Animal model of conjunctival primary acquired melanosis.

A condition clinically identical to human conjunctival primary acquired melanosis (PAM) was induced in 16 of 20 Dutch (pigmented) rabbits after weekly topical 60-microliters applications of a 1% solution of 7,12-dimethylbenz[a]anthracene (DMBA) in acetone. Pigment stippling appeared in the conjunctiva as early as 5 weeks after the initial carcinogen application. Confluent patches of flat pigmentation appeared over the palpebral conjunctiva 18 weeks after the onset of treatment and showed progressive lateral enlargement and darkening. Histologically, a spectrum of changes from increased melanin production and melanocytic hyperplasia without atypia (resembling the human condition of PAM without atypia) through atypical melanocytic hyperplasia (resembling human PAM with atypia) was identified. The development of this model permits further investigations to explore and explain the clinically observed phenomenon of waxing and waning of PAM and its promotion to conjunctival malignant melanoma.

Pericyte response during choriocapillaris atrophy in the rabbit.

The rabbit and rat choriocapillaris atrophies in response to experimental destruction of the retinal pigment epithelium by intravenous injection of sodium iodate. This provides a convenient model of capillary atrophy. We have observed that pericytes are spared during this process; the atrophy is due to loss of endothelium only. Extensive examination of thin sections obtained 1 day to 11 weeks after administration of iodate showed that pericytes retained their normal relationship to the remnant capillary basement membrane left behind as the endothelial tube atrophied. This was most conspicuously manifested in their retention of processes longitudinally disposed along the sleeves of remnant basement membrane. The processes retained bundles of actin filaments that had dense regions along them and inserted into subplasmalemmal densities at basement membrane attachment sites, i.e. they had the characteristics of stress fibers. The pericytes did not phagocytose the debris of endothelial necrosis, in spite of their known phagocytic abilities. Necrotic endothelial cells were eliminated by sloughing into the capillary lumen. The observations support the idea that the function of pericytes in the choriocapillaris, the major source of nutrition for the retinal photoreceptors, resides in their contractility, and that pericytes do not remove necrotic endothelium during capillary atrophy.

Transition from normal to hypersecretory bronchial mucus in a canine model of bronchitis: changes in yield and composition.

Density-gradient analysis was used to follow the transition from normal to hypersecretory bronchial mucus in a model of bronchitis induced in dogs by chronic exposure to SO2 gas. Aspirates of saline bronchial lavage were obtained by fiberoptic bronchoscopy from dogs before, during a 6- to 9-month exposure period to SO2 gas, and during a recovery period of similar duration. Prior to SO2 exposure, aspirates from all animals had a low yield of nondialyzable macromolecules (15 +/- 6 mg/aspirate) and similar composition. Specifically, epithelial glycoprotein of typical buoyant density was not detected; rather a glycoconjugate of higher buoyant density with features of both proteoglycan and glycoprotein was identified. Neutral lipids were predominant with lesser amounts of phospholipids; no glycolipids were detected. During the SO2 exposure period, aspirates from five of the eight dogs contained components similar in buoyant density to human bronchitic glycoprotein. Glycoprotein isolated from the canine aspirates was similar to glycoprotein isolated from human chronic bronchitic sputum, having the same carbohydrate composition and range of oligosaccharide size. Further, during and after SO2 exposure some aspirates contained appreciable amounts of glycolipids. These data demonstrate substantial similarities in composition between normal human and canine mucus and in mucus isolated from dogs with chronic airway inflammation induced by repeated irritant exposure and from human patients with chronic bronchitis.

Hyperthyroxinemic mice have reduced natural killer cell activity. Evidence for a defective trigger mechanism.

Natural killer (NK) activity of peripheral blood lymphocytes from hyperthyroxinemic patients (Graves' disease or thyroxine (T4)-treated) is severely depressed. In order to study the relationship of thyroid hormone to NK activity, a model for hyperthyroxinemia was induced in mice by addition of T4 to the drinking water. Control mice were hypothyroid (fed propylthiouracil) or normal. Serum T4 levels were elevated (within 2 wk) in mice fed thyroid hormone. Six weeks after initiation of the diets, in vitro NK activity was undetectable in the peripheral blood, spleen, or lung mononuclear cell populations harvested from hyperthyroxinemic mice. Control mice had NK activity within the normal range. Spleen cells from mice fed thyroid hormone and control mice were tested for their ability to release lytic factors (natural killer cytotoxic factors). Lymphoid cells were incubated for 20 hr with unlabeled Yac-1 cells. Supernatants were tested for their capacity to lyse 51Cr-labeled Yac-1 cells in a 20-hr chromium release assay. Unlike controls, supernatants from hyperthyroxinemic spleen cells incubated with Yac-1 targets were unable to lyse 51Cr-Yac-1 cells. The NK cells from the mice fed T4 synthesized lytic factors because nonspecific stimuli, such as 12-O-tetradecanoyl phorbol-13-acetate and the calcium ionophore A23187, induced release of lytic factors capable of lysing Yac-1 targets into the media. These data support the hypothesis that excess thyroid hormone interferes with the triggering mechanism used by NK targets to cause release of lytic molecules from NK cells.

Chronic relapsing necrotizing encephalomyelitis produced by myelin basic protein in mice.

Chronic relapsing experimental autoimmune encephalomyelitis is commonly seen in a number of species after a single injection of whole white matter in adjuvant but not after inoculation with myelin basic protein, the major encephalitogen of central myelin. In the present report on large groups of SJL mice, we describe a form of chronic relapsing experimental autoimmune encephalomyelitis with destructive lesions after a single inoculation of myelin basic protein in complete Freund's adjuvant. This condition was studied for up to 19 months postinoculation and was characterized by a relapsing-remitting or a chronic progressive course, usually with a prolonged latent period. Higher doses of 400 and 800 micrograms of myelin basic protein were more effective in inducing this condition than were lower doses of 100 and 200 micrograms. Large lesions were apparent in the white matter. These comprised widespread destruction and Wallerian degeneration with some demyelination towards the margins. Demyelination was an initial, albeit transient, event which was subsequently masked by nerve fiber destruction. Polymorphonuclear leukocytes were early and prominent components of the inflammatory infiltrate and together with macrophages appeared to be involved in the lysis of myelin and axons. Thus, despite the clinical similarities, these features contrast the model with the more purely demyelinative lesions of chronic relapsing experimental autoimmune encephalomyelitis in other species and multiple sclerosis in man.

Mandibulofacial dysostosis (Treacher Collins syndrome): a new proposal for its pathogenesis.

Acute exposure to 400 mg/kg 13-cis retinoic acid (13-cis RA, isotretinoin, Accutane) on the ninth day postfertilization in mice (a time that corresponds to the fourth week postfertilization in humans) results in malformations that characterize mandibulofacial dysostosis (MFD, Treacher Collins syndrome). Deficiencies in the infraorbital region and in the mandibular ramus and condyle, abnormalities of the secondary palate, and external ear malformations were observed. Light and scanning electron microscopic analyses of affected embryos illustrate that within 12 hours of maternal 13-cis RA treatment, markedly excessive (possibly premature) cell death occurs in regions where some of the cells are normally destined to undergo programmed cell death. Previous studies with retinoids have shown that they labilize lysosomal membranes and expand and strengthen regions of programmed cell death. Of particular interest for this study was cell death occurring in the dorsal (proximal) aspects of the maxillary and mandibular prominences of the first visceral arch, the second visceral arch, and the first visceral cleft, areas that correspond to the locations of the first and second arch ectodermal ("ganglionic") placodes and first closing membrane, respectively. The derivatives of this region are those that are severely affected in MFD. As described in previous reports from this laboratory, 13-cis RA is known to interfere with neural crest cells, resulting in major craniofacial malformations. However, the exposure times involved were earlier than those described herein. It is hypothesized that effects on the first and second arch ectodermal placodal cells at a time following the release from the neural folds of neural crest cells into the developing cranial region are of great significance in the pathogenesis of MFD. This is in contrast to the prevailing hypothesis that these malformations are the direct result of a primary interference with neural crest cells.

Hemosiderosis in rodents and the effect of acetohydroxamic acid on urinary iron excretion.

Previously reported animal models of hemosiderosis fall short of simulating the human disease state of transfusion-induced hemosiderosis. An explanation for this was found by reexamining heat-treated red blood cell (rbc) loading in the mouse. After 18 intraperitoneal transfusions, each equal to two-thirds of mouse rbc volume, liver Fe reaches a level of 0.3% Fe (dry weight), which is far below the 2%-8% found in heavily transfused patients. Using the easily synthesized chelate compound ferric acetohydroxamate, Fe(AHA)3, liver Fe levels in the rat of up to 2% were achieved after 38 intraperitoneal injections. Fe was distributed in both the reticuloendothelial (RE) system and parenchymal cells, as ascertained by light microscopy. No definite histological or biochemical abnormalities could be demonstrated in loaded rats. Cardiac Fe was approximately doubled. Thus, chelate loading, while producing Fe liver levels similar to those of humans with hemosiderosis, may still be of limited usefulness in studying long-term sequelae. On the other hand, this model can be used in determining responses to chelating agents. To explore this, Fe stores were first labeled by giving 59Fe as 59Fe(AHA)3 prior to loading. In animals loaded to 0.7% liver Fe (calculated), desferroxamine, at a dose of 400 mg/kg, induced a 20-fold rise in urinary Fe. This was duplicated by AHA at a dose of 800-1600 mg/kg. It is concluded that Fe(AHA)3-loaded rats are a potentially useful model of hemosiderosis and that further studies are needed to determine whether AHA can be effective in the treatment of transfusion-induced hemosiderosis.

Hemiparkinsonism in monkeys after unilateral internal carotid artery infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Infusion of MPTP (0.2-0.8 mg/kg) into the right internal carotid artery of monkeys produces toxin-induced injury to the right nigrostriatal pathway with sparing of other dopaminergic neurones on the infused side and with negligible or little injury to the opposite, untreated side. There are contralateral limb dystonic postures, rigidity, and bradykinesia, but the animals are able to eat and maintain health without drug treatment. Spontaneous motor activity is attended by circling towards the injured side, whereas treatment with L-DOPA/-carbidopa or apomorphine stimulates circling towards the intact side. Dopamine and dopamine metabolite levels are normal in the left caudate and putamen, but markedly depressed on the right (MPTP-treated) side. This animal hemiparkinsonian model will be useful in studies of volitional movement control, drug treatments of Parkinson's disease, and functional efficacy of brain tissue implants.

An endocrine experimental model for myofibrillar disarray as found in hypertrophic cardiomyopathy.

The various etiologic suggestions for hypertrophic cardiomyopathy in man have been reviewed and experimental studies for endogenous pathways have been investigated experimentally. Intramuscular administration of triac to adult rats resulted in severe myocardial hypertrophy but no disarray. When studying the effect of triac on the myocardium of developing rats profound changes of disarray as well as hypertrophy were produced, mimicking the ultrastructural changes of hypertrophic cardiomyopathy in man. By using a variety of compounds with beta-adrenergic blocking action or predominantly membrane stabilizing properties or agonist action together with triac, the site where triac exerts its effect has been shown to be the cell membranes. A mechanism for production of cellular disarray has been delineated. Extrapolating to man, these experiments lend support to the suggestion that an endogenous hormonal mechanism may be operative in some patients with hypertrophic cardiomyopathy.

High steady-state levels of uric acid produced in rats by dietary training and potassium oxonate.

To reduce the inherent variability in serum uric acid levels of animals allowed ad libitum exposure to food containing potassium oxonate and uric acid, male Sprague-Dawley rats were trained to eat their daily food allotment in a 1.25-hr period each morning. After training the rats were fed a food mixture containing 5% potassium oxonate and 2% uric acid (w/w each). Serum blood levels of uric acid reached a steady state within 2 hr; these levels were maintained for an additional 4 hr. It is believed that the stomach emptying rate is a zero-order process under these experimental conditions.

Lymphocytosis induced by polyanions in rats.

More than twenty different polymers, mostly polyanions, were tested in rats for their ability to mobilize lymphocytes into the peripheral blood within 2--3 h. The various polyanions differed in basic structure, in side-chain (size and type), in molecular weight and configuration and in amount and type of anionic charge along the molecule. Neutral polymers and a polycation were also tested for comparison. Some of the polyanions were found to be very effective, others less so and some completely ineffective. Some were also toxic. The basic polymer to which the others were compared was polymethacrylic acid (PMAA), an already recognized mobilizing agent. The best agents were the heparinoids, sulphated polyanions, and the best of these, causing a 3--4-fold increase, were dextran sulphate and polyvinyl sulphuric acid (PVSA). Heparin, although the strongest anticoagulant, was the weakest mobilizer. Some factors that appear capable of modifying mobilization to varying degrees were molecular weight, size and configuration, sulphate content and mode of administration. In the case of PVSA, the smaller molecular weight substance gave a more prolonged lymphocytosis in blood. The high molecular weight substance gave a peak after 2 h, slightly earlier than with PMAA (2--3 h). The administration of protamine chloride to the rat (i.v.) caused an immediate reversal of mobilization, following a course to control values which was essentially identical to the normal decline, only earlier. Dextran sulphate of low molecular weight seems to be the polyanion of choice in subsequent mobilization experiments dealing with determination of the specific mononuclear cell type being mobilized. The single factor that all mobilizing polyanions have in common is a negative molecular charge. It is not yet known exactly how this charge induces the mobilization of mononuclear cells, nor what causes the variability in effectiveness among polyanions.

[Experimental studies on disseminated intravascular coagulation (author's transl)]. / Tierexperimentelle Untersuchungen zum Problem der disseminierten intravaskulären Gerinnung

Generalized disseminated intravascular coagulation (DIC) was produced in rats by thrombin infusion (550 NIH U thrombin/kg/h). Changes in the clotting system and morphological alterations were studied with consideration to early lesions. Furthermore, the influence of the antifibrinolytic agent Pamba and the fibrinolytic enzyme ocrase on DIC are studied. Microthrombi and degenerative alterations are increased in parenchymatous organs after thrombin infusion with Pamba is applied, they are decreased when ocrase is given. Alterations are most prominent in the lungs revealing the appearance of a "shock lung". Semiquantitative morphological examinations of lung, liver, kidney and adrenal glands were performed by counting microthrombi. There are marked differences between the test groups.

Comparison of human polycystic and medullary cystic kidney disease with diphenylamine-induced cystic disease.

Because of difficulty in obtaining cystic human kidneys for functional and morphologic study, animal models are receiving increasing attention. Most prominent among them is the renal cystic disease that is induced in rats through feeding of the antioxidant, diphenylamine, The present study examined the morphology of adult polycystic and medullary cystic kidney disease in man using scanning electron microscopy and compared them with diphenylamine-induced cystic kidney disease in rats. Diphenylamine nephropathy was induced by feeding rats 1 per cent diphenylamine for 12 to 18 months. All types of cystic disease showed changes in the renal corpuscles, including dilation of Bowman's space, podocyte fusion and degeneration, and basement membrane thickening. Cysts were noted along the entire nephron in polycystic and diphenylamine-induced cystic disease but only along the collecting ducts in medullary cystic disease. Cysts in polycystic disease were large and lined by flattened epithelium. Collecting duct cysts in diphenylamine cystic disease were lined by cells of irregular size and shape suggestive of cell hypertrophy and/or hyperplasia, whereas cysts of medullary cystic disease were lined by flattened epithelium except where the nephron entered or left the cyst. Cast material was generally found in the cysts of diphenylamine-induced and polycystic kidney disease but not medullary cystic disease. Atrophic glomeruli and tubules were found in all three diseases. Complete tubular obstruction was found in none; however, larger cysts frequently impinged on adjacent tubules and narrowed their lumina. The results of this study show that, in the terminal stages, cellular as well as gross morphologic differences exist between polycystic and medullary cystic kidney disease and that diphenylamine-induced cystic disease more closely resembles human polycystic than medullary cystic kidney disease.