Tensions generated in a lateral fabellotibial suture model. Comparison of methods of application of tension, fixation of tension and suture material.

OBJECTIVES: To compare suture tension on a simulated lateral fabellotibial suture model using various methods of application of tension, fixation, and suture materials. METHODS: Veterinarians constructed simulated lateral fabellotibial suture constructs on a tying stand with a force sensor. Participants used combinations of 45 kg test monofilament nylon, metric 7 braided polyethylene, crimps, crimper, or knots, with their choice of instruments to secure the constructs. The tension in completed constructs was measured and comparisons were made between nylon and polyethylene, the use of crimps compared to knots, and the use of a mechanical distractor compared to hand tightening techniques. A value of p <0.05 was considered significant. RESULTS: Fifty-eight veterinarians created 72 lateral suture constructs. Final tensions generated ranged from 1.4-171.0N. The median tension of nylon sutures (43.9N ± 44.7N) was significantly greater than polyethylene sutures (9.5 N ± 19.6N). The median tension of constructs secured with crimps (62.8N ± 42.4N) was significantly greater than constructs secured with knots (11.8 N ± 14.8N). The mechanical distractor generated significantly higher median tension (78N ± 50.4N), compared to methods without the device (18.6 N ± 25.1N). CLINICAL SIGNIFICANCE: There was a large variability in the tension generated in simulated lateral fabellotibial constructs. Veterinarians who used nylon, crimps, and the mechanical tensioner generated constructs with greater tensions.

Oncolytic effects of fatty acids in mice and rats.

Intrahepatic implants of M114 carcinoma in B6D2F1/J mice were treated by intraperitoneal injection of 30 mg sodium caprylate (octanoate), and implants of Nb2 lymphoma in Nb rats were treated with 300 mg tricaprylin orally. After 4-11 h extensive damage to tumor cells was evident microscopically whereas liver cells were unaffected. Tumors in mice treated once daily from the fourth to eighth day after implantation were obliterated. Subcutaneous implants of hepatoma Nb10L in Nb rats treated transdermally with a caprylic acid preparation underwent similar damage. Fatty acids can cause lysis of tumor cells with little damage to normal tissue in certain situations. This action is not related to mitotic activity and represents a novel mode of antitumor action.

An effect of corticosteroids on thymocytes not mediated by macromolecule synthesis.

Rat thymocytes were incubated for 2 min at 37 degrees C and the cells then broken by osmotic shock in 1.5 mM MgCl2 and the nuclei harvested. Treatment with 50 nM dexamethasone for 2 min resulted in about one third of nuclei showing abnormalities in appearance, in shape and density. This was not prevented by prior incubation for 10 min with actinomycin D and cycloheximide, but was when nuclei were isolated in the presence of anions larger than F- and Cl-, including I-, Br-, SO = 4 and citrate identical to. Subsequent addition of Cl- ion, however, resulted in development of abnormalities in steroid-treated nuclei. It is concluded that the steroid induces a mechanism resulting in influx of chloride ion leading to nuclear edema, which is not mediated by processes involving synthesis of macromolecules.

Increased cholesteryl ester content in liver of mice fed lipid emulsion diets high in polyunsaturated fats.

Mice were fed a liquid diet containing different fat sources for 6 days and several biochemical parameters in the liver were examined. Mice fed diets containing Nutralipid or Liposyn as 45% of total calories had 30.5 +/- 2.5 and 25.8 +/- 3.7 nmol cholesteryl esters per milligram liver protein, respectively, as compared with 13.14 +/- 2.4 for those fed regular mouse food and 13.7 +/- 2.45 for those fed an emulsion containing mostly triolein as fat source. A similar increase in liver cholesteryl esters resulting from estrogen treatment has been proposed as the basis for changes resulting in decreased bile flow in the rat. It is suggested that the high content of polyunsaturated fatty acids in nutrient emulsions might be responsible for cholestasis sometimes observed in patients receiving these preparations. This is further supported by the observation that, as in the case of estrogen treatment, the cholesteryl ester level returned to normal when mice were treated with the detergent Triton WR-1339.

Effect of the dietary fatty acid component on the release of 14C-taurocholate, 14C-bilirubin, alkaline phosphatase, and aspartate transaminase by isolated rat liver cells.

Rats were fed liquid diets for 7 days containing either triolein or Liposyn, which is rich in linoleic acid, as fat sources, and liver cell suspensions were prepared following collagenase perfusion. The release from isolated cells of alkaline phosphatase and aspartate transaminase during a 3-hr incubation did not differ. The uptake and release of 14C-taurocholate during a brief incubation was lower but not significantly in Liposyn-fed rats (0.1 greater than p greater than 0.05): the uptake was 9.74 +/- 1.58 vs 16.7 +/- 3.3 nmol/mg protein in triolein-fed rats; the release was 3.17 +/- 0.65 vs 5.35 +/- 1.01 nmol/mg protein in triolein-fed rats. The uptake of 14C-aminolevulinic acid was similar in both groups, but release of 14C-bilirubin during a 30-min incubation was 5,420 +/- 1010 in the Liposyn group vs 12,030 +/- 2,200 dpm/mg protein in the triolein group (p = 0.02). It is concluded that a diet high in linoleic acid decreases bilirubin release in isolated liver cells consistent with the ability of this diet to cause cholestasis in vivo.

Role of anions in the lymphocytolytic action of corticosteroids and fatty acids.

Rat thymocytes were exposed in vitro to the corticosteroid dexamethasone, 10 nM, for 10 min, or to oleic acid, 500 nM for 2 min. This results in cytolysis after 6 hr, if incubation is continued. Instead, the cells were centrifuged, the supernatant fluid decanted, and the cells subjected to osmotic shock in 1.5 mM MgCl2. The naked nuclei were incubated at 37 degrees C and examined by light and electron microscopy. Nuclear edema was evident early, and most nuclei showed damage with variation in shape and size and distinct folds, which was maximal by 1-2 hr as a result of these treatments. This was true also if nuclei were incubated in MgF2 or Mg(NO3)2 but not in MgBr2, MgI2, MgSO4 or Mg-citrate. Spleen lymphocyte nuclei showed similar damage but only after incubation with 20 microM oleic acid, and not at all with corticosteroids. The effects of both steroid and fatty acid, even at greatly increased concentrations, were inhibited by tri-n-butyl tin chloride, 10 microM, and by 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid, sodium salt, 10 microM, both of which block chloride ion transport. It is concluded that the cytolytic effects of both corticosteroids and free fatty acids involve influx of chloride ion resulting in nuclear edema, which subsequently leads to fragmentation of chromatin, karyorrhexis and, ultimately, cytolysis.

Inactivation of corticosteroids in intestinal mucosa by 11 beta-hydroxysteroid: NADP oxidoreductase (EC 1.1.1.146).

Activity of the enzyme 11 beta-hydroxysteroid:NADP oxidoreductase (EC 1.1.1.146) in human intestinal mucosa was determined by incubating scraped mucosa with 3H-cortisone and 14C-cortisol; these steroids were then extracted, separated chromatographically, and the radioactivity assayed to determine simultaneously both reductase and dehydrogenase activities. This was the only significant metabolic alteration which the substrate underwent. Only two cases had slight (5 and 13%) reductase activity. In 35 patients, 16 male and 19 female, including seven cases of Crohn's disease, three ulcerative colitis, five diverticulitis, two undergoing surgery for repair of injuries and 18 for carcinoma of colon or rectum, cortisol was converted to cortisone in 15 min with a wide range of values distributed uniformly up to 85% dehydrogenation, with a mean of 42%. When tissue homogenates were fortified with coenzymes, excess NADPH lowered dehydrogenase activity 81%; excess NADP increased dehydrogenase activity 2-fold in three cases. It is possible that a value is characteristic of an individual but perhaps more likely enzyme activity varies with metabolic events involving changes in the coenzyme levels in mucosa, and a random sampling might be expected to yield such a distribution of values. In any event, where activity is high most of the cortisol is inactivated within minutes. It is suggested that synthetic corticoids which escape such metabolic alteration might, except during pregnancy, prove superior in the treatment of conditions such as inflammatory bowel disease.

Decreased incorporation of 14C-glucosamine relative to 3H-N-acetyl glucosamine in the intestinal mucosa of patients with inflammatory bowel disease.

The synthesis of glycoproteins was investigated in intestinal mucosa from patients with inflammatory bowel disease (IBD) and from those with various other conditions. The incorporation into acid-insoluble macromolecules of the amino sugar glucosamine, the first and committed metabolite in the biosynthetic sequence, and its immediate derivative, N-acetyl glucosamine was determined. Tissue was incubated with 1-2 nmol 14C-glucosamine and 3H-N-acetyl glucosamine and the simultaneous incorporation of both isotopes was measured. Bowel tissue from areas microscopically uninvolved in active disease process was examined. Values for the incorporation of both substrates into acid-soluble constituents were similar for both IBD and non-IBD groups, as was also the incorporation of 3H into acid-insoluble constituents. The incorporation of 14C, however, when expressed relative to that of 3H in each individual patient, i.e., 14C/3H, was distinctly different in IBD cases. In 26 non-IBD samples this ratio ranged from 0.04-0.26 with a mean of 0.097 +/- 0.009. In nine cases of Crohn's disease values ranged from 0.013-0.06 with a mean of 0.039 +/- 0.011 (p less than 0.01); in nine cases of ulcerative colitis values were 0.007-0.06 with a mean of 0.031 +/- 0.006 (p less than 0.01). It is concluded that the step involving the N-acetylation of the amino sugar is relatively deficient in patients with IBD and this could reduce the synthesis of the glycoprotein cover which protects the mucosa from damage by bowel contents.

Spin labelling studies of cytolysis induced by fatty acids.

Rat thymocytes, spleen lymphocytes and isolated nuclei were incubated with fatty acids and then labelled with 5-doxylstearic acid and 12-doxylstearic acid. The ESR spectra only in the case of 5-doxylstearic acid showed changes which were demonstrable only under those conditions which resulted in cytolysis. Thymocytes in medium with 10% serum showed the effect at 10 microM, splenic lymphocytes at 100 microM. The effect was maximal at 2 min and was not enhanced by higher concentrations. The uptake of fatty acid by spleen cells required to cause this change was determined using 14C-oleic acid, to be 0.6 mumol/g tissue. This quantity is less than that required (label:lipid ratio less than 1:10) to produce major perturbations in membranes. Free fatty acids of C-8 to C-18 produced the effect, but not esters or amides. It was concluded that free fatty acids induce changes proximal to the polar region of membrane lipids which, if not progressive and essential to the ultimate process of lysis, are at least indicative of impending cell death at an early time.

Effects of dexamethasone on fetal mouse development.

The in vitro incorporation of 14C-leucine, 3H-uridine and 3H-thymidine into the acid-insoluble components of mouse fetal liver, gut, heart and lung as followed on gestational days 14, 16 and 19. Liver and gut showed a similar pattern: the incoporation of all three substrates decreased steadily until day 19, falling from one tenth to one fifth the values on day 14. Heart and lung decreased by half after day 16. Injection of mothers 16 h earlier with 200 microgram dexamethasone decreased in vitro incorporation on day 14 into liver and gut, respectively, of leucine by 38 and 37% and of thymidine by 67 and 56%. In heart and lung, only thymidine was decreased at this time by 61 and 33%, respectively. Values for uridine were affected significantly only in liver. Thymidine incorporation appeared to be the most sensitive parameter reflecting corticosteroid action and liver the most sensitive tissue. Treated fetuses were born at the usual time and weight gain did not differ from controls over the first 42 days neonatally. It is concluded that dexamethasone accelerates the development of many processes in fetal tissues which are induced by corticosteroids.

Glycogen deposition in fetal mouse tissues and the effect of dexamethasone.

Glycogen in fetal mouse tissues was isolated and determined using the anthrone method on gestational days 14-19. Fetal liver showed rapid glycogen deposition after gestational day 17, reaching a value of 46.7 mg/g wet weight on day 19. Heart and placenta showed sizable glycogen stores earlier, with peak values of 21.7 and 12.8 mg/g, respectively, on day 16 which declined thereafter. Lung maintained approximately 10 mg/g days 16-19; gut and brain contained approximately 2 and 4 mg/g, respectively. days 14-19. After injection of dexamethasone into mothers on gestational day 15.5, 16 h prior to assay, the glycogen content of liver increased approximately tenfold, while placental glycogen declined. This indicates that corticosteroids merely accelerate the pattern observed in the normal course of gestation.

Corticosterone metabolism and the incorporation of leucine, uridine, and thymidine into fetal mouse brain.

The change in the pattern of biotransformation of [14C]corticosterone in fetal mouse brain between gestational day 14 and 17 increased the proportion of unchanged hormone from 9-75%. A sharp decrease in the in vitro incorporation of [14C]leucine, [3H]uridine, and [3H]thymidine into incubated brain coincided with this change and continued until day 19, when the incorporation of the 3 substrates had fallen to 9, 54, and 16%, respectively, of that on day 14. Injection of dexamethasone reduced values on day 14 to those normally found on days 15-18. Enzymes which metabolize corticosteroids regulate their activity in specific tissues; these data suggest a hormonal influence on developing brain.

Corticosteroids, ornithine decarboxylase activity and the incorporation of leucine, uridine, and thymidine into mouse placenta.

The in vitro incorporation into mouse placenta of [14C]leucine, [3H]uridine, and [3H]thymidine fell between gestational days 14 and 19 by 61, 30 and 72%, respectively; ornithine decarboxylase activity fell by 75%. Injection of dexamethasone resulted on day 14 in values normally found between days 15 and 18. These changes coincided with rising activity in fetal liver of the enzyme 11 beta-hydroxysteroid:NADP oxidoreductase, which reduced the abundant 11-dehydrocorticosterone to the active hormone, corticosterone. These events appear to be part of a spectrum of effects produced by corticosteroids on fetal growth at this time, probably mediated primarily by fetal liver.

Maternal-fetal relationships in corticosteroid metabolism.

Corticosterone in fetal mouse tissues after injection of mothers with 14C-corticosterone was determined by acetylation with 3H-acetic anhydride and cocrystallization to constant specific activity. The corticosterone content of whole fetal tissue varied between gestational days 13 and 17 from 641 to 300 ng/g. Specific activity of fetal hormone remained essentially constant; after a 15-min pulse this was as much as one-quarter that of maternal hormone. Placenta, head and liver showed distinctly different patterns which changed during this time, with a decrease in conversion of corticosterone to 11-dehydrocorticosterone. A sharp increase occurred in the activity of fetal liver 11beta-hydroxysteroid:NADP oxidoreductase activity. This mitochondrial enzyme, pH optimum 6, KM = 33 micrometer, reduced the metabolite, raising the relative amount of corticosterone in the fetus from 16 to 91%. 1 day after removal of maternal adrenals both maternal and fetal corticosterone were normal, indicating ability of fetal adrenals to function. Maternal corticosterone, however, crossed the placenta readily and it is considered likely that, normally, the maternal hormone predominates. Regardless of origin, corticosterone is maintained by enzymatic conversion in a distinct manner in different tissues.

Metabolism of natural and synthetic corticosteroids in relation to their effects on mouse fetuses.

14C-glucose uptake by mouse fetuses was reduced by doses of dexamethasone, 200 mug or more, which over 2 days caused fetal death. Uptake by strain C57B1/6J greater than A/J greater than SWV. Corticosterone or cortisol, 4 mg, caused neither reduced uptake nor fetal death. These differences occurred despite similar maternal hyperglycemia in all cases. Recovery of 3H-steroid after 15 min, total and unchanged steroid per gram fetal tissue was: corticosterone 1.7 and 0.3%; dexamethasone 0.12 and 0.06% of the dose. Rapid metabolism of corticosterone apparently prevents accumulation in fetal tissue sufficient to evoke a response under these conditions.

Glucocorticoid receptors and lymphocytolysis in normal and neoplastic lymphocytes.

When cells of the thymus or mouse leukemias P288 and L1210 are exposed in vitro to the potent synthetic glucocorticoid, 3H-Triamcinolone acetonide, the steroid enters the cells passively and binds to macromolecules in the cytoplasm. At 37 degrees C this hormone-receptor complex enters the nucleus and associates with the chromatin. The association with chromatin occurs not only in the corticosteroid-sensitive rat thymocytes and mouse tumors P288 and P1798S but also in the corticosteroid-resistant mouse tumors L1210 and P1798R. An apparent correlation, although not absolute, exists between the content of glucocorticoid-binding macromolecule and the sensitivity of the lymphocytes studied to the lytic effect of glucocorticoids; the sensitive cells having more receptor than the resistant cells. The process of lysis is attributed to the release from the much larger stores of triglyceride in thymus and sensitive lymphoma cells, of a large pool of FFA which causes focal damage to the nuclear membrane resulting in karyorrhexis and, subsequently, to cytolysis. Resistance is attributed to the capacity for preventing the accumulation of greater than about 0.5 fmole FFA/cell. Resistant cells induced to accumulate greater amounts, even for a few minutes, ultimately undergo lysis. Most effective in accomplishing this are branched chain fatty acids of C-8 and higher, which block FFA metabolism, causing accumulation which results in cytolysis.