Morphological changes to endothelial and interstitial cells and to the extra-cellular matrix in canine myxomatous mitral valve disease (endocardiosis).

Morphological and functional changes in endothelial and interstitial cells are considered central to myxomatous degeneration of the canine mitral valve (endocardiosis). The aim of this study was to describe and quantify changes in valve endothelial cells (VECs), interstitial cells (VICs) and the extra-cellular matrix (ECM) of the sub-endothelial zone of diseased valves using a combination of transmission electron microscopy, stereology and computer-aided image analysis. Marked degradation of the endothelium was evident in diseased valves, which coincided with significant degradation of the local ECM (P<0.001). There were decreases and increases in the numbers of VECs and VICs, respectively, in diseased valves, with particular accumulation of VICs subjacent to the valve surface (P<0.01). Overall, VICs were more pleomorphic than VECs in both normal and diseased valves, but for VECs, the degree of pleomorphism was significantly different in diseased valves (P<0.0001). The findings of the study confirm that canine myxomatous mitral valve disease is associated with marked endothelial damage, with attendant proliferation of subjacent activated myofibroblasts. The fact that similar endothelial changes are present in normal valves suggests these processes not only contribute to valve pathology, but may also represent life-long valve remodelling.

The betaine-GABA transporter (BGT1, slc6a12) is predominantly expressed in the liver and at lower levels in the kidneys and at the brain surface.

The Na(+)- and Cl(-)-dependent GABA-betaine transporter (BGT1) has received attention mostly as a protector against osmolarity changes in the kidney and as a potential controller of the neurotransmitter GABA in the brain. Nevertheless, the cellular distribution of BGT1, and its physiological importance, is not fully understood. Here we have quantified mRNA levels using TaqMan real-time PCR, produced a number of BGT1 antibodies, and used these to study BGT1 distribution in mice. BGT1 (protein and mRNA) is predominantly expressed in the liver (sinusoidal hepatocyte plasma membranes) and not in the endothelium. BGT1 is also present in the renal medulla, where it localizes to the basolateral membranes of collecting ducts (particularly at the papilla tip) and the thick ascending limbs of Henle. There is some BGT1 in the leptomeninges, but brain parenchyma, brain blood vessels, ependymal cells, the renal cortex, and the intestine are virtually BGT1 deficient in 1- to 3-mo-old mice. Labeling specificity was assured by processing tissue from BGT1-deficient littermates in parallel as negative controls. Addition of 2.5% sodium chloride to the drinking water for 48 h induced a two- to threefold upregulation of BGT1, tonicity-responsive enhancer binding protein, and sodium-myo-inositol cotransporter 1 (slc5a3) in the renal medulla, but not in the brain and barely in the liver. BGT1-deficient and wild-type mice appeared to tolerate the salt treatment equally well, possibly because betaine is one of several osmolytes. In conclusion, this study suggests that BGT1 plays its main role in the liver, thereby complementing other betaine-transporting carrier proteins (e.g., slc6a20) that are predominantly expressed in the small intestine or kidney rather than the liver.

Donkey dental anatomy. Part 2: Histological and scanning electron microscopic examinations.

Ten normal cheek teeth (CT) were extracted at post mortem from donkeys that died or were euthanased for humane reasons. Decalcified histology was performed on three sections (sub-occlusal, mid-tooth and pre-apical) of each tooth, and undecalcified histology undertaken on sub-occlusal sections of the same teeth. The normal histological anatomy of primary, regular and irregular secondary dentine was found to be similar to that of the horse, with no tertiary dentine present. Undecalcified histology demonstrated the normal enamel histology, including the presence of enamel spindles. Scanning electron microscopy was performed on mid-tooth sections of five maxillary CT, five mandibular CT and two incisors. The ultrastructural anatomy of primary and secondary dentine, and equine enamel types-1, -2 and -3 (as described in horses) were identified in donkey teeth. Histological and ultrastructural donkey dental anatomy was found to be very similar to equine dental anatomy with only a few quantitative differences observed.

Donkey dental anatomy. Part 1: Gross and computed axial tomography examinations.

Post-mortem examination of 19 donkey skulls showed that donkeys have a greater degree of anisognathia (27% width difference between upper and lower jaws) compared to horses (23%). Teeth (n=108) were collected from 14 skulls and examined grossly and by computed axial tomography (CAT). A greater degree of peripheral enamel infolding was found in mandibular cheek teeth (CT) compared to maxillary CT (P<0.001). A significant increase in peripheral cementum from the apical region to the clinical crown was demonstrated in all CT (P<0.0001). All donkey CT had at least five pulp cavities with six pulp cavities present in the 06s and 11s. A new endodontic numbering system for equid CT has been proposed. A greater occlusal depth of secondary dentine (mm) was present in older donkeys (>16 years) than in the younger (<15 years) donkeys studied. Based on gross and CAT examinations, donkey dental anatomy was shown to be largely similar to that described in horses.

Use of a topical disinfectant as part of a hoof care programme for horses with diseases of the hoof capsule.

Twenty-three horses with persistent hoof horn defects were treated topically with a hoof disinfectant as part of a hoof care programme for a year. The active ingredients of the disinfectant were a poloaximer-iodine complex, ethylenediamine dihydriodide, isopropyl alcohol and propylene glycol. Hoof trimmings were taken at the start of the study and every six weeks, and examined by scanning and transmission electron microscopy. At the beginning of the study all the horn samples contained large numbers of bacteria, and samples from eight of the horses also had fungal hyphae intermingled with the bacteria. After the application of the hoof disinfectant and adjustments to their diet, there were rapid improvements in the gross appearance of the feet of all the horses; some of them improved within two to three weeks and by 12 weeks the horn quality of all the horses had greatly improved.

Structure of peripheral cementum of normal equine cheek teeth.

The structure of peripheral cementum of mandibular and maxillary cheek teeth was studied by gross observation, light microscopy, and scanning and transmission electron microscopy. Teeth from four horses aged 4, 8, 17, and 30-years-old were examined. The cementum of the occlusal surface was thicker on the buccal aspect when compared with the lingual aspect of mandibular teeth. The cementum of the occlusal surface was thicker on the palatal aspect when compared with the buccal aspect of maxillary teeth. There was no peripheral cementum on the interdental aspects of either mandibular or maxillary cheek teeth. In the adult tooth, cementum covered the crown and root surfaces. The peripheral cementum of the gingival region showed a dramatic increase in thickness compared with cementum covering the alveolar portion of the embedded tooth. At a microscopic level, three layers (primary, secondary, and tertiary) were identified within cementum. The increase in cementum in the gingival region was of tertiary cementum. All three layers contained vital cementocytes. There was a well-developed vascular system within the cementum. Nerve bundles were also present. Three cementoblast profiles were identified at the junction of the cementum with the periodontal ligament. The results reported in this paper show that cementum is a dynamic vital tissue. These findings should contribute to a greater understanding of the etiology and pathogenesis of cemental caries and periodontal disease in the horse.

Hypertonic activation of the renal betaine/GABA transporter is microtubule dependent.

BACKGROUND: Epithelial cells in the renal inner medulla accumulate osmolytes such as betaine to maintain normal cell volume during prolonged extracellular hypertonic stress. Betaine accumulation is the result of activation of transcription of the BGT1 transporter gene followed by increased betaine transport. METHODS: We studied the possible role of microtubules in this adaptive mechanism using renal cells in culture. RESULTS.: In cultured renal cell lines [Madin-Darby canine kidney (MDCK) and mouse inner medullary collecting duct (mIMCD-3)], up-regulation of BGT1 activity was maximal after 24 to 30 hours in growth medium made hypertonic (510 mOsm/kg) by the addition of sucrose or NaCl. Up-regulation was reversed within 24 to 36 hours after returning cells to isotonic medium. Both cycloheximide (20 micromol/L) and nocodazole (20 micromol/L) blocked the hypertonic up-regulation of BGT1. Nocodazole was partially effective even when added 16 to 20 hours after the switch to hypertonic medium. Recovery from nocodazole action was rapid, and there was full activation of BGT1 transport within three to six hours after nocodazole removal, suggesting rapid trafficking to the cell surface once microtubules repolymerized. Hypertonic activation of BGT1 transport was detected in an isolated membrane fraction and was blocked by cycloheximide but not by nocodazole. Confocal microscopy confirmed the increased abundance of BGT1 proteins in the plasma membrane of hypertonic cells and showed that BGT1 remained intracellular during nocodazole treatment. CONCLUSIONS: Hypertonic activation of BGT1 in renal cells requires de novo protein synthesis and microtubule-dependent trafficking of additional transporters to the cell surface. The apparent resistance of membrane BGT1 to nocodazole blockade is likely due to the presence in the membrane fraction of an increased intracellular pool of active BGT1 transporters.

The effect of feeding grass silage in early pregnancy on claw health during first lactation.

Two groups of eight Holstein-Friesian heifers were fed either a grass-silage-based diet (S) or one based on meadow hay supplemented with 1.8 kg/day barley concentrate mix (H) during cubicle housing as young stock (and in early pregnancy). Lameness and claw lesion development were monitored from approximately four weeks before until 20 weeks after first calving. No significant difference was found between S and H for claw conformation or horn growth and wear. Both groups showed net wear immediately after calving. The prevalence of poor locomotion and the extent of lesion development 20 weeks after calving (when they were highest) were significantly (P< 0.05) higher in S than H. It was concluded that feeding grass silage to young stock may deleteriously affect subsequent claw health and that this risk factor requires further study.

Bone adaptation to a polyester fiber anterior cruciate ligament replacement.

A series of polyester fiber ACL implants was studied in ovine stifle joints up to 2 years postimplantation. The implants were linked to the bone-tunnel wall by oriented fibrous tissue. Cross-sections of the tunnels showed bone ingrowth among the implant fibers at 2 years. A human trial of the Apex implant yielded a series of retrievals, some associated with gross bone-tunnel enlargement. There was no evidence of bone ingrowth in the human implants. It was hypothesized that-tunnel enlargement resulted from fretting at the implant-tissue interface in response to cyclic loads in use.

Failure mechanisms of polyester fiber anterior cruciate ligament implants: A human retrieval and laboratory study.

It has been hypothesized that ACL implant failure is often caused by bone impingement in knee extension following malplacement of the tibial tunnel. This study examined polyethylene terephtalate fiber ACL implants retrieved from a clinical study, and, to confirm the hypothesis, also set up a laboratory study intended to duplicate the failure mechanism. SEM and TEM examination of 25 ruptured implants gave details of fiber failure morphology, with shearing into longitudinal fibrils, followed by rupture, when the fibrils burst apart. Cadaver joints were run in a knee simulator, with deliberately impinging ACL implants. SEM examination of implants abraded in the knee in vitro showed identical fiber damage patterns, thus confirming the impingement hypothesis.

Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury.

In renal tubular epithelial cells, oxidant injury results in several metabolic alterations including ATP depletion, decreased Na+K+ATPase activity, and altered intracellular sodium and potassium content. To investigate the recovery of LLC-PK1 cells following oxidant injury and to determine if recovery can be accelerated, we induced oxidant stress in LLC-PK1 cells with 500 microM hydrogen peroxide for 60 min. Identical cohorts of oxidant-stressed cells were incubated in recovery medium without epidermal growth factor (EGF) or recovery medium containing 25 ng EGF per ml. ATP levels, Na+K+ATPase activity in whole cells, Na+K+ATPase activity in disrupted cells, and intracellular sodium and potassium ion content were determined at 0, 5, 24, 48, and 72 h following oxidant injury in each cohort of cells. In oxidant-stressed cells recovering in medium without EGF, ATP levels, Na+K+ATPase activity, and intracellular ion content improved but continued to remain substantially lower than control values at all time points following oxidant stress. In cells recovering in medium with EGF, ATP levels, Na+K+ATPase activity, and the intracellular potassium-to-sodium ratio were significantly higher at nearly all time points than values in cells recovering in medium alone. In cells recovering with added EGF, Na+K+ATPase activity had improved to control levels, whereas ATP levels and intracellular ion content approached control values by 72 h following oxidant stress. We conclude that oxidant-mediated ATP depletion, altered Na+K+ATPase activity, and intracellular ion content remain depressed for several d following oxidant stress and that EGF accelerated recovery of LLC-PK1 cells from oxidant injury.

Differential activation of system A and betaine/GABA transport in MDCK cell membranes by hypertonic stress.

Accumulation of osmolytes by renal cells is due in part to increased uptake via specific transporters. These include amino acid transport system A and the betaine/GABA transporter (BGT1). Transport changes have been characterized using intact cells which makes the intracellular mechanisms difficult to determine. In this study the hypertonic upregulation of system A and BGT1 was studied directly at the membrane level in Madin-Darby canine kidney (MDCK) cells. Both system A and BGT1 transport systems were detected in an isolated membrane fraction containing plasma membranes. System A transport was increased in membranes prepared from cells after 6 h hypertonic stress (449 mosmol/kg) but BGT1 activity was minimal and not different from isotonic controls. The increase in system A was blocked by inhibitors of RNA and protein synthesis. BGT1 transport was induced in membranes prepared after 24 h hypertonicity. At this time system A activity in the membrane fraction remained increased, unlike the downregulation observed in intact MDCK cells. We conclude that differential upregulation of system A and BGT1 by hypertonic stress is due to intrinsic changes in these transporters at the membrane level. In contrast, the downregulation of system A in intact cells when hypertonicity is prolonged for 24 h is likely due to the action of an intracellular repressor that is not present in the isolated membranes.

Claw lesions in dairy cattle: methods for assessment of sole and white line lesions.

Claw lesions are a major cause of lameness in dairy cattle. Analysis of the development of lesions is aided by numerical representation of their significance. Using data from observations on 31 heifers at 9 weeks post-calving, 5 lesion scoring method were compared. These were: (1) number of lesions; (2) severity; (3) adjusted severity; (4) size (measured by a novel technique involving image analysis of distal view photographs) and (5) size multiplied by adjusted severity (combined score). Relationships between scores for sole and white line lesions and between different claws within a cow were investigated. The small size but high clinical significance of severe lesions means that severity must be weighted if combined with size in a score. Sole and white line lesions showed a moderate but significant correlation in terms of severity but none in terms of size. The highest correlation between scores for a single claw (the right hind outer) and the remaining claws was found for adjusted severity of sole lesions.

A permeability barrier in the dorsal wall of the equine hoof capsule.

The permeability barrier in the dorsal wall of the equine hoof capsule was studied by means of horseradish peroxidase (HRP) in 0.9 N saline solution as a water soluble tracer. Section were treated with 3'3'-diaminobenzidine tetrachloride (DAB) and before dissection the quality of the horn of feet from 10 horses was assessed and given a subjective grade as either good or poor. Blocks of tissue from each horse were left in either an oven at 60 degrees C or in water for 2 weeks before treatment in HRP, sectioning and DAB solution. Regions observed were i) outer surface, ii) outermost layers of the horn, iii) cut edge of the outer layer, iv) inner layer of horn, v) cut edge of the inner layer and vi) laminae. Horn deemed to be normal horn and of good 'quality' showed very slight penetration of HRP 3-5 cell layers deep in the outer layer. The cut edge of the outer layer of the wall of the 'normal' horn also showed minimal penetration of HRP through the intercellular spaces. The cut edge of the inner layers of the wall of normal, good quality horn showed penetration of the tracer up to 20 cell layers deep, with HRP in both the intercellular spaces and within the cells. In contrast, sections of horn from horses with brittle feet showed deep cracks in the outer surface into which the HRP had penetrated. Good quality horn showed no change in the position of the permeability barrier after soaking in water for 14 days, but the brittle horn showed an increase in permeability to HRP. In brittle horn, reaction product was seen deep within the section in the intercellular spaces of the intertubular horn only. Placing horn in an oven had no effect on the permeability barrier. The permeability barrier of the dorsal wall of the equine hoof capsule differs with the layer of the wall. Horn considered to be of poor quality had a weaker permeability barrier than horn of good quality.

Hyperosmotic stress up-regulates amino acid transport in vascular endothelial cells.

Cultured vascular endothelial cells take up L-proline by sodium-dependent transport. Cells incubated in medium made hyperosmotic by addition of sucrose showed a dose-dependent increase in Na+/proline cotransport. Studies with alpha-(methylamino)isobutyric acid revealed that the up-regulation was specific for amino acid transport system A. Up-regulation was blocked by actinomycin D and cycloheximide, indicating roles for gene transcription and protein synthesis. Up-regulation was maximum after five to six hours of hyperosmotic treatment, but returned to control levels when osmotic stress was maintained for 24 hours. The decline at 24 hours was accompanied by a significant increase in Na+/gamma-aminobutyric acid cotransport. The activity of this system, which also transports betaine, remained unchanged after just five hours of hyperosmotic stress. Inclusion of betaine in the hyperosmotic medium reduced up-regulation of system A. Na/Pi cotransport also was up-regulated by five hours of hyperosmotic stress. Up-regulation of system A, but not Na/Pi cotransport, was detected in isolated membrane fractions indicating that increased activity of this membrane transport system may be one mechanism by which vascular endothelial cells accumulate amino acids. The amino acids may act as organic osmolytes to help maintain normal cell volume during the early phase of hyperosmotic stress.

A light microscopic and ultrastructural examination of calcified dental tissues of horses: 1. The occlusal surface and enamel thickness.

Gross and microscopic examinations were undertaken on 46 cheek (molar and premolar) and 4 incisor equine teeth that were fractured, or sectioned either with a lathe or diamond saw. Specimens were examined without treatment, after decalcification or acid etching, utilising light, and scanning and transmission electron microscopy. In some horses, the occlusal surface of the teeth were covered with an organic pellicle. The occlusal surface of the underlying equine enamel contained different wear patterns, including polished areas, local fractures, wedge-shaped pits, striations and depressions. Occlusal dentine showed depressions whose depth was related to its occlusal surface area, with larger surface areas having deeper depressions. The thickness of equine enamel varied greatly throughout its folds in the transverse plane, and was thickest in areas where folds were parallel to the long axis of the maxilla and mandible. Enamel thickness remained constant in the longitudinal plane (throughout the length of the tooth). Peripheral enamel was more deeply infolded in lower than in upper cheek teeth and this appeared to compensate for the absence of infundibula (deep, cup-like enamel indentations that are partially filled with cement) in the lower cheek teeth.

A light microscopic and ultrastructural examination of calcified dental tissues of horses: 2. Ultrastructural enamel findings.

Ultrastructural examinations of defined 3 equine enamel types termed equine (Eq.) Types 1, 2 and 3 enamel, according to the transverse appearance of their enamel prisms and the amount and appearance of their interprismatic enamel. Eq. Type 1 enamel contained alternating rows of oval shaped prisms and thick interprismatic enamel plates, and was found adjacent to the amelodentinal junction. Eq. Type 2 enamel consisted of circular, 'keyhole' to 'horseshoe' shaped prisms with little or no interprismatic enamel and was located adjacent to the amelocemental junction. Eq. Type 3 enamel was composed of rounded prisms surrounded by large amounts of interprismatic enamel and was inconsistently present in a thin layer at the amelodentinal and amelocemental junctions. Prism decussation was seen in the thickest peripheral enamel of the upper cheek teeth but was present throughout incisor enamel therefore making incisors highly resistant to cracking. Scanning electron microscopic examination showed enamel crystals to be cylindrical shaped on transverse section; however, on transmission electron microscopic examination these crystals had shapes, ranging from near oval to rectangular and formed small subunits, with crystals diverging from each other at various angles.

A light microscopic and ultrastructural examination of calcified dental tissues of horses: 3. Dentine.

Ultrastructural examinations of equine dentine found that dentinal tubules extended from the amelodentinal junction towards the pulp forming primary curvatures. The number of dentinal tubules/unit area and their diameters increased significantly from the amelodentinal junction towards the pulp cavities, particularly in regular secondary dentine, but irregular secondary (tertiary) dentine contained no dentinal tubules. Dentinal tubules contained odontoblast processes that appeared to extend as far as the amelodentinal junction, but due to iatrogenic loss during specimen preparation, odontoblasts were seldom found in regular secondary dentine. In primary dentine, the dentinal tubules were surrounded by large amounts of peritubular dentine that increased in diameter from the amelodentinal junction towards the junction of primary and secondary dentine. The site of the dentinal tubule within the peritubular dentine varied at different dentinal sites. Peritubular dentine was present in primary dentine only and was surrounded by a thin layer of intertubular dentine. When acid etched, peritubular dentine gave dentine a honeycomb appearance adjacent to the junction of primary and secondary dentine.