Laminitis is a debilitating disease of equidae that has major welfare and economic consequences for the equine industry worldwide. The frequency of this disease in equids is estimated to be 1.5% to 34%.1 Laminitis has typically been considered to develop secondary to systemic inflammation or gastrointestinal tract disease2–4 or uneven weight distribution in limbs.5 More recently, results of other studies6,7 have indicated that endocrine disturbances (ie, endocrinopathic laminitis) account for most cases of laminitis evaluated at first-opinion practices in Europe and the United States.6,7 Such endocrinopathies include pituitary pars intermedia dysfunction and hyperinsulinemia attributable to equine metabolic syndrome. Recently, laminitis has been induced in horses and ponies with a pEHC technique.8,9 Hyperinsulinemia in the absence of systemic inflammation and hyperglycemia consistently occurs in animals with laminitis that is associated with pituitary pars intermedia dysfunction or equine metabolic syndrome.10–12
The laminar region of the hoof in equids holds the distal phalanx and the hoof wall together; it consists of epidermis connected to underlying dermis by a basement membrane. The surface area of this dermal-epidermal junction is greatly increased by complex primary and secondary infoldings, called laminae. In an anatomically normal hoof, there are 550 to 600 PEL, each with 150 to 200 SEL.13 Regardless of disease etiology, the laminar structure in horses with laminitis weakens, which leads to stretching of laminae because of ground reaction forces, pull of the deep digital flexor tendon on the palmar or plantar aspect of the distal phalanx, and pressure that develops along the dorsal aspect of the hoof wall during breakover (ie, the period between the point at which the heel leaves the ground and the toe leaves the ground at the end of the stance phase of the stride).14 Studies of laminitis have typically involved histologic examination of hoof lamina samples obtained from horses that received an overload of carbohydrate (starch or oligofructose). However, results of studies15–18,a in which a pEHC technique was used indicate substantial differences between endocrinopathic and nonendocrinopathic forms of laminitis. In all animals with laminitis, considerable elongation of SEL develops; however, severe basement membrane damage may be the most prominent pathological finding for horses with laminitis induced with carbohydrate overload.15 For horses that undergo a pEHC technique, basement membrane damage is minimal and frequently localized to SEL of the axial aspects of PEL.8,9 Additionally, in ponies and horses with insulin-induced laminitis, increased apoptotic cell death and mitotic and proliferative activity have been detected, but these factors have only been quantified for horses.8,19
Insulin is a peptide hormone secreted by pancreatic β cells. Secretion of insulin is mainly regulated in response to plasma glucose concentration, but compensatory hyperinsulinemia develops when target cells have insulin resistance. After binding to its receptor, insulin may have metabolic effects via the phosphatidylinositol 3-kinase (PI-3K) pathway or mitogenic and growth-related effects via the mitogen-activated protein kinase pathway. During a state of insulin resistance and hyperinsulinemia, the mitogen-activated protein kinase pathway is the dominant pathway.20,21 Insulin resistance in human keratinocytes alters patterns of proliferation and differentiation.22 Proliferative effects of insulin may cause inappropriate laminar keratinocyte proliferation, resulting in the elongation of SEL detected for equids with laminitis induced by means of a pEHC technique; however, the relative contributions of cell proliferation and stretching to elongation of SEL have not been determined.23,b We hypothesized that elongation of SEL is attributable to cell stretching rather than simple addition of cells of the same size. Our objective was to measure morphological features of the SEL of ponies with insulin-induced laminitis and to quantify and localize changes in cell death and proliferation. Given that stretching of cells would indirectly indicate a reduction in resistance to mechanical forces of weight bearing, another objective was to determine differences in histomorphometric variables of SEL between forefeet and hind feet; in all previous studies of laminitis in ponies and horses, only forefeet have been evaluated, to the authors' knowledge.
Materials and Methods
Animals—All experimental procedures were performed in another study,24 and archived samples collected during that study were used in the present study. The experimental protocol was approved by the Animal Ethics Committee of the University of Queensland, which monitors compliance with the Animal Welfare Act (2001) and The Code of Practice for the care and use of animals for scientific purposes.25 Eight healthy ponies (mean ± SE age, 6.3 ± 1.7 years; body weight, 244.1 ± 32 kg) with no known history of laminitis and no evidence of previous laminitis detected during examination of the hooves were included in the study. Ponies included 6 geldings, 1 sexually intact male, and 1 mare. By means of a randomization procedure, 4 of the ponies were allocated to a treatment group and 4 to a control group. Laminitis was induced with a pEHC technique. Treatment group ponies received recombinant human insulinc and glucose IV. Control group ponies received an equivalent volume of saline (0.9% NaCl) solution IV. For treatment group ponies, 1 jugular venous catheter was used for the simultaneous administration of insulin at a fixed rate and glucose at a variable rate to maintain euglycemia (blood glucose concentration, 5 mmol/L). A catheter in the other jugular vein was used to collect blood samples for monitoring of blood glucose and insulin concentrations. After collection of baseline blood samples, insulin (45 mU/kg in 50 mL of saline solution as a bolus followed by 6 mU/min/kg for the duration of the experiment) was administered IV. Intravenous administration of 50% glucose solutiond was initiated at a mean ± SE rate of 24.4 ± 3.0 mmol/min/kg.26 The glucose administration rate was adjusted when blood glucose concentrations differed from the target concentration (5 mmol/L) by > 1 mmol/L. Control ponies received saline solution (14.7 mL/min/kg for 72 hours, which was the mean rate of fluid administration for treatment group ponies during the same period).
Obel grade 2 laminitise occurred 50.3 ± 5.3 hours (mean ± SE) after initiation of insulin and glucose administration in treatment group ponies, at which time insulin and glucose administration was stopped. All treatment group ponies received phenylbutazone (4.4 mg/kg, IV or PO) at the time of completion of insulin and glucose administration and were euthanized within 6 to 12 hours. Control group ponies were euthanized after 72 hours of saline solution administration. All ponies were euthanized with an overdose of barbiturate.
Hoof tissue sample collection and histologic examination—The distal aspect of all 4 limbs of each pony was disarticulated at the metacarpophalangeal or metatarsophalangeal joint within 10 minutes after death and sectioned with a band saw, as previously described.15 A sagittal section of hoof at the midpoint between the coronary band and the ground-bearing surface on the dorsal aspect was obtained. This section was then dissected to obtain 5-mm2 tissue samples, extending from the stratum medium to the dermal connective tissue. All hoof tissue samples were fixed in neutral-buffered 10% formalin for 24 hours, processed with routine histologic methods, embedded in paraffin wax, sectioned (thickness, 5 μm), and stained with H&E or by means of the PAS method.
Histomorphometry—Sections of hoof tissue samples were histologically examined by 2 of the authors (NPK and JPK) with a light microscope. Digital images were obtained with image capture softwaref and a digital microscope camera.g Images of PAS-stained sections from each of the 4 feet of all 8 ponies were obtained at 40× magnification and then merged (for each section) with photo-editing softwareh for measurements of PEL. Then, images from each examined region (axial [adjacent to the distal phalanx], middle, and abaxial [adjacent to the stratum medium of the hoof wall]) of each of 10 randomly selectedi PEL were obtained at 200× magnification for measurements of SEL (Figures 1 and 2). All the measurements were performed with the aid of image analysis software.j
Photomicrograph indicating measurement of PEL length (a) and width (b) and the locations of abaxial (c), middle (d), and axial (e) PEL regions. For PEL length measurements, a continuous line was drawn from the axial tip of the PEL to the level of the base (where it joined the adjacent PEL). The PEL width was measured at the middle of each PEL by drawing a straight line that extended to the tips of SEL on each side. PDL = Primary dermal lamina. PAS stain; bar = 500 μm.
Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.161
Photomicrograph indicating measurement of SEL length (a), width (b), and angle (c) in the abaxial region of a PEL. For SEL length measurements, a continuous line was drawn from the tip of each SEL to the base at the junction with the keratinized axis of the associated PEL. The SEL width measurements were performed at the midpoint of each SEL by drawing a straight line. The angle between the PEL axis and the SEL axis was measured with an angle tool in computer image analysis software. PAS stain; bar = 200 μm.
Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.161
The length of each PEL was measured with a drawing toolk from the tip (axial) to the base (abaxial; Figure 1). The width of a PEL was measured at a location 50% along the length of the measurement line for that lamina; this measurement was determined from the tips of SEL on one side to the tips of SEL on the other side. The SEL length, width, and angle were measured for 10 randomly selected SEL (5 from the left side and 5 from the right side) at 200× magnification from each of the 3 examined regions of PEL (Figure 2). The regions were located 10%, 50%, and 90% along the PEL length, corresponding to the axial, middle, and abaxial regions, respectively.16 The lengths of SEL were measured from the tip to the base of such lamina at the junction with the keratinized axis of associated PEL. The widths of SEL were measured from basement membrane to basement membrane at the longitudinal midpoint of each SEL (identified along a straight line). The angle between axes of PEL and those of SEL was measured with the angle tool in the image analysis software.j The number of epidermal cell nuclei per SEL was counted from an SEL base to the tip for 10 randomly selected SEL from each of the 3 regions of PEL (as for measurement of the other SEL variables). The number of epidermal cell nuclei per micrometer of an SEL was calculated by dividing the number of cell nuclei by the SEL length (for an individual SEL). Percentage differences between the control and treatment group ponies were calculated for each variable (PEL length and width; SEL length, width, and angle; cell number per SEL; and cell number per micrometer of an SEL).
Quantification of apoptotic and proliferative cells in SEL—Apoptosis of SEL cells was confirmed on the basis of morphological criteria, including cytoplasmic rounding, nuclear shrinkage, chromatin condensation and fragmentation, and formation of apoptotic bodies.27 Some of the apoptotic bodies had been phagocytosed by neighboring epithelial cells (as expected). The number of apoptotic cells per 200× field was counted for each of the 3 regions (axial, middle, and abaxial) of evaluated PEL (n = 10/hoof) in H&E-stained sections (Figure 3).
Photomicrograph of a representative SEL of a pony with insulin-induced laminitis. Notice the numerous rounded, hypereosinophilic apoptotic epithelial cells and large numbers of apoptotic bodies, some of which contain nuclear fragments. Some of the apoptotic bodies have been phagocytosed by adjacent, viable epithelial cells (arrow). H&E stain; bar = 50 μm.
Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.161
Immunolocalization of proliferating cells was performed with a mouse monoclonal antibody (Ki-S2) to the TPX2 proteinl; TPX2 is a nuclear protein (repp86/p100) associated with cell proliferation that is specific for S, G2, and M phases of the cell cycle. Unlike the more commonly used immunohistochemical proliferation markers Ki-67 and topoisomerase-Iiα antigens, TPX2 does not label cells in the G1 phase of the cell cycle, thereby avoiding inclusion of cells in damage-induced G1 arrest.28 In addition, because TPX2 is required for mitotic spindle formation, the antibody to that protein can label dividing cells that do not contain mitotic figures, which makes it superior to conventional staining techniques for detection of mitosis.29 The TPX2 immunolabeling was performed with an automated stainer and a horseradish peroxidase system.m Antigen retrieval was achieved by heating sections in a digital electric pressure cookern in sodium citrate buffer (pH, 6; 125°C for 1 minute and 40 seconds). This antibody to the TPX2 protein has been validated for use with equine tissue samples by means of Western blot analysis and immunolabeling of positive control (epidermal) tissues. Primary antibodies were diluted at an optimal ratio (1:800) in antibody-dilution solution.m Positive control tissue samples were human and equine skin samples. For negative reagent control samples (to exclude nonspecific binding identified by the detection system), mouse IgG was used instead of the primary antibody. Sections were stained with chromagen 3.3′-diaminobenzidine tetrahydrochloridem twice for 5 minutes (with an intervening Tris-buffered saline with Tween wash) followed by counter-staining with Mayer hematoxylin (30 seconds). Five PEL on each slide were randomly selected, and the total number of cells with positive results for TPX2 in each region (axial, middle, and abaxial) was counted by one of the authors (MMN) with the assistance of image analysis software.j
Statistical analysis—Comparisons of variables between ponies in treatment and control groups were performed for PEL length and width; SEL length, width, and angle; cell number per SEL; cell number per micrometer of SEL; and number of apoptotic and proliferative cells per 200× field. Results are reported as median and IQR values. Statistical analysis was performed with computer software.o Values of all variables, except PEL width, were nonnormally distributed; therefore, a nonparametric Kruskal-Wallis test with Bonferroni adjustment was used for pairwise comparisons. Values of P < 0.05 were considered significant.
Results
Histomorphometry—No significant differences were detected in PEL length or width between right and left hooves for forefeet or hind feet; therefore, measurements for right and left hooves were pooled. Significant differences were detected in PEL length between forefeet and hind feet for control group ponies; therefore, all variables were analyzed separately for forefeet and hind feet. For treatment and control group ponies, PEL length was significantly (P = 0.015 for both groups) greater in forefeet than hind feet. For control group ponies, the median PEL length and width were 3,566 μm (IQR, 2,940 to 4,060 μm) and 295 μm (IQR, 263 to 341 μm) in forefeet, respectively, and 3,267 μm (IQR, 2,929 to 3,587 μm) and 302 μm (IQR, 271 to 347 μm) in hind feet, respectively. For treatment group ponies, the median PEL length and width were 4,105 μm (IQR, 3,754 to 4,504 μm) and 294 μm (IQR, 249 to 330 μm) in forefeet, respectively, and 3,769 μm (IQR, 3,225 to 4,278 μm) and 314 μm (IQR, 270 to 366 μm) in hind feet, respectively. The PEL length for forefeet and hind feet were significantly (P < 0.001) longer (mean percentage difference, 15%) in treatment group ponies than they were in control group ponies (Figure 4). Median SEL length, width, and angle and percentage differences between groups for each evaluated region in forefeet and hind feet were summarized (Table 1). For control group ponies, abaxial region SEL were significantly longer (P = 0.003) and narrower (P < 0.001) and had a smaller angle toward the PEL axis (P = 0.002) in forefeet than they did in hind feet. In treatment group ponies with experimentally induced laminitis, SEL were longer, were narrower, and had a smaller angle in all 3 examined laminar regions in all feet, compared with control ponies; this change was most prominent in the middle region (P < 0.001).
Photomicrographs of laminar tissue of a control pony (A) and a pony with insulin-induced laminitis (B). Notice that the PEL (and associated SEL) of the laminitic pony are substantially longer than those of the control pony. H&E stain; bars = 500 μm.
Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.161
Median (IQR) SEL length, width, and angle and percentage difference between control ponies (n = 4) and ponies with insulin-induced laminitis (treatment; 4) in axial, middle, and abaxial laminar regions in forefeet and hind feet.
| Axial | Middle | Abaxial | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Variable | Control | Treatment | Difference (%) | Control | Treatment | Difference (%) | Control | Treatment | Difference (%) |
| Forefeet | |||||||||
| Length (μm) | 151.0 (116.5–193.0) | 284.4 (189.5–405.8)* | 88 | 209.3 (154.5–288.8) | 435.9 (331.1–534.5)* | 108 | 221.0 (177.4–279.2) | 294.8 (226.9–374.2)* | 33 |
| Width (μm) | 33.9 (28.0–42.1) | 22.6 (18.0–30.6) | 50 | 25.5 (20.9–30.6) | 17.3 (14.7–21.3)* | 47 | 24.3 (20.8–28.7) | 21.6 (18.7–25.0)* | 13 |
| Angle (°) | 75.5 (49.9–103.0) | 21.0 (15.8–29.0)* | 260 | 37.1 (24.8–55.3) | 18.0 (14.7–23.2)* | 106 | 35.4 (26.2–45.1) | 25.3 (20.5–34.9) | 40 |
| Hind feet | |||||||||
| Length (μm) | 149.4 (111.6–199.0) | 360.9 (255.0–509.3)* | 142 | 198.2 (146.0–267.8) | 461.9 (346.4–569.9)* | 133 | 206.0 (160.8–249.8) | 287.0 (228.4–355.4)* | 39 |
| Width (μm) | 31.0 (25.3–38.8) | 21.8 (16.7–27.9)* | 42 | 25.4 (21.9–29.8) | 16.4 (14.3–19.6)* | 55 | 26.6 (22.6–31.3) | 19.9 (15.9–25.1)* | 34 |
| Angle (°) | 66.9 (46.2–94.9) | 21.4 (16.7–29.6)* | 213 | 39.1 (28.8–54.2) | 19.4 (15.4–24.2)* | 102 | 38.4 (30.5–45.4) | 24.4 (19.5–29.8)* | 57 |
One hundred SEL length, width, and angle measurements were determined for each laminar region in each foot of the ponies.
Values are significantly (P < 0.05) different between treatment and control group ponies.
For control group ponies, there were significantly (P < 0.001) more epidermal cell nuclei per SEL in forefeet (abaxial and middle laminar regions) than in hind feet. For treatment group ponies, there were no significant differences between forefeet and hind feet regarding the number of epidermal cell nuclei per SEL. Significantly greater numbers of cell nuclei per SEL were detected in axial (P < 0.001) and middle regions (P = 0.003) of forefeet laminae and axial and middle (P < 0.001) and abaxial (P = 0.001) regions of hind feet laminae than in control pony SEL (Figure 5). The number of epidermal cell nuclei per SEL in treatment group ponies was a median percentage of 6% (forefeet) and 17% (hind feet) higher than values for control ponies.
Bar charts of numbers of epidermal cell nuclei per SEL (A and B) and epidermal cell nuclei per micrometer of SEL (C and D) in axial, middle, and abaxial laminar regions in forefeet (A and C) and hind feet (B and D) of treatment group ponies with insulin-induced laminitis (black bars; n = 4) and control ponies (gray bars; 4). The upper boundary of each bar indicates the median value; the error bar indicates the upper quartile of the IQR. *Values are significantly (P < 0.05) different between the treatment and control group.
Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.161
No significant differences were detected for control group ponies regarding numbers of cell nuclei per micrometer of SEL between forefeet and hind feet. For both forefeet and hind feet, significantly more cell nuclei per micrometer were detected in the axial region versus other regions of laminae. The number of cell nuclei per micrometer of SEL was significantly (P < 0.001) lower for treatment group ponies versus control ponies in all laminar regions in all feet (Figure 5). The number of cell nuclei per micrometer of SEL in treatment group ponies was 75% (forefeet) and 77% (hind feet) lower than the values for control ponies.
Apoptosis and cellular proliferation—Apoptotic cells and cells with positive results for TPX2 were rarely detected in hoof tissue samples obtained from control group ponies, with no significant differences detected among regions (Figure 6). Numbers of apoptotic cells were significantly (P < 0.001) higher in treatment group ponies versus control ponies in abaxial and middle regions of laminae in forefeet and hind feet. Numbers of cell nuclei with positive results for TPX2 were significantly higher for treatment group ponies in forefeet for axial regions (P = 0.001) and in hind feet for abaxial (P = 0.049), middle (P = 0.017), and axial (P = 0.043) regions.
Bar charts of numbers of apoptotic (A and B) and proliferative (positive results for TPX2; C and D) cells per hpf (200×) in axial, middle, and abaxial laminar regions in forefeet (A and C) and hind feet (B and D) of ponies with insulin-induced laminitis (black bars; n = 4) and control ponies (gray bars; 4). The upper boundary of each bar indicates the median value; the error bars indicate the upper quartile of the IQR. *Values are significantly (P < 0.05) different between the treatment and control group ponies.
Citation: American Journal of Veterinary Research 75, 2; 10.2460/ajvr.75.2.161
Discussion
The results of the present study indicated a significantly lower number of cell nuclei per micrometer of SEL in all laminar regions in forefeet and hind feet for treatment group ponies versus control group ponies, without evidence of laminar disruption. These findings suggested that such cells in treatment group ponies were longer than those in control group ponies. The percentage increase in total SEL cell number (approx 12%) was considerably smaller than the percentage decrease in cell number per micrometer (approx 75%) in treatment group ponies versus control ponies. These findings suggested that cell elongation was likely the primary mechanism for laminar stretching during laminitis. The relationship between such lesions and hyperinsulinemia in equids with laminitis was not determined. The finding that numbers of both apoptotic and proliferating SEL cells were higher in treatment group ponies than they were in control group ponies suggested a complex relationship between cell stretching and alteration in cell cycles. The temporal relationship between these processes cannot be determined by means of histologic examination of tissue samples obtained at 1 time; however, the spatial localization of such changes may suggest potential relationships. For example, in the hind feet, compared with the forefeet, the extent of cellular elongation was the same, but the increase in total numbers of cells forming the SEL, compared with control ponies, was greater, and proliferating cells were detected in all SEL regions (abaxial, middle, axial). Conversely, total SEL cell number was not significantly higher in the abaxial region of the forefeet of treatment group ponies versus control group ponies, and apoptosis was more prominent than proliferation. Differences in biomechanics between hind feet and forefeet attributable to differences in weight-bearing loads may have caused such variation in results among anatomic locations. Our finding that the greatest numbers of apoptotic cells were located in abaxial and middle laminar regions in all feet of treatment group ponies might also have been attributable to mechanical factors. Mechanical stress induces apoptosis of multiple cell types, including epithelial cells.30 The abaxial region SEL were longer than those in other regions in control ponies. Therefore, if long SEL have a lower tolerance for mechanical forces versus short SEL, the small amount of elongation of SEL in the abaxial region may have been sufficient to cause cell death. Additionally, the thick keratin cores of PEL attached to and between rows of SEL in abaxial regions might have limited elongation and contributed to mechanical stress. The micromechanics of PEL and SEL have not been determined at this level of anatomic detail, to the authors' knowledge.
The SEL cell proliferation detected in treatment group ponies in this study could have been a response to cell death, as develops during epithelial homeostasis and wound-healing processes, rather than aberrant proliferation of laminar epidermal cells.31 In hind feet of treatment group ponies with experimentally induced laminitis in this study, numbers of apoptotic and proliferating cells in all regions were higher than those in hind feet of control group ponies; this finding indicated such cells in treatment group ponies had a faster cell death– proliferation cycle versus that of control group ponies. However, the severity of these processes varied among laminar regions in forefeet of treatment group ponies, with increased apoptosis detected in cells of middle and abaxial regions and increased proliferation in axial regions. In horses, approximately 58% of body weight is supported on forefeet; therefore, mechanical forces on the laminar region of feet are likely greater in forefeet than they are in hind feet and lesions may be more severe in forefeet.32 The potential complexity of the accelerated cell death–proliferation cycle during laminitis is also suggested by comparison of results between horses and ponies. In the forefeet of Standardbreds, for which a higher mechanical load would be expected versus that of ponies, increased numbers of proliferating cells are detected in axial and abaxial laminar regions at 24 and 48 hours after induction of laminitis (middle laminar regions were not analyzed in that study), whereas numbers of apoptotic cells are lower at those times versus earlier times.19 The proliferative response of laminar cells may continue after the rate of apoptotic cell death has decreased, and that rate of apoptotic cell death may decrease beginning in axial laminar regions. Evaluation of equids with other types of experimentally induced laminitis and those with naturally developing disease at various times (acute and chronic) may provide further information regarding how the 3 evaluated cellular events (stretching, apoptosis, and proliferation) progress during laminitis in forefeet and hind feet.
Cell stretching might also cause cell entry into the cell cycle, as has been determined for other cell types.30,33,34 The SEL elongation was most pronounced in middle laminar regions of treatment group ponies with induced laminitis in the present study; however, cells might have different mechanostat set points (a range of tissue tensions within which the balance of internal cytoskeletal tension and external tension can be maintained) among regions. For example, fibroblasts obtained from equine digital tendons that are exposed to different cyclic mechanical strains in vivo have different proliferative responses to the same strain in vitro.35 Changes in cell shape in response to tension alter activities of various cell factors, including those of cell cycle regulators.36 Differences in cell cycle entry and cell death responses of SEL cells to various amounts of stretching among laminar regions are unknown.
The results of the present study regarding SEL elongation, narrowing, and alteration in orientation (relative to PEL) in all 4 feet of ponies with insulin-induced laminitis seemed to be primarily attributable to cell stretching. The relationships among cell stretching, proliferation, and apoptosis in the various evaluated laminar regions seemed to be complex, and the mechanisms by which hyperinsulinemia induced or facilitated such processes are not known, to the authors' knowledge.
ABBREVIATIONS
| IQR | Interquartile range |
| PAS | Periodic acid–Schiff |
| pEHC | Prolonged euglycemic-hyperinsulinemic clamp |
| PEL | Primary epidermal lamina |
| SEL | Secondary epidermal lamina |
Visser M. Investigation of proteolysis of the basement membrane during the development of equine laminitis. PhD thesis, University of Queensland, St Lucia, Brisbane, QLD, Australia, 2008.
de Laat M, Kyaw-Tanner M, Patterson-Kane J, et al. Insulin and the insulin-like growth factor system: a novel theory for hyperinsulinemic laminitis pathogenesis (abstr). J Equine Vet Sci 2011;31:581–582.
Humulin R, Eli-Lilly Australia, West Ryde, NSW, Australia.
Baxter Healthcare Pty Ltd, Old Toongabbie, NSW, Australia.
Obel N. Studies on the histopathology of acute laminitis. PhD dissertation, Almqvist and Wiksells Boktryckeri AB, Uppsala, Sweden, 1948.
Cell⁁D, version 2.8, Olympus Soft Imaging Solutions, Münster, Germany.
Olympus DP71, Olympus Imaging America Inc, Center Valley, Pa.
Adobe Photoshop CS5, version 12.0 × 64, Adobe Systems Inc, San Jose, Calif.
Research Randomizer, version 4.0, Geoffrey C. Urbaniak and Scott Plous. Available at: www.randomizer.org/form.htm. Accessed Apr 18, 2013.
ImageJ, version 1.4.3, National Institutes of Health, Bethesda, Md. Available at: rsbweb.nih.gov/ij/index.html. Accessed Apr 18, 2013.
Wacom Intuos4, Wacom Europe, Krefeld, Germany.
TPX2/ab32795, Abcam, Cambridge, England.
Dako, Glostrup, Denmark.
Menarini Antigen Access Unit, A. Menarini Diagnostics Ltd, Winnersh-Wokingham, Berkshire, England.
PASW Statistics, version 18 for Windows, IBM Corp, New York, NY.
References
1. Wylie CE, Collins SN, Verheyen KL, et al. Frequency of equine laminitis: a systematic review with quality appraisal of published evidence. Vet J 2011; 189: 248–256.
2. Garner HE, Coffman JR, Hahn AW, et al. Equine laminitis of alimentary origin: an experimental model. Am J Vet Res 1975; 36: 441–444.
3. Galey FD, Whiteley HE, Goetz TE, et al. Black walnut (Juglans nigra) toxicosis: a model for equine laminitis. J Comp Pathol 1991; 104: 313–326.
4. van Eps AW, Pollitt CC. Equine laminitis induced with oligofructose. Equine Vet J 2006; 38: 203–208.
5. Virgin JE, Goodrich LR, Baxter GM, et al. Incidence of support limb laminitis in horses treated with half limb, full limb or transfixation pin casts: a retrospective study of 113 horses (2000–2009). Equine Vet J Suppl 2011;(40):7–11.
6. Karikoski NP, Horn I, McGowan TW, et al. The prevalence of endocrinopathic laminitis among horses presented for laminitis at a first opinion/referral equine hospital. Domest Anim Endocrinol 2011; 41: 111–117.
7. Donaldson MT, Jorgensen AJ, Beech J. Evaluation of suspected pituitary pars intermedia dysfunction in horses with laminitis. J Am Vet Med Assoc 2004; 224: 1123–1127.
8. Asplin KE, Patterson-Kane JC, Sillence MN, et al. Histopathology of insulin-induced laminitis in ponies. Equine Vet J 2010; 42: 700–706.
9. de Laat MA, van Eps AW, McGowan CM, et al. Equine laminitis: comparative histopathology 48 hours after experimental induction with insulin or alimentary oligofructose in Standardbred horses. J Comp Pathol 2011; 145: 399–409.
10. McGowan CM, Frost R, Pfeiffer DU, et al. Serum insulin concentrations in horses with equine Cushing's syndrome: response to a cortisol inhibitor and prognostic value. Equine Vet J 2004; 36: 295–298.
11. Frank N, Geor RJ, Bailey SR, et al. Equine metabolic syndrome. J Vet Intern Med 2010; 24: 467–475.
12. McFarlane D. Equine pituitary pars intermedia dysfunction. Vet Clin North Am Equine Pract 2011; 27: 93–113.
13. Pollitt CC. Anatomy and physiology of the inner hoof wall. Clin Tech Equine Pract 2004; 3: 3–21.
14. Wilson A, Weller R. The biomechanics of the equine limb and its effect on lameness. In: Ross MW, Dyson SJ, eds. Diagnosis and management of lameness in the horse. 2nd ed. St Louis: Elsevier Saunders 2011;277.
15. Pollitt CC. Basement membrane pathology: a feature of acute equine laminitis. Equine Vet J 1996; 28: 38–46.
16. Morgan SJ, Hood DM, Wagner IP, et al. Submural histopathologic changes attributable to peracute laminitis in horses. Am J Vet Res 2003; 64: 829–834.
17. French KR, Pollitt CC. Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study. Equine Vet J 2004; 36: 230–235.
18. van Eps AW, Pollitt CC. Equine laminitis model: lamellar histopathology seven days after induction with oligofructose. Equine Vet J 2009; 41: 735–740.
19. de Laat MA, Patterson-Kane JC, Pollitt CC, et al. Histological and morphometric lesions in the pre-clinical, developmental phase of insulin-induced laminitis in Standardbred horses. Vet J 2013; 195: 305–312.
20. Kim JA, Montagnani M, Koh KK, et al. Reciprocal relationships between insulin resistance and endothelial dysfunction: molecular and pathophysiological mechanisms. Circulation 2006; 113: 1888–1904.
21. Cusi K, Maezono K, Osman A, et al. Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle. J Clin Invest 2000; 105: 311–320.
22. Buerger C, Richter B, Woth K, et al. Interleukin-1β interferes with epidermal homeostasis through induction of insulin resistance: implications for psoriasis pathogenesis. J Invest Dermatol 2012; 132: 2206–2214.
23. de Laat MA, Pollitt CC, Kyaw-Tanner MT, et al. A potential role for lamellar insulin-like growth factor-1 receptor in the pathogenesis of hyperinsulinaemic laminitis. Vet J 2013; 197: 302–306.
24. Asplin KE, Sillence MN, Pollitt CC, et al. Induction of laminitis by prolonged hyperinsulinaemia in clinically normal ponies. Vet J 2007; 174: 530–535.
25. National Health and Medical Research Council. Australian code of practice for the care and use of animals for scientific purposes. 7th ed. Canberra, ACT, Australia: National Health and Medical Research Council, Australian Government, 2004.
26. DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 1979; 237: E214–E223.
27. Meyers RK, McGavin MD. Cellular and tissue responses to injury. In: Zachary JF, McGavin MD, eds. Pathologic basis of veterinary disease. 4th ed. St Louis: Mosby Elsevier 2007;16–32.
28. Rudolph P, Knüchel R, Endl E, et al. The immunohistochemical marker Ki-S2: cell cycle kinetics and tissue distribution of a novel proliferation-specific antigen. Mod Pathol 1998; 11: 450–456.
29. Heidebrecht HJ, Buck F, Steinmann J, et al. p100: a novel proliferation-associated nuclear protein specifically restricted to cell cycle phases S, G2, and M. Blood 1997; 90: 226–233.
30. Mayr M, Li C, Zou Y, et al. Biomechanical stress-induced apoptosis in vein grafts involves p38 mitogen-activated protein kinases. FASEB J 2000; 14: 261–270.
31. Greenhalgh DG. The role of apoptosis in wound healing. Int J Biochem Cell Biol 1998; 30: 1019–1030.
32. Hood DM, Wagner IP, Taylor DD, et al. Voluntary limb-load distribution in horses with acute and chronic laminitis. Am J Vet Res 2001; 62: 1393–1398.
33. Nguyen HT, Hsieh MH, Gaborro A, et al. JNK/SAPK and p38 SAPK-2 mediate mechanical stretch-induced apoptosis via caspase-3 and -9 in NRK-52E renal epithelial cells. Nephron Exp Nephrol 2006; 102: 49–61.
34. Ning QM, Sun XN, Zhao XK. Role of mechanical stretching and lipopolysaccharide in early apoptosis and IL-8 of alveolar epithelial type II cells A549. Asian Pac J Trop Med 2012; 5: 638–644.
35. Goodman SA, May SA, Heinegård D, et al. Tenocyte response to cyclical strain and transforming growth factor beta is dependent upon age and site of origin. Biorheology 2004; 41: 613–628.
36. Chicurel ME, Chen CS, Ingber DE. Cellular control lies in the balance of forces. Curr Opin Cell Biol 1998; 10: 232–239.
