The effects of Strongylus vulgaris parasitism on eosinophil distribution and accumulation in equine large intestinal mucosa.

REASONS FOR PERFORMING STUDY: Eosinophilic granulocytes have been associated with parasite or immune-mediated diseases, but their functions in other disease processes remain unclear. Cause and timing of eosinophil migration into the equine gastrointestinal mucosa are also unknown. OBJECTIVE: To determine the effects of intestinal parasitism on eosinophils in equine large intestinal mucosa. METHODS: Large intestinal mucosal samples were collected from horses and ponies (n = 16) from the general veterinary hospital population, ponies (n = 3) raised in a parasite-free environment, ponies experimentally infected with 500 infective Strongylus vulgaris larvae and treated with a proprietary anthelmintic drug (n = 14), and a similar group of ponies (n = 7) that received no anthelmintic treatment. Total eosinophil counts and eosinophil distribution in the mucosa were determined by histological examination. A mixed model analysis was performed and appropriate Bonferroni adjusted P values used for each family of comparisons. P<0.05 was considered significant. RESULTS: There was no difference in large intestinal mucosal eosinophil counts and eosinophil distribution between ponies infected with S. vulgaris and those raised in a parasite-free environment. Experimental infection with S. vulgaris, with or without subsequent anthelmintic treatment, did not change eosinophil counts, and counts were similar to those for horses from the general population. CONCLUSIONS: Migration of eosinophils to the equine large intestinal mucosa appears to be independent of exposure to parasites. Large intestinal mucosal eosinophils may have more functions in addition to their role in defence against parasites.

Age-related morphometry of equine calcified cartilage.

Although there are many studies in the equine literature focused on articular diseases and the aetiology of osteoarthritis, few have concentrated on normal articular structures and how they change with age. The objective of this investigation was to study the thickness and morphology of the calcified cartilage layer of the distal metacarpus over a range of ages. A parasagittal slab of bone was sectioned from the region of sesamoid contact on the medial condyle of the metacarpi from 34 horses. The slab of bone was preserved, dehydrated and embedded, undecalcified, in methylmethacrylate and then stained with toluidine blue. Six repeatable fields of interest from the distal aspect of each metacarpus were digitised and examined to determine the morphology of the calcified cartilage layer. The thickness of the calcified cartilage, range 88-426 microm, was estimated using a method of integration. The results indicate an age-related influence on the thickness of the calcified cartilage layer, generally increased in older horses. While this finding is significant, perhaps more importantly a positional relationship was also identified, indicating that pressures endured by different regions within a joint may dictate morphological development of the tissues. This study has begun to lay the groundwork to determine whether the calcified layer of the hyaline cartilage could be involved in the development of osteoarthritis.

Rehabilitation of dogs with surgically treated cranial cruciate ligament-deficient stifles by use of electrical stimulation of muscles.

OBJECTIVE: To determine effect of electrical muscle stimulation (EMS) on rate and degree of return to function of the limb and development of degenerative joint disease (DJD) after surgical creation and subsequent stabilization of the cranial cruciate ligament (CrCL)-deficient stifle. ANIMALS: 12 clinically normal adult large (19.5 to 31.5 kg) dogs. PROCEDURE: Dogs were anesthetized, and the right CrCL was severed via arthrotomy, destabilizing the stifle. After 3 weeks, the stifle was surgically stabilized. Three weeks later, 6 dogs were subjected to an EMS treatment protocol for the thigh muscles. At 5, 9, 13, and 19 weeks after stifle destabilization, treated (n = 6) and control (n = 6) dogs were evaluated for return of stifle function. Gross and histologic evaluations of the stifles were performed at 19 weeks after stifle destabilization. RESULTS: Treated dogs had significantly (P = 0.001) better lameness score than did control dogs. There was less palpable crepitation of the stifle in treated dogs (P = 0.06); treated dogs also had significantly (P = 0.01) fewer radiographic signs of bone changes. Thigh circumference was significantly (P = 0.02) larger in treated dogs. There was less gross cartilage damage (P = 0.07) in the EMS-treated dogs, but more medial meniscal damage (P = 0.058, cranial pole; P = 0.051, caudal pole). CONCLUSIONS: Improved lameness scores, larger thigh circumference, and decreased radiographically apparent bony changes observed for the treated group of dogs support the hypothesis that dogs treated by EMS after surgical stabilization of the CrCL-deficient stifle had improved limb function, with less DJD, than did dogs treated with the currently accepted clinical protocol of cage rest and slow return to normal activity. However, results of force plate evaluation did not support the hypothesis. Increased meniscal damage in dogs treated by EMS may be cause for concern.

Correlation between anatomic features and low-field magnetic resonance imaging of the equine metacarpophalangeal joint.

OBJECTIVE: To expand our current knowledge and to establish limits of correlation between signal intensities of the magnetic resonance (MR) image and actual macroscopic and microscopic anatomic features of the imaged structures of the equine metacarpophalangeal joint (MCPJ). SAMPLE POPULATION: The right MCPJ was obtained from 4 adult horses that were euthanatized for reasons unrelated to the musculoskeletal system. PROCEDURE: The distal portion of the right forelimbs was collected from 4 equine cadavers. The bones were drilled to provide fixed reference points and examined by MR imaging. After imaging, the joints were sectioned for gross and histologic inspection. The MR images were aligned and correlated with digitized gross and histologic images to identify tissue types. RESULTS: Comparison of the images resulted in identification of different bone types, articular cartilage, and soft tissue structures of the equine MCPJ. CONCLUSION: Results provided relevant information regarding the appearance of the imaged tissues of the equine MCPJ. CLINICAL RELEVANCE: Although MR imaging does not have current clinical applications for equine practitioners, its wide acceptance as the imaging modality used for most human musculoskeletal derangements may aid in developing more realistic applications in equine medicine.

Effects of aging on the endodontic system, reserve crown, and roots of equine mandibular cheek teeth.

OBJECTIVE: To document age-related changes in the morphology of the endodontic system, reserve crown, and roots of equine mandibular cheek teeth. DESIGN: Equine mandibular cheek teeth from horses of various ages were compared, using radiography, x-ray computed tomography, and histologic examinations. SAMPLE POPULATION: 48 right hemi-mandibles from horses 2 to 9 years old. PROCEDURE: Hemi-mandibles were radiographed, imaged by computed tomographic reconstruction, and reformatted. Histologic examination was used to identify and correlate tissue types. RESULTS: Permanent mandibular cheek teeth of the horse, at the time of eruption, consisted of an exposed crown and a reserve crown with a widely dilated apex. The endodontic system consisted of 5 or 6 pulp horns that connected to an expansive pulp in the reserve crown, which was confluent with the primordial pulp bulb surrounding the tooth's apex. At the time of eruption, mandibular cheek teeth did not have a distinct pulp chamber, roots, or evidence of root formation. However, within 2 years after eruption, mesial and distal roots and a pulp chamber were present. A distinct pulp chamber, communicating with the pulp horns and both root pulp canals, was identifiable for 4 to 5 years from the time of root formation. The endodontic system of cheek teeth, 6 to 8 years after eruption, consisted of 2 unattached compartments, made up of a root canal, pulp chamber, and 2 or 3 pulp horns. CLINICAL RELEVANCE: The age-related morphologic changes in equine mandibular cheek teeth have important implications for application of endodontic therapy in horses.

Bone composition and histological analysis of young and aged rats fed diets of varied calcium bioavailability.

OBJECTIVE: The effects of varied calcium bioavailability on bone development (young animal) and bone maintenance (aged animal) were evaluated by the application of bone composition and histologic/histomorphometric techniques. METHODS: Groups of male young (3-4 weeks old) and aged (24-month-old) rats were fed nutritionally-complete but calcium-restricted (0.15%) diets in which calcium was derived from spinach or nonfat dry milk (NFDM). A 0.5% calcium casein-based diet group was included as a comparison at the National Research Council's requirement level. All animals were pair-fed to the spinach-fed group. At the end of 60 days, lumbar vertebrae, tibia and femurs were harvested for bone histomorphometric and bone composition analyses. RESULTS: Histological analysis of the fifth lumbar vertebral body of the young spinach-fed group showed retarded development and mineralization of trabecular bone. Histomorphometry revealed that the spinach-fed group seemed to respond to the lower bioavailable calcium source by reducing the mineralization of bone matrix and stimulating bone resorption with a resultant decrease in bone volume (0.027, 0.059, 0.068 mm3 in spinach, NFDM and 0.5% calcium casein-based diet groups, respectively). The aged animals, however, appeared to respond to the reduction in available dietary calcium by mobilizing skeletal reserves, with a subsequent decrease in bone volume (0.057, 0.060, 0.073 mm3 in spinach, NFDM and 0.5% calcium casein-based diet groups, respectively). CONCLUSION: This study demonstrates that a low bioavailable calcium source, spinach, suppresses calcium deposition in bones of young animals resulting in a reduction in both quality and quantity of bone. Moreover, despite the stable structure of the mature bone, a low bioavailable calcium source may also compromise the skeletal integrity of the aged animal.

Alterations in calcium intake on peak bone mass in the female rat.

This study compared the effect of a calcium deficit or surfeit on bone growth and development in the early phase of peak bone mass attainment with the late phase of peak bone mass attainment using the female Sprague-Dawley rat as a model. Groups of weanling animals were fed one of three nutritionally complete but calcium-altered diets (0.25%, 0.5%, or 1.0% calcium) for 8 weeks. Animals within each diet group were then rerandomized into one of the above diets and fed until 37 weeks of age. Each group contained five rats. In addition, three groups that received the 0.25% calcium diet for the first 8 weeks remained on the diet until week 20 when they were further randomized into one of the three diet groups and fed until 37 weeks of age. Results of this experiment indicate that increasing the calcium intake after adolescence (12-weeks-old) of those female rats consuming a low calcium diet will not substantially alter the adult bone volume of the metaphyseal region of the proximal tibia. Further, low calcium intakes through adolescence retard and prolong longitudinal bone growth. In contrast, however, those rats fed a diet providing calcium either at (0.5%) or twice the National Research Council's requirement level through adolescence had greater tibial bone volume as an adult when fed diets containing 1.0% calcium after this time period. It appears that the mechanism for this increase involves both a protection from resorption and an increase in bone formation/mineralization. This study is the first to show that low calcium intakes through adolescence have a nonreversible, deleterious effect on peak bone mass, whereas higher intakes promote greater peak bone mass and provide potential protection from age-related bone loss.

Development of a teaching model for surgical endodontic access sites in the dog.

A dolicocephalic canine skull was used to develop a teaching and study model for surgical endodontic access sites of the maxillary and mandibular canine teeth, the maxillary fourth premolar and the mandibular first molar teeth. Coronal endodontic access sites were created, and endodontic files were inserted through the access preparation into the root canal system. The teeth were radiographed to determine the ideal surgical access points. Barium markers were placed over these points and each tooth was radiographed again to confirm the appropriate location of the marker. Surgical accesses to the apices were created by drilling through the barium markers and underlying bone. Following localization of the apices, apicoectomies were performed, exposing the apical portion of the pulp canal. The maxillary and mandibular canine teeth, the maxillary fourth premolar tooth, and the mandibular first molar tooth and surrounding bone were harvested from a similar dolicocephalic dog following euthanasia. The specimens were embedded in methylmethacrylate, sectioned, and mounted on slides to reveal the structures located around the surgical access sites.

Histological appearance of naturally occurring canine physeal fractures.

The Salter-Harris classification system is widely used to describe the anatomical appearance of and predict the prognosis for physeal fractures in canine clinical patients. Salter and Harris classified experimentally induced physeal fractures on radiographic and histological appearance, however, the good prognosis afforded Salter-Harris type I and II fractures in experimental animals has been questioned for the canine patient. Twelve naturally occurring physeal fractures from five traumatized dogs, who were euthanatized at the request of their owners, and one resected femoral head were examined histologically. Ten of the 13 physeal fractures disrupted the cells in the proliferative zone. The histological appearance of growth plate disruption in the injured animal correlates more closely with the clinical observations of growth retardation than the experimental observation of continued growth after fracture through the hypertrophic zone. The results of this study indicated that considerable damage to the physeal cartilage occurred during the traumatic incident in most of these clinical animal patients.

Organoapatites: materials for artificial bone. III. Biological testing.

This article reports on the in vivo testing of new artificial bone materials we have termed "organoapatites." These materials consist of mineral networks in which organic polymers are intimately dispersed by nucleation and growth of apatite crystals from a mother liquor containing the organic substances. Organoapatites were tested as implants in adult canine cortical bone for periods in the range from 12-35 weeks and fluorochromes were used in the model to investigate the kinetics of bone growth or repair. The analysis of histological samples was carried out using histomorphometric methods as well as fluorescence microscopy. Results showed excellent apposition of poly(amino acid) organoapatites with mineralized bone and fibrous encapsulation when a synthetic polyelectrolyte was the only organic component. This observation suggests that the molecularly dispersed organic dopant amounting to only 2-3% by weight of the microstructure can play a critical role in the tissue response to the implant. Relative to apatite controls, organoapatites were also found to have greater resistance to fragmentation in vivo and those containing amino acid units revealed interfacial bioerosion accompanied by regeneration of mineralized tissue. Design of organoapatite compositions and microstructures may therefore be useful in achieving the specific rate of biological response which is clinically desired.

Intraosseous pressure and pathologic changes in horses with navicular disease.

Navicular bone intraosseous pressure, gross pathologic, histologic, and histochemical data were collected from 8 horses with navicular disease and 4 control horses. Simultaneous navicular bone intraosseous, medial palmar arterial, and saphenous venous pressures were measured for the left and right forelimbs of each horse under general anesthesia. Gross pathologic evaluation included grading of changes on the flexor surface of the navicular bone. Safranin-O-fast green-stained sections were used for histologic-histochemical grading of the hyaline articular and fibrocartilage surfaces of the navicular bones. Hematoxylin and eosin-stained sections were used for morphologic evaluation of the marrow spaces of navicular bones. Mean navicular bone intraosseous pressure for horses with navicular disease was significantly (P < 0.001) higher than that for controls. Differences in medical palmar arterial or saphenous venous pressures were not significant between groups. The median flexor surface gross pathologic and histologic-histochemical fibrocartilage scores for horses with navicular disease were significantly (P < 0.001) more severe than those for control horses. The histologic-histochemical hyaline cartilage scores for control horses and those for horses with navicular disease were not significantly different. Fibrosis of the marrow spaces beneath the flexor cortex of horses with navicular disease was more pronounced than that of control horses.

Evaluation of canine cortical bone graft remodeling.

A stable cortical bone fracture model was developed to evaluate the remodeling rate of cortical bone grafts. Samples of cortical bone were harvested with a trephine and press fit into predrilled holes in the femoral diaphyses of four live dogs. The percentages of new bone, unremodeled graft bone, porosity, forming bone surface area, and resorbing bone surface area were determined morphometrically and compared in cortical autografts, cortical allografts sterilized with 84% ethylene oxide (EO), and allografts sterilized with 12% EO. The host-graft interfaces healed without formation of fibrous tissue or cartilage, indicating a stable fracture surface. The amount of new bone formed in cortical autografts and allografts sterilized with 84% EO was significantly greater than the amount of new bone in allografts sterilized with 12% EO. There was no significant difference between the amounts of new bone formed in the allografts sterilized with 84% EO and the cortical autografts. No significant differences were detected in percentages of porosity or bone surface areas.

Bone composition and histology of young growing rats fed diets of varied calcium bioavailability: spinach, nonfat dry milk, or calcium carbonate added to casein.

Bone composition and histology were evaluated in young growing rats fed nutritionally complete but calcium-restricted (0.15%) diets in which calcium was derived from spinach, nonfat dry milk (NFDM), or CaCO3 added to casein. Groups of male weanling rats were pair-fed for 28 d. A 0.5% calcium casein-based diet group fed ad libitum was included to provide a comparison of normal bone structure and composition. Bone growth and bone ash were depressed in spinach-fed rats. Total bone tibia calcium in 0.5% calcium casein-based, 0.15% calcium casein-based, NFDM and spinach diet groups were 64.0, 29.2, 30.7 and 13.8 mg, respectively. All other measured bone mineral levels were also lower, except for potassium. Femur hydroxyproline concentrations were 1.2, 1.6, 1.6 and 2.1% in 0.5% calcium casein-based, 0.15% calcium caseinbased, NFDM and spinach diet groups, respectively. Bone histomorphometry indicated gross underdevelopment and compromised mineralization of trabecular bone of spinach-fed rats. For the first time, it has been demonstrated with histologic techniques that calcium from the low bioavailable source, spinach, compromises both the quantity and quality of bone. In contrast, when calcium is fed to growing animals at levels below the National Research Council requirement but from a highly bioavailable source (i.e., NFDM and CaCO3), there is only a reduction in bone quantity.

Radiographic and histologic effects of chondroitinase ABC on normal canine lumbar intervertebral disc.

Twelve dogs were divided into two equal groups and given lumbar intradiscal injections of 10, 50, or 100 U/ml of chondroitinase ABC reconstituted in sodium acetate buffer. Radiographs of the lumbar spine were made before and after surgery in both groups. Additional films were made at 5 days after surgery in Group I and at 7, 14, and 21 days after surgery in Group II. All spaces injected with 50 or 100 U/ml chondroitinase ABC demonstrated significant radiographic narrowing in both groups compared with uninjected control and buffer injected discs (P less than 0.001). Discs injected with 10 U/ml of chondroitinase ABC showed increased narrowing over time from 7 to 21 days (P less than 0.05). A zone of safranin O depletion was present in the ventral anulus fibrosus adjacent to the nucleus pulposus in all treated discs, indicating proteoglycan loss. All histologic effects of chondroitinase ABC were confined to intervertebral disc tissues. Chondroitinase ABC appears to be effective for chemonucleolysis in dogs.

Lymphatic drainage of epidural space in rabbits.

Epidural lymphatic drainage was examined using epidural catheterization and an infusion of india ink. The india ink was drained quickly through a connecting lymphatic vessel from the nerve root foramen to the anterior lumbar lymphatic vessels. Many kinds of epidurally administered agents may be drained in this manner.

The effects of chondroitinase ABC on the rabbit intervertebral disc. A roentgenographic and histologic study.

A series of intradiscal injections of chondroitinase ABC was performed in 31 young adult rabbits. Roentgenograms were taken immediately postinjection and were repeated at two, three, five, six, seven, or nine days after injection. All had roentgenographic evidence of disc space narrowing at all stages after the injection. Histologic evaluation of safranin-O fast green-stained sections indicated degradation of proteoglycans in the annulus fibrosus of chondroitinase ABC-injected discs. The cells of the chondroitinase ABC-injected discs, the end plates, and the growth-plate areas of adjacent vertebral bodies appeared unaffected.

Canine intervertebral disks: correlation of anatomy and MR imaging.

Because the structure, maturation, and degeneration of canine intervertebral disks resemble those of humans, the authors developed a model of acute intervertebral disk degeneration in dogs. Herniated disks of 18 dogs were examined with magnetic resonance (MR) imaging and then sectioned with a freezing microtome. On the basis of their morphologic appearance in the freezing microtome sections, most of the lumbar intervertebral disks could be categorized as one of six types. Each type has characteristic features and a distinctive appearance on MR images. Findings of this study--including decreased signal intensity from the intervertebral disk, altered signal intensity in contiguous bone marrow, bulging of the anulus fibrosus, herniation of the nucleus pulposus, and contrast enhancement after intravenous administration of gadolinium diethylenetriaminepentaacetic acid (DTPA)--resemble observations from human clinical studies. Classification of degenerating disks and identification of MR imaging characteristics of each type may improve the interpretation of MR images and recognition of early disk degeneration in humans.

Lysosomal changes in renal proximal tubular epithelial cells of male Sprague Dawley rats following decalin exposure.

A histochemical stain for acid phosphatase served as a marker for lysosomal alterations in renal tubular cells associated with male rat hyaline droplet nephropathy. Morphometric analysis and quantitative histochemistry were used to compare the size and acid phosphatase stain reaction of lysosomes in tubular epithelial cells of treated and control animals. Decalin exposure increased the size and significantly (p less than 0.01) reduced the acid phosphatase stain intensity of individual lysosomes. However, there was no significant different (p greater than 0.05) between the acid phosphatase stain intensity of treated and control animals when analyzed on a whole cell basis. The increase in size of the lysosomes without a proportional increase in whole cell acid phosphatase stain intensity indicates a dilution or a failure to accommodate in the acid phosphatase concentration (stain intensity/microns 2) per lysosome. All acid phosphatase stain reaction product was contained within intact lysosomes, mitigating against the hypothesis of lysosomal enzyme leakage as the cause of cell death in decalin-induced alpha 2U globulin nephropathy.

A new method for preparation of undecalcified bone sections.

A new method for preparation of sections of undecalcified bone is described. Samples of ovine bone were embedded in methylmethacrylate and thick-sectioned with a cutoff machine or commercial band saw. Composite slides were prepared by gluing white acrylic to glass using cyanoacrylate glue. Bone sections were glued to the composite slide and then surface polished by grinding or ultramilling. The polished surface of the section was then etched and stained. The techniques described in this paper reduce the time spent grinding or milling sections and improve resolution of surface-stained features of undecalcified bone sections.