Temporal Protection of Chick Embryo against Endotoxin by Isoproterenol, Polymyxin B Sulfate, and Hydrocortisone

Abstract

The lethal effect of endotoxin in the 10-11 day chick embryo could be prevented by the administration of hydrocortisone ( 1 ) , isoproterenol (2), or polymyxin B sulfate (3) when endotoxin and the protectorant were given simultaneously. Since the properties of these agents differ, a variance in protection could be anticipated with a change in the time between the administration of endotoxin and protectorant. To preclude a nonspecific effect of in vitro mixing of endotoxin and a protectorant, separate administration vas desirable. This is a further report on lethality studies in the chick embryo when the three aforementioned agents were administered at varying time intervals before and after the administration of LD50-100 dose of endotoxin.Methods. White leghorn fertile eggs (Ghostley Pearl, Anoka, Minnesota) were used throughout. Ten-day eggs were candled and a large vein was exposed by removal of the overlying shell. After a 60-min interval which enhanced the exposed vessel's resistance to the trauma of injection, a drop of sterile mineral oil was placed on the membrane for better visualization of the vessel when transilluminated. The same vessel or a similarly prepared one was used for the second intravenous injection. A total of 1.5 minims was injected each time. Eggs with traumatic bleeding were discarded. The eggs were placed in a humidified incubator set at 99.5OF between the first and second injection and were returned to the incubator after the second injection for 18-24 hr, at which time the viability of embryos was determined by candling. All nonviable embryos and those with questionable viability were examined grossly.Only those embryos with clear evidence of intra-embryo hemorrhage were recorded as experimental deaths.Materials. Endotoxin: a single lot of Esch erichia coli endotoxin prepared in our laboratory as previously described ( 5 ) was used in all experiments. Pro tec torants : sterile in jectable isoproterenol hydrochloride (Isuprel) ,1 polymyxin B sulfate,2 and hydrocortisone sodium succinate (Solu-Cortef ) were used throughout. Each 10 pg of Isuprel contained 6 pg of lactic acid, 90 pg of sodium lactate, 350 pg of sodium chloride and 50 pg of sodium bisulfite. Each 25 pg of hydrocortisone sodium succinate contained 0.2 pg of anhydrous sodium biphosphate, 0.62 pg of methyl paraben, 0.02 pg of propyl paraben and 2.2 pg of exsiccated sodium phosphate. These additives with the respective drugs were not independently controlled. Incubator : a single stage humidity and temperature controlled incubator was used to house the developing and treated embryos? Syringes: disposable sterile, nontoxic, nonpyrogenic, mono jet 501-M 1 cc syringes graduated in minims with a 27-gauge needle were used for all in jections.6Results. A total of 25 experiments with appropriate saline controls were carried out. Each experiment compared endotoxin with one or more protectorants used independently and with saline control at one or more time intervals. The results in the Tables represent the sum total of embryos in each...

Effect of Polymyxin B Sulfate on Endotoxin Activity in a Gram-Negative Septicemia Model

“…Those that did not have a definitive gel within 24 hr were judged to have negligible or no endotoxin activity. A standard curve was constructed using The antibiotic polymyxin B sulfate has been shown to neutralize known concentrations of Escherichia coli 0127B8 endotoxin; the endotoxin lethality in chick embryos and adrenalectomized mice times obtained were read off the standard curve and were calcu- (8,13,15) and to prevent endotoxin-induced leukopenia, throm-lated to represent micrograms of endotoxin activity per milliliter bocytopenia, disseminated intravascular coagulation, and renal of plasma. Each new lot of lysate was tested and was considered cortical necrosis in rabbits (3,14) and the fall in cardiac output, active if a solid gel formed between 2.5 and 4.0 min after the mean arterial pressure, and systemic vascular resistance in dogs addition of 1.0 pg E. coli endotoxin (0127:B8, Difco Laboratories, (12).…”

“…Quantitative colony counts using heparinized whole blood were performed by pour plate technique on 5% blood agar plates his investigation and other reports have shown that ~o l~m~x i n (h4icro-~ech Diagnostics, Tucson, AZ). The plates were incubated B sulfate neutralized endotoxin effects in animals given purified at 370 and 24 hr later the small, mucoid gray, nonhemolytic endotoxin, dead endotoxin-containing organisms, and gram-nega-colonies were counted, tive septicemic animals (3,6,8,(12)(13)(14)(15). The antiendotoxin effect ~~~~~i~i~~d plasma was obtained by ofthe blood of the antibiotic appears to be separate from its antimicrobial at 150 at 40 for 10 min, ~h~ plasma was divided equally and effects.…”

Prevention of the Generalized Shwartzman Reaction and Endotoxin Lethality by Polymyxin B Localized in Tissues

“…Since bacterial endotoxin has been chiefly implicated in producing these complications, numerous attempts have been made to prevent or modify the toxic effect of endotoxin in various animal models. Polymyxin B sulfate administered simultaneously with endotoxin has been shown to reduce endotoxin lethality in chicken embryos (7,15) and adrenalectomized mice (13) and to prevent the generalized Shwartzman reaction in rabbits (5,14). Sodium colistimethate has similar activity but is less effective than polymyxin B (4,15).…”

Inactivation of Endotoxin by Polymyxin B

“…A 10,000-fold range in the relative susceptibility of different endotoxins to inactivation by polymyxin B was found. The endotoxin most susceptible to polymyxin B was derived from an organism resistant to polymyxin B by disk sensitivity testing, suggesting that the bacteriocidal and endotoxin detoxifying properties of polymyxin need not be directly related.Recent studies have demonstrated the capacity of polymyxin B to protect animals from a variety of toxic effects of endotoxin, including the generalized Schwartzman reaction (5, 23), diffuse intravascular coagulation (6, 7), renal cortical necrosis (6, 7), leukopenia (6, 7), and death (6,12,21,23). The limulus gelation test, developed by Levin and others (13,14), has provided a useful in vitro means to assess an endotoxin activity which corresponds with in vivo indices of toxicity (3,15,24).…”