Filamentous Structure of Hard ß-Keratins in the Epidermal Appendages of Birds and Reptiles.

The structures of avian and reptilian epidermal appendages, such as feathers, claws and scales, have been modelled using X-ray diffraction and electron microscopy data, combined with sequence analyses. In most cases, a family of closely related molecules makes up the bulk of the appendage, and each of these molecules contains a central ß-rich 34-residue segment, which has been identified as the principal component of the framework of the 3.4 nm diameter filaments. The N- and C-terminal segments form the matrix component of the filament/matrix complex. The 34-residue ß-rich central domains occur in pairs, related by either a parallel dyad or a perpendicular dyad axis, and form a ß-sandwich stabilized by apolar interactions. They are also twisted in a right-handed manner. In feather, the filaments are packed into small sheets and it is possible to determine their likely orientation within the sheets from the low-angle X-ray diffraction data. The physical properties of the various epidermal appendages can be related to the amino acid sequence and composition of defined molecular segments characteristic of the chains concerned.

Ability of hydroxypropyl chitosan nail lacquer to protect against dermatophyte nail infection.

The development of a topical agent that would strengthen the nail, improve the natural barrier, and provide better drug penetration to the nail bed is needed. In this study, we examined the effects of a hydroxypropyl chitosan (HPCH)-based nail solution using a bovine hoof model. Following application of the nail solution, changes in the hardness of the hoof samples were measured using the Vickers method. Tensile and flexural strengths were tested by stretching or punching the samples, respectively. The ultrastructure was examined using scanning electron microscopy (SEM), and samples stained with periodic acid-Schiff (PAS) stain were used to determine the fungal penetration depth. The comparators included 40% urea and 70% isopropyl alcohol solutions. The HPCH nail solution increased hoof sample hardness in comparison to the untreated control sample (mean, 22.3 versus 19.4 Vickers pyramid number [HV]). Similarly, the HPCH solution increased the tensile strength (mean, 33.07 versus 28.42 MPa) and flexural strength (mean, 183.79 versus 181.20 MPa) compared to the untreated control. In contrast, the comparators had adverse effects on hardness and strength. SEM showed that the HPCH solution reduced the area of sample crumbling following abrasion compared to the untreated control (7,418 versus 17,843 pixels), and the PAS-stained images showed that the HPCH solution reduced penetration of the dermatophyte hyphae (e.g., penetration by Trichophyton mentagrophytes was <25 µm at day 9 versus 275 µm in the untreated control). Unlike chemicals normally used in cosmetic treatments, repeated application of the HPCH nail solution may help prevent the establishment of new or recurring fungal nail infection.

Scanning electron microscopy and fungal culture of hoof horn from horses suffering from onychomycosis.

Horn samples were taken from the hooves of eight horses with clinical signs of equine onychomycosis in at least one hoof capsule. None of the horses had a documented mycological history. The predominant alterations of the horn capsules were sand cracks, white line disease, brittleness (especially around the nail holes), parakeratosis and bruising. The horn samples were stored in sterile tubes for transportation and transferred onto Sabouraud Dextrose Agar and dermatophyte test agar for mycological examination within 6 h. Fungal cultures were incubated for 30 days at room temperature. Fungal identification was based on colonial morphology and microscopic examination of conidia. Horn samples were also stored at -80°C until used for scanning electron microscopy (SEM). The fungal culture revealed that the hoof horn from all eight horses was infected with keratinophilic fungi. The keratinopathogenic fungi Trichophyton spp and Scopulariopsis brevicaulis were also detected in six horses. SEM revealed severe alterations of the horn structure in horn samples infected with keratinopathogenic fungi compared to horn samples from a sound hoof. The most evident changes were deterioration of the tubular structure of the horn wall, disruption of the horny layers, superficial lysis of cornified cells and the presence of fungal elements. Samples without dermatophyte or Scopulariopsis infection, in contrast, were similar to healthy hoof horn.

Carbohydrate alimentary overload laminitis.

In acute laminitis, the suspensory apparatus of the distal phalanx fails at the lamellar dermal/epidermal interface. A grading system for the histopathology of laminitis is based on the consistent pattern of histologic changes to the secondary epidermal lamellae, basal cells, and basement membrane that occur as carbohydrate-induced laminitis develops. The actual trigger factors of carbohydrate-induced laminitis remain unidentified.

Different polypeptides form the intermediate filaments in bovine hoof and esophageal epithelium and in aortic endothelium.

Polypeptides that form 10-nm filaments in vitro were isolated from three different bovine tissues: the viable portion of the hoof epithelium, the epithelium of the esophagus, and cultured endothelial cells derived from aorta. The seven polypeptides from hoof, the two from esophagus, and the one from endothelial cells were different with respect to mobility in SDS polyacrylamide gels and/or limited proteolytic digestion. Peptide maps of the different filament-forming polypeptides (FFP's) showed that none of the smaller FFP's was a fragment of any of the larger FFP's. Several isomobile fragments were found in the peptide maps of different FFP's, suggesting that they might contain regions of amino acid sequence homology. We present a hypothesis that suggests how the different 10-nm filament-forming proteins may be related.

Equine laminitis: ultrastructural lesions detected in ponies following hyperinsulinaemia.

REASONS FOR PERFORMING STUDY: Anatomical changes in the hoof lamellar tissue induced by prolonged hyperinsulinaemia have not been described previously. Analysis of the induced lesions may promote understanding of hyperinsulinaemic laminitis pathogenesis and produce clinical benefit. OBJECTIVES: To use light and transmission electron microscopy (TEM) to document hoof lamellar lesions in ponies clinically lame after prolonged hyperinsulinaemia. METHODS: Nine clinically normal, mature ponies were allocated randomly to either a treatment group (n = 5) or control group (n = 4). The treatment group received insulin via a modified, prolonged euglycaemic hyperinsulinaemic clamp technique (EHCT) and were subjected to euthanasia when clinical signs of Obel grade II laminitis occurred. The control group was sham treated with an equivalent volume of 0.9% saline and killed at 72 h. Lamellar tissues of the right front feet were harvested and processed for TEM. RESULTS: Lamellae from insulin treated ponies were attenuated and elongated with many epidermal basal cells (EBC) in mitosis. Unlike carbohydrate induced laminitis in horses there was no global separation at the lamellar dermal/epidermal interface among ponies. Sporadic EBC basement membrane (BM) separation was associated with the proximity of infiltrating leucocytes. In 2 ponies, the lamellar BM was thickened. The number of hemidesmosomes/microm of BM was decreased in all insulin treated ponies. CONCLUSIONS: Prolonged hyperinsulinaemia causes unique lamellar lesions normally characteristic of acute and chronic laminitis. Lamellar proliferation may be an insulin effect through its mitogenic pathway. Aberrant lamellar mitosis may lengthen and weaken the lamellar, distal phalanx attachment apparatus and contribute to the clinical signs that developed. POTENTIAL RELEVANCE: The study shows that insulin alone, in higher than normal circulating concentrations, induces profound, changes in lamellar anatomy. Medical control of insulin resistance and hyperinsulinaemia may ameliorate lesions and produce clinical benefit.

Ultrastructural morphology is distinct among primary progenitor cell isolates from normal, inflamed, and cryopreserved equine hoof tissue and CD105+K14+ progenitor cells.

The equine hoof dermal-epidermal interface requires progenitor cells with distinct characteristics. This study was designed to provide accurate ultrastructural depictions of progenitor cells isolated from inflamed tissue and normal tissue before and after cryopreservation and following selection of cells expressing both keratin (K) 14 (ectodermal) and cluster of differentiation (CD) 105 (mesodermal). Passage 3 cell ultrastructure was assessed following 2D culture and after 3D culture on decellularized hoof tissue scaffolds. Outcome measures included light, transmission electron, and scanning electron microscopy, immunocytochemistry, and CD105+K14+ cell trilineage plasticity. Cells from normal tissue had typical progenitor cell characteristics. Those from inflamed tissue had organelles and morphology consistent with catabolic activities including lysosomes, irregular rough endoplasmic reticulum, and fewer vacuoles and early endosomes than those from normal tissue. Cryopreserved tissue cells appeared apoptotic with an irregular cell membrane covered by cytoplasmic protrusions closely associated with endocytic and exocytic vesicles, chromatin aggregated on the nuclear envelop, abundant, poorly organized rough endoplasmic reticulum, and plentiful lysosomes. Cells that were CD105+K14+ were distinguishable from heterogenous cells by infrequent microvilli on the cell surface, sparse endosomes and vesicles, and desmosomes between cells. Cells expressed ectodermal (K15) and mesodermal (CD105) proteins in 2D and 3D cultures. Inflamed and cryopreserved tissue isolates attached poorly to tissue scaffold while normal tissue cells attached well, but only CD105+K14+ cells produced extracellular matrix after 4 d. The CD105+K14+ cells exhibited osteoblastic, adipocytic, and neurocytic differentiation. Ultrastructural information provided by this study contributes to understanding of equine hoof progenitor cells to predict their potential contributions to tissue maintenance, healing, and damage as well post-implantation behavior.

Equine laminitis: ultrastructural lesions detected 24-30 hours after induction with oligofructose.

REASONS FOR PERFORMING STUDY: The pathology of equine laminitis has been well-documented 48 h after dosing with oligofructose when clinical lameness and lamellar disintegration is well advanced. Further analysis of the earliest lesions, by collecting lamellar samples at the first sign of foot lameness after oligofructose dosing is required in order to increase understanding of the disease. OBJECTIVES: To investigate lamellar epidermal hemidesmosome damage and basement membrane dysadhesion by transmission electron microscopy (TEM). METHODS: Eight clinically normal, mature Standardbred horses were divided randomly into 2 groups of 4. The treatment group were dosed with oligofructose (10 g/kg bwt) and subjected to euthanasia when shifting weight from one foot to other commenced and at the first sign of lameness during walking and turning. This occurred at 24 h in 3 horses and 30 h in one. The sham treatment control group were dosed with water and subjected to euthanasia after 48 h. Lamellar tissues of the front feet were harvested and processed for ultrastructural study using TEM. RESULTS: Examination by TEM showed excessive waviness of the basement membrane zone and pointed tips of some secondary epidermal lamellae, an ultrastructural lesion typical of laminitis. The average number of hemidesmosomes/microm of basement membrane was decreased and their distance from the centre of the lamina densa of the basement membrane was increased. CONCLUSIONS: Laminitis lesions are detectable 24 h after oligofructose administration. POTENTIAL RELEVANCE: Hindgut events occurring in the first 24 h after dosing have begun the destruction of the hoof lamellar interface. Prevention and treatment strategies should precede lameness if they are to be efficacious.

Comparative study of the claws of Pediculus humanus capitis between archaeological and modern specimens.

Metric data of the claws of archaeological specimens of Pediculus humanus capitis (dating between 1500 B.C. and A.D. 1500) and modern lice specimens coming from school children were analyzed and compared. Both sets of samples come from Arica in northern Chile. The overall sample is comprised of 14 archaeological specimens (6 females and 8 males) of Pediculus humanus capitis and 22 modern specimens (13 females and 9 males). All specimens were studied with scanning electron microscopy (SEM), uncoated, using variable pressure mode. The objective of this study was to metrically analyze the first couple of clutches of ancient and modern adult lice specimens (width and length of the tibio-tarsal claw and tarsus length) to test if morphological changes have taken place throughout time in these anatomical elements. We found that archaeological male and female specimens presented significant differences in the tibio-tarsal width (right and left). When comparing data between archaeological and modern male specimens, statistically significant differences were found in almost all the parameters studied, except for the right tarsal length. On the other hand, archaeological and modern female specimens showed no statistically significant change in the variables studied. In brief, our data suggest that modern male specimens have undergone a process of claw reduction, but females have maintained the same dimensions.

Microstructure and Hardness of Buffalo's Hoofs.

The aim of this study was to describe the microstructure of hoof capsules of the buffalo. In addition, the study emphasized the morphometric aspects of the horn tubules, the Vickers nanohardness of the dorsal and abaxial walls and sole of the digits of the thoracic and pelvic limbs of the buffalo. The abaxial wall in the thoracic and pelvic digits showed larger diameter of the horn tubules when compared to all dorsal wall and sole. In addition, the abaxial wall of the thoracic digits showed larger diameter of the horn tubules when compared with the pelvic digits. According to the three-dimensional microtomography, the dorsal wall was higher in density compared with the abaxial wall. The latter exhibited an intermediate density, while the sole showed the lowest density. The Vickers nanohardness test showed that there was no difference in hardness and resistance between the experienced regions. However, the elastic modulus was greater on the transversal section of the hoof capsule. In conclusion, the results of the current study show that modern technologies such as microtomography and subsequent imaging can be used to investigate details of the basic morphology in different regions of the buffalo's hoof.

Microtomographic Parameters and Nanoindentation of the Hoof of Girolando Cattle.

The aim of this study was to describe the microstructure of the pigmented and depigmented hoof capsule of Girolando cattle by bi- and tridimensional microtomography and nanoindentation, analysing the possible relation between these findings and the susceptibility of such animals to podal diseases. To carry out the microtomography and the nanoindentation, duplicated samples were collected from the dorsal wall, abaxial wall and pre-bulbar sole of the hoof capsule. Material collection was performed in 40 medial digits of thoracic limbs and 40 lateral digits of pelvic limbs. The bidimensional microtomography showed that the dorsal wall of the thoracic and pelvic limbs presented higher density, followed by the abaxial wall, and finally by the sole, with the lowest density. Moreover, the hoof capsule of cows of Girolando breed is a compact, non-porous material, and constituted by extratubular and intratubular keratin. By the tridimensional microtomography, it was possible to measure the angles of the corneal tubules in relation to the periople and the claws in the different regions of the hoof capsule, which were 90° for the dorsal wall, 55° for the abaxial wall and 70° for the sole. The tridimensional microtomography also showed corneal tubules of different diameters: 17, 51, 85, 119 and 153 µm. The nanoindentation test, when performed in different regions of the hoof capsule, did not reveal significant difference of Vickers hardness in the evaluated areas. However, we verified a larger elastic module of these regions on the transversal cut of the corneal tubules compared to the longitudinal cut.

Magnetic resonance microscopy of the equine hoof wall: a study of resolution and potential.

REASONS FOR PERFORMING STUDY: Obtaining magnetic resonance images of the inner hoof wall tissue at the microscopic level would enable early accurate diagnosis of laminitis and therefore more effective therapy. OBJECTIVES: To optimise magnetic resonance imaging (MRI) parameters in order to obtain the highest possible resolution of the structures beneath the equine hoof wall. METHODS: Magnetic resonance microscopy (MRM) was performed in front feet from 6 cadaver horses using T2-weighted fast spin echo (FSE-T2), and T1-weighted gradient echo (GRE-T1) sequences. RESULTS: In T2 weighted FSE images most of the stratum medium showed no signal, however the coronary, terminal and sole papillae were visible. The stratum lamellatum was clearly visible and primary epidermal lamellae could be differentiated from dermal lamellae. CONCLUSION: Most structures beneath the hoof wall were differentiated. Conventional scanners for diagnostic MRI in horses are low or high field. However this study used ultra-high field scanners currently not available for clinical use. Signal-to-noise ratio (S/N) increases as a function of field strength. An increase of spatial resolution of the image results in a decreased S/N. S/N can also be improved with better coils and the resolution of high field MRI scanners will increase as technology develops and surface array coils become more readily available. POTENTIAL RELEVANCE: Although MR images with microscopic resolution were obtained ex vivo, this study demonstrates the potential for detection of lamellar pathology as it occurs. Early recognition of the development of laminitis to instigate effective therapy at an earlier stage and may improve the outcome for laminitic horses. Clinical MR is now readily available at 3 T, while 4 T, 7 T and 9 T systems are being used for human whole body applications.

Pododermal angiogenesis and angioadaptation in the bovine claw.

Pododermal microvascularization has been suggested to play a key role in the physiological function of the bovine claw and in the pathogenesis of claw diseases. According to our working hypothesis, angiogenesis plays a central role in the physiological and pathological function of the claw and is induced by the pro-angiogenic vascular endothelial growth factor (VEGF). As a basis for further research, the aim of the present study was to examine the mechanisms of pododermal angiogenesis in the functional adaptation of the microvasculature of the claw in histological serial sections and microcorrosion casts of healthy juvenile and adult claws as well as pathologically altered claws. Scanning electron microscopy of microcorrosion casts allowed assessment of the 3D aspect of pododermal angiogenesis and angioadaptation, and was substantiated by a concomitant examination of a 3D in vitro model of angiogenesis based on cultured bovine microvascular endothelial cells. Particularly in the juvenile, but also in the adult claw, sprouting and intussusceptive angioadaptation was demonstrated and resembled the respective stages of in vitro angiogenesis. Evidence of angiogenic processes was also detected in the pathologically altered claws displaying symptoms of subclinical laminitis and/or the digital dermatitis complex. The detected angioadaptation was visible expression of the increased metabolic demands of the claw caused by the growing body weight load. Angiogenic remodeling of the pododermal angioarchitecture was also the connectional reparative principle in pathologically altered claws. Related research perspectives for prophylaxis and therapy of claw diseases are discussed.

Unilateral basement membrane zone alteration of the regenerated laminar region in equine chronic laminitis.

Between the laminar epidermis and the laminar dermis of laminar region (LR) in equine foot, it can be observed the basement membrane zone (BMZ), which is composed of a basement membrane and its accompaniments like the hemidesmosome and anchoring fibril. Alteration in the BMZ in equine laminitis is possibly related with not only development but also recovery outcome and recurrence of this disease. However, there is little known about the structure of the BMZ during the recovery phase of this disease. To assess the condition of the BMZ of LR affected by chronic laminitis, the tissue was examined in three cases at two weeks, four weeks and three months after the onset of laminitis, using pathological, immunohistochemical and electron microscopic techniques. Histologically in all laminitis cases, there was a regenerated laminar epidermis with proliferating keratinocytes between the Stratum medium and the dermis, but it included the undeveloped secondary epidermal laminae (ud-SELs) structure in one side of the primary epidermal laminae, especially in the part of the deep area of LR. Immunohistochemical results were positive for the anti-type IV collagen, anti-type VII collagen and anti-laminin 5 antibodies in the most BMZs. However, partial BMZs adjacent to the ud-SELs were negative for the anti-type VII collagen and anti-laminin 5 antibodies. Ultrastructurally, in the BMZ of the ud-SEL, the lamina densa and the lamina lucida were present. In contrast, the anchoring fibrils and the hemidesmosomes were either absent, or present at lower than normal levels. In conclusion, the present study indicated that the part of regenerated LR in chronic laminitis was not able to fully restore to construct the BMZ for a long time, especially in the unilateral side of laminar epidermis. It might be related with recurrence of this disease.

FOXN1 is critical for onycholemmal terminal differentiation in nude (Foxn1) mice.

Nude mice have a mutation in the transcription factor Foxn1(nu), resulting in downregulation of hair keratins. Although hair follicles develop normally, the hair fibers become structurally weak, curl, and break off at the surface. Nails in nude mice are deformed, based on alterations of the onychocyte differentiation process. Elemental microanalysis of the nail plate reveals marked decreases in sulfur concentrations in the nude mouse nail plates. Immunohistochemistry shows a lack of keratin 1 expression in terminally differentiating keratinocytes of the nail matrix. Instead, the typical differentiation process of the matrix is altered toward an epidermis-like differentiation pattern, comprising the production of filaggrin-containing keratohyalin granules in cells resembling those of the stratum granulosum, which are never observed in normally haired mice. The nail plate has diffuse basophilic stippling. It is thinner than normal, weak, and in most Foxn1(nu)/Foxn1(nu) mice breaks where it separates from the hyponychium. These studies indicate that the Foxn1(nu) mutated gene has effects beyond downregulating keratin expression, including changes in filaggrin expression, and is critical for normal onycholemmal differentiation. The nails of nude mice provide new insights into the molecular controls of onychocyte differentiation, and they offer a useful model to investigate the pathogenesis of nail hypergranulosis, a common feature in human nail diseases.

Fetal development of the segment-specific papillary body in the equine hoof.

Fetal development of the unique papillary body and its localized peculiarities in the equine hoof are described based on the study of 51 fetuses, nine newborn foals, and five adult horses. The shape and dimensions of the dermal papillae and lamellae have a formative influence on the structure and physical quality of the corneous hoof capsule with its horn tubules and lamellae. The size and arrangement of these horn structures determine the mechanical quality of hoof horn. Proper horn quality is a prerequisite for the various functions of the hoof capsule, such as protecting the living dermis supporting the hoof capsule, shock absorption, and formation of the suspensory apparatus of the distal phalanx. Development of the segment-specific papillary body is initiated by the increasing mitotic activity of the epidermal cells invaginating the dermal surface, thus forming dermal microridges. These microridges are transformed into single dermal papillae, which are arranged in rows, or enlarged to become primary and secondary dermal lamellae. The formation of a segment-specific papillary body enables the increasing keratinization ratio in the hoof epidermis and the formation of the characteristic tubular and lamellar horn responsible for the special mechanical properties of hoof horn.

The basement membrane at the equine hoof dermal epidermal junction.

In the equine hoof, the basement membrane connects the heavily keratinised hoof wall to the dense connective tissue of the distal phalanx, a region able to withstand considerable mechanical stress. This study investigated the properties of this important anatomical and physiological structure. In contrast to haematoxylin and eosin, the connective tissue stains, periodic acid Schiff, periodic acid silver methenamine and Azan showed good resolution of lamellar basement membrane. The lamellar basement membrane cross-reacted with mouse monoclonal antibodies raised against human laminin, thereby providing evidence that laminin is a component of the equine basement membrane. The ultrastructure of the equine hoof basement membrane was essentially the same as in other animals but appeared to have many anchoring fibrils and extensions of the lamina densa into the adjoining connective tissue, an arrangement interpreted to convey extra strength to the region. Large areas of the surface of the hoof wall basement membrane could be exposed to examination with the scanning electron microscope by treating tissue blocks with detergent/enzyme or sodium bromide. When epidermal lamellae were separated from their dermal counterparts the basement membrane stayed with the dermis and the dermal lamellae retained their natural shape despite the absence of an adjacent epidermis. The exposed surface of the lamellar basement membrane was generally smooth and unbroken, marked with small indentations and fine wrinkles. At the cut edges of the lamellae, a mesh of fine connective tissue fibres were attached to the inner surface of the basement membrane. The basement membrane of both toh coronary and terminal papillae was folded into numerous longitudinal ridges, all parallel to the long axis of the papillae.(ABSTRACT TRUNCATED AT 250 WORDS)

Basement membrane pathology: a feature of acute equine laminitis.

Thirty-two dorsal, mid-hoof wall, lamellar sections from 8 Standardbred horses, humanely killed 48 h after the administration of an alimentary carbohydrate overload, were sectioned and examined by light microscopy. Sections were stained with the connective tissue and basement membrane stains periodic acid-Schiff (PAS), Azan and periodic acid silver methanamine (PASM) and with routine haematoxylin and eosin (H&E). Lesions of the epidermal lamellae, attributable to laminitis, were graded in order of increasing severity from Grade N (normal), Grade 1 (mild), Grade 2 (moderate) to Grade 3 (severe and extensive). The grading system was based principally on changes to lamellar basement membrane (BM) which were clearly visible when the connective tissue stains PAS and PASM were used. Earliest changes were rounding of the basal cell nuclei and elongation of secondary epidermal lamellae (SELs). Secondary epidermal lamellae tips were pointed instead of round and the basement membrane had separated from the lamellae. In early Grade 1 lesions, this was obvious at the tips of the SELs where the BM had lifted to form teat-shaped bubbles. The absence of BM at the tips of secondary dermal lamellae, along with varying amounts of connective tissue, was considered a progression in severity and classified as Grade 2. Eventually, even the primary epidermal and primary dermal lamellae separated from each other and the empty shells of isolated BM, in what was once the tip of the primary epidermal lamella, signified that a global separation of the epidermal and dermal lamellae had occurred (Grade 3 lesion). The histopathological grading system correlated well with the degree of lameness at the time of euthanasia, (r2 = 0.94) and apparently described the severity of laminitis accurately. Disintegration of the BM and failure of its attachment to the basal cells of the epidermis appears to be one of the earliest pathological events to occur in acute laminitis and could be the change that initiates the collapse of the lamellar architecture. Histopathological diagnoses of laminitis are strengthened when based on sections stained with at least PAS, in addition to routine H&E and should exhibit evidence of the BM pathology described here.

Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study.

REASONS FOR PERFORMING STUDY: Light microscopical studies show that the key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. More precise knowledge of the damage occurring in the lamellar basement membrane zone may result if laminitis affected tissue is examined with the transmission electron microscope. This could lead to better understanding of the pathogenesis of lesions and the means of treatment or prevention. OBJECTIVES: To investigate the ultrastructure of acute laminitis as disease of greater severity is induced by increasing oligofructose (OF) dosage. METHODS: Three pairs of normal horses, dosed with OF at 7.5, 10 and 12.5 g/kg bwt via nasogastric intubation, developed laminitis 48 h later. Following euthanasia, their forefeet were processed for transmission electron microscopy. Lamellar basal cell hemidesmosome (HD) numbers and the distance between the basal cell plasmalemma and the lamina densa of the basement membrane were estimated and compared to control tissue. RESULTS: Increasing OF dosage caused greater HD loss and more severe laminitis. The characteristic separation of the basement membrane, cytoskeleton failure and rounded basal cell nuclei results from combined HD dysassembly and anchoring filament failure. CONCLUSIONS: Without properly assembled HDs, dysadhesion between the lamina densa of the basement membrane (BM) and epidermal basal cells occurs, emphasising the fundamental importance of HDs in maintaining attachment at the lamellar interface. Medical conditions that trigger lamellar matrix metalloproteinase (MMP) activation and/or compromise entry of glucose into lamellar basal cells appear to promote loss and failure of HDs and, therefore, laminitis development. POTENTIAL RELEVANCE: A correlation between lameness severity and escalating loss of lamellar HDs now exists. Therapy aimed at protecting the lamellar environment from haematogenous delivery of MMP activators or from glucose deprivation may control laminitis development.

Equine laminitis: glucose deprivation and MMP activation induce dermo-epidermal separation in vitro.

REASONS FOR PERFORMING STUDY: Acute laminitis is characterised by hoof lamellar dermal-epidermal separation at the basement membrane (BM) zone. Hoof lamellar explants cultured in vitro can also be made to separate at the basement membrane zone and investigating how this occurs may give insight into the poorly understood pathophysiology of laminitis. OBJECTIVES: To investigate why glucose deprivation and metalloproteinase (MMP) activation in cultured lamellar explants leads to dermo-epidermal separation. METHODS: Explants, cultured without glucose or with the MMP activator p-amino-phenol-mercuric acetate (APMA), were subjected to tension and processed for transmission electron microscopy (TEM). RESULTS: Without glucose, or with APMA, explants under tension separated at the dermo-epidermal junction. This in vitro separation occurred via 2 different ultrastructural processes. Lack of glucose reduced hemidesmosomes (HDs) numbers until they disappeared and the basal cell cytoskeleton collapsed. Anchoring filaments (AFs), connecting the basal cell plasmalemma to the BM, were unaffected although they failed under tension. APMA activation of constituent lamellar MMPs did not affect HDs but caused AFs to disappear, also leading to dermo-epidermal separation under tension. CONCLUSIONS: Natural laminitis may occur in situations where glucose uptake by lamellar basal cells is compromised (e.g. equine Cushing's disease, obesity, hyperlipaemia, ischaemia and septicaemia) or when lamellar MMPs are activated (alimentary carbohydrate overload). POTENTIAL RELEVANCE: Therapies designed to facilitate peripheral glucose uptake and inhibit lamellar MMP activation may prevent or ameliorate laminitis.