Morphologic and biochemical studies of canine mucopolysaccharidosis I.

This report presents the necropsy and biochemical findings on the first dog to die with alpha-L-iduronidase deficiency (mucopolysaccharidosis I, MPS I). Gross pathologic features, light- and electron-microscopic findings, and tissue enzyme, glycosaminoglycan (GAG), and sphingolipid levels are compared with the human disease counterpart and the previously described feline model. Results lend further support for the similarities of the canine disease and human MPS I.     

Immunohistochemical studies in acute and chronic canine chagasic cardiomyopathy

A major characteristic of Chagas' disease is a myocarditis constituted primarily of mononuclear cells, both during the acute and chronic phases of the disease. Using monoclonal antibodies and image analyses we have quantified canine CD8(+) T cells (caCD8(+) T cells), canine CD4(+) T cells (caCD4(+) T cells) and neutrophils in canine chagasic myocardiopathy induced by two strains isolated from the first human clinical case of Chagas' disease. We also evaluated the influence of tissue parasitism in the genesis of chronic myocarditis through immunohistochemistry. As in human myocarditis, there was a predominance of T lymphocytes in the inflammatory infiltrate in all animals studied. In the dogs inoculated with strain Berenice 78 (Be78) and necropsied during the acute phase of infection, we found 58% caCD8(+) and 42% caCD4(+) T cells. In chronically infected animals, 53% of T cells were represented by caCD8(+) and 47% were caCD4(+) T cells. Since normal canine lymphoid organs are constituted by 70-80% caCD4(+) T cells and 20-30% caCD8(+) T cells our results indicate a higher proliferation of caCD8(+) T cells in dogs inoculated with the Be78 strain. In chronic myocarditis induced by the Berenice 62 (Be62) strain, caCD8(+) cells constituted 33% of the T cells and 67% were caCD4(+) T cells, a proportion similar to that found in normal canine lymphoid organs. Since the Be78 strain induces greater loss of myocardiocytes than strain Be62, we believe that the caCD8(+) T cells, among other factors, can be important in the genesis of these lesions. Amastigote nests and immunohistochemically labelled Trypanosoma cruzi antigen were not found in dogs necropsied during the chronic phase. The absence of the parasite in the myocardium suggests the involvement of other mechanisms in the genesis of the inflammatory process.     

Morphologic studies in the skeletal dysplasias.

Considerable progress has been made in the delineation of the genetic skeletal dysplasias, a heterogeneous group of disorders, that consist of over 80 distinct conditions. Morphologic studies have added a further dimension to the delineation of these conditions, their diagnosis, and the investigation of their pathogenetic mechanisms. In certain diseases, the morphologic alterations are characteristic and pathognomonic. In others only nonspecific alterations are observed, whereas in still other disorders growth-plate structure is essentially normal. Histologic, histochemical, and electronmicroscopic studies of growth-plate cartilage have provided new insights into the complexity of morphogenetic events in normal growth through the demonstration of morphologic defects in the genetic disorders of skeletal growth. As yet, very little is known of the biochemical abnormalities underlying the morphologic abnormalities. However, the great variety of morphologic findings points to a number of different pathogenetic defects in the synthesis, release, and assembly of connective tissue macromolecules and in the cells involved in growth-plate metabolism.     

Virus-induced animal model of osteosarcoma in the rat: Morphologic and biochemical studies.

Osteosarcomas were produced by the intratibial inoculation of New Zealand black rats with Moloney sarcoma virus (MSV) at 1 day and 4 days of age. Radiographic evidence of osteosarcoma development was first demonstrated at 10 to 15 days postinoculation in both groups. Subsequent radiographic and light and electron microscopic evaluation of tumor-bearing rats demonstrated that osteosarcomas in rats inoculated at Day 4 of age were more osteoproliferative osteosarcomas than those in rats inoculated on Day 1. Rats inoculated at 4 days of age lived longer, had more slowly growing osteosarcomas, and developed a consistent tumor-associated cachexia compared to tumor-bearing rats inoculated at Day 1. Both groups of rats had a 93% metastasis rate involving either sublumbar lymph nodes, lungs, or both. Tumor-bearing rats inoculated at 4 days of age had consistent elevations in both urinary hydroxyproline excretion (HOP/CR) and serum alkaline phosphatase levels, and in serum calcium levels at some time points. The high tumor incidence after a short latent period and the morphologic and biochemical similarities between the MSV-induced murine osteosarcoma and the osteosarcoma in human beings makes this discrete tumor and a valuable animal model for the evaluation of new therapeutic regimens.     

Cytogenetic studies in a canine malignant melanoma.

In a 13-year-old, male rough-haired Dachshund with a malignant melanoma, cytogenetic evaluation of tumour cells showed hyperdiploidy (79 to 81 chromosomes) in 50 per cent of the metaphases. Several bi-armed chromosomes (centric fusions, one isochromosome and two unidentified markers) were observed.     

Morphologic and morphometric characteristics of hypertrophic cardiomyopathy

Seventy endomyocardial biopsies of the left and right ventricular wall taken from 23 patients during catheterization of the heart cavities have been studied. There were three groups of patients: those with hypertrophic cardiomyopathy (HCMP) (7 patients), those with dilated cardiomyopathy (11) and 5 patients with chronic myocarditis. The most characteristic features of HCMP were as follows: 1) muscle fiber hypertrophy, 2) hypertrophy of cardiomyocyte nuclei, 3) perinuclear cardiomyocyte nimbus, 4) disorganization of muscle fibers, 5) the decrease of the nucleus-cytoplasmic ration. The degree of these parameters was scored 0 to 3. Maximum total score was 15 (100%). If histological index of HCMP was higher that 60%, the diagnosis of HCMP was considered to be confirmed. The method possesses a high sensitivity (87.5%) and specificity (95%) and can be used in the diagnosis and differential diagnosis of HCMP.     

A perfused canine right bundle branch-septal model for electrophysiological studies.

As documented in the literature, isolated ventricular muscle preparations perfused with protein-free media become edematous and deteriorate. We hypothesized that the addition of colloid oncotic pressure to the perfusion medium might affect favorably the electrical characteristics and survival of an isolated right bundle branch-septal muscle preparation perfused via the anterior septal artery. Perfusion with protein-free Tyrode solution produced edema formation and a loss of transmembrane potential of Purkinje and muscle cells within 5.9 +/- 0.40 h. Addition of bovine albumin, 4 g/100 ml, to the Tyrode solution or perfusion with horse serum greatly reduced the formation of edema. Furthermore, intact electrical activity continued for more than 9 +/- 0.31 h (P less than 0.001). The preparations showed a greater diastolic potential and action potential amplitude during perfusion than during superfusion with Tyrode solution. In conclusion: 1) a viable, perfused septal muscle preparation was developed for electrophysiological studies, and 2) colloid oncotic pressure in the perfusion medium is essential to preserve the integrity of the capillary circulation and to eliminate edema formation and tissue death.     

Streptozotocin-induced pancreatic insulitis in mice. Morphologic and physiologic studies.

Pancreatic insulitis and diabetes mellitus were induced in Charles River CD-1 mice with five subdiabetogenic injections of streptozotocin. Plasma glucose and immunoreactive insulin levels were measured and animals were sacrificed at intervals for morphologic studies of pancreatic islets and measurements of extractable pancreatic immunoreactive insulin. Light microscopy revealed striking insulitis, 5 to 6 days after streptozotocin injections, with cell necrosis and eventual islet atrophy due to beta-cell necrosis, and numerous type C viruses within many of the surviving beta-cells. Light microscopic immunoperoxidase stains of islet cell hormones and electron microscopy identified relatively increased numbers of alpha- and delta-cells within the atrophic islets 6 and 12 months after streptozotocin injections. Plasma glucose, plasma immunoreactive insulin, and extractable pancreatic immunoreactive insulin measurements documented the persistence of profound hyperglycemia, as well as the reduction of plasma and pancreatic immunoreactive insulin levels. Immunofluorescence studies demonstrated the absence of circulating islet cell antibodies during both the acute and chronic stages of the syndrome. The pathogenesis of this model of insulin-deficient diabetes is believed to be a cell-mediated autoimmune reaction directed against pancreatic beta-cells altered by subdiabetogenic injections of streptozotocin. The importance of the increased number of type C viruses within surviving beta-cells remains obscure.     

Morphologic and physiologic studies of three dematiaceous pathogens.

Uncertainty in identifying a dematiaceous mold from a human bronchial washing precipitated a study of Dactylaria gallopava (Cooke) Bhatt et Kendrick and its relationship to Scolecobasidium constrictum Abbott. Morphologic and physiologic studies were conducted with representative isolates and subcultures derived from the isolates used to prepare the type specimens of these two fungi, as well as a third dark mold, Ochroconis constricta (Abbott) de Hoog et von Arx. All test isolates were morphologically similar in that two-celled, cylindrical blastoconidia with a rhexolytic mode of dehiscence were the predominant and most consistent anamorphic structures. Sympodial development of the conidiophore was too variable to distinguish the isolates. Based on their morphologic similarity, we propose a new combination within the genus Dactylaria as amended by Bhatt and Kendrick, D. constricta (Abbott) Dixon et Salkin. However, D. constricta could be differentiated into two groups on the basis of physiologic tests--a subgroup in which isolates grow at 37 and 45 degrees C, give a delayed positive gelatin reaction, and are inhibited on Mycosel agar; and a second subgroup in which isolates grow on Mycosel agar and give a rapid positive gelatin reaction, but do not grow at 37 or 45 degrees C. We recommend that D. constricta be viewed as a potential pathogen when isolated from clinical specimens.     

Cytogenetic studies in a canine mammary tumor

In a 9-year-old female dog (Basset Artesian Normand) with a mammary adenocarcinoma, cytogenetic evaluation of tumor cells showed a chromosome number of 76 in most metaphases (95%). The following abnormalities were found: symmetric metacentric chromosomes 1 and 6, centric fusion 3/38, a marker X-chromosome (Xmar) and a biarmed small marker chromosome (mar). In the remaining metaphases (5%) there were additional biarmed marker chromosomes present.     

Morphologic studies of hepatocytes entrapped in hollow fibers of a bioartificial liver

A bioartificial liver cartridge was prepared by inoculating porcine hepatocytes into the inner space of hollow fibers of a hemodialyzer. The hepatocytes formed rod shaped cell aggregates during in vitro perfusion culture within 1 day. Morphologic examination was carried out on the aggregates by optical and electron microscopy. Each hepatocyte was in direct contact with adjacent cells and a bile canaliculus-like structure was occasionally seen between hepatocytes. High magnification observation showed that the canaliculus was separated from the remainder of the intercellular space by a tight junction. These facts suggest that the hepatocytes formed functionally associated cell aggregates with a compact morphology not unlike hepatocyte spheroids. These structures were well maintained for 7 days in culture, and then the amorphous area in the aggregates and the nonviable cell number increased with lengthening culture period. The bioartificial liver maintained the ability to metabolize lidocaine, ammonia, and galactose for 7 days and then deteriorated with time.     

Development and validation of a canine T-cell-dependent antibody response model for immunotoxicity evaluation

A T-cell dependent antibody response (TDAR) model to evaluate compounds for potential immunotoxicity in dogs has not been reported. The objective of these studies was to develop and validate a dog TDAR model using the T-cell dependent antigen, keyhole limpet hemocyanin (KLH). Studies were conducted to determine the appropriate dose of KLH, immunization route and kinetics of the antibody response to KLH in the dog. To validate the sensitivity of this method, we investigated the TDAR to KLH in the dog with a known immunosuppressive drug, cyclosporine (Neoral). The results of this study demonstrate that a robust primary IgM and IgG response to KLH can be generated in dogs and the IgG response was sensitive to cyclosporine treatment.     

Morphologic studies of bladder tumors treated with hematoporphyrin derivative photochemotherapy.

The morphologic changes that occurred in transplanted rat bladder tumors after treatment with hematoporphyrin derivative (HPD) and/or phototherapy were investigated. Transitional cell bladder tumors were initiated subcutaneously in male F344 rats by injection of AY27 cells. When tumors reached 1 cm in diameter, the rats received either HPD (10 mg/kg body weight) photochemotherapy, HPD only, phototherapy only, or no treatment. Tumors were sampled immediately (0 time), 1/2, 1, 2, 4, and 24 hours after phototreatment for light and electron microscopy. Tumors receiving HPD-photochemotherapy displayed progressive injury to both tumor cells and endothelial cells. Early changes (0-2 hours) included focal tumor and endothelial cell vacuolation and swelling as well as sloughing of tumor cells into papillary spaces. Tumor cells and endothelial cells displayed vacuolization and damage to cell mitochondria immediately after phototreatment. Intercellular spaces also increased in size. Lethally injured cells were apparent in papillary spaces. At 4 hours after phototherapy, tumor cells and endothelial cells exhibited extensive cell damage, including mitochondrial destruction, endoplasmic reticulum swelling, polyribosome disaggregation, and plasma membrane blebbing. By 24 hours after phototherapy, the majority of cells within the tumor were necrotic. Untreated tumors and those treated with phototherapy-only did not exhibit these changes. Tumors that received HPD only exhibited focal areas of cell swelling and focal mitochondrial vacuolization in both tumor and endothelial cells. These changes, unlike the HPD-light-treated group did not progress and were reversible.     

Evaluation of metallic personalized hemiarthroplasty: a canine patellofemoral model.

The purpose of this study was to characterize the response of articular cartilage to weight bearing against a metallic personalized hemiarthroplasty prosthesis. Ten dogs each underwent surgery in which an elastomeric replica of the left femoral patellar groove was made. Using this replica, a 0.5-mm-thick prosthesis was cast in Co-Cr alloy and subsequently the surface was polished to a mirror finish which had a center line average roughness value in the range of human hemiarthroplasty implants. A second surgery was performed to resurface the left trochlea with this prosthesis. Five animals were sacrificed at 3 months and 5 at 6 months. Cartilage damage occurred primarily in the distal region of the patella, and was especially evident at 6 months. Mechanical indentation tests conducted on patellar cartilage in a saline bath at 37 degrees C indicated both increased deformation and residual deformation in the affected areas, indicative of degenerative change. Areas of fibrillation with a depletion of proteoglycans were identified histologically. These areas were only superficial at 3 months but became more extensive at 6 months. Rheological analysis of the synovial fluid of tests joints indicated that a decrease in viscosity occurred from 3 to 6 months, an additional indicator of progressive degeneration. This novel implant model showed that even if a metallic hemiarthroplasty implant had an identical geometry as the joint surface being replaced and had a reasonably smooth surface, cartilage degeneration inevitably resulted.