William G. Cioffi

Female Sex Hormones Regulate Macrophage Function After Trauma-Hemorrhage and Prevent Increased Death Rate From Subsequent Sepsis

ObjectiveTo determine whether reduction of circulating female sex hormones by ovariectomy causes suppression of macrophage (M&phgr;) function after trauma-hemorrhage and increases susceptibility to subsequent sepsis. Summary Background DataStudies indicate that immune functions are markedly depressed in males but not in proestrus females after trauma-hemorrhage. Although male sex steroids are immunosuppressive, it remains unknown whether female sex hormones are immunoprotective after trauma-hemorrhage. MethodsCirculating female sex hormones were reduced by ovariectomy of 8-week-old female CBA/J mice. Two weeks afterward, ovariectomy and proestrus sham-ovariectomy mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhagic shock (35 ± 5 mm Hg for 90 minutes, then resuscitated) or sham operation. Two hours afterward, splenic and peritoneal M&phgr; and Kupffer cells were isolated and cytokine production was assessed. In a second series of experiments, animals were subjected to sepsis by cecal ligation and puncture at 24 hours after trauma-hemorrhage or sham operation, and survival was assessed. ResultsRelease of interleukin-1 and interleukin-6 by splenic and peritoneal M&phgr; from proestrus mice was maintained after trauma-hemorrhage, whereas release of interleukin-1 and interleukin-6 by M&phgr; from ovariectomized mice was depressed by approximately 50%. In contrast, trauma-hemorrhage resulted in a fourfold increase of Kupffer cell release of tumor necrosis factor-alpha in ovariectomized females and a fivefold increase in plasma concentrations of tumor necrosis factor-alpha. Release of tumor necrosis factor-alpha and plasma concentrations were unchanged in proestrus mice under such conditions. When proestrus and ovariectomized animals were subjected to sepsis by cecal ligation and puncture at 24 hours after trauma-hemorrhage or sham operation, ovariectomized mice had a significantly higher death rate than proestrus mice. ConclusionsThese findings suggest that female sex hormones play a critical role in maintaining immune responses after trauma-hemorrhage by suppressing the elaboration of tumor necrosis factor-alpha and prevent the increased lethality from subsequent sepsis. Thus, female sex hormones may be a useful adjunct in preventing trauma-induced immunodepression and increased susceptibility to subsequent sepsis.

Estradiol administration after trauma-hemorrhage improves cardiovascular and hepatocellular functions in male animals.

ObjectiveTo determine whether female sex steroids have any salutary effects on the depressed cardiovascular and hepatocellular functions following trauma and hemorrhage in male animals. Summary Background DataStudies indicate that gender difference exists in the immune and cardiovascular responses to trauma-hemorrhage, and that male sex steroids appear to be responsible for producing immune and organ dysfunction, but it remains unknown if female sex steroids produce any salutary effects on the depressed cellular and organ functions in males following trauma and hemorrhage. MethodAdult male Sprague-Dawley rats underwent a midline laparotomy (i.e., trauma induction), and were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of the maximum bleed-out volume was returned in the form of Ringer’s lactate (RL). Animals were then resuscitated with RL at 4 times the shed blood over 60 minutes. 17&bgr;-Estradiol (50 &mgr;g/kg) or an equal volume of vehicle was injected subcutaneously 15 minutes before the end of resuscitation. The maximal rate of ventricular pressure increase or decrease (±dP/dtmax), cardiac output, and hepatocellular function (i.e., maximal velocity and overall efficiency of in vivo indocyanine green clearance) were assessed at 24 hours after hemorrhage and resuscitation. Plasma levels of interleukin (IL)-6 were also measured. ResultsLeft ventricular performance, cardiac output, and hepatocellular function decreased significantly at 24 hours after trauma-hemorrhage and resuscitation. Plasma levels of IL-6 were elevated. Administration of 17&bgr;-estradiol significantly improved cardiac performance, cardiac output, and hepatocellular function, and attenuated the increase in plasma IL-6 levels. ConclusionAdministration of estrogen appears to be a useful adjunct for restoring cardiovascular and hepatocellular functions after trauma-hemorrhage in male rats.

Flutamide : a novel agent for restoring the depressed cell-mediated immunity following soft-tissue trauma and hemorrhagic shock

Recent studies indicate beneficial effects of androgen depletion in male mice, before trauma-hemorrhage on cell-mediated immunity following soft-tissue trauma and hemorrhagic shock. Nonetheless, it remains unknown whether androgen receptor blockade following the insult has any salutary effects. To study this, male C3H/HeN mice were either sham-operated or subjected to soft-tissue trauma (i.e., 2.5 cm midline laparotomy) followed by hemorrhagic shock (blood pressure 35 +/- 5 mmHg for 90 min) and then adequately resuscitated (shed blood and lactated Ringers). Immediately after the completion of resuscitation, as well as 24 and 48 h thereafter, the animals received either vehicle, 10 mg/kg body weight (BW) flutamide or 25 mg/kg BW flutamide subcutaneously. At 72 h after resuscitation, all animals were killed. The spleens and peritoneal macrophages (M phi) were then harvested and cultures established to determine IL-2 and IL-3 release, splenocyte proliferative capacity, as well as splenic and peritoneal M phi IL-1 release. Moreover, plasma testosterone and corticosterone levels were measured. Our results indicate that trauma-hemorrhage resulted in significant depression of splenocyte and M phi functions in vehicle-treated and animals receiving 10 mg/kg BW flutamide. Treatment with 25 mg/kg BW flutamide following trauma-hemorrhage, however, resulted in levels of cytokine release which were comparable with those found in sham-operated animals. No significant alterations in plasma corticosterone and testosterone levels were observed in any of the experimental groups. These findings indicate that short-term therapy of males with the androgen receptor blocker, flutamide at 25 mg/kg BW, following trauma-hemorrhage has protective effects on immune functions. This protective effect is dose dependent, since 10 mg/kg BW flutamide did not produce significant salutary effects. Thus, flutamide represents a novel and safe agent for improving the depressed functions in male trauma patients suffering severe blood loss.

Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis.

Apoptosis and inflammation play an important role in the pathogenesis of direct/pulmonary acute lung injury (ALI). However, the role of the Fas receptor-driven apoptotic pathway in indirect/nonpulmonary ALI is virtually unstudied. We hypothesized that if Fas or caspase-8 plays a role in the induction of indirect ALI, their local silencing using small interfering RNA (siRNA) should be protective in hemorrhage-induced septic ALI. Initially, as a proof of principle, green fluorescent protein-siRNA was administered intratracheally into transgenic mice overexpressing green fluorescent protein. Twenty-four hours after siRNA delivery, lung sections revealed a significant decrease in green fluorescence. Intratracheally administered Cy-5-labeled Fas-siRNA localized primarily in pulmonary epithelial cells. Intratracheal instillation of siRNA did not induce lung inflammation via toll-like receptor or protein kinase PKR pathways as assessed by lung tissue interferon-alpha, tumor necrosis factor-alpha, and interleukin (IL)-6 levels. Mice subjected to hemorrhagic shock and sepsis received either Fas-, caspase-8-, or control-siRNA intratracheally 4 hours after hemorrhage. Fas- or caspase-8-siRNA significantly reduced lung tissue Fas or caspase-8 mRNA, respectively. Only Fas-siRNA markedly diminished lung tissue tumor necrosis factor-alpha, IL-6, IL-10, interferon-gamma, IL-12, and caspase-3 activity. Fas-siRNA also preserved alveolar architecture and reduced lung neutrophil infiltration and pulmonary epithelial apoptosis. These data indicate the pathophysiological significance of Fas activation in nonpulmonary/shock-induced ALI and the feasibility of intrapulmonary administration of anti-apoptotic siRNA in vivo.

Testosterone and/or low estradiol: normally required but harmful immunologically for males after trauma-hemorrhage.

BACKGROUND Previous studies indicate that after severe hemorrhage, immune functions are markedly depressed in males, whereas females do not show any depression. Although androgen depletion by castration of mice before soft-tissue trauma and hemorrhagic shock prevents the depression of cell-mediated immunity, it remains unknown whether testosterone per se is responsible for producing immune depression. METHODS Female C3H/HeN mice were pretreated with 5alpha-dihydrotestosterone (DHT) or vehicle for 20 days. The mice then underwent soft-tissue trauma (laparotomy) and hemorrhagic shock (blood pressure 35+/-5 mm Hg for 90 minutes) followed by adequate fluid resuscitation (shed blood and lactated Ringers solution) or sham operation. Two groups of nontreated male C3H/HeN mice were included as controls: one group was subjected to hemorrhagic shock followed by resuscitation, and the second group underwent only sham operation. At 24 hours after trauma-hemorrhage and resuscitation, animals were killed, macrophages harvested from the peritoneum and spleen, and their ability to release interleukin (IL)-1 and IL-6 was evaluated. Plasma DHT, estradiol, and corticosterone levels were measured by radioimmunoassay. RESULTS Treatment of female mice with DHT produces a significant increase in DHT levels that was comparable with those seen in nontreated male mice. Alternatively, estradiol levels in female mice were significantly depressed by DHT treatment to levels comparable with those observed in control males. In the vehicle-treated female mice, no depression of the macrophage function was evident after trauma hemorrhage. In contrast, testosterone-treated female mice that had experienced hemorrhage showed significant depression in splenic and peritoneal macrophage IL-1 and IL-6 production, comparable with the values seen in macrophages from male mice that had experienced hemorrhage. CONCLUSIONS These findings indicate that pretreatment of female mice with DHT depresses macrophage function after trauma-hemorrhage, which mimics the changes seen in normal male mice subjected to trauma-hemorrhage. We propose, therefore, that high testosterone and/or low estradiol levels are responsible for producing the immune depression in male mice after trauma-hemorrhage. Testosterone receptor blocking agents, e.g., flutamide, and/or estradiol administration should thus be useful adjuncts for preventing immune depression in male trauma patients.

Transfer Times to Definitive Care Facilities Are Too Long: A Consequence of an Immature Trauma System

Objective:The purpose of this study was to review our experience with interfacility transfers to identify problems that could be addressed in the development of a statewide trauma system. Background:The fundamental tenet of a trauma system is to get the right patient to the right hospital at the right time. This hinges on well-defined prehospital destination criteria, interfacility transfer protocols, and education of caregivers. Patients arriving at local community hospitals (LOCs) benefit from stabilization and transfer to trauma centers (TCs) for definitive care. However, in the absence of a formalized trauma system, patients may not reach the TC in a timely fashion and may not be appropriately treated or stabilized at LOCs prior to transfer. Methods:Our facility is a level I TC and regional referral center for a compact geographic area without a formal trauma system. The Trauma Registry was queried for adult patients admitted to the trauma service between January 1, 2001 and March 30, 2003. Patients were divided into 2 groups: those received directly from the scene (DIR) and those transferred from another institution (TRAN). Medical records were reviewed to elucidate details of the early care. Data are presented as mean ± SEM. Continuous data were compared using Student t test, and categorical data using χ2. Transfer times were analyzed by one-way ANOVA. Results:A total of 3507 patients were analyzed. The TRAN group had a higher Injury Severity Score (ISS) (17.5 versus 11.0, P < 0.05), lower Glasgow Coma Score (GCS) (13.3 versus 14.1, P < 0.05), lower initial systolic blood pressure (SBP) (130 versus 140, P< 0.05), and higher mortality (10% versus 79%, P < 0.05) than the DIR group. The average time spent at the LOC was 162 ± 8 minutes. The subgroup of patients with hypotension spent an average of 134 minutes at the LOC, often receiving numerous diagnostic tests despite unavailability of surgeons to provide definitive care. Severe head injury (GCS = 3) triggered more prompt transfer, but high ISS was underappreciated and did not result in a prompt transfer in all but the most severely injured group (ISS > 40). Some therapeutic interventions were initiated at the LOCs, but many were required at the TC. A total of 23 (8%) TRAN patients required critical interventions within 15 minutes of arrival; mortality in this group was 52%. Mortality among those requiring laparotomy after transfer was 33%. Conclusions:All but the most severely injured patients spend prolonged periods of time in LOCs, and many require critical interventions upon arrival at the TC. It is unreasonable to expect immediate availability of surgeons or operating rooms in LOCs. Thus, trauma system planning efforts should focus on 1) prehospital destination protocols that allow direct transport to the TC; and 2) education of caregivers in LOCs to enhance intervention skill sets and expedite transfer to definitive care.

Gastrointestinal Complications in Patients Undergoing Heart Operation: An Analysis of 8709 Consecutive Cardiac Surgical Patients

Introduction:Gastrointestinal (GI) complications following heart operation may be life-threatening. Systematic analysis of risk factors to allow early identification of patients at risk for GI complication may lead to the development of strategies to mitigate this complication as well as to optimize management after its occurrence. Methods:Of 8709 consecutive patients undergoing heart operation during 7 years (1997–2003), 46 (0.53%) developed GI complications requiring surgical consultation. Preoperative, intraoperative, and postoperative predictors of complication and death were identified and compared with a control group. Results:Significant (P < 0.05) preoperative predictors of complication were prior cerebrovascular accident (CVA), chronic obstructive pulmonary disease (COPD), type II heparin-induced thrombocytopenia, atrial fibrillation, prior myocardial infarction, renal insufficiency, hypertension, and need for intra-aortic balloon counter-pulsation. The most frequent serious GI complication was mesenteric ischemia, which developed in 31 (67%) patients. Twenty-two (71%) of these patients were explored, and 14 (64%) died within 2 days of heart operation. Of the 9 patients with mesenteric ischemia who were not explored, 7 (78%) died within 3 days of heart operation. Other complications included diverticulitis (5), pancreatitis (4), peptic ulcer disease (4), and cholecystitis (2). The mortality rate in this group of other diagnoses was lower (40%), and death occurred later (32 days) after heart operation (P = 0.03 compared with mesenteric ischemia). Predictors of death from GI complication included New York Heart Association (NYHA) class III and IV heart failure, smoking, chronic obstructive pulmonary disease, history of syncope, aspartate aminotransferase (AST) >600U/L, direct bilirubin >2.4mg/dL, pH < 7.30, and the need for >2 pressors. Conclusions:The most common catastrophic GI complication after cardiac surgery is mesenteric ischemia, which is frequently fatal. This complication may be a result of atheroembolization, heparin-induced thrombocytopenia, or hypoperfusion. Techniques to reduce the occurrence of and/or preemptively diagnosis postcardiotomy mesenteric ischemia are necessary to decrease its associated mortality.

Sex steroids regulate pro- and anti-inflammatory cytokine release by macrophages after trauma-hemorrhage

Studies indicate that macrophage immune responses in males are depressed after trauma-hemorrhage, whereas they are enhanced in females under such conditions. Nonetheless, the involvement of male and female sex steroids in this gender-dependent dimorphic immune response after trauma-hemorrhage remains unclear. To study this, male C3H/HeN mice were castrated and treated with pellets containing either vehicle, 5α-dihydrotestosterone (DHT), 17β-estradiol, or a combination of both steroid hormones for 14 days before soft tissue trauma (i.e., laparotomy) and hemorrhagic shock (35 ± 5 mmHg for 90 min followed by adequate fluid resuscitation) or a sham operation. Twenty-four hours later the animals were killed, plasma was obtained, and Kupffer cell and splenic and peritoneal macrophage cultures were established. For DHT-treated mice, we observed significantly decreased releases of the proinflammatory cytokines interleukin 1β (IL-1β) and IL-6 by splenic macrophage (-50 and -57%, respectively) and peritoneal macrophage (-51 and -52%, respectively) cultures after trauma-hemorrhage compared with releases by cultures of cells from mice subjected to a sham operation; in contrast, responses of splenic and peritoneal macrophage cultures from other groups subjected to trauma-hemorrhage did not change significantly. In addition, only DHT-treated animals exhibited increased Kupffer cell IL-6 release (+634%). The release of IL-10 in DHT-treated hemorrhaged animals was increased compared with that in sham-operated animals but was decreased in estrogen-treated mice under such conditions. These results suggest that male and female sex steroids exhibit divergent immunomodulatory properties with respect to cell-mediated immune responses after trauma-hemorrhage.

Implementation of a Tertiary Trauma Survey Decreases Missed Injuries

BACKGROUND Missed injuries (MIs) adversely affect patient outcome and damage physician/institutional credibility. The primary and secondary surveys are designed to identify all of a patients injuries and prioritize their management; however, MIs are prevalent in severely injured and multisystem trauma patients, especially when the patients condition precludes completion of the secondary survey. We hypothesized that implementation of a routine tertiary trauma survey (TS) would reduce the incidence of MIs in a Level I trauma center. METHODS In mid 1999, a TS form was created and TS documentation was mandated on all trauma intensive care unit (TICU) patients within 24 hours of admission. Patient data, including TS documentation and injury patterns, were concurrently recorded in an institutional trauma registry. Data were compared for patients admitted in 1997 to 1998 (PRE period) and 2000 to 2001 (POST period) using chi or Students test. RESULTS MIs decreased from 2.4% to 1.5% overall, and from 5.7% to 3.4% in TICU patients, after TS implementation. Patients with MIs were slightly older (49 vs. 45 years; > 0.05) and had higher Injury Severity Scores (21 vs. 10; < 0.05) than patients without MIs. Sixty percent of MI patients had brain injuries, 56% were admitted to the TICU, and 26% went directly from the emergency department to the operating room. The large majority of MIs in the POST period were detected in patients not undergoing timely TS. CONCLUSION ICU patients-particularly brain injury victims and those undergoing emergent surgical procedures-appear to be at highest risk for MI. Implementation of a standardized TS decreased MIs by 36% in our Level I trauma center, and more timely TS would likely have further reduced MIs. A TS should be routine in trauma centers.

Testosterone: the culprit for producing splenocyte immune depression after trauma hemorrhage

Studies indicate that, whereas immune functions in males are depressed, they are enhanced in females after trauma hemorrhage. Moreover, castration of male mice (i.e., androgen depletion) before trauma hemorrhage prevented the depression of cell-mediated immunity. Nonetheless, it remains unknown whether or not testosterone per se is responsible for producing the immune depression. To study this, female C3H/HeN mice ( n = 7 animals/group) were pretreated with 5α-dihydrotestosterone (DHT) or vehicle for 19 days, then subjected to laparotomy (e.g., trauma) and hemorrhagic shock (blood pressure 35 ± 5 mmHg for 90 min) followed by fluid resuscitation or sham operation. Nontreated males underwent either trauma hemorrhage or sham operation. Twenty-four hours thereafter, splenocyte immune functions as well as plasma DHT, estradiol, and corticosterone levels were measured. DHT-pretreated females had significantly ( P < 0.05) increased DHT levels, comparable to those seen in males. Conversely, estradiol levels in such females were similar to control males. Splenocyte proliferation as well as interleukin-2 and interleukin-3 release were not depressed in vehicle-treated females, whereas it was in DHT-treated females after trauma hemorrhage, comparable to hemorrhaged males. Thus high testosterone and/or low estradiol levels appear to be responsible for producing splenocyte immune depression in males after trauma hemorrhage. Agents that block testosterone receptors or increase estradiol levels may therefore be helpful in improving depressed immune functions in male trauma patients.Studies indicate that, whereas immune functions in males are depressed, they are enhanced in females after trauma hemorrhage. Moreover, castration of male mice (i.e., androgen depletion) before trauma hemorrhage prevented the depression of cell-mediated immunity. Nonetheless, it remains unknown whether or not testosterone per se is responsible for producing the immune depression. To study this, female C3H/HeN mice (n = 7 animals/group) were pretreated with 5-dihydrotestosterone (DHT) or vehicle for 19 days, then subjected to laparotomy (e.g., trauma) and hemorrhagic shock (blood pressure 35 +/- 5 mmHg for 90 min) followed by fluid resuscitation or sham operation. Nontreated males underwent either trauma hemorrhage or sham operation. Twenty-four hours thereafter, splenocyte immune functions as well as plasma DHT, estradiol, and corticosterone levels were measured. DHT-pretreated females had significantly (P < 0.05) increased DHT levels, comparable to those seen in males. Conversely, estradiol levels in such females were similar to control males. Splenocyte proliferation as well as interleukin-2 and interleukin-3 release were not depressed in vehicle-treated females, whereas it was in DHT-treated females after trauma hemorrhage, comparable to hemorrhaged males. Thus high testosterone and/or low estradiol levels appear to be responsible for producing splenocyte immune depression in males after trauma hemorrhage. Agents that block testosterone receptors or increase estradiol levels may therefore be helpful in improving depressed immune functions in male trauma patients.