The cost of self-imposed regulatory burden in animal research.

U.S. federal regulations and standards governing the care and use of research animals enacted in the mid- to late 1980s, while having positive effects on the welfare and quality of the animals, have resulted in dramatic increases in overall research costs. In addition to the expenses of housing and caring for animals according to the standards, establishing the requisite internal compliance bureaucracies has markedly driven up costs, in both institutional monetary expenditures and lost research effort. However, many institutions are increasing these costs even further through additional self-imposed regulatory burden, typically characterized by overly complex compliance organizations and unnecessary policies and procedures. We discuss the sources of this self-imposed burden and recommend strategies for avoiding it while preserving an appropriate focus on animal well-being and research success.

Effects of cage size and enrichment on reproductive performance and behavior in C57BL/6Tac mice.

The authors examined the effects of cage size and enrichment on mouse breeding performance and behavior. Breeding trios of C57BL/6Tac mice were housed in cages of two different sizes ('standard' and 'large' cages with 82 in(2) and 124 in(2) floor space, respectively). Half of the cages of each size contained four enrichment items (Nestlet, plastic tunnel, nylon rings and running wheel), whereas the remaining cages had no enrichment. The authors measured the following reproductive parameters: litter size, number of pups that survived to weaning age, average pup weights at 21 d after birth and number of days between births of litters. A subset of weaned male and female pups from each cage size and enrichment condition completed a suite of behavioral tests. Pups raised in large cages weighed less than those raised in standard cages. Enrichment and cage size had certain behavioral effects, which were dependent on gender and behavioral measure. Male pups born in enriched cages showed more anxiety-like behavior and less exploration than did males born in non-enriched cages. Though being raised in enriched or large cages did not clearly improve pups' performance in behavioral tests, enrichment (regardless of cage size) did significantly benefit reproductive performance; pups from non-enriched cages weighed less than pups from enriched cages, and fewer survived to weaning age.

Foreword and Introduction to This Issue on Contemporary Safety Topics in Animal Research.

Institutions with animal care and use programs are obligated to provide for the health and well-being of the animals, but are equally obligated to provide for safety of individuals associated with the program. The topics in this issue of the ILAR Journal, in association with those within the complimentary issue of the Journal of Applied Biosafety, provide a variety of contemporary occupational health and safety considerations in today's animal research programs. Each article addresses key or emerging occupational health and safety topics in institutional animal care and use programs, where the status of the topic, contemporary challenges, and future directions are provided.

The effect of cage size on reproductive performance and behavior of C57BL/6 mice.

Scientific research has yet to conclusively determine the optimal cage size for mice. The authors examined the effect of cage size on mouse breeding performance and on offspring behavior, which can serve as indications of overall well-being. They housed breeding trios of C57BL/6Tac mice in standard or large individually ventilated cages and measured four reproductive parameters: litter size; litter survival to weaning age; average pup weight at 7, 14 and 21 days; and the number of days between litter births. They investigated the behavior of a subset of male and female pups from parents housed in cages of each size in the elevated plus maze test, the open field assay and the acoustic startle test. Cage size had no significant effect on any of the reproductive parameters measured and few or inconsistent effects on behavior in weaned pups.

A pathologist's perspective of the somatoform disorders.

OBJECTIVE: The object of this study was to revisit the evidence that somatoform disorders should be classified as mental and not physical disorders, as viewed through the eyes of a pathologist rather than a psychiatrist. METHODS: An academic clinical pathologist was asked to review recent widening in the concepts of physical disease and to comment on whether these changes made the criteria for designating somatoform disorders as mental disorders more workable or less workable. RESULTS: The review showed that somatoform disorders share many characteristics with certain types of physical conditions, especially those with multiorgan manifestations and multifactor causes. The criteria used to separate somatoform disorders from physical diseases are now less clear than when this distinction was first proposed. CONCLUSION: Discussions for reviewing the status of the somatoform disorders for DSM-V need input from a wide range of medical disciplines besides psychiatry.

Cholesteryl ester transfer protein expression prevents diet-induced atherosclerotic lesions in male db/db mice.

OBJECTIVE: Accompanying more atherogenic lipoprotein profiles and an increased incidence of atherosclerosis, plasma cholesteryl ester transfer protein (CETP) is depressed in diabetic obese patients compared with nondiabetic obese counterparts. The depressed levels of CETP in the plasma of diabetic obese individuals may contribute to the development of an atherogenic lipoprotein profile and atherogenesis. We have examined the effect of CETP expression on vascular health in the db/db model of diabetic obesity. METHODS AND RESULTS: Transgenic mice expressing the human CETP minigene were crossed with db/db strain, and 3 groups of offspring (CETP, db, and db/CETP) were placed on an atherogenic diet for 16 weeks. The proximal aorta was then excised and examined for the presence of atherosclerotic plaques. In db mice, 9 of 11 had intimal lesions with a mean area of 26 098+/-7486 microm2. No lesions greater than 1000 microm2 were observed in db/CETP or CETP mice. CETP-expressing mice had lower circulating cholesterol concentrations than db mice. Fractionating plasma lipids by FPLC indicated that the difference in total cholesterol was primarily attributable to differences in VLDL and LDL. CONCLUSIONS: The expression of human CETP in db/db mice prevented the formation of diet-induced lesions, suggesting an antiatherogenic effect of CETP in the context of diabetic obesity.

Overcoming severe renal ischemia: the role of ex vivo warm perfusion.

BACKGROUND: The ability to effectively utilize kidneys damaged by severe (2 hr) warm ischemia (WI) could provide increased numbers of kidneys for transplantation. The present study was designed to examine the effect of restoring renal metabolism after severe WI insult during ex vivo warm perfusion using an acellular technology. After warm perfusion for 18 hr, kidneys were reimplanted and evaluated for graft function. METHODS: Using a canine autotransplant model, kidneys were exposed to 120 min of WI. They were then either reimplanted immediately, hypothermically machine perfused (4 degrees C) for 18 hr with Belzer's solution, or transitioned to 18 hr of warm perfusion (32 degrees C) with an acellular perfusate before implantation. RESULTS: Warm perfused kidneys with 120 min of WI provided life-sustaining function after transplantation, whereas the control kidneys immediately reimplanted or with hypothermic machine perfusion did not. The mean peak serum creatinine in the warm perfused kidneys was 3.7 mg/dl, with the mean peak occurring on day 2 and normalizing on day 9 posttransplant. CONCLUSIONS: These results indicate that 18 hr of ex vivo warm perfusion of kidneys is feasible. Furthermore, recovery of renal function during warm perfusion is demonstrated, resulting in immediate function after transplantation. The use of ex vivo warm perfusion to recover function in severe ischemically damaged kidneys could provide the basis for increasing the number of transplantable kidneys.

Aprotinin restores the adhesive capacity of dysfunctional platelets.

The post-operative coagulopathy associated with cardiopulmonary bypass (CPB) is known to be predominantly related to platelet dysfunction. The use of the serine protease inhibitor aprotinin dramatically reduces CPB associated hemorrhage and is thought to act primarily through the inhibition of plasmin without directly influencing platelets. Our data indicate that there is a direct effect of aprotinin on platelet adhesion, which has not been previously reported. We found that when aprotinin was added to blood samples with poorly adhesive platelets, platelet adhesion significantly increased as measured by the percent coverage of denuded arterial segments in the Baumgartner perfusion chamber. In preliminary experiments using expired platelet concentrates or fresh whole blood, the addition of aprotinin induced a positive increase of 22+/-7.5 and 14+/-6.2 percentage point in platelet adhesion, respectively. A simulated CPB model that recirculated a unit of anticoagulated whole blood for 2 h was used (n=14) to induce a platelet adhesion defect similar to that seen in clinical CPB. At initiation of recirculation, platelet adhesion was 55+/-9.5% but dropped to 13+6.5% coverage after 2 h simulated CPB. The addition of aprotinin to the post-recirculation samples induced a significant restoration of platelet adhesion back to 38+/-11% coverage. When epsilon amino-caproic acid with soybean trypsin inhibitor was added to post recirculation samples, there was no similar effect on adhesion scores. To compare these findings with surgical CPB, we collected one blood sample at the beginning and two at the end of CPB from each of seven open-heart patients. Aprotinin was added to one of each of the post-CPB samples. Platelet adhesion at the onset of surgical CPB was only 39+/-11% in this patient group but dropped to 7+/-7% by the end. Similar to the model, the addition of aprotinin post-CPB restored adhesion to 29+/-11%. These results suggest some action of aprotinin other than its antiplasmin effect, and that platelet adhesion in general can be promoted by aprotinin.

Limited efficacy of calcium and magnesium in a porcine model of hydrofluoric acid ingestion.

OBJECTIVE: This investigation evaluated the effectiveness of calcium and magnesium in treating oral hydrofluoric acid (HF) poisoning. METHODS: The controlled laboratory investigation used anesthetized pigs. Subjects received HF via NG tube, titrated to abolish electrocardiographic abnormalities. The untreated group received saline infusion. The treatment group received serial injections of calcium chloride (CaCl2) and magnesium chloride (MgCl2). A third group received oral infusions of Calcium fluoride (CaF2). We measured heart rate, QRS interval, pH, bicarbonate, calcium, magnesium, and potassium. The Wilcoxon Rank Sum test was used to compare intra- and inter-subject differences. RESULTS: Fatality occurred in all pigs receiving HF. Compared to the untreated group, trends for the treatment group were toward a larger amount of HF to produce fatality (83.1 +/- 17.5 grams vs. 37.7 +/- 16.1 grams, p = 0.08), to cause QRS prolongation (72.5 +/- 25.8vs. 33.8 +/- 14.9 grams, p = 0.08), and to lower potassium at mortality (4.9 +/- 0.7 vs. 8.7 +/- 2.7 mEq/L, p = 0.08). No major changes in calcium (-1.0 +/- 0.7 mEq/L) or magnesium (0.4 +/- 0.6 mEq/L) occurred in the untreated group. Tachycardia developed in all pigs and ventricular arrhythmias occurred in 2 of 3 pigs of both groups [CaF2 administration caused no QRS prolongation or ventricular arrhythmias and had no effect on laboratory parameters]. CONCLUSION: CaCl2 and MgCl2 replacement delayed but did not prevent fatality and QRS prolongation. Although this result suggests Ca++ and Mg++ may be beneficial in the treatment of systemic HF toxicity, factors other than hypocalcemia and hypomagnesemia play a role in toxicity.

Immunologic response enhances atherosclerosis-type 1 helper T cell (Th1)-to-type 2 helper T cell (Th2) shift and calcified atherosclerosis in Bacillus Calmette-Guerin (BCG)-treated apolipoprotein E-knockout (apo E-/-) mice.

Although immunocompetent hosts develop protective type 1 helper T cell (Th1) responses in mycobacterial infections, seroepidemiologic studies show that patients with atherosclerosis commonly express high antibody titers against mycobacterial heat shock protein (HSP) 65 and may develop a nonprotective type 2 helper T cell (Th2) response and advanced disease. These studies were undertaken to define mycobacterial dose requirements and kinetics for development of antibodies to HSP65, the Th1 to Th2 shift of immune response, and calcified atherosclerotic lesion development in the apo E-/- mouse. Fourteen-week apo E-/- female mice were treated intraperitoneally (ip) with heat-killed M. bovis Bacillus Calmette-Guerin (BCG), and 14 days later, cross-sections from the ascending aortas were stained for measurement of lesion size and calcium deposition. At 14 days, 0.01-mg BCG induced Th1 responses against HSP65. In contrast, 1-mg BCG induced splenic PGE2-releasing macrophages with a Th1-to-Th2 shift of responses to HSP65, which was PGE2-dependent. Treatment with 1-mg BCG significantly lowered bone density with increases in marrow osteoclastogenesis and development of calcified lesions in the aorta. At 14 days, 0.01-mg BCG induced Th1-dependent HSP65 responses and did not advance atherosclerosis. In contrast, for 1-mg BCG, a PGE2-dependent Th1-to-Th2 shift of responses to HSP65 and evidence of bone resorption are associated with advanced calcified atherosclerotic lesions.

Induction of cytolytic anti-Gal antibodies in alpha-1,3-galactosyltransferase gene knockout mice by oral inoculation with Escherichia coli O86:B7 bacteria.

Naturally occurring antibodies against [Gal alpha-1,3-Gal] structures (anti-Gal antibodies) are the primary effectors of human hyperacute rejection (HAR) of nonhuman tissue. Unlike most mammals, humans lack a functional alpha-1,3-galactosyltransferase (GalT) gene and produce abundant anti-Gal antibodies, putatively in response to GalT(+) enteric bacteria. GalT knockout (KO) mice have been generated as a small animal model of HAR but inconsistently express anti-Gal antibodies. We hypothesized that enteric exposure of GalT KO mice to live GalT(+) bacteria would produce cytolytic anti-Gal antibodies. Naive mice lacking anti-Gal antibodies were orally immunized with 10(10) live GalT(+) Escherichia coli O86:B7 bacteria and assayed for anti-Gal antibody titer, isotype, and cytolytic activity. Fecal samples were tested for E. coli O86:B7 prior to and after inoculation. In two separate experiments, 77 to 100% (n = 31) of mice developed serum anti-Gal immunoglobulin G (IgG; titer, 1:5 to 1:80) and/or anti-Gal IgM antibodies (titer, 1:5 to 1:1,280) 14 days postinoculation. Induced anti-Gal antibodies caused complement-mediated cytolysis of GalT(+) target cells, with extensive cytolysis observed consistently at serum IgM titers of >/=1:320. Absorption with synthetic [Gal alpha-1,3-Gal] inhibited both antibody binding and cytolysis. E. coli O86:B7 was recovered from stool samples from 83 to 94% of inoculated mice but not from naive mice, thus confirming enteric exposure. These findings demonstrate that oral inoculation with E. coli O86:B7 is a novel and effective method to induce cytolytic anti-Gal antibodies in GalT KO mice and support the premise that enteric exposure to GalT(+) bacteria induces anti-Gal antibodies in humans. These studies also suggest a role for GalT KO mice in elucidating anti-Gal responses in microbial immunity.

Comparative healing of surgical incisions created by a standard "bovie," the Utah Medical Epitome Electrode, and a Bard-Parker cold scalpel blade in a porcine model: a pilot study.

A comparative study of skin incision healing using a standard "bovie" and a new design electroscalpel, Utah Medical Products Epitome Electrode (Midvale, UT), was conducted in a porcine model. Wounds were evaluated objectively at 14 and 28 days after surgery using wound bursting strength measurements and histologic wound scoring. Each electrosurgical device was compared with wound healing of cold scalpel incisions as the gold standard using the same criteria. Statistical differences of healing between the bovie and the Epitome indicating preferential healing for the Epitome wounds were demonstrated for bursting strength at 14 days (p = 0.002). Comparisons of the measured "zone of coagulation necrosis" produced by the electroscalpels demonstrated significantly decreased thermal tissue damage favoring the Epitome (p = 0.0003). Greater differences in wound healing favoring the cold scalpel occurred in comparisons of bovie with cold scalpel than Epitome with cold scalpel, and overall results demonstrated healing for the Epitome wounds closely approximated that for cold scalpel. The authors conclude that this new generation electroscalpel provides measurable improvements in incisional wound healing compared to established electrosurgical technology.