Arthur Saltzman

Regional suppression, therapy after onset and prevention of relapses in experimental allergic encephalomyelitis by mitoxantrone ☆

Mitoxantrone, a new antineoplastic anthracenedione drug, suppressed the development of experimental allergic encephalomyelitis (EAE) in rats when administered during the incubation period. One dose of mitoxantrone had a favorable effect on EAE even when withheld until after the onset of clinical signs. In a model of relapsing EAE, a single dose during a remission prevented the expected relapse. Mitoxantrone had a particularly favorable effect when administered at the site of inoculation of the encephalitogenic antigen and adjuvant, indicating the importance of its interaction with the regional draining lymph nodes. These results support the possible clinical use of mitoxantrone for immunosuppression.

Inhibition of experimental allergic encephalomyelites by lithium chloride: specific effect or nonspecific stress?

Intraperitoneal injections of lithium chloride inhibited development of experimental allergic encephalomyelitis (EAE) in rats. The degree of inhibition was inversely related to the intensity of the immunologic stimulation, which depended on the choice of adjuvant. High dosage of lithium, the intraperitoneal route, and an intermittent schedule of administration were required to achieve suppression of EAE. This suggested that the immunosuppression was a toxic effect, mediated, at least in part, through the hypothalamic-pituitary-adrenal cortex axis. This hypothesis was supported by evidence that the lithium treatment schedule, especially the intermittent schedule, had caused adrenal cortical activation with thymolysis in intact rats and that it was lethal to adrenalectomized rats. Lithium also inhibited EAE produced by adoptive immunization, thus implicating the efferent arm of the immune response. A single dose of lithium was effective in this form of EAE, even in adrenalectomized rats. Therefore lithium affects EAE by both specific immunosuppressive and nonspecific adrenocortical-dependent mechanisms.

ARE UREA AND CREATININE UREMIC TOXINS IN THE RAT

Urea and creatinine are not generally considered to be important uremic toxins despite evidence from dialysis experiments to the contrary, and despite striking elevations of these nitrogenous waste products in uremia. In order to study this problem in acute uremia, we used a new dietary method for prolonging the survival of bilaterally nephrectomized rats. Urea or creatinine were injected on three successive days starting one day after the inception of uremia. Urea or creatinine injections shortened the survival time of acutely uremic rats, and increased the involution of thymus and spleen. The extra urea, but not creatinine, increased the serum osmolality. These data indicate that urea and creatinine are toxic in the acutely uremic rat. Hypertonicity of the serum may contribute to the toxicity of urea. Additional mechanisms of toxicity and additional toxins are not excluded.

Carbohydrate Diet Prolongs Survival of Rats with Acute Uremia after Bilateral Nephrectomy

Seymour Levine, MD, Nathan S. Kline Institute for Psychiatric Research, 140 Old Orangeburg Road, Orangeburg, NY 10962 (USA) 3.8 + 0.8(4) 4.8 ± 0.4 (4) 8.4 ± 0.7(12) 8.2 ± 0.6 (32) 2.9 ± 0.8(8) 3.9 ± 0.8(12) 5.5 ± 1.2 (16) 6.4 ± 0.8(28) Average number of days after the second nephrectomy ± standard deviation (number of rats in parentheses). Special diets started when the first nephrectomy was done (7 days before second nephrectomy). Antibiotics in drinking water started 3 days before the second nephrectomy. Dear Sir, Removal of both kidneys is used to elucidate renal functions, whether excretory, metabolic or endocrine. Bilateral nephrectomy also provides a model for the study of uremic toxicity [1], dialysis [2] and pharmacokinet-ics without the influence of renal excretion. However, the brief survival of anephric rats, usually 2 or 3 days, limits its usefulness [3-15]. In this communication we report prolongation of survival to 6-9 days. The survival of bilaterally nephrecto-mized rats has been prolonged by dietary means in the past. Pretreatment with potassium-deficient diets retarded the development of lethal hyperkalemia [5, 11,13]. Also, low-protein and/or high-carbohydrate diets reduced the elevation of blood ammonia seen in uremia by reducing the source of exogenous and endogenous nitrogenous precursors. We have carried the dietary approach to its limit by feeding a protein-free, potassium-free, exclusively carbohydrate diet, namely, pure sucrose. Not only was this diet more effective for prolonging survival, but sucrose cubes are readily available, cheaper than specialized commercial diets, highly palatable and useable in conventional pellet feeders. Female Sprague-Dawley rats, 100-200 g in weight, or male Lewis rats, weighing 200-300 g, were anesthetized with ketamine hy-drochloride 50 mg/kg and acepromazine ma-leate 5 mg/kg intramuscularly. The right kidney was removed through incisions in the midline dorsal skin and the right costoverte-bral angle muscle. At the same time, the Purina Formulab 5008 Chow was replaced by a potassium-deficient diet (purified rodent diet AIN-93G, modified by omission of Rodent chow Potassium-deficient diet Sucrose Sucrose + oil potassium salts from the mineral mix [16], potassium content 29 mg/kg, from Dyets Inc., Bethlehem, Pa., USA, No. 113510), by sucrose cubes (Domino Dots, Domino Sugar Corp., New York, N.Y., USA) or oil-treated cubes as specified in table 1. Seven days later, the left kidney was removed. No change was made in the diet, and tap water was freely available at

Feeding sugar overnight maintains metabolic homeostasis in rats and is preferable to overnight starvation

Rats are often starved overnight for many different reasons. Overnight starvation causes loss of body and liver weights, depletion of liver glycogen, decrease of blood glucose and loss of amino acids because of gluconeogenesis. Providing pure sucrose cubes as the sole overnight nutrient is a simple, inexpensive way to empty the gastrointestinal (GI) tract, while minimizing liver changes and preventing decrease of blood glucose and loss of amino acids. Adding sugars to the overnight drinking water as the sole nutrient has the same beneficial effects, provided the type of sugar and its concentration allow for sufficient intake and provided hyponatremia is avoided. Feeding sucrose cubes or sugar solutions will empty the gastrointestinal tract as effectively as starvation. In all instances, simple precautions against coprophagy and pica should be taken in order to secure optimal benefit.

Effects of coprophagy on serum urea and the weight of the gastrointestinal tract of fed or fasted rats

Coprophagy can be minimized by fitting rat cages with metal grids which allow faecal pellets to pass through to the floor of the cage. When bedding was omitted overnight, the extent of coprophagy could be estimated from the weight of the droppings on the cage floor or the weight of the gastrointestinal (GI) tract removed from rats housed with or without grids. The effect of coprophagy was also demonstrated by the elevation of serum urea nitrogen in rats that consumed faeces. Therefore, precautions against coprophagy, or their absence, should be specified in all experimental protocols and reports.

Parenteral Aluminum Compounds Produce a Local Toxic Myopathy in Rats: Importance of the Anion

Aluminum lactate, injected in rats, produced skeletal muscle necrosis of diaphragm and abdominal wall subjacent to peritoneal surfaces. Deeper muscle cells (distal from inoculum) were less severely affected. Ultrastructural studies of diaphragm revealed inoculum coating collagen fibrils, aggregating next to muscle basal lamina and localized within phagocytes. Aluminum lactate penetrated lymphatic vessels and caused reactive changes on the pleural as well as peritoneal surfaces of diaphragm. In contrast, injection of aluminum citrate did not produce myopathy. Also, mixtures of aluminum lactate with aluminum citrate, sodium citrate, or another chelating agent failed to produce myopathy. Therefore, the regional myopathy produced by the lactate salt provides a model for in vivo cytotoxicity of aluminum in which anionic binding is a critical determinant.

Acute uremia produced in rats by nephrotoxic chemicals is alleviated by protein deficient diet.

Rats injected with mercuric chloride develop an acute renal tubular necrosis with uremia, which is frequently lethal. Pretreatment for 3 or 7 days with a protein-free diet reduces the mortality, the clinical signs (tremor), and the severity of renal tubular necrosis, and ameliorates the uremic chemical findings in the serum. Similar results followed injection of a nephrotoxic amino acid, d-serine, after pretreatment with a protein-free diet. Indirect evidence suggests that induction of metallothionein may be involved, at least in the experiments with mercury. Acute uremia produced by nephrotoxic chemicals may be useful for further studies of the role of nutrition in uremia, while avoiding the surgical procedures and prolonged observations required for the “remnant kidney” models.

Distribution of Small Intestinal Lesions in Anaphylaxis of Rats

Hemorrhage and congestion were not uniformly distributed along the small intestine during systemic anaphylaxis in rats. Duodenum had little hemorrhage and congestion and the terminal ileum had even less. Maximum involvement occurred in jejunum of 13% of rats, in ileum of 47%, and in both jejunum and ileum of 40%. Sequential scoring along the entire length of small intestine revealed different patterns of distribution with more than one peak of intensity in some of the rats. Within a lesioned area, banding was common except where hemorrhage and congestion were so severe as to obliterate all patterns. Banding represented a gradient of injury with respect to the vascular supply. The pale stripes (less severe hemorrhage) contained the penetrating vessels derived from the terminal mesenteric arcades. The dark stripes (more severe hemorrhage) did not contain such vessels. In addition, the mesenteric side of the intestine was less affected than the antimesenteric side. This constituted a second gradient that might also be related to the distance from the vascular supply. In both instances, proximity to larger blood vessels had a protective effect. These two gradients and the variable sites and patterns of distribution have not been described previously in intestinal anaphylaxis. Gradients and the variability of the distribution of lesions in intestinal anaphylaxis should be considered in experiments on pathogenesis and altered function.

The Hyperacute Form of Allergic Encephalomyelitis Produced in Rats Without the Aid of Pertussis Vaccine

The hyperacute form of experimental allergic encephalomyelitis (EAE), characterized by a short incubation period, severe paralysis, high mortality, and abundant polymorphonuclear leukocytes and fibrin in the lesions, was produced in rats without the use of pertussis vaccine (previously considered an essential requirement) or Freunds adjuvant. Carbonyl iron or mineral oil without mycobacteria were effective adjuvants and whole rat spinal cord was the best antigen. Hyperacute EAE was produced in this manner in some Lewis rats, most dark agouti (DA) rats and most F1 hybrids of these two strains. Clinical signs were earlier in onset and more severe in the DA strain than in the Lewis strain in all adjuvant-antigen combinations that were tested. Dark agouti rats developed clinical signs in six days, histological lesions in five days, and localized EAE lesions could be induced in four days. The data support the hypothesis that hyperacute type lesions (neutrophils and fibrin) can be caused by an exceptionally strong immune response to neural antigen, whether that response is engendered by a particular adjuvant (pertussis vaccine) or by an unusual degree of genetic susceptibility (DA rats).