Streptozotocin-induced diabetes mellitus in pigs

We induced, as a precondition for a pancreas transplant, insulin-dependent diabetes mellitus in 67 Yorkshire Landrace pigs by administering streptozotocin. A dosage of 150 mg/kg body weight gave rise to a long-lasting diabetes mellitus that persisted with time (follow-up period: 26 weeks). Consecutive measurements of serum glucose and plasma insulin, before and up to 30 hours after administering streptozotocin, revealed triphasic behavior: initial hyperglycemia (1st to 3rd hour), pronounced hypoglycemia (12th to 18th hour), then hyperglycemia (22nd hour on). IVGTTs done 1 to 7 days after administering streptozotocin revealed a reduction of the K-value (glucose disappearance rate) from 0.3 (day 2) to 0.07 (day 4). Immunohistochemical studies revealed a complete loss of all beta-cells, concomitantly with a relative increase in glucagon- and somatostatin-positive cells. We also observed a complete loss of pp (pancreatic polypeptide)-positive cells. Diabetes induced by streptozotocin at 150 mg/kg body weight is complete and permanent; our mortality rate was 0%. Given the high morbidity rate after pancreatectomy, streptozotocin should be the method of choice for inducing diabetes mellitus in pigs.

Supplemental oxygen, but not supplemental crystalloid fluid, increases tissue oxygen tension in healthy and anastomotic colon in pigs

BACKGROUND: Low tissue oxygen tension is an important factor leading to the development of wound dehiscence and anastomotic leakage after colon surgery. We tested whether supplemental fluid and supplemental oxygen can increase tissue oxygen tension in healthy and injured, perianastomotic, and anastomotic colon in an acutely instrumented pig model of anastomosis surgery. METHODS: Sixteen Swiss Landrace pigs were anesthetized (isoflurane 0.8%-1%) and their lungs ventilated. The animals were randomly assigned to low fluid treatment ("low" group, 3 mL x kg(-1) x h(-1) lactated Ringer's solution) or high fluid treatment ("high" group, 10 mL/kg bolus, 18 mL x kg(-1) x h(-1) lactated Ringer's solution) during colon anastomosis surgery and a subsequent measurement period (4 h). Two-and-half hours after surgery, tissue oxygen tension was recorded for 30 min during ventilation with 30% oxygen. Three hours after surgery, the animals' lungs were ventilated with 100% oxygen for 60 min. Tissue oxygen tension was recorded in the last 30 min. Tissue oxygen tension was measured with polarographic Clark-type electrodes, positioned in healthy colonic wall, close (2 cm) to the anastomosis, and in the anastomosis. RESULTS: In every group, tissue oxygen tension during ventilation with 100% oxygen was approximately twice as high as during ventilation with 30% oxygen, a statistically significant result. High or low volume crystalloid fluid treatment had no effect on colon tissue oxygen tension. CONCLUSIONS: Supplemental oxygen, but not supplemental crystalloid fluid, increased tissue oxygen tension in healthy, perianastomotic, and anastomotic colon tissue.

The immediate and sustained effects of volume challenge on regional blood flows in pigs

BACKGROUND: The postoperative assessment of volume status is not straightforward because of concomitant changes in intravascular volume and vascular tone. Hypovolemia and blood flow redistribution may compromise the perfusion of the intraabdominal organs. We investigated the effects of a volume challenge in different intra- and extraabdominal vascular beds. METHODS: Twelve pigs were studied 6 h after major intraabdominal surgery under general anesthesia when clinically normovolemic. Volume challenges consisted of 200 mL rapidly infused 6% hydroxyethyl starch. Systemic (continuous thermodilution) and regional (ultrasound Doppler) flows in carotid, renal, celiac trunk, hepatic, and superior mesenteric arteries and the portal vein were continuously measured. The acute and sustained effects of the challenge were compared with baseline. RESULTS: Volume challenge produced a sustained increase of 22% +/- 15% in cardiac output (P < 0.001). Blood flow increased by 10% +/- 9% in the renal artery, by 22% +/- 15% in the carotid artery, by 26% +/- 15% in the superior mesenteric artery, and by 31% +/- 20% in the portal vein (all P < 0.001). Blood flow increases in the celiac trunk (8% +/- 13%) and the hepatic artery (7% +/- 19%) were not significant. Increases in regional blood flow occurred early and were sustained. Mean arterial and central venous blood pressures increased early and decreased later (all P < 0.05). CONCLUSIONS: A volume challenge in clinically euvolemic postoperative animals was associated with a sustained increase in blood flow to all vascular beds, although the increase in the celiac trunk and the hepatic artery was very modest and did not reach statistical significance. Whether improved postoperative organ perfusion is accompanied by a lower complication rate should be evaluated in further studies.

Norepinephrine to increase blood pressure in endotoxaemic pigs is associated with improved hepatic mitochondrial respiration

ABSTRACT: INTRODUCTION: Low blood pressure, inadequate tissue oxygen delivery and mitochondrial dysfunction have all been implicated in the development of sepsis-induced organ failure. This study evaluated the effect on liver mitochondrial function of using norepinephrine to increase blood pressure in experimental sepsis. METHODS: Thirteen anaesthetized pigs received endotoxin (Escherichia coli lipopolysaccharide B0111:B4; 0.4 mug/kg per hour) and were subsequently randomly assigned to norepinephrine treatment or placebo for 10 hours. Norepinephrine dose was adjusted at 2-hour intervals to achieve 15 mmHg increases in mean arterial blood pressure up to 95 mmHg. Systemic (thermodilution) and hepatosplanchnic (ultrasound Doppler) blood flow were measured at each step. At the end of the experiment, hepatic mitochondrial oxygen consumption (high-resolution respirometry) and citrate synthase activity (spectrophotometry) were assessed. RESULTS: Mean arterial pressure (mmHg) increased only in norepinephrine-treated animals (from 73 [median; range 69 to 81] to 63 [60 to 68] in controls [P = 0.09] and from 83 [69 to 93] to 96 [86 to 108] in norepinephrine-treated animals [P = 0.019]). Cardiac index and systemic oxygen delivery (DO2) increased in both groups, but significantly more in the norepinephrine group (P < 0.03 for both). Cardiac index (ml/min per.kg) increased from 99 (range: 72 to 112) to 117 (110 to 232) in controls (P = 0.002), and from 107 (84 to 132) to 161 (147 to 340) in norepinephrine-treated animals (P = 0.001). DO2 (ml/min per.kg) increased from 13 (range: 11 to 15) to 16 (15 to 24) in controls (P = 0.028), and from 16 (12 to 19) to 29 (25 to 52) in norepinephrine-treated animals (P = 0.018). Systemic oxygen consumption (systemic VO2) increased in both groups (P < 0.05), whereas hepatosplanchnic flows, DO2 and VO2 remained stable. The hepatic lactate extraction ratio decreased in both groups (P = 0.05). Liver mitochondria complex I-dependent and II-dependent respiratory control ratios were increased in the norepinephrine group (complex I: 3.5 [range: 2.1 to 5.7] in controls versus 5.8 [4.8 to 6.4] in norepinephrine-treated animals [P = 0.015]; complex II: 3.1 [2.3 to 3.8] in controls versus 3.7 [3.3 to 4.6] in norepinephrine-treated animals [P = 0.09]). No differences were observed in citrate synthase activity. CONCLUSION: Norepinephrine treatment during endotoxaemia does not increase hepatosplanchnic flow, oxygen delivery or consumption, and does not improve the hepatic lactate extraction ratio. However, norepinephrine increases the liver mitochondria complex I-dependent and II-dependent respiratory control ratios. This effect was probably mediated by a direct effect of norepinephrine on liver cells.

Effect of Supplemental Oxygen versus Dobutamine Administration on Liver Oxygen Tension in dPP-Guided Normovolemic Pigs

Background: Difference in pulse pressure (dPP) confirms adequate intravascular filling as a prerequisite for tissue perfusion. We hypothesized that both oxygen and dobutamine increase liver tissue oxygen tension (ptO(2)). Methods: Eight anesthetized pigs received dPP-guided fluid management. Hepatic pO(2) was measured with Clark-type electrodes placed subcapsularly, and on the liver surface. Pigs received: (1) supplemental oxygen (F(i)O(2) 1.0); (2) dobutamine 2.5 mug/kg/min, and (3) dobutamine 5 mug/kg/min. Data were analyzed using repeated-measures ANOVA followed by a Tukey post-test for multiple comparisons. ptO(2 )measured subcapsularly and at the liver surface were compared using the Bland-Altman plot. Results: Variation in F(i)O(2) changed local hepatic tissue ptO(2) [subcapsular measurement: 39 +/- 12 (F(i)O(2) 0.3), 89 +/- 35 mm Hg (F(i)O(2) 1.0, p = 0.01 vs. F(i)O(2) 0.3), 44 +/- 10 mm Hg (F(i)O(2) 0.3, p = 0.05 vs. F(i)O(2) 1.0); surface measurement: 52 +/- 35 (F(i)O(2) 0.3), 112 +/- 24 mm Hg (F(i)O(2) 1.0, p = 0.001 vs. F(i)O(2) 0.3), 54 +/- 24 mm Hg (F(i)O(2) 0.3, p = 0.001 vs. F(i)O(2) 1.0)]. Surface measurements were widely scattered compared to subcapsular measurements (bias: -15 mm Hg, precision: 76.3 mm Hg). Dobutamine did not affect hepatic oxygenation. Conclusion: Supplemental oxygen increased hepatic tissue pO(2) while dobutamine did not. Although less invasive, the use of surface measurements is discouraged.

Reactivity of human natural antibodies to endothelial cells from Galalpha(1,3)Gal-deficient pigs

BACKGROUND: Xenoreactive human natural antibodies (NAb) are predominantly directed against galactose-alpha(1,3)galactose (Gal). Binding of immunoglobulin (Ig) G and IgM NAb activates porcine endothelial cells (pEC) and triggers complement lysis responsible for hyperacute xenograft rejection. In vitro, IgG NAb induce human natural killer (NK) cell-mediated lysis of pEC by antibody-dependent cell-mediated cytotoxicity (ADCC). The present study examined the levels of anti-porcine NAb in a large number of individuals and addressed the functional role of non-Gal anti-porcine NAb. METHODS: Sera from 120 healthy human blood donors were analyzed for the presence of anti-porcine NAb by flow cytometry using porcine red blood cells (pRBC), lymphoblastoid cells (pLCL), and pEC derived from control or Gal-deficient pigs. Xenogeneic complement lysis was measured by flow cytometry using human serum and rabbit complement. ADCC was analyzed by chromium-release assays using human serum and freshly isolated NK cells. RESULTS: Human IgM binding to pRBC was found in 93% and IgG binding in 86% of all samples. Non-Gal NAb comprised 13% of total IgM and 36% of total IgG binding to pEC. NAb/complement-induced lysis and ADCC of Gal-deficient compared to Gal-positive pEC were 21% and 29%, respectively. The majority of anti-Gal and non-Gal IgG NAb were of the IgG2 subclass. CONCLUSIONS: The generation of Gal-deficient pigs has overcome hyperacute anti-Gal-mediated xenograft rejection in nonhuman primates. Non-Gal anti-porcine NAb represent a potentially relevant immunological hurdle in a subgroup of individuals by inducing endothelial damage in xenografts.

Effect of two bioabsorbable barrier membranes on bone regeneration of standardized defects in calvarial bone: a comparative histomorphometric study in pigs

BACKGROUND: The effect of two different bioabsorbable collagen membranes on bone regeneration was assessed in standardized, membrane-protected calvarial defects in pigs. METHODS: Two standardized defect types (6 x 6 x 6 mm and 9 x 9 x 9 mm) were produced in the calvaria of pigs: empty defects without a membrane (group 1; eight defects per size); defects filled with deproteinized bovine bone mineral (DBBM) without a membrane (group 2; eight defects per size); defects filled with DBBM and covered by a collagen membrane (group 3; eight defects per size); and defects filled with DBBM and covered by a cross-linked collagen membrane (CCM) (group 4; eight defects per size). Sacrifice took place 16 weeks after surgery, and the following parameters were analyzed: descriptive histology; semiquantitative histology (SQH), assessing bone regeneration in the whole defect area; and histomorphometric analysis of the percentage of bone and DBBM in the regenerated area at three different depth levels of the defect. RESULTS: Using SQH, both membrane types resulted in significantly better bone regeneration compared to groups 1 and 2, irrespective of the defect size (P <0.005), with no difference between the two membranes. In the histomorphometric analysis, the layer immediately below the surface exhibited a significantly higher percentage of bone in groups 3 (27%) and 4 (36%) versus the two other groups for the 9 x 9 x 9-mm defects. No such differences were apparent for the 6 x 6 x 6-mm defects or the other two depth levels (bottom and middle layer) for either defect size. CONCLUSIONS: The two collagen membranes tested significantly enhanced bone regeneration, especially in the superficial level of the calvarial bone defects. The prototype CCM did not provide any further advantage in the present animal model.

Antibiotic resistance and genetic diversity in Staphylococcus aureus from slaughter pigs in Switzerland

Nasal carriage of Staphylococcus aureus was evaluated in pigs at slaughterhouse. The nasal cavities of 304 pigs from 54 herds were screened. Eighty-nine percent of the farms harbored pigs that were colonized with S. aureus. Among them, no MRSA were found, indicating a low prevalence. However, pigs were found to harbor S. aureus, which displayed resistance to penicillin (blaZ) (62.5%), tetracycline [tet(M)] (33.3%), streptomycin (strpS194) (27%), clindamycin [erm(B)] (4.1%), erythromycin [erm(B)] (4.1%), kanamycin (4.1%), chloramphenicol (catpC194) (2%) and gentamicin [aac(6')-Ie-aph(2')-Ia] (2%). The S. aureus isolates mainly belong to Ridom spa type t034 (31.3%), t208 (14.6%) and t899 (12.5%). These pig-associated spa types have not yet been detected in hospitalized human patients in Switzerland. Surveillance programs are now necessary at both inland and import levels to rapidly detect and suppress the emergence of MRSA in pigs in Switzerland.