James L. Ferguson

Steroid hormone alterations following induction of chronic intraperitoneal sepsis in male rats.

The influence of sepsis on male reproductive function in chronic animal models has not been extensively investigated. On the basis of earlier clinical studies, it was hypothesized that chronic intraperitoneal (i.p.) sepsis in rats would modulate the circulating levels of steroid reproductive hormones. Male Sprague-Dawley rats (300-375 g) were randomized to septic and nonseptic groups. Sepsis was induced with cecal slurry (200 mg/kg/5 mL 5% dextrose in water (D5W); i.p.) in septic rats, while nonseptic rats received only sterile D5W. The rats (n = 8-12) were catheterized to measure systemic hemodynamics and to collect blood at 0, 12, 24, and 48 h after induction of sepsis/sham sepsis. A separate group of normal rats was included to serve as an unoperated control group. The plasma concentration of corticosterone, progesterone, and testosterone in serum was determined using radioimmunoassay. The heart rate was significantly increased at t = 12, 24, and 48 h following induction of sepsis. However, septic rats did not display any significant alterations in the mean arterial pressure and pulse pressure. Basal circulating concentrations of serum corticosterone, progesterone, and testosterone were 356 +/- 124 ng/mL, 2.37 +/- 1.03 ng/mL, and 1.88 +/- .29 ng/mL, respectively, in the unoperated rats. At t = 0 h there was a significant increase in the levels of corticosterone in septic rats and in the levels of progesterone in both septic and nonseptic rats. The elevations in the concentrations of corticosterone and progesterone returned to basal values after 24 and 48 h. The septic animals had significantly decreased levels of testosterone at t = 24 and 48 h as compared with basal values and nonseptic groups. Our model of sepsis produced a time-dependent decrease in levels of testosterone, an end product of male steroidogenesis. This, along with unchanged levels of corticosterone and progesterone at 24 and 48 h following sepsis, indicates that separate mechanisms for steroidogenesis regulating synthesis of these steroid hormones (progesterone and testosterone) occur with sepsis. It is concluded that in our chronic septic rat model, induction of i.p. sepsis produced dysfunction in steroidogenesis, which selectively affected the synthesis of testosterone.

The Effects Of Diaspirin Cross-linked Hemoglobin In Sepsis

We tested the hypothesis that diaspirin cross-linked hemoglobin (DCLHb; Baxter Healthcare Corp.) would improve blood pressure, organ perfusion, and mortality during sepsis. Rats were catheterized to assess general hemodynamics (protocol 1) or regional blood flow (protocol 2). Sepsis was induced by intraperitoneal introduction of a cecal slurry (100 mg/kg). In protocol 1, rats received either 100 or 250 mg/kg DCLHb, or albumin at 1, 2, or 4 h after sepsis induction. Hemodynamics were recorded at these times and daily for 72 h. DCLHb increased blood pressure, prevented 72 h leukocytosis, and reduced mortality, but the timing of DCLHb administration was crucial. In protocol 2 only moribund septic animals received 100 mg/kg DCLHb or iso-oncotic albumin i.v. Hemodynamics and regional organ blood flows were measured at baseline, immediately before and after treatment, and at 24 h. DCLHb immediately increased blood pressure with no changes in cardiac output, heart rate, or regional perfusion. DCLHb increased regional perfusion to vital areas at 24 h (compared to albumin group). Distribution of cardiac output in albumin-treated rats was significantly skewed toward skeletal muscle at a time when cardiac output was significantly lower as compared with DCLHb treated animals. In conclusion, DCLHb safely elicited a pressor response, and improved regional perfusion to selected tissues. However, DCLHb benefits were best obtained when given within a specific time frame.

BMP-2 but not VEGF or PDGF in fibrin matrix supports bone healing in a delayed-union rat model.

Treatment of delayed bone healing and non‐unions after fractures, osteotomies or arthrodesis still is a relevant clinical challenge. Artificially applied growth factors can increase bone healing and progressively gain importance in clinical routine. The aim of this study was to determine the effects of rhPDGF‐BB, rhVEGF‐165, and rhBMP‐2 in fibrin matrix on bone healing in a delayed‐union rat model. Thirty‐seven rats underwent a first operation where a standardized femoral critical size defect was created. A silicone spacer was implanted to impair vascularization within the defect. At 4 weeks the spacer was removed in a second operation and rhPDGF‐BB, rhVEGF‐165, or rhBMP‐2 were applied in a fibrin clot. Animals in a fourth group received a fibrin clot without growth factors. At 8 weeks fibrin bound rhBMP‐2 treated animals showed a significantly increased union rate and bone volume within the defect compared to the other groups. Single application of fibrin bound rhPDGF‐BB and rhVEGF‐165 failed to increase bone healing in our atrophic non‐union model.

Effect of NG-nitro-L-arginine methyl ester on testicular blood flow and serum steroid hormones during sepsis.

ABSTRACT Production of nitric oxide (NO) via NO synthase (NOS) has been implicated in the regulation of steroidogenesis in normal physiology and septic pathophysiology. The hypothesis that blockade of NOS by NG-nitro-L-arginine methyl ester (L-NAME) would affect testicular blood flow and circulating levels of steroid reproductive hormones was tested. Male Sprague-Dawley rats (350–450 g) were randomized to septic and nonseptic groups. Sepsis was induced with an intraperitoneal (i.p.) injection of a cecal slurry (200 mg/kg in 5 mL 5% dextrose in water (D5W)) in rats, while nonseptic rats received only sterile D5W. The rats (n = 6 per group) were catheterized in the jugular vein, left ventricle (via right carotid artery), and tail artery to determine blood flow and systemic hemodynamics and to collect blood at 24 h after induction of sepsis/sham sepsis. After baseline (24 h post-cecal slurry challenge) measurement, L-NAME (.50 mg/ kg-min) was infused through the jugular vein for 10 min, blood flow was determined using a radioactive microsphere technique, and blood samples were collected. The serum concentrations of corticosterone, progesterone, and testosterone were determined using radioimmunoassay. Plasma concentrations of NO byproducts (NOx) were determined using the Greiss reaction. After 24 h, heart rate, testicular blood flow, and NOx levels were significantly increased, whereas the serum concentration of testosterone was significantly decreased in the septic group as compared with the nonseptic group. However, serum concentrations of progesterone and corticosterone at 24 h after induction of sham-sepsis or sepsis were not statistically different. Infusion of L-NAME significantly reduced the testicular blood flow and serum NOx levels in septic rats as compared with their baseline values. The administration of L-NAME significantly increased the concentration of testosterone in nonseptic and septic rats as compared with their respective basal values. However, testosterone levels in septic rats were still significantly lower than in nonseptic rats. The results of this study indicate that the synthesis of NO through NO synthase may play a role in the regulation of testicular blood flow and the serum levels of testosterone, associated with chronic peritoneal sepsis in the rat.

Splanchnic vascular control during sepsis and endotoxemia.

Endotoxemia and sepsis often result in circulatory derangements which manifest as perfusion maldistributions. It has been widely accepted that the splanchnic circulation decreases in perfusion during advanced septic or endotoxemic states. Impaired perfusion of splanchnic organs may result not only in organ dysfunction but also exacerbations of polymicrobial bacteremia due to intestinal mucosal leakage. Consequently, evaluation of the splanchnic mechanisms of vasoregulation and how perfusion is maintained is vital to any topic concerning the management of the septic patient.

Elevated coronary endothelin-1 but not Nitric oxide in diabetics during CABG

BACKGROUND After coronary artery bypass grafting procedures, a higher incidence of morbidity and mortality has been reported in diabetic patients. We tested whether coronary artery bypass grafting in diabetics affects the endothelin-1 and nitric oxide coronary effluent profile during reperfusion. METHODS Twenty-one consecutive patients (9 with type II diabetes mellitus, 12 non-diabetics) underwent coronary artery bypass grafting by one surgeon. The two groups did not differ in preoperative ejection fraction, Parsonnet score, number of vessels bypassed, or cross-clamp time. Each patient was treated in the same intraoperative manner with single atrial, aortic, and antegrade and retrograde cardioplegia (CPL) cannulas. Cold CPL arrest was by antegrade and retrograde infusion of modified Buckberg CPL solution. Warm CPL solution was infused before reperfusion. Coronary sinus blood samples were obtained for estimation of endothelin-1 and nitrite plus nitrate before CPL arrest and at 1 and 15 minutes after each of 2 reperfusion periods. RESULTS In diabetics, endothelin-1 was significantly increased at all reperfusion times as compared with non-diabetics. Nitrite plus nitrate levels were significantly higher in patients with diabetes than in those without, but did not change with time in either of the groups. CONCLUSIONS Reperfusion after CPL during coronary artery bypass grafting procedure can trigger the release of endothelin-1 in patients with diabetes mellitus. This may favor increased vascular tone or positive inotropic responses after coronary artery bypass grafting and may contribute to significant cardiovascular consequences in diabetic patients.

Effect of aminoguanidine on plasma nitric oxide by-products and blood flow during chronic peritoneal sepsis.

We hypothesized that plasma nitric oxide (NO), generated via inducible NO synthase (iNOS) or endothelial constitutive NO synthase and measured via its by-products NO2- and NO3- (NO2- + NO3- = NOx) would increase and remain elevated during chronic peritoneal sepsis. We further hypothesized that treatment with aminoguanidine (AG; 50 mg/kg), a selective iNOS inhibitor, would decrease NO production and alter blood flow. Sprague Dawley rats were randomized to septic and nonseptic groups. Septic rats received an intraperitoneal cecal slurry (200 mg of cecal material/5 mL 5% dextrose-H2O/kg); control rats received sterile 5% dextrose-H2O (5 mL/kg) only. Plasma NOx and hemodynamics were measured 0, 4, 12, 24, and 48 h after sepsis or sham induction. We also examined the effect of AG, an iNOS inhibitor, on plasma NOx levels and tissue blood flow at 24 h. Septic rats uniformly displayed signs of sepsis, including lethargy, piloerection, and diarrhea. NOx levels were significantly elevated compared with controls at 4, 12, 24, and 48 h (p ≤ .05). Septic rats also demonstrated hypotension (t = 12, 24, and 48 h) and tachycardia (t = 4, 12, 24, and 48 h). The infusion of AG (50 mg/kg intravenously for 30 min) at 24 h significantly decreased plasma NOx in septic animals. Plasma NOx concentrations returned to basal levels by 90 min after infusion of AG. In addition, blood flow studies demonstrated that AG treatment in nonseptic rats resulted in a significant decrease in blood flow to the stomach, skin, and adipose tissue, whereas AG infusion did not significantly alter the regional perfusion profile in septic animals. Furthermore, treatment with AG did not significantly alter mean arterial pressure in either group; however, nonseptic animals exhibited a decrease in stroke volume, and septic animals demonstrated an increase in heart rate. In contrast to the rise and fall of NOx levels in endotoxemia, this study demonstrates that the initial rise is sustained during 48 h of peritoneal sepsis. This sustained increase in NOx levels in this model correlated with the observable signs of systemic infection and may relate to enhanced iNOS activity. AG infusion demonstrated variable effects on regional tissue blood flow profiles in septic and nonseptic animals and attenuated the increase in plasma NOx levels in septic animals, an index of iNOS activity.

Evidence that adenosine contributes to maintenance of hepatosplanchnic blood flow during peritoneal sepsis in rats.

Sepsis induced derangements in hepatosplanchnic perfusion can contribute to organ damage and death. Adenosine, a common and potent metabolic vasodilator, has not been evaluated as a mechanism for maintenance of blood flow during sepsis. We tested the hypothesis that adenosine receptor blockade would cause a decrease in hepatosplanchnic blood flow during intraperitoneal (i.p.) sepsis in the rat. Rats (250–350 g) were catheterized for hemodynamic and blood flow measurements with tracer microspheres. Sepsis was induced with an i.p. injection of cecal material (150 mg/kg in D5W; 5 mL/kg), and baseline measurements were taken 24 h later. Animals then received either the adenosine receptor antagonist 8-PTH (10 mM, 1.5 mL/kg), its vehicle (1.5 mL/kg) or normal saline (1.5 mL/kg), intravenously, and measurements were repeated 1 and 10 min later. There was a significant increase in hepatosplanchnic portal resistance in septic animals given 8-PTH, with no change in mean arterial blood pressure (MAP) or heart rate. Regionally, there was a significant decrease in gastric, small intestinal, cecal, and pancreatic blood flow when compared with vehicle. Adenosine receptor blockade caused a significant reduction in hepatosplanchnic blood flow during sepsis, suggesting that maintenance of splanchnic blood flow during sepsis involves receptor mediated adenosine actions.

A comparative biomechanical evaluation of hernia mesh fixation by fibrin sealant.

BACKGROUND The atraumatic fixation of meshes by fibrin sealant (FS) has been established for both open and laparoscopic techniques of hernia repair. This study was performed to evaluate the use of FS in hernia mesh fixation with different polymerization speed (thrombin concentrations), using commercial hernia meshes, and in two techniques, transabdominal preperitoneal mesh placement (TAPP) and intraperitoneal mesh placement (IPOM). MATERIALS AND METHODS A median laparotomy was performed in a pig model and hernia meshes were placed in IPOM and TAPP techniques. After mesh fixation with FS using thrombin concentrations of 4 and 500 IU/mL, maximum shear force before failure was measured at 5, 60, and 120 min. RESULTS At both thrombin concentrations and in all meshes in which the technique was used, the TAPP method tended to show higher maximum force levels at failure than did the IPOM method. In both TAPP and IPOM techniques and in all meshes, the 4 IU/mL thrombin concentration FS was superior to the 500 IU/mL thrombin concentration sealant. CONCLUSIONS Although both thrombin concentrations are suitable for mesh fixation, lower concentrations allow slower polymerization and better sealant diffusion leading to higher maximum force levels at failure. The TAPP method was biomechanically superior to the IPOM method. There were no major differences between mesh products.

Adenosine and nitric oxide regulate regional vascular resistance via interdependent and independent mechanisms during sepsis

Objective Adenosine receptor blockade increases regional resting vascular resistance during sepsis. In healthy subjects, part of adenosine’s actions are mediated via stimulation of nitric oxide synthase. Because nitric oxide synthase activity is thought to be a major contributor to altered vascular tone in sepsis, we tested the hypothesis that some of the nitric oxide–mediated resting regional resistance during sepsis is secondary to endogenous adenosine stimulation of nitric oxide synthase. Design Prospective, randomized, controlled experiment. Setting Shock-trauma and basic science laboratory. Subjects Male Sprague-Dawley rats. Interventions Twenty-four hours after sepsis or sham induction, rats were separated into two groups (n = 6 to 10 in each group). Group 1 received a 10-min infusion of the adenosine antagonist 8-sulfophenyltheophylline (0.9 mg/kg·min) followed by a 10-min infusion of l-nitro-arginine-methyl ester (0.5 mg/kg·min). Group 2 similarly received l-nitro-arginine-methyl ester followed by 8-sulfophenyltheophylline in the presence of l-nitro-arginine-methyl ester. Measurements and Main Results Hemodynamic and blood flow measurements (microspheres) were made before infusions, 10 mins after the administration of each single-agent infusion, and 10 mins after combined-agent infusions were administered. No significant resistance alterations were observed in nonseptic rats. In septic rats, adenosine receptor blockade alone increased hepatosplanchnic and skeletal muscle vascular resistance, but no further increases were seen when l-nitro-arginine-methyl ester was added. Nitric oxide synthase inhibition alone increased hepatosplanchnic and skeletal muscle vascular resistances. When 8-sulfophenyltheophylline was added to the infusion, skeletal muscle vascular resistance increased significantly more than with l-nitro-arginine-methyl ester alone, but there were no further increases in hepatosplanchnic resistance. Renal and adipose vascular resistances increased with l-nitro-arginine-methyl ester infusions, and 8-sulfophenyltheophylline produced no effect. Conclusions During sepsis, nitric oxide caused resting vasodilation independent of adenosine in the renal and adipose vasculature. In the hepatosplanchnic circulation, there is reciprocal adenosine-nitric oxide interaction in maintaining resting regional resistance. Skeletal muscle displayed a dual adenosine-mediated (nitric oxide–independent) and nitric oxide–mediated (adenosine receptors required) interaction to regulate resting resistance during sepsis. These data indicate that in the hepatosplanchnic and skeletal muscle vasculature, all of the resting nitric oxide–mediated vasodilation is secondary to endogenous adenosine action, but in adipose and renal vasculature, resting nitric oxide mediated vasodilation is independent of adenosine. Endogenous adenosine also appears to play a significant role in determining resting skeletal muscle resistance that is independent of nitric oxide synthase during sepsis.