Deficient Rabbits Research Articles

Effects of Endotoxin in Cortisone-Treated Rabbits with a Hereditary Deficiency of the Sixth Component of Complement (C6 Deficiency)

Endotoxin was infused into normal rabbits and C6 deficient rabbits prepared with cortisone for the generalized Shwartzman reaction. Endotoxin produced profound granulocytopenia and moderate thrombocytopenia in both normal and C6 deficient rabbits. In normal rabbits endotoxin consistently produced extensive intravascular clotting. In C6 deficient animals endotoxin resulted in intravascular clotting of variable extent. In one group of eight C6 deficient rabbits mean fibrinogen levels fell 0.67 g per 1 over 6 hrs after endotoxin and four of eight animals developed a generalized Shwartzman reaction. In a second group of seven C6 deficient rabbits mean fibrinogen level fell only 0.17 g per 1 over 6 hrs and one animal developed a generalized Shwartzman reaction. Values for mean fibrinogen consumption, calculated from plasma fibrinogen levels and rate of disappearance of 25I-fibrinogen, were as follows: normal animals infused with saline, 10 mg per kg; C6 deficient animals infused with endotoxin, 58 mg per kg. Fibrinogen consumption after endotoxin was found to be related to granulocyte levels prior to endotoxin, which determined the number of granulocytes disappearing from the blood after endotoxin. The data indicate that C6 deficiency in the rabbit does not prevent intravascular clotting and the generalized Shwartzman reaction.

Purification and characterization of xanthine oxidase from livers of vitamin E deficient rabbits

Xanthine oxidase which increases in activity during vitamin E deficiency was purified from livers of deficient rabbits. The procedure incorporates preparative sucrose gradient centrifugation and yields a homogeneous preparation on acrylamide gel electrophoresis. The purified enzyme exhibits a pH optimum of 8.1 and K m value of 22 μM. Gel filtration chromatography gave the molecular weight of 280 000. Acrylamide gel electrophoresis in the presence of sodium dodecylsulphate reveals two types of subunits of molecular weights 52 000 and 99 000.

The Role of Complement in Endotoxin‐Induced Disseminated Intravascular Coagulation: Studies in Congenitally C6‐Deficient Rabbits

Summary.The effect of intravenous infusion of endotoxin on the activation of intravascular coagulation and on the generation of glomerular microclots was studied in rabbits congenitally deficient in the sixth component of complement (C6). Endotoxin infusion induced the formation of circulating soluble fibrin and caused a drop in leucocyte and platelet counts, a decrease in factor‐V and factor‐VII activities and a prolongation of the thromboplastin time as well as the partial thromboplastin time. These haematological changes are indicative of consumption coagulopathy and were similarly distinctive in C6‐deficient as in normal rabbits infused with endotoxin. Furthermore, renal glomerular microclots formed in 90% of the C6‐deficient and in 80% of the normal rabbits after endotoxin administration. The haematological as well as the histological results of these experiments demonstrate that the sixth component of complement and the later complement components C7–9 are not essential to the initiation of intravascular coagulation by endotoxin and the manifestation of the generalized Shwartzman reaction. The data make it likely that the release of platelet factor 3 does not represent the trigger mechanism of intravascular coagulation since disseminated intravascular coagulation could be induced in C6‐deficient rabbits although the platelets of these animals are incapable of releasing platelet factor 3 activity after platelet‐endotoxin interaction.

Abnormal platelet response to thromboplastin infusion in rabbits deficient in the sixth component of complement.

SummaryThe slow infusion into normal rabbits of a low dose of thromboplastin (tissue factor), generally insufficient to cause thrombosis or consumption of coagulation factors, produced thrombocytopenia and a reduction in total serum complement activity. In contrast, the administration of thromboplastin to rabbits genetically deficient in C6 resulted in small increments in platelet number. Reconstitution of the complement system of C6 deficient rabbits with purified C6 restored the capacity to develop thrombocytopenia following thromboplastin administration. These studies demonstrate that thromboplastin can act on platelets through the participation of the complement system and that C6 is essential for this reaction.

Release of 51Cr-Endotoxin from Bacteria as an Assay of Serum Bactericidal Activity

Abstract In order to facilitate the study of normal serum on Gram-negative bacteria we have developed a bactericidal assay based on the release of 51Cr from labeled bacteria. Release of radioactivity was shown to be mediated by complement and paralleled the bactericidal action of sera measured by change in colony-forming units. Serum from the C6 deficient rabbits and serum that had been treated to inactivate complement did not release 51Cr. The rate of release of 51 Cr was decreased by absorption of serum with Bentonite but the maximum percentage released was not decreased. 51Cr was not released from serum resistant bacteria. The 51Cr appears to be associated with the lipopolysaccharide fraction of the bacteria. Since this structure is the target for the bactericidal reaction, the release of 51Cr is a direct measure of the effect of complement on the bacteria. Under most conditions this reaction leads to cell death so that the release of 51Cr can be used as an assay of bactericidal activity. It was shown that the assay was feasible with Escherichia coli, Salmonella and Hemophilus influenza B.

Biochemical changes in progressive muscular dystrophy. X. Studies on the biosynthesis of protein and RNA in cellular fractions of the skeletal muscle of normal and vitamin E deficient rabbits.

Present investigation was undertaken to elucidate the metabolism of protein and nucleic acids in the skeletal muscle of control and vitamin E deficient rabbits. In order to attain this objective, the content and synthesis of protein and RNA were measured in the homogenate and various subcellular fractions of the skeletal muscle of control and vitamin E deficient rabbits. Our results indicate a possibility of reduction in the muscle volume in the dystrophic muscle since the content of DNA and RNA did not change in such muscles. The protein content, however, decreased considerably in the dystrophic muscle.Incorporation studies indicated a considerable increase in the synthesis of protein and RNA. It was also observed that the excretion of radioactivity was much higher in the urine of dystrophic animals. These findings indicate a rapid turnover of protein in the dystrophic muscle.The sucrose density gradient analysis of polysomal material from the dystrophic muscle indicated a lowering in the concentration of heavier polysomes synthesizing myosin and a considerable increase in the region of lighter polysomes synthesizing tropomyosin. From these findings it is speculated that vitamin E may play an important role in the synthesis of specific muscle proteins. Addition of 250 μg of poly-U restored the polysomal content of dystrophic muscle to that of control, leading to the hypothesis that vitamin E deficient rabbit muscle may be lacking in the messenger RNA content.

The behaviour of complement and platelets in lethal endotoxin shock in rabbits.

The <i>in vivo</i> behaviour of chromium-labelled platelets was studied in normal, C6 deficient and C3-9 depleted rabbits following the injection of a potentially lethal dose of an endotoxin <i>(S. marcescens).</i> The majority of labelled platelets were acutely destroyed in normal rabbits and only a small proportion returned briefly to the circulation. All normal rabbits subsequently died. In marked contrast, labelled platelets survived normally in C3-9 depleted rabbits following injection of the endotoxin and no C3-9 depleted rabbit died. In C6 deficient rabbits, platelets were acutely sequestered but destroyed to only a variable degree. C6 deficient rabbits, in which platelet recovery following early sequestration was greater than 65%, survived subsequently, whereas C6 deficient rabbits, in which platelet recovery was less than 45%, died. Platelet factor 3 (PF3) activation by endotoxin was also studied both <i>in vivo</i> and in platelet rich plasma <i>in vitro</i>. A major degree of PF3 activation occurred in normal rabbits, a minor degree in C6 deficient rabbits and none in C3-9 depleted rabbits. A direct association was found between complement status, PF3 activation, platelet survival and subsequent mortality following endotoxin injection.

Host Defense Against Bacterial Endotoxemia-Contribution of The Early and Late Components of Complement to Detoxification

Abstract The precise mechanisms and tissue sites involved in the in vivo detoxification of endotoxin remain unresolved. Although a number of studies have indicated that circulating plasma is a principal site of endotoxin inactivation (1–3), it was believed that the complement system was not required for detoxification of endotoxin (1). However, a recent report which demonstrated the increased susceptibility of C6 deficient rabbits to the injection of bacterial endotoxin suggests that complement may be important (4). Endotoxin can activate the complement system either by the classical mechanism proceeding through the activation of C1, C4 and C2 to activate C3, or via the alternate or bypass pathway proceeding through another series of proteins, as yet incompletely defined, to enter the complement sequence directly at the C3 step (5–7). In this study a further attempt is made to evaluate the role of complement in the detoxification of endotoxin, with particular reference to the contribution of the early (C1, C4 and C2) and late acting (C3–C9) complement components.

The Requirement for Serum Complement in the Detoxification of Bacterial Endotoxin

Abstract The in vitro detoxification of bacterial endotoxin requires both the fifth (C5) and and sixth (C6) components of complement and proceeds in the presence of 0.01 M EDTA. Addition of purified C5 or C6 to the genetically deficient serum corrects the defect in detoxification of endotoxin. Mice lacking C5 show a greatly increased susceptibility to the lethal effects of endotoxin and C6 deficient rabbits, in contrast to normal rabbits, fail to develop an increased detoxifying capacity in serum as a result of recent exposure to endotoxin. While human plasma is more effective than serum in detoxifying endotoxin, this cannot be completely ascribed to the reduction in levels of cations. The mechanism by which the complement system participates in the detoxification of endotoxin is peculiarly different from the usual sequential pathways of the complement system. In broad biologic terms, these findings suggest a previously unrecognized, protective function of the complement system.

A Blood Coagulation Abnormality in C6 Deficient Rabbits and Its Correction by Purified C6

Abstract In this communication we report a coagulation abnormality in rabbits genetically deficient in the sixth component of complement which is characterized by prolonged whole blood clotting time and deficient prothrombin consumption. The mean glass clotting time in five C6 deficient rabbits was 38 min (range: 34 to 41 min) as opposed to a mean of 24 min (range: 20 to 31 min) in five normal rabbits. Clotting times in plastic tubes varied from 140 to over 400 min in C6 deficient animals, whereas the range for the normal rabbits was 70 to 90 min. Prothrombin consumption measured in plastic tubes for 1 hr at 37°C varied from 0% to 10% in six deficient rabbits, whereas the mean for six normal rabbits was 72.5% with a range of 62% to 79%. Prothrombin and partial thromboplastin times were normal in the deficient animals as were activities of the classical clotting factors II, V, VII, VIII, IX, X, XI and XII.

Complement and Immunologic Reactions in Vivo

Abstract The role of complement in immunologic inquiry of the glomerulus has been studied. Sheep antibody to rabbit renal basement membrane (ARBM), when injected into rabbits, induced glomerulitis and proteinuria dependent in part upon the participation of host neutrophils. Neutrophil depletion diminished the level of proteinuria but the degree varied from donor to donor and with the time of immunization of the sheep. Fractionation of the ARBM on DEAE-Sephadex revealed different zones of antibody activity: in the fast γ region, antibody was obtained that was greatly dependent upon neutrophils for its action, while in the slow γ regions, antibody was found that produced glomerular injury not dependent upon neutrophils. The injury produced by the neutrophil independent antibody was not affected by C3 depletion with cobra venom factor (CoF) and occurred equally well in C6 deficient rabbits.

Effects of hydrocortisone acetate and estradiol in normal and adrenalectomized salt deficient rabbits.

These experiments were undertaken to determine the involvement of the adrenocortical hormones in the inhibition by estrogens of the polyuric-polydipsic syndrome induced by salt deficiency in the male rabbit. Daily measurements were made of body weight, water intake and urinary volume, Na† and K† during 10 consecutive days of food deprivation in normal and adrenalectomized male rabbits receiving 17β-estradiol (100 µg/day) and/or hydrocortisone acetate (Fac, 2.5 mg/day) for the first 5 days. Normal males, when deprived of food, developed the typical polyuria and polydipsia which were accompanied by a marked rise in urinary Na†. The administration of Fac further enhanced significantly the fluid exchange during the first 5 days. The administration of estradiol completely inhibited the polyuriapolydipsia, and this effect per sisted into the second 5 days. Estradiol administration to the Fac treated rabbits not only abolished the enhancement due to Fac but depressed water intake and urine volume to values less ...

Vitamin A deficiency and glycolipid sulfation

The relationship between vitamin A deficiency and the formation of the sulfatides in brain was investigated in vivo in rats of various ages. By varying litter size and by pair feeding, the control animals had body weights similar to those of the deficient rats. No significant differences in the concentrations or in the total amounts of sulfatides were detected in the brains of vitamin A deficient or control rats, weanling or adult, or in most of the preweanling rats studied at the period of rapid myelination. Except for relatively small, malnourished, vitamin A deficient preweanling rats, variation in body weight within animals of the same age group, did not by itself influence the content of brain sulfatides. Vitamin A deficient animals also showed equal or greater incorporation of (35)S into sulfatides compared to controls. Deficient rabbits were able to incorporate (35)S into the sulfatides of brain and ocular tissues to the same or greater extent as did control animals. Thus, vitamin A deficiency does not interfere in the sulfation of glycolipids or perhaps generally in the processes of biological sulfation as has been proposed previously.

Dilution of phsophate in the plasma of tocopherol deficient rabbits.

L'etude de la cinetique de la dilution du phosphate dans le plasma des lapins deficients enα-tocopherol a mis en evidence que, chez ces animaux, la vitesse de disparition du phosphate du plasma ainsi que l'espace de phosphate se trouvent augmentes.

The regulatory function of vitamine E

1. 1. A rise in the creatine phosphokinase (CPK) of skeletal muscle of vitamin-E-deficient rabbits precedes the appearance of significant amounts of enzyme in the blood. 2. 2. Injected rabbit muscle CPK is catabolized in normal, deficient, and treated rabbits with a half-time of 1.5 hr. 3. 3. Treatment of deficient rabbits having elevated serum CPK values with α-tocopherol or ethionine results in a sharp decline of CPK activity in plasma with a half-time of 12 hr. Ethionine has no effect on the assay method for CPK. 4. 4. It is suggested that vitamin E deficiency may represent a state of excessive synthesis of catabolic and redundant enzymes which is repressed by vitamin E and inhibited by ethionine. If the hereditary muscular dystrophies are caused by a mutation in a regulatory gene, the similarity of these disorders to vitamin E deficiency and the ineffectiveness of α-tocopherol as a therapeutic agent is explained.

Cardiovascular Lesions Associated with Experimental Copper Deficiency in the Rabbit

Copper deficiency was induced in Dutch Belted rabbits by feeding from weaning a milk-sucrose diet supplemented with vitamins, certain minerals and amino acids and fed in an agar gel form. Reduced growth, lowered hemoglobin and hematocrit levels, and achromotrichia, alopecia and dermatosis were observed in deficient rabbits. Grossly, calcific nodules were observed in the heart of some deficient animals. Histologically, the myocardial lesions consisted of necrosis of cardiac muscle fibers with dystrophic calcification, but without a significant inflammatory reaction. Elastin defects consisted of loss of elastic fibers and calcification and fragmentation of the internal elastic membrane in muscular and elastic arteries. Calcification of the media was observed in some muscular arteries. Lesions were not observed in the coronary arteries. Penicillamine and British anti-Lewisite did not significantly accentuate the copper deficiency. However, supplementation of a diet containing 3 ppm copper with 1% ascorbic acid apparently induced a copper deficiency producing signs and lesions observed in animals made deficient by feeding a diet containing 2 ppm copper. There was little evidence of a deficiency in the rabbits receiving 3 ppm copper. These preliminary studies suggest that the copper requirement of the rabbit is approximately 3 ppm.

The ultrastructure of the choroid plexus in hydrocephalic offspring from vitamin A deficient rabbits.

The fine structure of the lateral choroid plexus in vitamin A deficient hydrocephalic New Zealand white rabbits was observed with light and electron microscopy. A vitamin A deficient diet was fed to all rabbits, but controls in addition received 12,500 I. U. of vitamin A powder weekly. The offspring of does thus maintained were studied. The rabbits were decapitated; the left lateral choroid plexus was exposed, and subjected to the fixative within 90 seconds. Conventional techniques of fixation, embedding, and sectioning were employed. Suitable sections were carbon-filmed after being placed upon copper grids and viewed with a modernized RCA EMU-2A electron microscope. Light microscopy disclosed blood extravasation in the choroid plexus of the hydrocephalic rabbits. In addition, the electron microscope revealed the following: 1. weakened areas between endothelial cells comprising the capillaries with platelet thrombi often associated, 2. “free” red and white blood cells, thrombocytes, and precipitated plasma proteins located in the subarachnoid space, 3. large cisternae between the plasma membranes of the “stretched” ependymal cells, 4. an apparent increase in the number of parallel and whorled forms of endoplasmic reticulum, and 5. an apparent increase in inclusion bodies associated with the arrays of endoplasmic reticulum. A hypothesis is set forth concerning the production of hydrocephalus in vitamin A deficiency.

Localization of Renin in Juxtaglomerular Cells of Rabbit and Dog Through the Use of the Fluorescent-Antibody Technique

Canine antisera to rabbit and hog renin were coupled to several fluorescein dyes. The antiserum was immunologically adsorbed once each with rabbit-liver and with bone-marrow powder, thereby removing excess dye and eliminating nonspecific staining. Mounted frozen-dried sections of kidneys from sodium- deficient rabbits (in which hyperplasia and hypergranulation of juxtaglomerular [JG] cells were present), from control rabbits, from a sodium-deficient dog, and from sodium-deficient rats were treated with the adsorbed labeled antiserum. Ultraviolet microscopy (direct technique) revealed an intense yel low-green fluorescence sharply limited to the granules of the juxtagloinerular cells in all sections studied from kidneys of rabbit and dog. JG granules in rats did not fluoresce, an observation in accord with the species spe cificity of renin. The indirect ("sandwich") method was also employed (with adsorbed, labeled, rabbit antiserum to canine globulin), and although slight staining of gloinerular epithelium resulted, that in the JG granules was far more intense. In our hands, the faint glornerular staining was not blocked by prior treatment with unlabeled renin. Staining of JG granules in any kidney paralleled the intensity of JG granulation by light microscopy and the amount of extractable renin in the same kidney. JG-granule staining (direct and indirect techniques) was blocked by neutralization of the antirenin with renin. Heterogenous anti sera (to insulin; to human gamma globulin) failed to stain. Other rabbit tissues (heart, lung, liver) similarly treated with labeled antirenin never stained. If this work can be repeated with immunologically pure renin, the evidence presented here, together with previously published studies from this and other laboratories, will establish beyond any reasonable doubt that the source of renin in the kidney is the juxtaglomerular cell, as postulated first by Goormagh tigh nearly a quarter of a century ago.

A method for electrocardiogram wave-pattern estimation. Example: left ventricular hypertrophy.

Canine antisera to rabbit and hog renin were coupled to several fluorescein dyes. The antiserum was immunologically adsorbed once each with rabbit-liver and with bone-marrow powder, thereby removing excess dye and eliminating nonspecific staining. Mounted frozen-dried sections of kidneys from sodium- deficient rabbits (in which hyperplasia and hypergranulation of juxtaglomerular [JG] cells were present), from control rabbits, from a sodium-deficient dog, and from sodium-deficient rats were treated with the adsorbed labeled antiserum. Ultraviolet microscopy (direct technique) revealed an intense yel low-green fluorescence sharply limited to the granules of the juxtagloinerular cells in all sections studied from kidneys of rabbit and dog. JG granules in rats did not fluoresce, an observation in accord with the species spe cificity of renin. The indirect ("sandwich") method was also employed (with adsorbed, labeled, rabbit antiserum to canine globulin), and although slight staining of gloinerular epithelium resulted, that in the JG granules was far more intense. In our hands, the faint glornerular staining was not blocked by prior treatment with unlabeled renin. Staining of JG granules in any kidney paralleled the intensity of JG granulation by light microscopy and the amount of extractable renin in the same kidney. JG-granule staining (direct and indirect techniques) was blocked by neutralization of the antirenin with renin. Heterogenous anti sera (to insulin; to human gamma globulin) failed to stain. Other rabbit tissues (heart, lung, liver) similarly treated with labeled antirenin never stained. If this work can be repeated with immunologically pure renin, the evidence presented here, together with previously published studies from this and other laboratories, will establish beyond any reasonable doubt that the source of renin in the kidney is the juxtaglomerular cell, as postulated first by Goormagh tigh nearly a quarter of a century ago.

Phosphate metabolism in nutritional muscular dystrophy and hyperthyroidism.

Summary1) Rabbits and rats were made Vit. E-deficient by feeding a purified diet. Control animals received the same diet supplemented with Vit. E. Hyperthyroidism was induced in rats by giving subcutaneous injections of sodium thyroxinate. Control and experimental animals from each group were injected with P32 in dilute HCl and specific activities of plasma inorganic phosphate, and skeletal muscle inorganic phosphate, creatine phosphate, and ATP phosphate determined. The S. A. of the plasma inorganic phosphate was unchanged by Vit. E deficiency and was decreased by hyperthyroidism. Skeletal muscle inorganic phosphate S. A. was increased by Vit. E deficiency and the difficulties in interpreting these data were discussed. The S. A. of phosphates of creatine phosphate and ATP phosphate were high in both Vit. E deficient rabbits and hyperthyroid rats. The significance of these findings was discussed in relation to turnover of creatine phosphate and ATP. 2) These experiments indicate that there is an elevated ...