates the inflammatory response in peritoneal macrophages by affecting the set point of LPS-responsiveness. 28.2. Beta Adrenergic Antagonists in Sepsis: Effects on Survival in A Murine Model. R. S. Friese, J. Weller, P. Rhee; University of Arizona: College of Medicine Department of Surgery, Tucson, AZ Introduction: Distinct from their actions on the cardiovascular system, beta-blockers also have profound effects on modulation of the inflammatory response and metabolism. However, sepsis-induced myocardial depression has resulted in clinical avoidance of beta-blockers as treatment in severe sepsis. Potential benefits of beta-blockade in severe sepsis include blunting activation of the sympathetic nervous system resulting in decreased myocardial oxygen demand as well as modulation of the immune response. the purpose of this study was to examine the effect of beta-1 selective blockade on survival after endotoxemia. We hypothesized that beta-1 selective blockade initiated after septic insult would prolong survival in a murine model. Methods: 812 week old C57BL/6J male mice underwent intra-peritoneal injection of 12.5mg/kg of lipopolysaccharide (LPS). Four hours post LPS injection anesthetized animals underwent cannulation of the right external jugular vein with placement of an osmotic pump (Alzet; Cupertino, CA) in the subcutaneous space of the back. Pumps were primed to continuously deliver a beta-1 selective antagonist (esmolol hydrocholide; delivered at 6.7ug/kg/min) or an equal volume of normal saline (control). Primary outcome was survival at 120 hours (5 days) post LPS injection. KaplanMeier survival analysis with log-rank test was utilized to explore for mortality differences between groups. Significance was defined as p<0.05. Results: Mean pre-protocol weight was 25.6 + 2.2 mg. Continuous beta-1 selective antagonist infusion resulted in increased survival at five days post LPS injection. (Figure) Mean survival after LPS injection with beta blockade was 70.4 + 36.4 hours (n¼24). While mean survival after LPS injection without beta blockade was 48.1 + 25.4 hours (n¼23). Hazard ratio for mortality with beta-1 selective blockade is 0.40 (95% CI; 0.20-0.79). Conclusions: Continuous infusion of
a beta-1 selective antagonist initiated after septic insult improves survival at five days in a murine model. Our findings support the use of beta-1 selective blockade in the treatment of sepsis. Further studies exploring mechanisms for this improved survival are warranted.
28.3. Single Nucleotide Polymorphisms and Type of Steroid Impact Functional Response of the Human Glucocorticoid Receptor. A. C. Baker,1,2 V. W. Chew,2 K. Tung,1 D. Lim,2 T. L. Green,2 A. Amini,1,2 K. Cho,2 D. G. Greenhalgh1,2; 1University of California Davis Medical Center, Sacramento, CA; 2Shriners Hospitals for Children Northern California, Sacramento, CA Introduction: Clinical trials evaluating the use of steroids in septic shock have shown variable outcomes, yet little insight has developed as to why some patients improve with steroids and others do not. Studies have implicated polymorphisms of the human glucocorticoid receptor (hGR) coding sequence as a possible cause of altered steroid response. We previously identified a combination of 3 single nucleotide polymorphisms (SNP) of the hGR coding sequence that altered response. in order to further evaluate the role of hGR variations in humans, we further hypothesize that hGR polymorphisms along with type of steroid will influence the stress response and may lead to individualized tailoring of steroid therapy. Methods: Total RNA was isolated from 97 healthy human blood samples and surveyed for the hGR gene. the National Center for Biotechnology Information hGRa sequence was used as a reference for comparison. Two unique SNPs we previously studied, and shown to result in decreased hGRa activity in the absence of steroid (A214G and T962C), were selected for further evaluation with various steroids. Derivative hGRa isoform constructs which isolated the non-synonymous SNPs were created (A214G-hGRa and T962C-hGRa). Luciferase activity was measured to assess functional response of HEK293 cells transfected with these hGR isoforms using a reporter assay in the presence of graded concentrations of 0.9% saline, hydrocortisone(HYD), methylprednisolone(MPS) and dexamethasone(DEX). Significance was determined using ANOVA. Results: Each isoform had a unique dose-response curve to each steroid with a peak activity depending on concentration and type of steroid. the presence of either SNP A214G or T962C within hGRa, resulted in a decreased response when compared to hGRa when stimulated with HYD at a concentration range from 1x106 mM to 1 mM (P<.05). the same decreased response compared to hGRa occurred for the SNPs with DEX stimulation, but at a much lower concentration range than HYD (1x109 mM to 1x106 mM). in the presence of MPS, the presence of SNP A214G resulted in greater activity when compared to hGRa at a concentration range of 1x106 mM to 1x102 mM (P<.05), whereas the presence T962C resulted in activity equivalent to hGRa (Figure). Conclusions: SNPs alter the response of the hGR in both the presence and absence of steroids. in addition, different steroids appear