Perioperative Cannabinoids Significantly Reduce Postoperative Opioid Requirements in Patients Undergoing Coronary Artery Bypass Graft Surgery.

Background Opioids, commonly used to control pain associated with surgery, are known to prolong the duration of mechanical ventilation and length of hospital stay. A wide range of adjunctive strategies are currently utilized to reduce postoperative pain, such as local and regional nerve blocks, nerve cryoablation, and adjunctive medications. We hypothesized that dronabinol (a synthetic cannabinoid) in conjunction with standard opioid pain management will reduce opioid requirements to manage postoperative pain. Methods Sixty-eight patients who underwent isolated first-time coronary artery bypass graft surgery were randomized to either the control group, who received only standard opioid-based analgesia, or the dronabinol group, who received dronabinol (a synthetic cannabinoid) in addition to standard opioid-based analgesia. Dronabinol was given in the preoperative unit, before extubation in the ICU, and after extubation on the first postoperative day. Preoperative, intraoperative, and postoperative parameters were compared under an IRB-approved protocol. The primary endpoints were the postoperative opioid requirement, duration of mechanical ventilation, and ICU length of stay, and the secondary endpoints were the duration of inotropic support needed, left ventricular ejection fraction (LVEF), and the change in LVEF. This study was undertaken at Northwest Medical Center, Tucson, AZ, USA. Results Sixty-eight patients were randomized to either the control group (n = 37) or the dronabinol group (n = 31). Groups were similar in terms of demographic features and comorbidities. The total postoperative opioid requirement was significantly lower in the dronabinol group [39.62 vs 23.68 morphine milligram equivalents (MMEs), p = 0.0037], representing a 40% reduction. Duration of mechanical ventilation (7.03 vs 6.03h, p = 0.5004), ICU length of stay (71.43 vs 63.77h, p = 0.4227), and inotropic support requirement (0.6757 vs 0.6129 days, p = 0.7333) were similar in the control and the dronabinol groups. However, there was a trend towards lower durations in each endpoint in the dronabinol group. Interestingly, a significantly better preoperative to postoperative LVEF change was observed in the dronabinol group (3.51% vs 6.45%, p = 0.0451). Conclusions Our study found a 40% reduction in opioid use and a significantly greater improvement in LVEF in patients treated with adjunctive dronabinol. Mechanical ventilation duration, ICU length of stay, and inotropic support requirement tended to be lower in the dronabinol group, though did not reach statistical significance. The results of this study, although limited by sample size, are very encouraging and validate our ongoing investigation.

Physiological and microvascular responses to hemoglobin concentration-targeted hemolytic anemia in rats.

Hemolytic anemia (HA) is reduced blood oxygen-carrying capacity resulting from the depletion of red blood cells. Treatment for severe cases involves transfusion to improve oxygen delivery (Do2), which carries risk. In humans, a total hemoglobin (tHb) concentration of 8 g/dL is severe, and <7 g/dL indicates transfusion. Some evidence suggests that compensatory mechanisms maintaining Do2 are not compromised until <5 g/dL rendering transfusion at 7 g/dL premature. A Sprague-Dawley rat model of phenylhydrazine-induced HA was assessed over decreasing tHb for a Do2 decompensation point. Three groups (100, 50, or 25% tHb, equating to 16.4, 7.4, or 3.2 g/dL) were generated. Cardiopulmonary, blood chemistry, and oxygenation parameters were measured under anesthesia. Vasoconstrictive responsiveness to phenylephrine was assessed in the exteriorized spinotrapezius. For 50% tHb, cardiopulmonary parameters, Do2, and lactate levels were similar to those for 100% tHb. Enhanced vasoconstriction occurred with 50% tHb (P < 0.0001), not 25% tHb. The 25% group showed decreases in cardiopulmonary parameters, Do2, and lactate levels compared with the 100% and 50% groups (P < 0.05). Do2 showed a positive correlation with lactate levels at 25% tHb, but decompensation, defined by peripheral hypoxia, was not reached. This is the first study relating Do2 to tHb in rats. A 50% reduction in tHb was supported by vascular compensation, whereas 25% tHb levied the cardiopulmonary system. A decompensation point was not identified. A rising need for treatment as tHb levels decline below 8 g/dL is evident, but, as compensatory mechanisms remain intact as tHb approaches 3.2 g/dL in rats, a transfusion limit of 5 g/dL in healthy patients is supported.NEW & NOTEWORTHY Early, chronic compensation to severe hemolytic anemia is vascular, switching to cardiopulmonary support as hemoglobin levels decline. Oxygen delivery does not correlate with serum lactate level until total hemoglobin is reduced by 75%.

Prehospital whole blood resuscitation prevents coagulopathy and improves acid-base status at hospital arrival in a nonhuman primate hemorrhagic shock model.

BACKGROUND: Hemorrhage remains the primary cause of preventable death in civilian and military trauma. The Committee on Tactical Combat Casualty Care recommends prehospital (PH) resuscitation with whole blood (WB). However, 6% hetastarch in lactated electrolyte (HEX) and crystalloids are more commonly available and used for PH resuscitation in military and civilian environments, respectively. The mechanistic benefits of PH WB resuscitation have not been well studied and remain to be elucidated. STUDY DESIGN AND METHODS: The aim of this study was to evaluate the differences in simulated PH WB and HEX resuscitation, specifically with regards to coagulation, physiologic, and metabolic outcomes to better elucidate the mechanistic benefits of WB. In a randomized study, the physiologic, coagulation, and metabolic responses to simulated PH WB (n = 12) or HEX (n = 12) were evaluated in a nonhuman primate model of severe polytraumatic hemorrhagic shock. RESULTS: Notable findings included 1) equivalence of shock reversal between simulated PH WB and HEX treatment groups as determined by hemodynamics and base deficit and 2) prevention of coagulopathy at simulated hospital arrival with initial WB resuscitation as determined by viscoelastic and plasmatic coagulation assays. CONCLUSION: The major benefit of WB, as compared to HEX, in simulated PH resuscitation appears to be prevention of coagulopathy at hospital arrival. Both fluids effectively reversed shock in this model, implying that efficacious provision preload (cardiac output support and hence oxygen delivery) and coagulation proteins (prevention of coagulopathy) are mechanisms underlying WB's effectiveness in early resuscitation of hemorrhagic shock.

Whole blood mitigates the acute coagulopathy of trauma and avoids the coagulopathy of crystalloid resuscitation.

INTRODUCTION: The contributions of type and timing of fluid resuscitation to coagulopathy in trauma remain controversial. As part of a multifunctional resuscitation fluid research effort, we sought to further characterize the coagulation responses to resuscitation, specifically as compared to whole blood. We hypothesized that early whole blood administration mitigates the acute coagulopathy of trauma by avoiding the coagulopathy of CR resuscitation. METHODS: Anesthetized rhesus macaques underwent polytraumatic, hemorrhagic shock, then a crossover study design resuscitation (n = 6 each) with either whole blood first (WB-1st) followed by crystalloid (CR); or CR-1st followed by WB. Resuscitation strategies were the following: WB-1st received 50% shed blood in 30minutes, followed by twice the shed blood volume (SBV) of CR over 30minutes and one times the SBV CR over 60minutes, where CR-1st received twice the SBV of CR over 30minutes, followed by 50% of shed blood in 30minutes, and one times the SBV CR over 60minutes. Blood samples were collected at baseline, end-of-shock, end-of-first and end-of-second resuscitation stages, and end-of-resuscitation for assessment (thromboelastometry, platelet aggregation, and plasmatic coagulation factors). Statistical analyses were conducted using two-way analysis of variance ANOVA with Bonferroni correction and t-tests; significance was at p < 0.05. RESULTS: Survival, blood loss, hemodynamics, and shock duration were equivalent between the groups. Compared to baseline, parameters measured at first and second resuscitation stage time points directly following CR infusion revealed abnormalities in thromboelastometry (clot formation time, α angle, and maximum clot firmness), platelet aggregation response (to collagen, arachidonic acid, and adenosine diphosphate), and plasmatic coagulation (prothrombin time, anti-thrombin 3, and fibrinogen), while whole blood infusion resulted in stabilization or correction of these parameters following its administration. CONCLUSIONS: These data suggest that in the setting of trauma and hemorrhagic shock, the coagulation alterations begin before intervention/resuscitation; however, these are significantly aggravated by CR resuscitation and could perhaps be best termed acute coagulopathy of resuscitation. STUDY TYPE: Translational animal model.

Chronic Adrenergic Signaling Causes Abnormal RNA Expression of Proliferative Genes in Fetal Sheep Islets.

Intrauterine growth restriction (IUGR) increases the risk of developing diabetes in later life, which indicates developmental programming of islets. IUGR fetuses with placental insufficiency develop hypoxemia, elevating epinephrine and norepinephrine (NE) concentrations throughout late gestation. To isolate the programming effects of chronically elevated catecholamines, NE was continuously infused into normally grown sheep fetuses for 7 days. High plasma NE concentrations suppress insulin, but after the NE infusion was terminated, persistent hypersecretion of insulin occurred. Our objective was to identify differential gene expression with RNA sequencing (RNAseq) in fetal islets after chronic adrenergic stimulation. After determining the NE-regulated genes, we identified the subset of differentially expressed genes that were common to both islets from NE fetuses and fetuses with IUGR to delineate the adrenergic-induced transcriptional responses. A portion of these genes were investigated in mouse insulinoma (Min6) cells chronically treated with epinephrine to better approximate the ß-cell response. In islets from NE fetuses, RNAseq identified 321 differentially expressed genes that were overenriched for metabolic and hormone processes, and the subset of 96 differentially expressed genes common to IUGR islets were overenriched for protein digestion, vitamin metabolism, and cell replication pathways. Thirty-eight of the 96 NE-regulated IUGR genes changed similarly between models with functional enrichment for proliferation. In Min6 cells, chronic epinephrine stimulation slowed proliferation and augmented insulin secretion after treatment. These data establish molecular mechanisms underlying persistent adrenergic stimulation in hyperfunctional fetal islets and identify a subset of genes dysregulated by catecholamines in IUGR islets that may represent programming of ß-cell proliferation capacity.

Nonhuman Primate (Rhesus Macaque) Models of Severe Pressure-Targeted Hemorrhagic and Polytraumatic Hemorrhagic Shock.

BACKGROUND: We endeavored to develop clinically translatable nonhuman primate (NHP) models of severe polytraumatic hemorrhagic shock. METHODS: NHPs were randomized into five severe pressure-targeted hemorrhagic shock (PTHS) ±â€Šadditional injuries scenarios: 30-min PTHS (PTHS-30), 60-min PTHS (PTHS-60), PTHS-60 + soft tissue injury (PTHS-60+ST), PTHS-60+ST + femur fracture (PTHS-60+ST+FF), and decompensated PTHS+ST+FF (PTHS-D). Physiologic parameters were recorded and blood samples collected at five time points with animal observation through T = 24 h. Results presented as mean ±â€ŠSEM; statistics: log transformation followed by two-way ANOVA with Bonferroni multiple comparisons, Wilcoxon nonparametric test for comparisons, and the Friedmans' one-way ANOVA; significance: P < 0.05. RESULTS: Percent blood loss was 40% ±â€Š2, 59% ±â€Š3, 52% ±â€Š3, 49% ±â€Š2, and 54% ±â€Š2 for PTHS-30, PTHS-60, PTHS-60+ST, PTHS-60+ST+FF, and PTHS-D, respectively. All animals survived to T = 24 h except one in each of the PTHS-60 and PTHS-60+ST+FF groups and seven in the PTHS-D group. Physiologic, coagulation, and inflammatory parameters demonstrated increasing derangements with increasing model severity. CONCLUSION: NHPs exhibit a high degree of resilience to hemorrhagic shock and polytrauma as evidenced by moderate perturbations in metabolic, coagulation, and immunologic outcomes with up to 60 min of profound hypotension regardless of injury pattern. Extending the duration of PTHS to the point of decompensation in combination with polytraumatic injury, evoked derangements consistent with those observed in severely injured trauma patients which would require ICU care. Thus, we have successfully established a clinically translatable NHP trauma model for use in testing therapeutic interventions to trauma.

Whole blood and Hextend: Bookends of modern tactical combat casualty care field resuscitation and starting point for multifunctional resuscitation fluid development.

BACKGROUND: Hemorrhage is the leading cause of preventable death in traumatically injured civilian and military populations. Prehospital resuscitation largely relies on crystalloid and colloid intravascular expansion, as whole blood and component blood therapy are logistically arduous. In this experiment, we evaluated the bookends of Tactical Combat Casualty Care Guidelines recommendations of prehospital resuscitation with Hextend and whole blood in a controlled hemorrhagic shock model within non-human primates, as means of a multifunctional resuscitative fluid development. METHODS: In the nonhuman primate, a multiple injuries model was used, consisting of a musculoskeletal injury (femur fracture), soft tissue injury (15-cm laparotomy), and controlled hemorrhage to a mean arterial pressure of 20 mm Hg, demarcating the beginning of the shock period. Animals were randomized to prehospital interventions of whole blood or Hextend at T = 0 minutes, and at T = 90 minutes definitive surgical interventions and balanced sanguineous damage control resuscitation could be implemented. All animals were euthanized at T = 480 minutes. Data are expressed as mean ± SEM; significance, p < 0.05. RESULTS: No significant differences in survival (83% vs. 100%; p = 0.3), tissue perfusion (EtCO2 and StO2) or endpoints of resuscitation (base deficit, lactate, pH) between Hextend and whole blood were identified. Second, whole blood compared with Hextend demonstrated significantly earlier normalization of clot formation time, maximal clot firmness, and α angle. CONCLUSION: A future multifunctional resuscitative fluid including an asanguineous, oncotic, non-oxygen-carrying component to facilitate intravascular volume expansion, and a component with synthetic coagulation factors and fibrinogen to deter coagulopathy may show equivalence to whole blood. LEVEL OF EVIDENCE: N/A: Study type: translational animal model.

Tissue injury suppresses fibrinolysis after hemorrhagic shock in nonhuman primates (rhesus macaque).

BACKGROUND: Hypoperfusion is associated with hyperfibrinolysis and early death from exsanguination, whereas tissue trauma is associated with hypofibrinolysis and delayed death from organ failure. We sought to elucidate the effects of injury patterns on fibrinolysis phenotypes using a nonhuman primate (NHP) model. METHODS: NHPs were randomized to three injury groups (n = 8/group): 60 minutes severe pressure-targeted controlled hemorrhagic shock (HS); HS + soft tissue injury (HS+); or HS + soft tissue injury + femur fracture (HS++). Animals were resuscitated and monitored for 360 minutes. Blood samples were collected at baseline, end-of-shock, end-of-resuscitation (EOR), and T = 360 minutes for assessments of: severity of shock (lactate) and coagulation via prothrombin time, partial thromboplastin time, D-dimer, fibrinogen, antithrombin-III, von Willebrand factor, and viscoelastic testing (ROTEM). Results are reported as mean ± SEM; statistics: two-way analysis of variance and t-tests (significance: p < 0.05). RESULTS: Blood loss, prothrombin time, partial thromboplastin time, antithrombin-III, fibrinogen, and von Willebrand factor were equivalent among groups and viscoelastic testing revealed few differences throughout the study. D-dimer increased approximately threefold, at EOR in the HS group, and at T = 360 minutes in the HS+ and HS++ groups (p < 0.05). At EOR, in the HS group compared with the HS+ and HS++ groups; the D-dimer-lactate ratio was twofold greater (2.2 ± 0.3 vs. 1.1 ± 0.3 and 1.1 ± 0.2, respectively; p < 0.05) and tissue factor-activated fibrin clot 30-minute lysis index was lower (98 ± 1% vs. 100 ± 0% and 100 ± 0%, respectively; p < 0.05). CONCLUSION: NHPs in HS exhibit acute suppression of fibrinolysis in the presence of tissue injury. Additional assessments to more comprehensively evaluate the mechanisms linking tissue injury with the observed fibrinolysis shutdown response are warranted.

Islet adaptations in fetal sheep persist following chronic exposure to high norepinephrine.

Complications in pregnancy elevate fetal norepinephrine (NE) concentrations. Previous studies in NE-infused sheep fetuses revealed that sustained exposure to high NE resulted in lower expression of α2-adrenergic receptors in islets and increased insulin secretion responsiveness after acutely terminating the NE infusion. In this study, we determined if the compensatory increase in insulin secretion after chronic elevation of NE is independent of hyperglycemia in sheep fetuses and whether it is persistent in conjunction with islet desensitization to NE. After an initial assessment of glucose-stimulated insulin secretion (GSIS) at 129 ± 1 days of gestation, fetuses were continuously infused for seven days with NE and maintained at euglycemia with a maternal insulin infusion. Fetal GSIS studies were performed again on days 8 and 12. Adrenergic sensitivity was determined in pancreatic islets collected at day 12. NE infusion increased (P < 0.01) fetal plasma NE concentrations and lowered (P < 0.01) basal insulin concentrations compared to vehicle-infused controls. GSIS was 1.8-fold greater (P < 0.05) in NE-infused fetuses compared to controls at both one and five days after discontinuing the infusion. Glucose-potentiated arginine-induced insulin secretion was also enhanced (P < 0.01) in NE-infused fetuses. Maximum GSIS in islets isolated from NE-infused fetuses was 1.6-fold greater (P < 0.05) than controls, but islet insulin content and intracellular calcium signaling were not different between treatments. The half-maximal inhibitory concentration for NE was 2.6-fold greater (P < 0.05) in NE-infused islets compared to controls. These findings show that chronic NE exposure and not hyperglycemia produce persistent adaptations in pancreatic islets that augment ß-cell responsiveness in part through decreased adrenergic sensitivity.

Inflammatory Profile in Response to Uncontrolled Hemorrhage in a Non-Human Primate (Rhesus Macaque) Model.

BACKGROUND: Uncontrolled hemorrhage (UH), the leading cause of potentially survivable combat-related death, elicits a deleterious inflammatory response. Our group previously reported an increased secretion of pro-inflammatory cytokines in a novel non-human primate model of UH; however, to better understand the molecular profile of the inflammatory response to UH, we performed a comprehensive evaluation of inflammation at the proteomic and transcriptomic level. METHODS: Anesthetized rhesus macaques (n = 8) underwent UH by 60% left lobe hepatectomy T = 0 min. At T = 5 min, animals received 11 mL of 5% albumin followed by normal saline infusion to a total resuscitation volume of 20 mL/kg by T = 120 min. Blood (T = 0, 5, 20, 120, 480 min) was collected for qPCR and multiplex cytokine quantification. Results from each non-human primate (NHP) per time-point are shown. Statistical analysis by one-way ANOVA with repeated measures, P <0.05 was considered significant. RESULTS: Luminex analysis in serum revealed significant up-regulation compared with baseline of 8 cytokines/chemokines starting T = 120 min postinjury and significant down-regulation of 4 cytokines/chemokines as early as T = 20 min postinjury. Gene expression analysis in white blood cells uncovered 10 genes that were up-regulated greater than 3-fold compared with baseline and 29 genes that were down-regulated greater than 3-fold. CONCLUSION: The present study confirms the presence of systemic inflammation after UH at the proteomic and transcriptomic level providing insight into the inflammatory mediators that are involved as well as their kinetics following UH. The data demonstrates that NHP hemorrhage models may be suitable for evaluating therapeutics to control inflammation following hemorrhage.

Intrauterine growth-restricted sheep fetuses exhibit smaller hindlimb muscle fibers and lower proportions of insulin-sensitive Type I fibers near term.

Intrauterine growth restriction (IUGR) reduces muscle mass and insulin sensitivity in offspring. Insulin sensitivity varies among muscle fiber types, with Type I fibers being most sensitive. Differences in fiber-type ratios are associated with insulin resistance in adults, and thus we hypothesized that near-term IUGR sheep fetuses exhibit reduced size and proportions of Type I fibers. Placental insufficiency-induced IUGR fetuses were ∼54% smaller (P < 0.05) than controls and exhibited hypoxemia and hypoglycemia, which contributed to 6.9-fold greater (P < 0.05) plasma norepinephrine and ∼53% lower (P < 0.05) plasma insulin concentrations. IUGR semitendinosus muscles contained less (P < 0.05) myosin heavy chain-I protein (MyHC-I) and proportionally fewer (P < 0.05) Type I and Type I/IIa fibers than controls, but MyHC-II protein concentrations, Type II fibers, and Type IIx fibers were not different. IUGR biceps femoris muscles exhibited similar albeit less dramatic differences in fiber type proportions. Type I and IIa fibers are more responsive to adrenergic and insulin regulation than Type IIx and may be more profoundly impaired by the high catecholamines and low insulin in our IUGR fetuses, leading to their proportional reduction. In both muscles, fibers of each type were uniformly smaller (P < 0.05) in IUGR fetuses than controls, which indicates that fiber hypertrophy is not dependent on type but rather on other factors such as myoblast differentiation or protein synthesis. Together, our findings show that IUGR fetal muscles develop smaller fibers and have proportionally fewer Type I fibers, which is indicative of developmental adaptations that may help explain the link between IUGR and adulthood insulin resistance.

Adrenal Demedullation and Oxygen Supplementation Independently Increase Glucose-Stimulated Insulin Concentrations in Fetal Sheep With Intrauterine Growth Restriction.

In pregnancies complicated by placental insufficiency and intrauterine growth restriction (IUGR), fetal glucose and oxygen concentrations are reduced, whereas plasma norepinephrine and epinephrine concentrations are elevated throughout the final third of gestation. Here we study the effects of chronic hypoxemia and hypercatecholaminemia on ß-cell function in fetal sheep with placental insufficiency-induced IUGR that is produced by maternal hyperthermia. IUGR and control fetuses underwent a sham (intact) or bilateral adrenal demedullation (AD) surgical procedure at 0.65 gestation. As expected, AD-IUGR fetuses had lower norepinephrine concentrations than intact-IUGR fetuses despite being hypoxemic and hypoglycemic. Placental insufficiency reduced fetal weights, but the severity of IUGR was less with AD. Although basal plasma insulin concentrations were lower in intact-IUGR and AD-IUGR fetuses compared with intact-controls, glucose-stimulated insulin concentrations were greater in AD-IUGR fetuses compared with intact-IUGR fetuses. Interestingly, AD-controls had lower glucose- and arginine-stimulated insulin concentrations than intact-controls, but AD-IUGR and AD-control insulin responses were not different. To investigate chronic hypoxemia in the IUGR fetus, arterial oxygen tension was increased to normal levels by increasing the maternal inspired oxygen fraction. Oxygenation of IUGR fetuses enhanced glucose-stimulated insulin concentrations 3.3-fold in intact-IUGR and 1.7-fold in AD-IUGR fetuses but did not lower norepinephrine and epinephrine concentrations. Together these findings show that chronic hypoxemia and hypercatecholaminemia have distinct but complementary roles in the suppression of ß-cell responsiveness in IUGR fetuses.

Control of severe intra-abdominal hemorrhage with an infusible platelet-derived hemostatic agent in a nonhuman primate (rhesus macaque) model.

BACKGROUND: Hemorrhage remains the leading cause of potentially survivable trauma mortality. Recent reports indicate that injuries sustained in noncompressible anatomic locations (i.e., truncal and junctional) account for 86.5% of hemorrhage-related deaths. Infusible human platelet-derived hemostatic agents (hPDHAs) represent a promising strategy to reduce blood loss from noncompressible injuries. Here, we evaluate the hemostatic efficacy of a lyophilized hPDHA in a rhesus macaque model of severe, uncontrolled hemorrhage. METHODS: Hemorrhage was induced via laparoscopic 60% left-lobe hepatectomy in anesthetized rhesus macaques (T = 0 minute). Treatment infusion began with an 11-mL bolus (T = 5-6 minutes) of either 5% albumin solution (control; n = 8) or hPDHA (1.2 × 10(10) platelet equivalents, n = 8), followed by 2.8-mL/min 0.9% normal saline at T = 6-20 minutes. Resuscitation continued with normal saline (0.22 mL/kg/min) to a total volume of 20 mL/kg at T = 120 minutes, at which time surgical hemostasis was achieved and percent blood loss quantified. Animals were monitored until T = 480 minutes and then euthanized, and necropsy was performed with emphasis on intravascular and end-organ thrombi. Data are expressed as mean ± SEM; significance, p < 0.05. RESULTS: Both groups exhibited a ∼70% decrease in mean arterial pressure (MAP) from T = 0-5 minutes. Percent blood loss was 44.2 ± 3.9% in hPDHA animals, and 44.3 ± 3.3% in controls. Survival rates were 4 of 8 for hPDHA animals and 7 of 8 for controls. Regardless of treatment, percent blood loss was greater (p < 0.02) in nonsurviving animals (55 ± 2%, n = 5) compared with surviving animals (42% ± 3%, n = 11). No pathologic intravascular thrombi were observed in either group. CONCLUSION: The isolated administration of hPDHA did not significantly reduce blood loss; however, thrombocytopenia was not present in the model, and clinically, platelets would be administered in combination with plasma. Mortality was not statistically different between groups (p = 0.14) but was related to blood loss. Future studies should consider the use of hPDHA in combination with additional therapeutics (e.g., factors) and a model that incorporates thrombocytopenia or platelet dysfunction.

Rapid assessment of shock in a nonhuman primate model of uncontrolled hemorrhage: Association of traditional and nontraditional vital signs to mortality risk.

BACKGROUND: Heart rate (HR), systolic blood pressure (SBP) and mean arterial pressure (MAP) are traditionally used to guide patient triage and resuscitation; however, they correlate poorly to shock severity. Therefore, improved acute diagnostic capabilities are needed. Here, we correlated acute alterations in tissue oxygen saturation (StO2) and end-tidal carbon dioxide (ETCO2) to mortality in a rhesus macaque model of uncontrolled hemorrhage. METHODS: Uncontrolled hemorrhage was induced in anesthetized rhesus macaques by a laparoscopic 60% left-lobe hepatectomy (T = 0 minute). StO2, ETCO2, HR, as well as invasive SBP and MAP were continuously monitored through T = 480 minutes. At T = 120 minutes, bleeding was surgically controlled, and blood loss was quantified. Data analyses compared nonsurvivors (expired before T = 480 minutes, n = 5) with survivors (survived to T = 480 minutes, n = 11) using repeated-measures analysis of variance with Bonferroni correction. All p < 0.05 was considered statistically significant. Results were reported as mean ± SEM. RESULTS: Baseline values were equivalent between groups for each parameter. In nonsurvivors versus survivors at T = 5 minutes, StO2 (55% ± 10% vs. 78% ± 3%, p = 0.02) and ETCO2 (15 ± 2 vs. 25 ± 2 mm Hg, p = 0.0005) were lower, while MAP (18 ± 1 vs. 23 ± 2 mm Hg, p = 0.2), SBP (26 ± 2 vs. 34 ± 3 mm Hg, p = 0.4), and HR (104 ± 13 vs. 105 ± 6 beats/min, p = 0.3) were similar. Association of values over T = 5-30 minutes to mortality demonstrated StO2 and ETCO2 equivalency with a significant group effect (p ≤ 0.009 for each parameter; R(2) = 0.92 and R(2) = 0.90, respectively). MAP and SBP associated with mortality later into the shock period (p < 0.04 for each parameter; R(2) = 0.91 and R(2) = 0.89, respectively), while HR yielded the lowest association (p = 0.8, R(2) = 0.83). CONCLUSION: Acute alterations in StO2 and ETCO2 strongly associated with mortality and preceded those of traditional vital signs. The continuous, noninvasive aspects of Food and Drug Administration-approved StO2 and ETCO2 monitoring devices provide logistical benefits over other methodologies and thus warrant further investigation.

Fetal adrenal demedullation lowers circulating norepinephrine and attenuates growth restriction but not reduction of endocrine cell mass in an ovine model of intrauterine growth restriction.

Placental insufficiency is associated with fetal hypoglycemia, hypoxemia, and elevated plasma norepinephrine (NE) that become increasingly pronounced throughout the third trimester and contribute to intrauterine growth restriction (IUGR). This study evaluated the effect of fetal adrenal demedullation (AD) on growth and pancreatic endocrine cell mass. Placental insufficiency-induced IUGR was created by exposing pregnant ewes to elevated ambient temperatures during mid-gestation. Treatment groups consisted of control and IUGR fetuses with either surgical sham or AD at 98 days gestational age (dGA; term = 147 dGA), a time-point that precedes IUGR. Samples were collected at 134 dGA. IUGR-sham fetuses were hypoxemic, hypoglycemic, and hypoinsulinemic, and values were similar in IUGR-AD fetuses. Plasma NE concentrations were ~5-fold greater in IUGR-sham compared to control-sham, control-AD, and IUGR-AD fetuses. IUGR-sham and IUGR-AD fetuses weighed less than controls. Compared to IUGR-sham fetuses, IUGR-AD fetuses weighed more and asymmetrical organ growth was absent. Pancreatic ß-cell mass and α-cell mass were lower in both IUGR-sham and IUGR-AD fetuses compared to controls, however, pancreatic endocrine cell mass relative to fetal mass was lower in IUGR-AD fetuses. These findings indicate that NE, independently of hypoxemia, hypoglycemia and hypoinsulinemia, influence growth and asymmetry of growth but not pancreatic endocrine cell mass in IUGR fetuses.

Placental insufficiency decreases pancreatic vascularity and disrupts hepatocyte growth factor signaling in the pancreatic islet endothelial cell in fetal sheep.

Hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGFA) are paracrine hormones that mediate communication between pancreatic islet endothelial cells (ECs) and ß-cells. Our objective was to determine the impact of intrauterine growth restriction (IUGR) on pancreatic vascularity and paracrine signaling between the EC and ß-cell. Vessel density was less in IUGR pancreata than in controls. HGF concentrations were also lower in islet EC-conditioned media (ECCM) from IUGR, and islets incubated with control islet ECCM responded by increasing insulin content, which was absent with IUGR ECCM. The effect of ECCM on islet insulin content was blocked with an inhibitory anti-HGF antibody. The HGF receptor was not different between control and IUGR islets, but VEGFA was lower and the high-affinity VEGF receptor was higher in IUGR islets and ECs, respectively. These findings show that paracrine actions from ECs increase islet insulin content, and in IUGR ECs, secretion of HGF was diminished. Given the potential feed-forward regulation of ß-cell VEGFA and islet EC HGF, these two growth factors are highly integrated in normal pancreatic islet development, and this regulation is decreased in IUGR fetuses, resulting in lower pancreatic islet insulin concentrations and insulin secretion.

Myoblasts from intrauterine growth-restricted sheep fetuses exhibit intrinsic deficiencies in proliferation that contribute to smaller semitendinosus myofibres.

Intrauterine growth restriction (IUGR) reduces skeletal muscle mass in fetuses and offspring. Our objective was to determine whether myoblast dysfunction due to intrinsic cellular deficiencies or serum factors reduces myofibre hypertrophy in IUGR fetal sheep. At 134 days, IUGR fetuses weighed 67% less (P < 0.05) than controls and had smaller (P < 0.05) carcasses and semitendinosus myofibre areas. IUGR semitendinosus muscles had similar percentages of pax7-positive nuclei and pax7 mRNA but lower (P < 0.05) percentages of myogenin-positive nuclei (7 ± 2% and 13 ± 2%), less myoD and myogenin mRNA, and fewer (P < 0.05) proliferating myoblasts (PNCA-positive-pax7-positive) than controls (44 ± 2% vs. 52 ± 1%). Primary myoblasts were isolated from hindlimb muscles, and after 3 days in growth media (20% fetal bovine serum, FBS), myoblasts from IUGR fetuses had 34% fewer (P < 0.05) myoD-positive cells than controls and replicated 20% less (P < 0.05) during a 2 h BrdU pulse. IUGR myoblasts also replicated less (P < 0.05) than controls during a BrdU pulse after 3 days in media containing 10% control or IUGR fetal sheep serum (FSS). Both myoblast types replicated less (P < 0.05) with IUGR FSS-supplemented media compared to control FSS-supplemented media. In differentiation-promoting media (2% FBS), IUGR and control myoblasts had similar percentages of myogenin-positive nuclei after 5 days and formed similar-sized myotubes after 7 days. We conclude that intrinsic cellular deficiencies in IUGR myoblasts and factors in IUGR serum diminish myoblast proliferation and myofibre size in IUGR fetuses, but intrinsic myoblast deficiencies do not affect differentiation. Furthermore, the persistent reduction in IUGR myoblast replication shows adaptive deficiencies that explain poor muscle growth in IUGR newborn offspring.

Enhanced insulin secretion responsiveness and islet adrenergic desensitization after chronic norepinephrine suppression is discontinued in fetal sheep.

Intrauterine growth-restricted (IUGR) fetuses experience prolonged hypoxemia, hypoglycemia, and elevated norepinephrine (NE) concentrations, resulting in hypoinsulinemia and ß-cell dysfunction. Previously, we showed that acute adrenergic blockade revealed enhanced insulin secretion responsiveness in the IUGR fetus. To determine whether chronic exposure to NE alone enhances ß-cell responsiveness afterward, we continuously infused NE into fetal sheep for 7 days and, after terminating the infusion, evaluated glucose-stimulated insulin secretion (GSIS) and glucose-potentiated arginine-induced insulin secretion (GPAIS). During treatment, NE-infused fetuses had greater (P < 0.05) plasma NE concentrations and exhibited hyperglycemia (P < 0.01) and hypoinsulinemia (P < 0.01) compared with controls. GSIS during the NE infusion was also reduced (P < 0.05) compared with pretreatment values. GSIS and GPAIS were approximately fourfold greater (P < 0.01) in NE fetuses 3 h after the 7 days that NE infusion was discontinued compared with age-matched controls or pretreatment GSIS and GPAIS values of NE fetuses. In isolated pancreatic islets from NE fetuses, mRNA concentrations of adrenergic receptor isoforms (α1D, α2A, α2C, and ß1), G protein subunit-αi-2, and uncoupling protein 2 were lower (P < 0.05) compared with controls, but ß-cell regulatory genes were not different. Our findings indicate that chronic exposure to elevated NE persistently suppresses insulin secretion. After removal, NE fetuses demonstrated a compensatory enhancement in insulin secretion that was associated with adrenergic desensitization and greater stimulus-secretion coupling in pancreatic islets.

Reductions in insulin concentrations and ß-cell mass precede growth restriction in sheep fetuses with placental insufficiency.

In pregnancy complicated by placental insufficiency (PI) and intrauterine growth restriction (IUGR), the fetus near term has reduced basal and glucose-stimulated insulin concentrations and reduced ß-cell mass. To determine whether suppression of insulin concentrations and ß-cell mass precedes reductions in fetal weight, which would implicate insulin deficiency as a cause of subsequent IUGR, we measured basal and glucose-stimulated insulin concentrations and pancreatic histology at 0.7 gestation in PI fetuses. Placental weights in the PI pregnancies were 40% lower than controls (265 ± 26 vs. 442 ± 41 g, P < 0.05), but fetal weights were not different. At basal conditions blood oxygen content, plasma glucose concentrations, and plasma insulin concentrations were lower in PI fetuses compared with controls (2.5 ± 0.3 vs. 3.5 ± 0.3 mmol/l oxygen, P < 0.05; 1.11 ± 0.09 vs. 1.44 ± 0.12 mmol/l glucose; 0.12 ± 0.01 vs. 0.27 ± 0.02 ng/ml insulin; P < 0.05). During a steady-state hyperglycemic clamp (~2.5 ± 0.1 mmol/l), glucose-stimulated insulin concentrations were lower in PI fetuses than controls (0.28 ± 0.02 vs. 0.55 ± 0.04 ng/ml; P < 0.01). Plasma norepinephrine concentrations were 3.3-fold higher (P < 0.05) in PI fetuses (635 ± 104 vs. 191 ± 91 pg/ml). Histological examination revealed less insulin area and lower ß-cell mass and rates of mitosis. The pancreatic parenchyma was also less dense (P < 0.01) in PI fetuses, but no differences were found for pancreatic progenitor cells or other endocrine cell types. These findings show that hypoglycemia, hypoxemia, and hypercatecholaminemia are present and potentially contribute to lower insulin concentrations and ß-cell mass due to slower proliferation rates in early third-trimester PI fetuses before discernible reductions in fetal weight.

Hypoxaemia-induced catecholamine secretion from adrenal chromaffin cells inhibits glucose-stimulated hyperinsulinaemia in fetal sheep.

Abstract Hypoxaemia elicits adrenergic suppression of fetal glucose-stimulated hyperinsulinaemia. We postulate that this effect is mediated by catecholamines, exclusively, from fetal adrenal chromaffin cells. To investigate this hypothesis, square-wave hyperglycaemic clamp studies were performed under normoxaemic (26 ± 0.9 mmHg) and hypoxaemic (14 ± 0.3 mmHg) steady-state conditions in near-term fetal sheep that had undergone either surgical sham or bilateral adrenal demedullation (AD), values mentioned are ± SEM. Under normoxaemic conditions plasma noradrenaline concentrations were lower in AD fetuses than in sham-operated fetuses (457 ± 122 versus 1073 ± 103 pg ml(-1), P < 0.05). Plasma insulin concentrations were not different at euglycaemia between shams (0.46 ± 0.07 ng ml(-1)) and AD fetuses (0.44 ± 0.04 ng ml(-1)) and increased (P < 0.05) with hyperglycaemia in both groups although to a lesser extent in AD fetuses (0.94 ± 0.19 ng ml(-1)) compared to shams (1.31 ± 0.15 ng ml(-1); P < 0.05). Hypoxaemia increased plasma adrenaline (26-fold) and noradrenaline (5-fold) in shams but elicited no change in AD fetuses. Under hypoxaemic conditions, euglycaemic plasma insulin concentrations were reduced (P < 0.05) in both sham and AD fetuses to 0.30 ± 0.05 ng ml(-1) and 0.27 ± 0.01 ng ml(-1) respectively, and the insulin response to hyperglycaemia was abolished in shams but not affected in AD fetuses (0.33 ± 0.06 versus 0.73 ± 0.02 ng ml(-1), P < 0.05). Hypoxaemia also induced hyperlactacaemia and hypocarbia to a greater extent in shams than in AD fetuses, indicating that catecholamines potentiate reductions in oxidative metabolism independently of insulin. These findings demonstrate that the fetal adrenal chromaffin cells are the source for acute hypoxaemia-induced elevations in fetal plasma catecholamines and suppression of glucose-stimulated hyperinsulinaemia, but other factors reduce plasma insulin at euglycaemia.