Virus-induced cholesterol crystals.

One of the crystal types induced in cell cultures by a new feline herpesvirus was identified as cholesterol by crystal structure, polarized light microscopy, and mass spectroscopy.

Lead inclusion bodies in osteoclasts.

Inclusion bodies occur frequently in the nuclei and rarely in the cytoplasm of osteoclasts in pigs with experimental lead poisoning. The light and electron microscope pictures of undemineralized sections are similar to those described for liver cord cells and renal tubular cells.

Tissue-specific regulation of shell gland calbindin D28K biosynthesis by estradiol in precociously matured, vitamin D-depleted chicks.

Provision of Ca2+ for egg shell calcification in the avian uterus [egg shell gland (ESG)] derives mostly from vitamin D-dependent intestinal Ca2+ absorption from the diet. Ca2+ absorption is strongly linked to the intestinal vitamin D-dependent calbindin D28K (D28K) concentration. The laying hen ESG also contains D28K, and again, Ca2+ transport into the shell appeared to be linked to the ESG D28K concentration. However, evidence is now presented that ESG D28K synthesis may be estradiol (E2) dependent and vitamin D independent under certain conditions. One-day-old female chicks fed a vitamin D-free diet for as long as 6 weeks and then repeatedly injected im with E2 for up to 3 more weeks developed frank rickets, but possessed precociously matured reproductive tracts. While the tiny presumptive ESGs of nonestrogenized vitamin D-depleted chicks were devoid of D28K, the highly developed ESG, including the isthmus, of estrogenized chicks contained D28K. The ESGs of nonestrogenized, vitamin D-replete chicks also exhibited no development or detectable D28K. Regardless of whether vitamin D depleted or replete, estrogenized chick ESG contained similar D28K and D28K mRNA concentrations. Immunohistochemical techniques showed that the endometrial cellular localization of both D28K and Ca(2+)-ATPase (Ca2+ pump) in estrogenized chicks was similar to that in mature laying hens. There was no trace of D28K, nor was there any stimulation of Ca2+ absorption, in duodenum of vitamin D-free, immature chicks regardless of E2 treatment. As expected, both D28K and D28K mRNA were present in vitamin D-replete chick duodenum. We conclude that in E2-treated chicks, ESG D28K gene expression may be vitamin D independent and E2 dependent. This is the first clear demonstration of hormone-dependent tissue-specific D28K gene expression in the chick.

Calcitonin, parathyroidectomy and modelling of bones in the growing rat.

After calcitonin injection, parathyroidectomy, or both, in young growing rats, broadening of the proximal femoral metaphysis with lack of normal concavity of the medial and lateral contours was observed radiographically. Histologically, this abnormal modelling was associated with retarded osteocytic osteolysis. Further manifestations of decelerated resorption included retention and extension of the chondroid core in the secondary spongiosa, retention of cartilage in the cortex and a large increase in the number of cementing lines.

Effects of exercise and polysulfated glycosaminoglycan on repair of articular cartilage defects in the equine carpus.

Our aim was to determine if mild to moderate postoperative exercise and intra-articular polysulfated glycosaminoglycan result in improved repair of large, experimentally induced osteochondral defects in a weight-bearing surface of equine joints. Arthroscopic debridement was used to produce full-thickness defects in a weight-bearing area of the radial carpal bones in 18 ponies. The ponies were randomly assigned to two groups balanced for age: nine animals in the exercise and nine in the no exercise group. Six ponies in each group were medicated weekly for 5 weeks with an intra-articular injection of polysulfated glycosaminoglycan in one middle carpal joint beginning at the time of operation. Walking (twice daily) was begun 6 days postoperatively, and by the twelfth week postoperatively the ponies were trotting for 25 min and walking for 15 min twice daily. At the time of the ponies' death, 17 weeks postoperatively, each defect had an average of 50-75% coverage with repair tissue. Exercised, medicated joints had a significantly smaller area of coverage with repair tissue than exercised, nonmedicated joints. Cartilaginous repair tissue from exercised ponies contained significantly more glycosaminoglycan and type-II collagen (r = 0.53, p < 0.05). The ratio of hydroxylysine to hydroxyproline was significantly lower and the ratio of collagen content to total protein was significantly higher in the repair tissue of medicated joints than in the repair tissue of nonmedicated joints; this is consistent with the presence of less type-II collagen in the repair tissue in medicated joints. We concluded that postoperative exercise was beneficial and that the immediate postoperative use of intra-articular polysulfated glycosaminoglycan was detrimental to the development of cartilaginous repair tissue in large osteochondral defects of equine joints.

The effects of phenylbutazone on the intestinal mucosa of the horse: a morphological, ultrastructural and biochemical study.

Phenylbutazone, a non-steroidal anti-inflammatory drug known to produce intestinal erosions, was administered intravenously (13.46 mg/kg bodyweight) to 12 horses which were killed after 24, 48, 72 and 96 h. Eight untreated horses served as controls. Annular erosions in the duodenum and mucosal necrosis in the colon were seen after 48 h which progressed in severity. The erosions were characterised by sloughing of the surface epithelium, subepithelial cleft and bleb formation, necrosis of the lamina propria, degeneration of the walls of subsurface capillaries and microthrombosis. Large numbers of neutrophils with abundant fibrin and cellular debris were present at the erosion sites. Ultrastructurally, there was swelling of the endothelium of capillaries and small vessels, and of pericyte and smooth muscle cytoplasm in arterioles. In capillaries and post capillary venules, the endothelium ranged from swollen to lysed and necrotic. Extensive extravasation of erythrocytes and oedema were seen. These lesions were not seen in the control horses. Phenylbutazone produces a microvascular injury associated with the formation of duodenal and colonic erosions in horses. The duodenal and colonic mucosa were assayed at 48 and 96 h for prostacyclin and PGE2. There was no statistically significant difference between prostaglandin levels in the mucosa of control and treated horses. It was concluded that there was no correlation between mucosal prostaglandin levels and intestinal erosions after 48 h.

Endocrine and histologic correlates of the dynamics of the metacarpal growth plate in growing rams.

The objective was to study control of mature size by characterizing metacarpal growth plate closure in relation to relevant bone growth-regulating hormones in two breeds exhibiting distinct differences in mature frame size. Thirty-four Suffolk and 34 Dorset ram lambs were slaughtered in pairs within breed at birth, weaning and monthly intervals until 420 d and then bimonthly to 600 d. Plasma growth hormone was depressed to undetectable levels due to the high-energy, ad libitum-fed diet. Plasma insulin-like growth factor-I (IGF-I) rose over the growth period from 116 ng/ml (newborn Suffolk) to a high of 451 ng/ml (420-d Dorset); it appeared to peak at approximately 400 d and then declined to a stable level. Dorsets consistently exhibited higher IGF-I levels. The thyroid hormones exhibited no apparent age association. An age-associated rise was detected for testosterone, but not for estradiol. Mature metacarpal lengths were estimated to be 147.2 and 127.4 mm for Suffolks and Dorsets, respectively. Ninety-five percent of mature length was attained in Suffolks by 226 d and in Dorsets by 165 d. Growth plates, however, did not begin to appear closed until 390 d and closure was not complete in all animals until 480 d, suggesting that metacarpal growth rate was dissociated temporally from growth plate closure. Although growth plate closure likely is controlled by the endocrine system, there were no apparent relationships between circulating hormones and growth plate width, age at closure or zonal divisions within the growth plate, suggesting that the growth plate experiences a very different hormonal environment than what can be measured in the circulating blood.

Bone lesions in growing swine fed 3% cement kiln dust as a source of calcium.

Cement kiln dust (CKD) samples of which have been reported to stimulate growth in cattle and sheep, was fed to weanling pigs in a 42-d experiment. CKD at levels of 1.5 and 3.0% was added to a corn-soybean meal-oats-whey-type diet containing inadequate amounts of Ca (.4% of the diet) to provide final dietary Ca levels of .85 and 1.3%. A diet containing 3% limestone (1.3% dietary Ca) served as a positive control. Body weight gain was depressed by 3.0% CKD, and histopathological lesions of the humerus, not typical of nutritional secondary hyperparathyroidism or rickets, were observed. The lesions detected were osteonecrosis, thinning of the cortex and reduction in the width of the epiphyseal cartilage. Width of the proximal epiphyseal plate and cortical index of the humerus (width of cortex divided by total diameter at narrowest point of diaphysis) were smaller in pigs fed 3% CKD than in pigs fed 3% limestone. CKD contained 2.3% A1, 15 ppm Cd and 110 ppm Pb, providing 690, .45 and 3.3 ppm of A1, Cd and Pb, respectively, in the diet containing 3% CKD. Kidney, liver and bone ash concentrations of these three minerals were not increased by CKD, and typical toxicity signs were absent. It is concluded that CKD may contain one or more factors that interfere with normal bone metabolism in growing pigs when the diet contains 3.0% CKD.

Response to dietary fat and cholesterol in young adult boars genetically selected for high or low plasma cholesterol.

Twenty-eight young adult boars (age 7 to 8 mo) genetically selected for four generations for high (HG) or low (LG) plasma cholesterol were studied to assess dietary and genetic effects and their interactions on cholesterol metabolism. Boars within a genetic group were paired according to their plasma cholesterol concentration at 8 wk of age and were fed for 15 wk (2.7 kg/d) one of two diets (16.5% fat and 1,156 mg of cholesterol/kg diet, HD; or 3.1% fat and 0 cholesterol diet, LD) in a 2 x 2 factorial arrangement. Plasma total cholesterol (P < .01) and HDL-cholesterol (P < .01) concentrations were higher in boars fed HS (P < .01) and in HG boars (P < .01). There was a trend toward a diet x genotype interaction for plasma HDL-cholesterol (P < .06). Plasma insulin concentration tended to be lower in boars fed HD (P < .09) or HG boars (P < .10). There was a diet x genotype interaction for plasma glucagon (P < .04) concentration and a trend toward an interaction for insulin (P < .07). After 15 wk, all boars were killed by electrocution and exsanguination and measurements were taken. Backfat thickness was greater (P < .01) and carcass length (P < .01) and weights of the four lean cuts (P < .02) were lower in LG than in HG boars. No microscopic atherosclerotic plaques were observed in aorta or coronary arteries. Cholesterol concentration in subcutaneous fat was lower (P < .04) in LG boars, suggesting that cholesterol content of depot fat can be reduced by genetic selection in swine.

Changes in articular cartilage after intra-articular injections of methylprednisolone acetate in horses.

Eight mature horses with no prior signs of joint disease or history of intra-articular therapy were treated with 8 weekly intra-articular injections of methylprednisolone acetate. Treatments were given at a dose of 120 mg/joint into the right radiocarpal and intercarpal joints, with the left joints as untreated controls. Articular cartilage samples were obtained at necropsy 1, 4, and 8 weeks after the last injection. Compared with controls, cartilage from injected joints had a loss of hematoxylin basophilia and decreased intensity of staining in safranin O fast green dye. Chondrocyte necrosis and hypocellularity were observed in all samples of cartilage from treated joints. Proteoglycan content and its rate of synthesis were reduced. There was a progressive loss of proteoglycan content, whereas proteoglycan synthesis increased somewhat 4 and 8 weeks after treatment. Collagen content was unchanged, but its rate of synthesis was markedly inhibited. Collagen synthesis did not recover, but remained decreased at 5 to 15% of the values from untreated cartilage. Water percentage was increased, but fibronectin content was not significantly different. A single injection of methylprednisolone acetate was also given into the right metacarpophalangeal joints of 3 of the 8 horses in this group, with the left joints serving as untreated controls. Sixteen weeks after the treatment, cartilage of the treated joints had a loss of histochemical staining and proteoglycan content was reduced to 50% of control values. The mean rate of proteoglycan synthesis and mean fibronectin content were increased, but the differences were not statistically significant (P greater than 0.05). Other variables were essentially unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

An experimental model of chronic renal disease in dogs by infusion of microspheres into the renal arterial circulation.

The feasibility of renal arterial infusion of nonbiodegradable microspheres as a model of chronic renal disease in dogs was evaluated. Resin-coated, styrene-divinyl benzene copolymer microspheres were infused into the kidneys of healthy adult Beagles by direct injections of both renal arteries in a single surgical procedure. Injections of 25-microns diameter microspheres had minimal effect on either the clinical status or serum values of the dogs. Histologic examination revealed the majority of the microspheres lodged within the capillary beds of the glomeruli, and little change to the kidneys. However, injections of 50-microns diameter microspheres caused significant increases in serum concentrations of urea nitrogen and creatinine. Histologically, the larger microspheres obstructed afferent arterioles and small arteries, which caused diffuse glomerular necrosis and nephron damage. With doses ranging from 1 to 3 million microspheres/dog, a correlation between the quantity of microspheres injected and severity of renal damage was observed. The optimal dose for producing a model of moderate renal disease was determined to be 1.8 million microspheres/dog (0.9 million microspheres/kidney). During long-term studies, microsphere-injected dogs fed a moderately restricted protein ration remained relatively azotemic, compared with control dogs on the identical ration. During the 5-month postsurgical period, the serum urea nitrogen concentration averaged 18.41 +/- 1.59 mg/dl (mean +/- SE) for the microsphere-injected dogs vs 9.31 +/- 0.38 for the control dogs (P less than 0.001). Similarly, the mean serum creatinine value was significantly higher (P = 0.020) for the microsphere-injected dogs, compared with the controls (1.23 +/- 0.12 mg/dl vs 0.94 +/- 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

Capacitively coupled electrical stimulation of bone healing in the horse: in vivo study with a Salter type IV osteotomy model with stainless steel surface electrodes.

The use of capacitively coupled low-voltage signals for stimulation of osteogenesis has been reported in a variety of animal models. Electrically induced osteogenesis was investigated with a capacitively coupled electric field on a radius (distal-lateral orientation) osteotomy model, in conjunction with internal fixation and postoperative loading. Twelve adult horses of either sex were allotted to 2 groups of 6; 1 group was given electrical stimulation and the other served as controls. A low-voltage high-frequency capacitively coupled electrical signal was locally and continuously applied to the electrically stimulated group for 60 days through external, bare stainless steel surface electrodes which were placed on the skin in circuit with a small, portable power source. Harness compatibility and stimulator and battery durability were excellent. However, stainless steel electrodes required a rigid maintenance schedule to maintain consistent current levels. Synovial fluid evaluation demonstrated intra-articular inflammation (decreased viscosity, hyaluronic acid, and increased protein concentration) 1 week postoperatively that generally improved during subsequent weeks and no distinction between groups was observed at 60 days. Radiographically, there was no difference in the appearance of the healing process of control and that of stimulated horses during the 60 days. Angiography showed bridging blood vessels in both groups. Uptake of a bone seeking radiopharmaceutical peaked at 3 weeks in both groups and was 1.92 +/- 0.6 cps/pixel/mCi and 1.26 +/- 0.40 csp/pixel/mCi for control and stimulated horses, respectively. At any given observation period, uptake in the lesion area was greater in the control group. Ultimate strengths of trabecular bone in 60-day control radii and stimulated radii were 12.64 +/- 3.013 and 9.60 +/- 3.95 MN/m2, and the flexural moduli of elasticity were 698.0 +/- 423 and 402.0 +/- 523 MN/m2, respectively. Porosity index was similar for all specimens. Gross, histologic, and microradiographic evaluations indicated that controls healed more efficiently than stimulated horses. A capacitively coupled applied voltage of 2.2 V RMS (mean) producing a current of 17.32 mA (mean) did not stimulate sufficient bone production in a metaphyseal osteotomy model to affect the mechanical properties of the bone or accelerate the healing process.

Effects of sodium hyaluronate on tendon healing and adhesion formation in horses.

Sodium hyaluronate reduces adhesions after tendon repair in rodents and dogs, and has been used in limited clinical trials in people. To evaluate its effect on tendon healing and adhesion formation in horses and to compare these effects with those of a compound of similar visco-elastic properties, a study was performed in horses, using a model of collagenase injection in the flexor tendons within the digital sheath. Eight clinically normal horses were randomly allotted to 2 groups. Adhesion formation between the deep digital flexor tendon and the tendon sheath at the pastern region was induced in the forelimbs of all horses. Using tenoscopic control, a 20-gauge needle was inserted into the deep digital flexor tendon of horses under general anesthesia and 0.2 ml of collagenase (2.5 mg/ml) was injected. The procedure was repeated proximally at 2 other sites, spaced 1.5 cm apart. A biopsy forceps was introduced, and a 5-mm tendon defect was created at each injection site. Group-A horses had 120 mg of sodium hyaluronate (NaHA) gel injected into the tendon sheath of one limb. Group-B horses had methylcellulose gel injected at the same sites. The contralateral limbs of horses in both groups served as surgical, but noninjected, controls. Horses were euthanatized after 8 weeks of stall rest. Ultrasonographic evaluation revealed improved tendon healing after NaHa injection, but no difference in peritendinous adhesion formation. Tendon sheath fluid volume and hyaluronic acid (HA) content were greater in NaHA-treated limbs. Gross pathologic examination revealed considerably fewer and smaller adhesions when limbs were treated with NaHA. However, significant difference in pull-out strengths was not evident between NaHA-treated and control limbs. Histologically, the deep digital flexor tendon from the NaHA-treated limbs had reduced inflammatory cell infiltration, improved tendon structure, and less intratendinous hemorrhage. Treatment with methylcullulose had no significant effect on tendon healing, adhesion size, quantity, or strength or on the volume and composition of the tendon sheath fluid. Sodium hyaluronate, administered intrathecally, appears to have a pharmaceutically beneficial action in this collagenase-induced tendinitis and adhesion model in horses.

Bilateral degenerative coxofemoral joint disease in a foal.

Bilateral degenerative coxofemoral joint disease and noninflammatory osteonecrosis in the femoral heads were diagnosed in a 5-month-old Standardbred colt. Cytologic evaluation and bacterial cultures of coxofemoral synovial fluid, and radiographic and pathologic examination of the coxofemoral joints were conducted. The cause was not determined; however, a thrombus found in association with 1 focus of osteonecrosis was suspected as an etiologic factor.

Clinical signs and diagnosis of osteogenesis imperfecta in three dogs.

When a young dog is evaluated for multiple fractures with minimal to no accompanying trauma, the primary differential diagnoses are metabolic disease, physical abuse, and osteogenesis imperfecta (OI). Of these, secondary hyperparathyroidism is most common, but if serum concentrations of ionized calcium, phosphorus, vitamin D, and parathormone are within reference ranges, OI must be considered. Osteogenesis imperfecta is a heritable disease characterized by brittle bones. Results of studies using cultured skin fibroblasts indicate that most cases of OI in human beings are caused by a mutation in a type-I collagen gene. Osteogenesis imperfecta was recently identified in 3 dogs. Radiographic findings included multiple fractures in various stages of healing and generalized osteopenia. Cultured fibroblasts from skin biopsy specimens were used to diagnose OI. Structural abnormalities were found in type-I collagen from each dog. This cell culture assay can be used to evaluate dogs with brittle bones.

Defects in collagen fibrillogenesis causing hyperextensible, fragile skin in dogs.

Two unrelated mixed-breed dogs were donated for studies of their fragile, hyperextensible skin. Breeding of these dogs to bitches with normal skin showed that half of their male and female offspring also had fragile, hyperextensible skin, indicating that the defect was transmitted as an autosomal dominant trait in both dogs. Electron microscopy showed distinct abnormalities in the packing of collagen into fibrils and fibers in affected skin. These packing defects in dermal collagen were identical in related dogs, but were slightly different in unrelated animals. A clinical test, the skin extensibility index, was used to quantitate the extensibility of affected and unaffected skin. This index ranged from 8% to 15% in normal dogs and from 17% to 25% in newborn pups and adult dogs with collagen packing defects. The tensile strength of dorsolateral thoracic skin of affected pups was only 5% to 10% of that of matched specimens of paired littermates. The hyperextensibility and fragility of skin were the only clinical signs, but radiographic and microradiographic studies revealed subclinical involvement of bone.

Nutritional secondary hyperparathyroidism in free-living fledgling American crows (Corvus brachyrhynchos brachyrhynchos).

From June 1994 to June 1996, 18 fledgling American crows (Corvus brachyrhynchos brachyrhynchos) from multiple locations on Long Island, New York, were presented with signs of metabolic bone disease characterized by folding fractures of the proximal tibiotarsus. Plasma alkaline phosphatase levels were elevated, and the calcium/phosphorus ratio and 25-hydroxycholecalciferol (25-(OH)D3) levels were decreased. The histopathologic diagnosis was parathyroid hyperplasia and generalized osteodystrophia fibrosa. A diet low in bioavailable calcium and/or vitamin D3 was the probable cause. Also, exposure to xenobiotics could have contributed to the depression of 25-(OH)D3 levels.