Studies of a synthetic substrate in canine globoid cell leukodystrophy.

Gal et al. ((1977) Clin. Chim. Acta 77, 53-59) reported the use of a new synthetic substrate, 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside for the diagnosis of human globoid cell leukodystrophy. Assay of beta-galactosidase in brain homogenates from normal, carrier, and globoid cell leukodystrophy-affected dogs utilizing this new substrate demonstrated overlapping activities. Instead of reflecting specific D-galactosyl-N-acylsphingosine galactohydrolase (EC 3.2.1.46), the 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside beta-galactosidase activity in canine brain is highly correlated with nonspecific 4-methylumbelliferyl beta-galactosidase. Optimization of the 2-hexadecanoyl-amino-4-nitrophenyl-beta-D-galactopyranoside assay system for canine brain and the use of varying concentrations of taurocholate or taurodeoxycholate in the assay mixture did not alter the lack of specificity. These results indicate a significant difference in the nature of the underlying defect in galactosylceramide beta-galactosidase in canine globoid cell leukodystrophy compared to human globoid cell leukodystrophy.

Quantitative evaluation of spinal cord lesions in canine globoid leukodystrophy.

Lesion distribution in the spinal cord was investigated by sampling an average of 26 spinal cord segments in each of ten dogs severely affected by canine globoid leukodystrophy (GLD). GLD lesions were quantified by subdividing spinal white matter into small unit areas and judging the quartile extent of lesion involvement for each unit area. Along the length of the spinal cord, lesions declined from cranial to caudal, decreasing precipitously in the midlumbar region. The lumbosacral region had the least amount of lesion, and, based on lesion per unit area, the thoracic region was most severely involved. The dorsal funiculus had the greatest concentration and the least dispersion of lesion generally amoung the three funiculi. White matter at the periphery of the spinal cord was involved earlier and more severely than white matter adjacent to gray matter. Amount of lesion per spinal cord could be estimated adequately by sampling one segment from each of five regions. Dogs could be divided into two groups based on rate of lesion development. Clinical signs did not correlate closely with total amount of lesion per spinal cord. Of the total amount of variability among dogs in amount of spinal pathologic involvement, 78% could be accounted for by a mathematical model expressing spinal cord pathologic involvement as a quadratic function of age at onset and duration of the clinical syndrome.

Immunocytochemical localization of serotonin in the rat periaqueductal gray: a quantitative light and electron microscopic study.

The distribution of serotonin-like immunoreactivity in five regions of the rodent midbrain periaqueductal gray (PAG) was studied by using light and electron microscopic immunohistochemistry in combination with quantitative analysis. Light microscopic analysis revealed the presence of serotonin-like immunoreactive cell bodies located in the ventrolateral and ventromedial regions of the caudal PAG and serotonin-like immunoreactive processes throughout the PAG. Ultrastructural analysis showed dendritic profiles that stained positively for serotonin primarily in ventral regions, although an occasional profile was seen dorsally. Numerous synaptic contacts between unstained axon terminals and ventral dendritic profiles were seen. Axonal profiles that contained reaction product were identified throughout the PAG, but were rarely observed to make any type of specialized contact. Ultrastructural quantification of serotonin-like immunoreactive processes indicated that the highest volume fraction of serotonin immunoreactivity occurred caudoventrally where stained processes constituted 2.6% of the neuropil volume. Rostroventrally stained processes constituted only 0.14% of the neuropil volume at the level of the posterior commissure. By contrast the amount of serotonin-like immunoreactivity found dorsally remained relatively constant at all rostrocaudal levels. Analysis of serotonin staining among PAG regions demonstrated the lowest overall volume fraction in the dorsal region and the highest overall volume fraction in the ventromedial region. No significant differences were observed between medial and lateral regions. A comparison of the results of light microscopic quantitative analysis of serotoninergic processes with electron microscopic quantitative analysis indicated that both techniques produce comparable results.

Galactocerebrosidase activity in canine globoid leukodystrophy.

In an investigation of canine globoid leukodystrophy, cerebroside beta-galactosidase activities were assayed in 24 brains from neonatal and older dogs and in 90 canine leukocyte pellets from nine samplings. The neonatal brains had significantly less enzyme activity than the brains of older dogs, which indicates a potential complication in making neonatal and fetal enzymatic diagnoses. For leukocytes, heterozygous activities averaged 51 percent and globoid leukodystrophy activities 18 percent of mean enzyme activity of normal leukocytes. Variability of leukocyte enzyme activities among the nine samplings was large, but within each sample, variability per genotypic category was moderate. A statistical model was developed to facilitate enzyme diagnosis in the dog and, by implication, in human globoid leukodystrophy and other sphingolipidoses.

Muscarinic cholinergic receptors in the rat deep cerebellar nuclei: a quantitative autoradiographic study.

The distribution of muscarinic cholinergic receptors within the rat deep cerebellar nuclei was analyzed using in vitro receptor binding of [3H]quinuclidinylbenzilate (QNB) in conjunction with autoradiography. The highest density of QNB binding sites occurred in the lateral cerebellar (dentate) nucleus. Interpositus nuclei displayed an intermediate density of muscarinic cholinergic binding sites with the posterior interpositus nucleus demonstrating higher binding than the anterior nucleus. The fastigial (medial) cerebellar nucleus exhibited the lowest levels of QNB binding among the four cerebellar nuclei. These results indicate that muscarinic cholinergic receptors are present in the deep cerebellar nuclei and that differences in receptor density occur among the four nuclear groups.

Canine globoid cell leukodystrophy. Part 1. Further ultrastructural study of the typical lesion.

The ultrastructural changes of typical lesions in canine globoid cell leukodystrophy (GLD) have been studied. The globoid cells were located in the cerebral parenchyma as well as in the perivascular Virchow--Robin space. Features suggestive of a passage of the globoid cells from the cerebral parenchyma to the Virchow--Robin space were also observed through the interruptions in the basal lamina. The globoid cells had numerous thin pseudopods and contained various cytoplasmic inclusions which have been described previously. Detailed studies of these inclusions suggest that they represented aggregates of filamentous or linear sub-unit structures. Typical oligodendroglial cells were found on only a few occasions. Both globoid cells and oligodendroglia contained myelin debris, dense bodies and honey-comb like inclusions composed of numerous small myelin figures. In a few instances, crystalline polygonal inclusions identical to those found in the globoid cells, were found in the cytoplasm of the cells which were, with reasonable certainty, identifiable as oligodendroglia. In less affected areas where myelin was still present, degenerating oligodendroglia, with or without recognizable inclusions, were frequently encountered. Astrocytes and endothelial cells contained concentric lamellar inclusions and dense bodies but did not contain the tubular inclusions as seen in globoid cells. The possible significance of the ultrastructural features in regard to the pathogenesis of the GLD have been discussed.

Quantitation of cardiac gross morphology during the development of FZ-induced cardiomyopathy in turkey poults.

Furazolidone (FZ) at a dose of 700 ppm was fed to turkey poults 2-5 weeks posthatch. At 3, 4, and 5 weeks of age, seven poults each were sacrificed from control and FZ-fed groups. A section of ventricular tissue 1 mm thick was excised from the heart distal to the base at a level one-fourth of the distance between the base and apex. Parameters estimated from tracings of each section were area and circumference of both right and left ventricular lumen profile, area and circumference of entire heart profile, area of right ventricular free wall profile, area of left ventricular and septal wall profile, and area of entire heart muscle profile. A major finding was a sequential increase in the area of the ventricular lumina in the FZ-fed poults. Differences between the control and FZ-fed poults were statistically significant for the right lumen profile at 3 and 5 weeks of age and for the left lumen profile at 4 and 5 weeks of age. The area of the heart muscle profile was decreased in FZ-fed poults, but this difference was statistically significant only at 4 weeks of age. Data suggest that the pathogenesis of FZ-induced cardiomyopathy involves a dilation of ventricular lumina resulting in increased intraventricular blood volume and altered ECG patterns. Relationships between gross morphology and ECGs may be seen only in terminal cases of several weeks duration. This explains the inability to correlate ECGs with gross morphologic changes in all poults observed at necropsy.

Increased vascular density associated with lesions of canine globoid leukodystrophy.

Two control dogs and 2 dogs that had globoid cell leukodystrophy (GLD) were studied to document vascular alterations associated with the leukodystrophic lesion. Spinal white matter was sampled by transverse and frontal planes of section from dorsal and ventral halves of the lateral funiculus of five spinal segments. Vessel profiles were classified as capillaries or larger vessels. GLD lesion was associated with an increase in capillary mean diameter. Vascular density, estimated by profiles per area, was compared in affected and control tissues. Capillary density was not significantly different, but the density of larger vessels was increased in proportion to the extent of GLD lesion. The increased density is presumed to be the result of lengthening or proliferation of vessels which had normal density prior to lesion formation.

Immunohistochemical morphometry of pancreatic endocrine cells in diabetic, normoglycaemic glucose-intolerant and normal cats.

The anatomical distribution and volume fractions of pancreatic A cells (glucagon), B cells (insulin) and D cells (somatostatin) were evaluated by an immunoperoxidase technique in 6 diabetic cats, 6 normoglycaemic glucose-intolerant cats and 6 normal control cats. Islets lacking A cells were observed in some sections from the right lobe of the pancreas which correlated with a significantly lower A cell volume fraction in the right pancreatic lobe. Endocrine cell volume fractions in normoglycaemic glucose-intolerant cats were not significantly different from controls. Thus, a reduction in B cell volume fraction was not necessary for the occurrence of impaired glucose tolerance in these cats. However, the reduction of B cell volume fraction in the 2 normoglycaemic glucose-intolerant cats with insular amyloidosis may in part explain the more severely impaired glucose tolerance previously observed in these cats. Insular amyloidosis in our feline diabetics, as in human type II diabetics, was associated with a significant decrease in A and B cell volume fractions. In both human type II and feline diabetes mellitus, however, the reduction in B cell mass does not appear sufficient alone to lead to diabetes mellitus. Therefore, amyloid replacement of functional endocrine cells does not appear to be the primary diabetogenic event in feline diabetes mellitus, but may contribute to progression of the condition due to loss of functional B cell reserves. We thus postulate that a B cell defect precedes deposition of islet amyloid and that these amyloid deposits may thus provide an important biochemical clue to specific B cell derangements occurring in adult-onset diabetics.

Malignant hyperthermia susceptibility: cardiac histomorphometry of dogs and young and market-weight swine.

The defect causing malignant hyperthermia has been proposed to involve cardiac as well as skeletal muscle. We tested the hypothesis that histomorphometric parameters for ventricular wall from malignant hyperthermia-susceptible swine and dogs were abnormal. Hearts were obtained from: mature dogs, age- and weight-matched young swine (89 +/- 15 days, 30 +/- 3 kg); and market-weight swine (102 +/- 10 kg). Using light microscopy, estimates were made for muscle nuclear dimensions and the volume-fraction of nuclei, sarcoplasm, blood vessels, and interstitial space. Cardiac maturation in both MH and normal swine was accompanied by decreased myocyte volume-fraction due to decreased nuclear volume-fraction and increased interstitial space volume-fraction. Sarcoplasm and vasculature volume-fraction were unchanged after maturation. Nuclear volume-fraction was slightly greater (p less than 0.05) in the right ventricle than the left for malignant hyperthermia and normal swine. Myocyte nuclear dimensions were generally similar among animals. Dogs and the oldest group of swine were not significantly different. Myocytes of all swine contained multiple nuclei, closely spaced in rows of 2 to 12. In contrast, most myocytes of mature dogs apparently contained one or two nuclei. Histomorphometric values were not significantly different between normal and malignant hyperthermia young swine and dogs. However, within the market-weight swine, volume-fraction for malignant hyperthermia myocytes and myocyte nuclei was decreased and interstitial space was increased compared to normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a calculolytic diet in female dogs with induced struvite urolithiasis.

The calculolytic effect of a diet designed to reduce the urine concentration of urea, P, and Mg was evaluated in female Beagles with induced urease-positive urinary tract infections and struvite urolithiasis and in female Beagles with induced sterile struvite urolithiasis. The reduced-protein calculolytic diet induced urolith dissolution in 5 of 6 infected dogs with struvite urolithiasis in 2 to 5 months (means = 14.4 weeks). At the end of 6 months, uroliths in comparable control dogs fed a maintenance diet were 5 times larger and 14 times heavier than at the beginning of the study. The calculolytic diet induced urolith dissolution in 6 of 6 noninfected dogs with struvite uroliths in 2 to 4 weeks (means = 3.3 weeks). Four uroliths in noninfected dogs fed the maintenance diet dissolved over a period of 2 to 5 months (means = 14 weeks). Urolith dissolution in dogs fed the calculolytic diet was associated with diet-induced diuresis, reduction in urine pH, reduction in urine concentration of urea ammonia, P, and Mg, and increase in urine titratable acidity. Consumption of the calculolytic diet was also associated with significant (P = less than 0.01) reduction in the serum concentration of urea and albumin and a significant (P = less than 0.01) increase in serum hepatic alkaline phosphatase activity. Concomitant occurrence of hydropic degeneration of hepatocytes indicated that these biochemical and morphologic changes were associated with dietary protein restriction.

Prevalence of atretic corpora lutea in ovaries of bovine fetuses.

Both ovaries from 88 bovine fetuses in the fifth month or later of gestation were studied histologically to determine the prevalence, origin, and time of appearance of atretic corpora lutea (ACL). Ovaries from 36 (41%) fetuses had ACL; fetuses < 6 months of gestation did not have ACL. Six fetuses had more than 25 ACL, but there was no apparent relationship between fetal age and number of ACL. Formation of ACL involved disintegration of the stratum granulosum of secondary follicles, concomitant with proliferation and invasion by vascularized elements of the theca. Fully developed ACL consisted of a large primary oocyte surrounded by a prominent zona pellucida and encased in a well-vascularized, largely thecal, fibrocellular wall. They measured approximately 0.5 to 1.0 mm in diameter. Empty, collapsed zona pellucidas were seen in many of the degenerating ACL.

Feline renal ultrasonography: quantitative analyses of imaged anatomy.

Maximal kidney dimensions from serial ultrasonograms were measured in 10 healthy cats. Because clinical ultrasonographic examination is often performed in conjunction with excretory urography, the effect of radiographic contrast medium-induced diuresis on dimensional change also was evaluated. Although a slight, statistically significant increase in size was observed (compared with kidneys scanned without contrast medium), this increase could not be detected on the basis of visual image evaluation alone.

Effects of various methods of preservation on the stability of uric acid in frozen canine urine.

OBJECTIVE: To evaluate the effects of dilution and alkalinization, separately and together, on the stability of uric acid in canine urine stored at -20 C. DESIGN: Prospective-controlled study. ANIMALS: 5 dogs with confirmed ammonium urate uroliths, 6 Beagles, and 6 mixed-breed dogs. PROCEDURE: Dogs were fed a 31.4% protein (dry weight), meat-based diet for 21 days, and urine samples were collected on day 22. Urine samples were preserved, using combinations of dilution and alkalinization, and divided into 1-ml aliquots for storage at -20 C for 1 to 12 weeks. Urine uric acid concentrations were measured, using high-performance liquid chromatography, on day of collection (baseline), and after 1, 2, 4, 8, and 12 weeks. RESULTS: Alkalinization did not have a significant effect on reproducibility of measurements of uric acid concentrations in urine; however, dilution did have a significant effect. Compared with baseline, uric acid concentrations in urine samples collected from dogs with ammonium urate uroliths and Beagles and diluted 1:10 or 1:20 with deionized water were not different after storage for 1 to 12 weeks. Uric acid concentrations in urine samples collected from mixed-breed dogs did not differ from baseline values during the 12-week storage period whether samples were undiluted or were diluted 1:10 or 1:20 with deionized water. CONCLUSIONS: Measurements of uric acid concentration are most reproducible in canine urine samples stored at -20 C for 1 to 12 weeks when samples are diluted 1:20 with deionized water. CLINICAL RELEVANCE: To ensure reproducibility of measurements of uric acid concentration in urine samples collected from dogs affected with urate uroliths, urine should be diluted 1:20 with deionized water. Alkalinization is not necessary, and is not recommended because of the additional step in processing and its potential to interfere with measurement of other urinary analytes.

Teratogenic effect of acetohydroxamic acid in clinically normal beagles.

Acetohydroxamic acid (AHA), a potent urease inhibitor used for treatment of infection-induced struvite urolithiasis, was teratogenic after administration of 25 mg of AHA/kg of body weight/day orally to 5 clinically normal Beagles from the onset of proestrus until parturition. Thirty pups exposed to AHA in utero developed anomalies of the skeletal system, heart, and ventral midline. Cardiac anomalies included atrial septal defects (20%), ventricular septal defects (3%), and atrial and ventricular septal defects (3%). Skeletal anomalies included coccygeal hemivertebrae and fused coccygeal vertebrae (50%), supernumerary vertebrae (67%), supernumerary ribs (50%), duplicated sternebrae (3%), and lumbar hemivertebrae (3%). Defects of the ventral midline of the abdominal wall occurred in 20% of AHA-exposed pups. Other abnormalities included retarded growth, high neonatal mortality, and a decreased number of circulating RBC, compared with those in 30 control pups born to 5 Beagles given a placebo. Adverse effects of AHA in pregnant Beagles were limited to morphologic alterations (Howell-Jolly bodies, spherocytes, and target cells) in a small number of circulating RBC. Slight neutrophilic leukocytosis and monocytosis occurred between 0 and 30 days of pregnancy in dogs given AHA, compared with those in controls. Seemingly, AHA did not influence fertility, conception rate, or length of gestation.

Clinical evaluation of cats with lower urinary tract disease.

In a prospective study, 141 cats with hematuria, dysuria, urethral obstruction, or combinations of these signs were evaluated by contemporary diagnostic methods and compared with 26 clinically normal cats (controls). Specific diagnosis was established in 45% (64/141) of cats affected with lower urinary tract disease (LUTD). Crystalline matrix plug-induced urethral obstruction was diagnosed in 21% (30/141) of affected cats, uroliths were identified in 21% (30/141) of affected cats, uroliths with concomitant bacterial urinary tract infection (UTI) were identified in less than 2% (2/141) of affected cats, and bacterial UTI alone was identified in less than 2% (2/141) of cats with LUTD. Viruses, mycoplasmas, and ureaplasmas were not isolated from urine samples collected from affected or control cats. Bovine herpesvirus 4 (BHV-4)-neutralizing antibodies were not detected in any serum sample obtained from cats with LUTD or from control cats. In contrast, BHV-4 antibodies were detected by an indirect immunofluorescent antibody (IFA) test in sera obtained from 31% (44/141) of cats with LUTD and 23% (6/26) of control cats. The prevalence of positive BHV-4 IFA test results in affected cats was not significantly different from that observed in control cats. Significant association was not apparent between positive BHV-4 IFA test results and clinical diagnosis, abnormal laboratory findings, or cat age. However, the number of male cats with BHV-4 IFA titer was significantly (P less than 0.02, chi 2 test) greater than that of female cats. Detection of BHV-4 antibodies in approximately 30% of affected and control cats indicates prior virus exposure. Further investigations are warranted to clarify the specific role of BHV-4 in cats with naturally acquired LUTD.

Applied anatomy and physiology of the feline lower urinary tract.

Paired ureters, urinary bladder, and urethra constitute the lower urinary tract. Oblique passage of ureters through the bladder wall results in compression of the distal ureter to preclude urine reflux. Ureters are anchored by longitudinal ureteral musculature that outlines the bladder trigone and extends into dorsal submucosa of the urethra as urethral crest. The urinary bladder can be divided into apex, body, and neck. The male urethra has penile and pelvic components, the latter is divisible into preprostatic, prostatic, and postprostatic regions. The muscle coat of the bladder-urethra forms three functional entities in craniocaudal series. These are the detrusor muscle (to effect voiding), internal urethral sphincter (smooth muscle for generating tonic resistance), and external urethral sphincter (striated urethralis m. for phasic and voluntary continence). The vesical neck is a transition region. It is part of the internal urethral sphincter by virtue of its histology and innervation, but it contains detrusor fascicles that pull it open during micturition. Viscous accommodation plus sympathetic reflex inhibition of the vesical wall allows the urinary bladder to greatly expand in volume with minimal increase of intravesical pressure, within limits. At low volumes continence can be maintained by passive resistive elements of the urethral outlet. As volume increases, sympathetic reflex activity is necessary for continence. The striated external urethral sphincter is reflexly contracted to counter abrupt elevations of intravesical pressure and to maintain continence voluntarily. The pelvic plexus conveys sympathetic and parasympathetic innervation to the urinary tract. The pudendal nerve supplies the urethra and urethralis muscle. Ureters are largely independent of innervation. Internal and external urethral sphincters are activated by spinal reflexes, sympathetic and somatic reflexes, respectively. Normal micturition (sustained detrusor contraction and sphincter inhibition) is a brainstem-driven reflex, involving a spino-bulbo-spinal pathway and a pontine micturition center that switches from urine storage to micturition. All of the reflexes depend on neural activity in tension mechanoreceptors of the bladder wall and sacral afferent fibers.