Intravenous Injection Of Horse Serum Research Articles

Histopathological Evaluation of Horse Serum-induced Immune Complex Vasculitis in Swine: Implication to Coronary Artery Lesions in Kawasaki Disease

Immune complex (IC) vasculitis can be experimentally induced in animal models by intravenous injection of horse serum (HS), and the findings of HS-induced IC vasculitis in swine were very similar to that of Kawasaki disease (KD). The IC mechanism may be involved in the pathogenesis of vasculitis in KD. Here, we studied the two-dimensional (2D) echocardiographic and histopathological findings of acute, subacute, and healing phases of vasculitis induced by two different types of HS, and the reproducibility of IC vasculitis in swine. Our study group consisted of 24 pure-bred landrace male piglets of 1.5-3 months of age. They were divided into three HS groups (n=17), namely, Group A (n=8) receiving gamma globulin-free HS, and Group B (n=6) receiving donor herd HS, three doses at 5-day intervals, and Group C (n=3) that received only one dose of donor herd HS on Day 1, and the saline group (n=7) that received three doses of intravenous normal saline (NS) at 5-day intervals. The 2D echocardiography was performed every 3-4 days, and all piglets were killed for histopathological studies at different dates from Days 2 to Day 60. All the HS groups developed rashes and demonstrated significant dilation (54-150%) of coronary arteries in Groups A and B; when compared (p<0.02) with 9-53% dilation in Group C and the saline group. Histopathological changes of test groups were asymmetric coronary vasculitis in various stages, whereas none of the piglets in the control group developed vasculitis. No significant difference in the echocardiographic and histopathological findings was observed among the piglets that received two types of HS. HS can induce IC vasculitis in swine. The rashes and 2D echocardiographic and histopathological studies of the acute to healing phases showed close similarities with KD, and it is concluded that swine may serve as a unique experimental model for IC vasculitis and for various therapeutic trials.

Urinary trypsin inhibitor improves peripheral microcirculation and bronchospasm associated with systemic anaphylaxis in rabbits in vivo.

Using intravital microscopy of the rabbit ear for quantitative studies of microvascular dynamics, we examined the impact of urinary trypsin inhibitor (UTI), a proteolytic enzyme inhibitor, on microvascular changes during immune complex-mediated anaphylaxis. A total of 50 rabbits, previously sensitized with horse serum, were anesthetized and mechanically ventilated with pentobarbital and isoflurane for the intravital microscopy. Rabbits were then challenged with intravenous injection of horse serum to induce systemic anaphylaxis. One minute after the challenge, each rabbit was randomly assigned to receive saline (group C), 50,000 units x kg(-1) of UTI (group U1), or 150,000 units x kg(-1) (group U2). There were no statistical differences between hemodynamic variables, including heart rate (HR), mean arterial pressure (MAP), and central venous pressure (CVP), among the survivors in each treatment group. Peak inspiratory pressure rose in all three groups but at a much higher rate in group C (P < 0.05). In contrast with the moderate effects of UTI on the above parameters, microscopic evaluation revealed a substantial difference among treatment groups: upon the initiation of anaphylaxis, the arteriole started to reduce in diameter, but UTI prevented vasoconstriction in the arteriole in a dose-dependent manner. Similar results were observed with blood flow velocity. Because flow rate was calculated as the product of blood flow velocity and vascular cross-sectional area proportional to the square of the vessel diameter, these results indicate that UTI preserves microvascular flow rate during anaphylaxis. Rabbit ear microcirculation is highly preserved in the UTI-treated groups during systemic anaphylactic shock.

Inducible osteonecrosis in a rabbit serum sickness model: deposition of immune complexes in bone marrow.

We established inducible osteonecrosis in a rabbit serum sickness model. Osteonecrosis with marrow necrosis could be induced by the intravenous injection of horse serum in two doses separated in time by a period of three weeks. In this model, osteonecrosis could be successfully produced in rabbit femoral metaphysis. The incidence of marrow necrosis was 45% (9 of 20 rabbits) and trabecular necrosis occurred in 6 of 20 rabbits (30%) at 7 days after the second injection of the horse serum. In bone marrow of the femoral metaphysis, extravasation of erythrocytes and the formation of micro-thrombi in arterioles were often observed in an early stage of the present model and both findings correlate well each other (p = 0.0001). Immune complexes could be demonstrated using immunohistochemistry in bone marrow of the femoral metaphysis as well as in glomeruli of the kidney. Extravasation of erythrocytes in bone marrow of the femoral metaphysis was observed in 8 of 12 (67%) cases with immune complex deposition in the sinusoidal space of the femoral metaphysis and in 12 of 21 (57%) cases with immune complex deposition in glomeruli of the kidney. Immune complex deposition both in the sinusoidal space of femoral bone marrow (p = 0.0385) and in glomeruli of the kidney (p = 0.0209) closely related to extravasation of erythrocytes and microthrombi in arterioles in the early stage of this model. Early microcirculatory injury (extravasation of erythrocytes and microthrombi in arterioles) adjacent to osteonecrosis could be induced by immune complex deposition in femoral bone marrow and might be predictable characteristics for the inducible osteonecrosis in the present serum sickness model. The important findings in this study were that early microcirculatory injury was closely related to the deposition of immune complexes in femoral bone marrow, and that early microcirculatory injury associated with immune complex deposition was located close to osteonecrotic regions.

The immune reaction to heterologous serum causes osteonecrosis in rabbits.

Osteonecrosis (ON) was produced experimentally in rabbits by intravenous injection of horse serum. Eighty adult rabbits were used: 16 were injected twice with isotonic saline (Group A), 24 were injected once with saline and once with horse serum (Group B), and 40 were injected twice with horse serum (Group C). Both femurs of each rabbit were obtained from 2 h to 7 weeks after the final injection an were subjected to histological examination. No pathological changes were seen in Groups A and B. In Group C, 5 of 15 rabbits (33%) showed ON (necrosis of trabecula and bone marrow) in the femoral metaphysis. In Group C, the early major pathological findings in bone marrow are extravasation of erythrocytes in sinusoidal spaces and microthrombi in small arteries and arterioles near the lesion of extravasation. Immune complexes were demonstrated in the kidney within 24 h of the final injection of horse serum. The present study suggests that immunological reaction associated with serum sickness may play an important role in inducible ON and this model will contribute toward clarifying the pathogenesis of ON.

STUDIES ON HEPATIC CIRCULATION IN ANAPHYLACTIC SHOCK WITH SPECIAL REFERENCE TO THE PLASMA CONCENTRATION OF CATECHOLAMINES

Hypersensitive reaction to penicillin, or anaphylactic shock is a clinical problem of increasing magnitude. Although hepatic circulation in secondary shock due to trauma or hemorrhage has been extensively studied in animals especially on its irreversibility, hepatic circulation in anaphylactic shock has not yet been fully understood. In this paper, hepatic circulation in anaphylactic shock is discussed in correlation with systemic hemodynamics and the plasma concentration of catecholamines in dogs. The mechanism of the change of hepatic circulation is also mentioned. I HEPATIC CIRCULATION IN ANAPHY-LACTIC SHOCK Systemic and hepatic circulatory dynamics were examined simultaneously, for the furtherclarification of the possible relationship between those two systems. Material and Methods The observations reported in this paper were made on sensitized dogs under thiopentobarbital sodium anesthesia.(1) Adult dogs were sensitized by horse serum. At subsequent intervals after the last sensitization, anaphylaxis were provoked by a intravenous injection of horse serum.(2) Hepatic blood flow were determined by the modified Frank's method. A catheter was introduced from the right creasing magnitude. jugular vein into the hepatic vein and advanced as far as possible. Blood flow from the catheter, which is proved to be proportional to total hepatic flow, was measured directly and then the sampling blood was returned through the left jugular vein. Small amounts of heparin were used to prevent blood clotting in the catheter. (3) Cardiac output was determined by the method of STEWERT-HAMILTON using radio-iodinated serum albumin (RISA). RISA was rapidly introduced into the left ventricle or ascending aorta through a catheter advanced from the carotid artery. Counting of radio-activity changes in blood, which was sampled from the femoral artery, were made by using the Well type scintillation counter. (4) Mean artery pressure was measured by means of a strain gauge manometer connected with a tube in the femoral artery. Portal vein pressure was obtained by a catheter advanced into the vessel from a splenic branch vein and by attaching it to a water manometer. Inferior vena cava pressure was measured at the level of the vena caval ostia of the hepatic vein. (5) Total peripheral resistance was estimated by dividing mean artery pressure by cardiac out put. Splanchnic resistance was also calculated by the same principle.

Changes in the Blood Pressure and Plasma Proteins during Serum Anaphylactic Shock

Purpose Recently there is an increasing incidence of penicillin shock. In this department a systematic research has been conducted for the last several years on the problem of the shock on the basis of a hypothesis that a shock due to penicillin must be something analogus to a serum anaphylactic shock encountered in animals. The problem assigned to this author in this systematic research was about the change in the blood circulation and plasma proteins during a serum anaphylactic shock in animals. Method s Materials used were about 50 dogs weighting 10-20kg. For the sensitization they were injected with horse serum in dose of 0.2 cc/kg subcutaneously on every other day, this injection alternating with the intravenous injection of horse serum in dose of 1.0cc/kg on every other day, and for the total period of 10 consecutive days. After the completion of the sensitization, 3 weeks were allowed, and, after this latent period, animals were thrown into anaphylactic shock by the rapid intravenous injection of the horse serum in dose of 2.5-5 cc/kg. The femoral arterial blood pressure was transmitted through a vinyl tubing to a mercury manometer and recorded on kymographion. Plasma proteins concentration was measured by a refracto- meter of Hitachi type and the protein fraction pattern was examined by the paper-electro-phoresis. Where the anti-shock agents were used, animals were treated as follows : in the chlorpromazine group, they were injected with chlorpromazine in dose of 2 mg/kg intramuscularly 40 minutes before the provocation of a shock ; in adrenalin group, animals were injected with adrenalin in dose of 2-3mg together with the physiological saline solution by the drip infusion after a shock has been provoked ; in the prednisone group this drug was injected intramuscularly in dose of 3mg/ kg in 3 divided doses, i.e., 24 hours, 8 hours, and immediately before the provocation of shock.

Studies on the Histamine-Content of the Blood and Tissues of the Rabbit During Anaphylactic Shock

Summary During acute anaphylactic shock in the rabbit, there occurs a marked decrease in the blood-histamine, a lesser decrease in the plasma-histamine and a leucopenia. There does not appear to be any correlation between these changes and the symptoms of anaphylaxis. There is a decrease in the histamine-content of the lung and spleen of the rabbit following the production of anaphylactic shock. In non-sensitized rabbits, there is little or no change in the histamine-content of the blood following the intravenous injection of horse serum. A leucopenia may occur. When egg albumin is injected intravenously, however, a decrease in both the blood-histamine and the white blood count occurs. Histamine-shock in the rabbit is characterized by a moderate decrease in the histamine-content of the blood and a marked increase in the histamine-content of the plasma. A moderate leucopenia also occurs. These findings then differ from those which occur in anaphylactic shock in this species. By superimposing anaphylaxis on histamine-shock in the rabbit, it may be shown that not only is the histamine-content of the blood diminished, but that there is also some mechanism for the removal of large amounts of histamine from the plasma. It is also shown that a sudden increase in the plasma-histamine may occur under these circumstances.

Vitamin C and Anaphylactic Shock in Dogs

Contradictory reports upon the effect of Vitamin C on the anaphylactic reaction of guinea pigs have appeared. Hochwald, Lemke, and Solomonica have reported protective effects, while Schwartz and Cisloghi and Van Niekerk have reported negative effects. Giroud and Giroud have reported protective effects in rabbits. To our knowledge no study of the effect of administered Vitamin C on the anaphylactic reaction in the dog has been reported. Eighteen dogs were sensitized to horse-serum. After incubationary periods of 15 to 20 days they were anesthetized and the blood-pressure reactions recorded to provide an objective record of the degree of shock produced by the intravenous injection of horse-serum. Four dogs were given 250 mg of cevitamic acid daily by mouth throughout the incubationary period, 5 dogs were given intravenous injections of 200 mg prior to the sensitization, a daily oral dose of 500 mg during the incubationary period and a final intravenous injection of 500 mg before the provocative injection of horse-serum, and 9 dogs were given intravenous injections of 100 mg per kilo during the last 45 minutes before the provocative injection of horse-serum. Sixteen animals showed anaphylactic reactions, of which 4 were rapidly fatal and 9 were quite severe. The distribution of the various grades of severity of shock were similar with that in a large number of controls. Through the courtesy of Professor C. J. Farmer, the concentration of Vitamin C in the blood was determined in some of the animals. We are thus, enabled to say that the blood concentration was elevated from normal levels of approximately 01.20 mg % to around 0.6 mg,% in the first group, that in the second group the blood concentration was moderately high at the time of sensitization (2.0 to 4.0 mg %), was maintained at fairly high levels during the incubationary period (around 1.0 mg %) and was considerably elevated (11.0 to 20.0 mg %) at the time of the provocative injection of horse-serum, and that in the third group the Mood concentration was above 20 mg % at the time of the provocative injection of horse-serum.

PHENOMENON OF LOCAL SKIN REACTIVITY TO BACTERIAL FILTRATES: EFFECT OF BACTERIAL FILTRATES INJECTED INTRAVASCULARLY UPON REACTIONS TO ANTIGEN + ANTIBODY COMPLEXES

Rabbits were sensitized by a single intravenous injection of horse serum 6 days prior to the experiments. In these rabbits there appeared hemorrhagic and necrotic reactions in sites of intradermal tests with horse serum when the tests were preceded by 1 hour, or followed 18 to 24 hours later by an intravenous injection of potent bacterial filtrates. The skin-preparatory and reacting potencies of the filtrates were titrated by means of the phenomenon of local skin reactivity to bacterial filtrate. The experiments demonstrated that the ability of bacterial filtrates to elicit reactions in the antigen injected sites in sensitized animals is strictly conditioned by their skin-preparatory potency and seems to be quite independent of their provocative potency. Combinations of animal protein antigens with homologous antisera, which are endowed with provocative potency but are totally devoid of skin-preparatory potency, fail to elicit reactions in antigen-injected sites. The various methods of elicitation of the phenomenon of local tissue reactivity are summarized and discussed in the text.

PRECIPITIN RESPONSE IN THE BLOOD OF RABBITS FOLLOWING SUBARACHNOID INJECTIONS OF HORSE SERUM

1. Rabbits which have received a single dose of normal horse serum in the subarachnoid space produce precipitins in the blood in greater abundance, of higher titer, and persisting longer than those in control rabbits which have received a similar injection intravenously. 2. Repeated subarachnoid injections of normal horse serum in rabbits induce precipitins in the blood early. These may appear in high titer as soon as 1 week after the initial injection, whereas in rabbits similarly treated intravenously no precipitins are found at this time. They may appear a few days afterward and reach a high titer. 3. No anaphylactic manifestations occurred in rabbits treated repeatedly with subarachnoid injections of normal horse serum when the precipitin content of the blood was high. 4. Anaphylactins, as determined by passive transfer of anaphylaxis, were demonstrated in sera with high precipitin content. 5. These experiments may explain clinical evidences of anaphylaxis, observed when an initial intravenous injection of horse serum followed a series of intraspinal injections of such serum.

The Vasomotor Depression in Canine Anaphylaxis

Abstract In an earlier paper (1) it was shown that the coagulability of the blood in anaphylactic dogs is due directly to a reaction on the part of the cells of the liver. At the same time the suggestion was offered that the vasomotor depression night be due to a local congestive reaction of the liver, leading to a depletion of the general circulation, and in consequence, to a marked fall in blood pressure. This view was based on the observation that in very acute shock in dogs the liver alone of the abdominal organs presents an intense grade of congestion. The following experiments are in support of that suggestion. A dog is sensitized actively by means of the intravenous injection of horse serum. After an interval of three weeks the animal is etherized and the abdomen is opened by a median epigastric incision.

THE EFFECTS OF SERUM TREATED WITH PARARABIN

Horse serum, kept for 2 hours at 38 degrees C. in the presence of one-fifth its volume of a suspension of 0.5 per cent of pararabin inphysiological salt solution and then freed by means of centrifugalization and filtration from the pararabin produced when injected in appropriate doses into a normal rabbit, a considerable and prolonged fall of the blood pressure, a distinct retardation in the coagulation of the carotid blood, and sometimes, in addition, acceleration of the respiratory rate and the expulsion of numerous scybala; that is, the various symptoms observed to occur after the intravenous injection of horse serum in sero-anaphylactized rabbits. If the horse serum is first heated for 30 minutes to 56 degrees C. and then treated with pararabin in the manner described above, the intravenous injection of this serum into a normal rabbit exerts no more action on the arterial pressure, the coagulability of the arterial blood, the respiratory rate, and the intestinal activity, than does normal horse serum when introduced into the vein of a normal rabbit.

THE EFFECTS OF SERUM TREATED WITH AGAR

The intravenous injection of horse serum, kept for 2 hours at 38 degrees C. in the presence of one-fifth of its volume of a suspension of 0.5 per cent agar in physiological salt solution and then separated from the agar by centrifugalization and filtration, produces in normal rabbits in adequate doses a considerable and prolonged fall in the blood pressure, expulsion of feces, a diminished coagulability in the carotid blood, and at times accelerated respiration; that is, the various symptoms observed after the intravenous injection of horse serum in a seroanaphylactized rabbit. Horse serum previously kept for 30 minutes at 56 degrees C. and then treated with agar in the manner described above, when injected intravenously into a normal rabbit will have no more effect on the arterial pressure, on the intestinal movement, on the respiratory rate, or the coagulation of arterial blood than the introduction of horse serum into the veins of a normal rabbit.

Anaphylatoxin and the mechanism of anaphylaxis

Precipitin is identical with the antibody effective in passive sensitization. This is demonstrated by injecting a guinea-pig with the precipitate formed by a mixture of horse serum with the serum of a rabbit immunized thereto. This guinea-pig, if tested after an interval of three days by the intravenous injection of horse serum, presents a violent, at times a fatal anaphylactic response.If the precipitating antibody is heated at 72° for one half hour, it loses its precipitating functions, but retains its sensitizing power, though somewhat diminished. Such heated precipitin, sometimes described as “precipitinoid,” is known to retain its combining power with antigen. One may conclude, therefore, that the combining power, but not the precipitating power, of antibody is essential in anaphylaxis.Precipitating antibody heated at 72° for one half hour has lost its capacity to bind complement in the presence of antigen. However, it still retains its sensitizing value. These facts are illustrated by the following ...

The cellular interpretation of anaphylaxis and immunity

It has been shown in previous communications that the serological evidence is not in harmony with the current view that anaphylaxis is due to a reaction between the antibody present in the blood and the introduced antigen. This contention has now been confirmed with the help of the graphic method introduced by Dale, by means of the following experiment. A guinea-pig which is passively sensitized by the injection of 0.3 cubic centimeters of the serum of a rabbit immunized against horse serum, may be killed on the following day by an intravenous injection of horse serum. The uterine preparation also, on the second day, responds typically to the administration of horse serum. If, however, the antigen is applied immediately after sensitization, no reaction occurs, either in vivo or in vitro. The same failure to react follows the injection of relatively enormous amounts of the immune rabbit's serum, as for example three cubic centimeters. Hence, it is apparent that the presence of immune bodies in the circulating blood does not suffice to make a guinea-pig hypersensitive, but that these antibodies must first be bound by the body cells. In the present paper I shall, furthermore, make a preliminary report upon a new method of studying the mechanism of anaphylaxis and immunity. Hitherto it has been customary to study these phenomena by means of the reaction to antigen, induced through the presence of antibody in the organism. The object sought by the method herein described is to permit of the identification of the antigen, as well as of the antibody, in the sensitized or immunized animal. The essential feature of this method consists in the use of an immune serum as antigen. To illustrate: if a guinea-pig be sensitized by means of large doses of the serum of a rabbit immunized against horse serum, it has been shown that the passive sensitization to horse serum persists over a period of two weeks.

DISTURBANCES OF THE HEART-BEAT IN THE DOG CAUSED BY SERUM ANAPHYLAXIS

Anaphylactic shock in the dog, caused by the intravenous injection of horse serum into sensitized animals, may produce definite cardiac disturbances which are revealed by the electrocardiogram. These cardiac changes consist of disturbances in conduction of the heart impulses, abnormalities in the ventricular contractions, and other unusual disturbances of the mechanism of the heart-beat. They come on very quickly after the injection of serum and may be of short duration, and are not obtained during anti-anaphylaxis. They are not the result of the marked fall in blood pressure which occurs, nor does the central cardiac inhibitory mechanism play a part in their production. These cardiac disturbances apparently are a definite primary expression of anaphylactic shock in the dog. The right ventricle seems to be more affected than the left.