Supporting-limb laminitis (SLL) is an important complication of painful limb conditions, such as fractures that cause alterations in normal weight-bearing patterns. In these cases, increased weight-bearing load on the contralateral limb is specifically thought to interfere with blood perfusion in the supporting limb (SL).1–3 This theory is supported by findings from recent in vivo experimental studies4,5 demonstrating evidence of lamellar ischemia in limbs subjected to modest increases in weight-bearing load over prolonged periods (48 to 92 hours). Interestingly, in clinical cases of SLL, histological evidence of laminitis is not confined to the overloaded (supporting) limb; it is often present in multiple limbs (including the primarily injured limb).3,6,7 This indicates that alterations in weight-bearing patterns other than simple increases in mechanical load may adversely affect the hoof lamellae. Lamellar microvascular perfusion in healthy horses is affected by limb load cycling, with increases in walking or static weight-shifting steps causing increases in microvascular perfusion and glucose concentration within the lamellae.8 It has been suggested that a lack of normal cyclic load, in the absence of increased weight-bearing load, could also compromise lamellar microvascular perfusion sufficiently to damage the lamellae.2,3
There are currently few published descriptions of the histology of naturally occurring SLL, and there are no published studies describing the effects of prolonged, experimentally induced alterations in weight-bearing load on the histological appearance of the lamellae. The primary objective of the current study was to describe the histological features of lamellae from limbs subjected to prolonged, preferential weight bearing (PWB), induced using a nonpainful experimental model.4 A secondary objective was to compare this to the lamellar histology of horses that had prolonged ambulatory restriction (without altered weight bearing) as part of the same study. We hypothesized that histological changes consistent with laminitis would be associated with PWB and restricted ambulation (RA).
Methods
Animals and experimental design
Middorsal hoof lamellar samples from 12 healthy Standardbred horses (10 geldings and 2 mares; mean ± SD age, 6.4 ± 2.5 years; mean ± SD body weight, 452.5 ± 26.8 kg) that were part of a previously published study4 examining the effects of prolonged PWB on lamellar perfusion and metabolism were used. A full description of the study methods specifically for the animal protocols has been previously published.4 Briefly, all horses were sound at the walk and had no gross or radiographic abnormalities of the feet at the commencement of the study. The horses had been recently retired (< 4 weeks) from racing in Queensland, Australia. All horses were confined to stocks for a 92-hour experimental period. One forelimb of each horse was instrumented with 2 microdialysis probes at the beginning of the experiment, and this was designated the SL. In 6 of the horses, a platform shoe on the contralateral forelimb (CF) was used to induce an increase in weight-bearing load on the SL that was equivalent to approximately 10% bodyweight (approx 50 kg extra load on the SL) for the duration of the 92-hour experiment. These horses were designated as the PWB group. The remaining horses were barefoot and had no further intervention besides being housed in stocks on rubber mats for 92 hours and were designated the RA group. A schematic representation of the experimental groups and limb designations is included in Figure 1. To prevent any potential discomfort associated with prolonged PWB, a continuous bupivacaine peripheral nerve block of the palmar nerves was performed in the SL in PWB horses as previously described.9 The peripheral nerve block was also performed in the microdialysis-instrumented limb of the RA horses (this limb was also designated as “SL” in the RA group for simplicity, although it was not subjected to increased load) in order to control for any effects of local anesthetic blockade on metabolism in the original study.4 After 92 hours, each horse was euthanized with pentobarbital sodium (20 mg/kg bodyweight, IV). A separate control group (CON) consisted of archived middorsal lamellar samples harvested and processed identically from 8 Standardbred horses (3 geldings and 5 mares; age, 3 to 6 years; bodyweight, 365 to 454 kg) with no overt lameness and grossly normal feet. These horses were from the same population (Standardbred horses recently [< 4 weeks] retired from racing in Queensland, Australia) and had no intervention besides tissue harvest as part of a separate study that was conducted in the same year. The project was approved by The University of Queensland Animal Ethics Committee (approval number: SVS/098/15/GJCRF). All animals were monitored continuously by the investigators.
Schematic representation of the animal groups. Both the preferential weight-bearing (PWB) and restricted ambulation (RA) groups were instrumented with a microdialysis probe in the supporting limb (SL) as part of a previously published study. In the PWB group, PWB was experimentally induced on the SL (increase in load equivalent to 10% bodyweight, approx 50 kg) using a platform shoe fitted to the contralateral forelimb (CF) for a 92-hour period. The RA group remained barefoot and had no further intervention besides being housed in stocks on rubber mats for 92 hours (no induced increase in load on the SL). The control group was healthy horses that had no intervention (the control SL and CF forelimbs were randomly assigned). Hind feet (H) samples were pooled for all groups.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.09.0268
Sample harvest and histochemical and immunohistochemical staining
Middorsal lamellar sections were fixed (10% neutral-buffered formalin), routinely processed, paraffin-embedded, sectioned, and stained with H&E and periodic acid–Schiff, then graded for laminitis pathology by a blinded, board-certified veterinary pathologist (JBE). Additional sections on charged slides were made for molecular stains (immunohistochemistry and immunofluorescence) described below. Each histochemically stained section was scored based on the estimated distribution (0, absent; 1, focal, < 25%; 2, frequent, 25% to 75%; 3, widespread, > 75% tissue affected) of 4 major pathological findings (epidermal basal cell [EBC] disorganization, elongation of secondary epidermal lamellae [SEL], basement membrane separation, and evidence of cell death) as previously described.10,11 Scores were added, giving a total for each section (maximum of 12). The primary epidermal lamellae (PEL) length, nonkeratinized PEL length, SEL length, SEL width, and distance from the keratinized axis to the secondary dermal lamellae (SDL; SDL retraction, a measure of basement membrane detachment) were measured as previously described.10 Measurements were made on 5 PELs from each section. Within each PEL, 5 SELs from each of the axial, middle, and abaxial regions were selected for the measurement of SEL length, SEL width, and SDL retraction. The axial measurements were made at the most axial point of the PEL where there was an adjacent keratinized axis. The abaxial measurements were made at the abaxial base of the PEL. The middle measurements were made approximately halfway between the axial and abaxial measurements. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL), a marker of fragmented DNA indicative of cell death, was performed on fixed sections using a commercial kit (DeadEnd Fluorometric TUNEL System; Promega) following the manufacturer's instructions. Immunohistochemical staining for caspase-3, an apoptosis-specific marker, was performed using a rabbit anti-cleaved caspase-3 antibody (catalog number ASP175; Cell Signaling Technology)8; dilution 1:100; incubated 2 hours at room temperature) similar to that previously described12 but with fluorescent labeling (donkey anti-rabbit IgG secondary antibody, Alexa Fluor 488; catalog number A21206, Invitrogen; dilution 1:300) and a nuclear counterstain (4',6-diamidino-2-phenylindole). After incubating the sections in 2.0% hydrogen peroxide in Tris-buffered saline for 10 minutes, antigen retrieval was achieved by heating to 105 °C for 15 minutes in a Tris-EDTA buffer solution (pH 9.0). Samples were treated with a blocking reagent (Background Sniper; Biocare) for 30 minutes to limit background fluorescence. Immunohistochemical staining for the cellular proliferation marker targeting protein for Xenopus kinesin-like protein (TPX2)13,14 was performed on forelimb sections only. After deparaffinization, hydration, and treatment with 3% hydrogen peroxide in Tris-buffered saline for 10 minutes, antigen retrieval was achieved by treating the sections for 5 minutes in 0.01 M citrate buffer (pH 6.0) at 125 °C. Subsequently, the sections were incubated with TPX2 primary antibody (TPX2/ab32795; Abcam), diluted 1:100, for 60 minutes. The sections were then incubated with MACH1 mouse probe (15 minutes; Biocare Medical) and polymer (30 minutes; Biocare Medical) at room temperature. Staining signaling was developed with diaminobenzidine chromogen substrate, followed by counterstaining with hematoxylin. All slides were scanned at 20X (Aperio FL; Leica Biosystems), with measurements and cell counts performed using open-source software (Qupath Version: 0.5.1).15
Statistical analysis
All analyses were conducted using Stata/MP, version 18 (StataCorp), with 2-sided tests of hypotheses and P < .05 as the criterion for statistical significance. Descriptive statistics are expressed as mean with SD for normally distributed variables and median (IQR) for other continuous variables. The normality of data was assessed using the Shapiro-Wilk test. Frequency counts and percentages were used for categorical variables (eg, signalment and others).
For the analysis of the histomorphometry variables (PEL length, nonkeratinized PEL length, SEL length, SEL width, and SDL retraction) and cell counts (TUNEL-, caspase-3-, and TPX2-positive cell counts), a mixed-effects linear regression model with the interaction between group (PWB, RA, or CON) and limb (SL, CF, or hind) was used as a fixed effect. Random effects were set on the level of the individual animal. The normal distribution of the residual for each of the outcomes was assessed using quantile normal plots. To permit for possible departures from normality for some of the outcomes, robust estimation of the variance was used. Post hoc analysis was performed on the model-adjusted means. All model-adjusted means and effects were reported with their respective 95% CIs. The least significant difference method was used to correct for multiple comparisons. For the histological scores, an ordered logistic mixed-effects regression model was used for analysis, with the fixed effect defined as the interaction of group (PWB, RA, or CON) and limb (SL, CF, or hind), and random effects were set at the level of the individual animal. Odds ratios are expressed as the likelihood of a PWB or RA limb section having a higher score compared to the corresponding CON fore- or hindlimb.
Results
Histomorphometry
Histomorphometry measurements are summarized in Supplementary Table S1. Graphical depictions with corresponding photomicrographs are presented in Figures 2 and 3. The PELs were longer in PWB SL compared to control SL; the mean (95% CI) difference was 0.53 mm (0.21 to 0.84); P < .001). Increases in the length of the nonkeratinized portion of the PEL were present in all PWB and RA limbs compared to their respective controls (P < .05); however, this was most pronounced in PWB SL limbs (646 µm [481 to 810] greater than control; P < .001). The SELs in PWB SL feet were also elongated (SEL length, 96 µm [41 to 151] greater than control; P = .001) and narrower (SEL width, 2.2 µm [0.2 to 4.3] less than control; P = .04) compared to control SL. The PWB hind feet SELs were also 40 µm (4 to 76) longer compared to control hind feet (P = .03). In contrast, PWB CF and both the RA forefeet had SELs that were wider than their respective control feet (P = .05). Only the PWB SL feet had an increase in SDL retraction (14 µm [2 to 26] greater than control; P = .02), consistent with BM detachment.
Graphical depictions and periodic acid–Schiff (PAS)-stained photomicrographs of primary epidermal lamellae (PEL) from control horses (CON) compared to PWB and RA horses show (A) increased total PEL length (arrows; scale bars = 500 μm) in the SLs of PWB compared to CON (P < .001). Increased length of the nonkeratinized axial regions (arrows) of the PEL are present in SL, CF and hind feet from both PWB and RA horses (B) compared to CON (P ≤ .02). Scale bars = 200 μm. *Significant difference (P < .05).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.09.0268
Graphical depictions and PAS-stained photomicrographs show (A) increased secondary epidermal lamellar (SEL) length (black arrows; scale bar = 50 μm) and (B) secondary dermal lamellar (SDL) retraction (yellow arrows; scale bar = 50 μm) in SLs of PWB horses compared to RA horses or CON. C—Secondary epidermal lamellar width (black bars) is reduced in SL of PWB horses but increased in SL of RA horses and CFs of both PWB and RA horses compared to CON; no differences were detected in the hindlimbs (HIND) among the cohorts. Scale bar = 50 μm. *Significant difference (P < .05).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.09.0268
Histological scores
Histological scores are summarized in Supplementary Table S2, and graphical depictions, including corresponding photomicrographs highlighting the most pertinent features, are presented in Figure 4. Overall, histologic lesions were compatible with mild laminitis, and although the most severe lesions were identified within the PWB SL feet, significant increases in total histologic scores were identified within both PWB forelimbs (SL and CF) and also the RA SL compared to control (P < .02). Specifically, lesions were characterized by EBC rounding and disorganization with nuclei oriented askew to the natural SEL axis, SEL elongation with wavered-to-tapered contours, and, most striking, epidermal keratinocyte cell death characterized by shrunken, hypereosinophilic bodies, with pyknotic and karyorrhectic debris concentrated along the keratinized axis identified within both PWB and RA groups. The PWB SL scored higher for EBC disorganization (OR, 693 [95% CI, 46 to 10,386]), elongation (OR, 99 [14 to 704]), necrosis (OR, 96 [95% CI, 5.4 to 1,714]), and the total score (OR, 7 [95% CI, 3 to 16]) compared to control feet and also had the highest median (IQR) total score (7 [95% CI, 6 to 9]) of all limbs. Although significantly increased scores for elongation and EBC disorganization were present in multiple PWB and RA limbs, only the PWB SL had a higher score for necrosis compared to control. Although BM separation was noted in SEL tips of some PWB supporting limbs, PWB contralateral forelimbs, and RA supporting limbs, their separation scores did not significantly differ compared to control (P > .3).
Graphical depictions and PAS-stained photomicrographs that highlight features of the (A) sum of semiquantitative lamellar scores and individual scores, including (B) epidermal basal cell (EBC) disorganization, characterized by rounded nuclei oriented askew to their normal axis (arrows; PWB and RA); (C) SEL elongation (black bars; PWB and RA); (D) epidermal necrosis (arrowheads; PWB and RA) concentrated along the keratinized axis (KA); and (E) basement separation (open arrows; PWB). Scale bars = 20 μm. *Significant difference from corresponding CON limb (P < .05).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.09.0268
Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, caspase-3, and TPX-2 staining
Tabulated cell counts and graphical depictions of cell counts with representative photomicrographs of corresponding, serially matched, molecular- and histochemical-stained sections are presented in Figure 5 and Table 1. Compared to control feet that had occasional TUNEL-positive cells, frequent TUNEL-positive cells were present in PWB supporting-limb sections as well as RA supporting-limb and CF sections. The TUNEL-positive cells had characteristic spindle-shaped nuclei compatible with parabasal keratinocytes and were concentrated adjacent to the keratinized axes, occasionally extending into the SELs, similar to the pattern of cell death morphologically identified within histochemical-stained sections (Figure 4). There was an approximately 10-fold increase in TUNEL-positive cell counts in the PWB SL compared to control SL (P = .002). Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling–positive cell counts in RA SL and RA CF were more than double those of their corresponding control feet (P = .04). The TUNEL-positive parabasal keratinocytes did not stain positive for caspase-3. Although there was a subjective increase in the number of caspase-3–positive cells adjacent to the keratinized axes of PELs in both PWB and RA forelimb sections, there was no significant difference in caspase-3–positive cell counts versus control for any of the limbs (P = .1). Targeting protein for Xenopus kinesin-like protein–positive cells were rare in control sections, whereas TPX2-positive EBCs peripheral to the keratinized axes were frequent in PWB and RA forelimb samples. The mean TPX2-positive cell count was more than 20-fold higher in PWB SL compared to control SL (P = .002) and approximately 4-fold higher in RA SL and RA CF compared to control (P = .001).
Graphical depictions correlated with photomicrographs of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) and caspase-3 immunofluorescence, targeting protein for Xenopus kinesin-like protein 2 (TPX2) immunohistochemical, and PAS-stained histochemical serial middorsal lamellar sections from CON and PWB and RA horses. A—Compared to occasional TUNEL-stained positive cells (green fluorescence) in CON (white arrows), PWB and RA horses show frequent spindle-shaped positive parabasal cells, concentrated along the KA and extending into SEL (white bars), that are most pronounced in SLs of the PWB group but also present within both the SL and CF of the RA group. Scale bars = 100 μm. B—In contrast, comparatively fewer cells are positive for cleaved caspase-3 (green fluorescence), although the location of positive parabasal cells concentrated along the KA within sections from PWB and RA horses is similar to TUNEL stains. Scale bars = 100 μm. C—TPX2, a marker for mitosis, is also increased in keratinocytes within PWB and RA sections relative to CON; however, staining is concentrated within basal cells of peripheral regions of SEL (brown DAB stain; black arrows). Scale bars = 100 μm. Periodic acid–Schiff histochemical sections highlight regions of parabasal cell death concentrated along the KA (black arrows) and occasional mitoses within elongated secondary epidermal lamellae (insets) corresponding to molecular stains above. *Significant difference (P < .05).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.09.0268
Histochemical cell counts for the 3 groups.
| Preferential weight bearing (n = 6) | Restricted ambulation (n = 6) | Control (n = 8) | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Support limb | Contralateral forelimb | Hind | Support limb | Contralateral forelimb | Hind | Support limb | Contralateral forelimb | Hind | |
| TUNEL | 45 (30–60); P = .002 | 5 (1–8); P = .9 | 4 (2–8); P > .9 | 10 (3–17); P = .04 | 10 (5–16); P = .02 | 3 (2–4); P = .1 | 4 (2–5) | 4 (2–6) | 4 (2–7) |
| Caspase-3 | 9 (0–18); P = .1 | 2 (0–4); P = .4 | 2 (1–3); P = .5 | 3 (1–4); P = .5 | 3 (2–3); P = .5 | 2 (2–3); P = .8 | 2 (2–3) | 4 (1–6) | 2 (1–3) |
| TPX-2 | 116 (46–186); P = .002 | 106 (4.8–218); P = .072 | – | 20 (12–28); P < .001 | 19 (0–27); P = .001 | – | 5 (3–6) | 5 (3–6) | – |
Values reported are mean (95% CI) positive cell counts per PEL.
PEL = Primary epidermal lamellae. TUNEL = Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling. TPX2= Targeting protein for Xenopus kinesin-like protein 2.
P values are for the comparisons to the corresponding control group limb.
Discussion
A modest increase in weight-bearing load on a single limb for 92 hours caused lamellar lesions similar to early lesions reported in models of acute sepsis-related and hyperinsulinemia-associated laminitis, including elongation of primary epidermal lamellae and SEL, EBC proliferation, and basement membrane detachment.10,16,17 However, the prominent TUNEL-positive, caspase-3–negative parabasal cell death, focused along the PEL keratinized axes (and extending into the SELs), was a unique finding. The contribution of ischemia, previously documented using microdialysis in these same PWB SL limbs4 (but not in models of sepsis-related laminitis18,19 or hyperinsulinemia-associated laminitis20), is a likely explanation for this atypical pathology. Another unexpected finding was the presence of similar (albeit less severe) histopathological lesions not only in other (non-SL) limbs from the PWB group but also in limbs from the RA group. These results indicate that subtle damage to the lamellae can occur with altered weight-bearing patterns (particularly a lack of normal ambulation) even in the absence of persistently increased weight-bearing load. This mirrors clinical SLL, where multiple limbs (including the primarily injured limb) are often affected,3,6,7 and increased weight-bearing load appears to compound the damage in the SL.
The role of cell death (specifically apoptosis) has been previously investigated using histochemical methods in both naturally occurring laminitis and in tissues obtained at different stages from experimentally induced, sepsis-related laminitis models. There was no increase in TUNEL-positive cells at developmental or acute timepoints in carbohydrate overload or black walnut extract models of sepsis-related laminitis.12,21 An increase in TUNEL-positive epidermal cells was documented in 4 horses with naturally occurring laminitis (< 7 days clinical duration and of unspecified cause) that were presented for euthanasia.12 The TUNEL-positive cells were mostly parabasal keratinocytes that did not stain positive for caspase-3 (a similar finding to the current study). Considering the duration of signs and advanced pathology in those natural cases, the cell death was likely a secondary event. In the current study, extensive TUNEL-positive staining of parabasal cells was present prior to any evidence of clinical lameness (ie, in the developmental phase) and prior to major structural change within the lamellae and therefore appears to be a primary mechanistic event. The relative lack of caspase-3 staining combined with the previously documented presence of ischemia (increased microdialysate lactate:pyruvate and reduced urea clearance in these same PWB SL feet4) suggests that these cells were undergoing nonapoptotic cell death, most likely necrosis.22 At odds with this was the presence of TUNEL-positive parabasal cells (albeit in lower numbers) in the RA group forelimbs: the RA SL limbs were used as normally weight-bearing controls in the previous microdialysis study4 (designated as the CON limbs), and they had no microdialysis evidence of ischemia. However, as noted in the previous study,23 the peak microdialysate lactate:pyruvate (42 [95% CI, 41 to 49]) in the RA SL limbs was high compared to normal values reported in organs, such as the brain (lactate:pyruvate < 25), consistent with molecular evidence suggesting that healthy lamellar tissue is relatively hypoxic.24 As previously documented with microdialysis, the act of walking dramatically increases microvascular perfusion and glucose delivery within the lamellae; however, static weight shifting (as occurred in horses confined to stocks in the current study) has a much less profound effect.8 Results from the current study suggest that the already low oxygen and nutrient balance within healthy lamellae can be further compromised by a lack of normal ambulation over prolonged periods. The resultant negative energy balance (together with other factors, including altered biomechanics) is likely a major contributor to cell stress and death. This may be exacerbated in cells furthest from the microvascular supply (eg, the parabasal cells close to the keratinized axes of PELs, particularly once there has been retraction of the SDLs). Together, these results suggest that the physical mechanisms of ambulation (as opposed to nonambulatory, static, weight-shifting events) may be necessary to maintain nutrient delivery and tissue homeostasis in the parabasal lamellar keratinocytes even in the absence of increased limb load. This has implications for developing strategies to prevent SLL in the clinic.
Significant increases in TPX-2–positive EBCs mirrored the significant increases in TUNEL-positive parabasal cells, indicating that basal cell proliferation was associated with parabasal cell death in the PWB SL and in both RA forelimbs. It is unclear whether this proliferation represents a secondary event (in response to cell stress and death in parabasal cells) or whether altered mechanostatic signaling was a primary stimulus for proliferation. Given the upregulation of inflammatory signaling in the PWB SL limbs (published previously in a separate report25) and the fact that the RA forelimbs were under normal weight-bearing load, it is more likely that cellular proliferation occurred in response to parabasal cell stress and death. Nonapoptotic cell death (or even excessive apoptotic cell death) has profound proinflammatory effects in epidermal tissues that also stimulate the epidermal cell proliferative response.26 Epidermal basal cell proliferation is also a feature of other forms of laminitis, especially hyperinsulinemia-associated laminitis, where it appears to occur downstream of other events, at least in experimental models.13 Regardless of the cause, EBC proliferation and inflammation are energy-consumptive processes that could exacerbate a preexisting energy crisis affecting lamellar epidermal keratinocytes.
Measurable elongation of the SELs was present only in the PWB SL and PWB hind feet. Interestingly, both of these limbs were subjected to increased mechanical load within the model: a 10% bodyweight–equivalent increase in load for the PWB SL and a 2% bodyweight–equivalent increase in load for the PWB hind feet. Such elongation of SELs (and narrowing as documented in PWB SL) is common to other forms of laminitis and is likely a consequence of mechanical deformation in the face of disruption to the cytoskeleton and cellular adhesions within the SELs.10,13 An additional consideration in the current study is whether concomitant elongation and narrowing of SDL, together with the measured increase in dermal retraction, could have contributed to the narrowing or collapse of capillaries between SELs. The apparently sudden onset of ischemia documented by microdialysis in individual PWB SL limbs in the previous study4 could be reflective of regional mechanical occlusion of capillaries by this mechanism. In contrast, SELs in limbs subjected to reduced load (PWB CF) or normal mechanical load (RA SL and CF) had significant widening of SELs without elongation. This may have been a consequence of active proliferation of basal cells in these limbs in the absence of any increase in mechanical load. An increase in the nonkeratinized PEL length was present in all PWB and RA limbs compared to controls and reflects elongation of the axial region of SELs (that closest to the distal phalanx) where mechanical strain on the lamellae is thought to be greatest. This is a common early finding in other forms of laminitis.10 In the current study, the increase in nonkeratinized PEL length was sufficient in PWB SL limbs to cause an overall increase in the total PEL length measurement.
The main limitation of the current study is that the PWB model used may not be representative of naturally occurring SLL as it lacks the components of overt pain and systemic inflammation (due to trauma or infection), which are often associated with a severe primary limb injury that could predispose a horse to developing SLL in the clinical situation. However, the current study does demonstrate that alterations in limb weight-bearing patterns alone can be damaging to the lamellae even in the absence of these other factors. Determining when or how these developmental/subclinical lamellar lesions translate into clinically and radiographically detectable acute laminitis requires further study. Although there are currently no studies using histochemical markers of cell death applied to naturally occurring SLL, there is evidence that lesions from multiple limbs of natural SLL cases share similar features to those documented here, including damage that is focused along the keratinized axes of PELs.3,6 There are clear limitations to studying natural SLL, including the fact that most lesions are advanced and chronic by the time of euthanasia and tissue harvest; however, the quantification of cell death and proliferation in tissues from multiple limbs in natural SLL cases is warranted. While we cannot definitively identify the dead cells concentrated along the keratinized axes as parabasal cells (without costaining for specific cytokeratins), we propose that given their anatomic location and morphology, these cells are likely to be predominantly parabasal cells (as opposed to basal cells). Another limitation is the relatively small number of horses used in the study as well as the fact that the analysis was performed on small regions of the middorsal lamellae, which may not be representative of changes elsewhere in the digit.
In conclusion, we propose that a lack of normal ambulation interfered with microvascular perfusion, causing cell stress and death, particularly of parabasal keratinocytes. Under conditions of increased limb load, widespread and rapid death of these parabasal cells caused secondary proliferation of EBCs, elongation of primary and secondary lamellae, and subsequent basement membrane detachment. Restricted ambulation with normal limb load distribution was associated with less frequent parabasal cell death and less profound proliferation of basal cells that compromised (but did not destabilize) the lamellae. Since alterations in lamellar perfusion and perturbation of the homeostatic balance between lamellar keratinocyte cell death and proliferation are likely to contribute to natural SLL, further histological characterizations correlated with molecular analyses of natural SLL in multiple limbs (and at different stages of disease) are needed. Strategies for the prevention of SLL should aim not only to reduce weight-bearing load on the SL but to also encourage normal ambulation in all limbs.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors would like to acknowledge Alycia Kowalski, Cade Torcivia, and Dawn Gray-Earle for technical contributions to this work.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the composition of this manuscript.
Funding
Funded by a Grayson Jockey Club Research Foundation Grant.
References
- 2.↑
van Eps AW, Burns TA. Are there shared mechanisms in the pathophysiology of different clinical forms of laminitis and what are the implications for prevention and treatment? Vet Clin North Am Equine Pract. 2019;35(2):379–398. doi:10.1016/j.cveq.2019.04.001
- 3.↑
van Eps A, Engiles J, Galantino-Homer H. Supporting limb laminitis. Vet Clin North Am Equine Pract. 2021;37(3):657–668. doi:10.1016/j.cveq.2021.08.002
- 4.↑
van Eps AW, Belknap JK, Schneider X, et al. Lamellar perfusion and energy metabolism in a preferential weight bearing model. Equine Vet J. 2021;53(4):834–844. doi:10.1111/evj.13356
- 5.↑
Gardner AK, van Eps AW, Watts MR, Burns TA, Belknap JK. A novel model to assess lamellar signaling relevant to preferential weight bearing in the horse. Vet J. 2017;221:62–67. doi:10.1016/j.tvjl.2017.02.005
- 6.↑
Cassimeris L, Engiles JB, Galantino-Homer H. Interleukin-17A pathway target genes are upregulated in Equus caballus supporting limb laminitis. PLoS One. 2020;15(12):e0232920. doi:10.1371/journal.pone.0232920
- 7.↑
Engiles JB, Galantino-Homer HL, Boston R, McDonald D, Dishowitz M, Hankenson KD. Osteopathology in the equine distal phalanx associated with the development and progression of laminitis. Vet Pathol. 2015;52(5):928–944. doi:10.1177/0300985815588604
- 8.↑
Medina-Torres CE, Underwood C, Pollitt CC, et al. The effect of weightbearing and limb load cycling on equine lamellar perfusion and energy metabolism measured using tissue microdialysis. Equine Vet J. 2016;48(1):114–119. doi:10.1111/evj.12377
- 9.↑
Driessen B, Scandella M, Zarucco L. Development of a technique for continuous perineural blockade of the palmar nerves in the distal equine thoracic limb. Vet Anaesth Analg. 2008;35(5):432–448. doi:10.1111/j.1467-2995.2008.00405.x
- 10.↑
De Laat M, Van Eps A, McGowan C, Sillence MN, Pollitt CC. Equine laminitis: comparative histopathology 48 hours after experimental induction with insulin or alimentary oligofructose in Standardbred horses. J Comp Pathol. 2011;145(4):399–409. doi:10.1016/j.jcpa.2011.02.001
- 11.↑
Karikoski NP, Patterson-Kane JC, Asplin KE, et al. Morphological and cellular changes in secondary epidermal laminae of horses with insulin-induced laminitis. Am J Vet Res. 2014;75(2):161–168. doi:10.2460/ajvr.75.2.161
- 12.↑
Faleiros RR, Stokes AM, Eades SC, Kim DY, Paulsen DB, Moore RM. Assessment of apoptosis in epidermal lamellar cells in clinically normal horses and those with laminitis. Am J Vet Res. 2004;65(5):578–585. doi:10.2460/ajvr.2004.65.578
- 13.↑
de Laat MA, Patterson-Kane JC, Pollitt CC, Sillence MN, McGowan CM. Histological and morphometric lesions in the pre-clinical, developmental phase of insulin-induced laminitis in Standardbred horses. Vet J. 2013;195(3):305–312. doi:10.1016/j.tvjl.2012.07.003
- 14.↑
Stokes SM, Belknap JK, Engiles JB, et al. Continuous digital hypothermia prevents lamellar failure in the euglycaemic hyperinsulinaemic clamp model of equine laminitis. Equine Vet J. 2019;51(5):658–664. doi:10.1111/evj.13072
- 15.↑
Bankhead P, Loughrey MB, Fernandez JA, et al. QuPath: open source software for digital pathology image analysis. Sci Rep. 2017;7(1):16878. doi:10.1038/s41598-017-17204-5
- 16.↑
Pollitt CC. Basement membrane pathology: a feature of acute equine laminitis. Equine Vet J. 1996;28(1):38–46. doi:10.1111/j.2042-3306.1996.tb01588.x
- 17.↑
de Laat MA, McGowan CM, Sillence MN, et al. Equine laminitis: induced by 48 h hyperinsulinaemia in Standardbred horses. Equine Vet J. 2010;42(2):129–135. doi:10.2746/042516409X475779
- 18.↑
Medina-Torres CE, Underwood C, Pollitt CC, et al. Microdialysis measurements of lamellar perfusion and energy metabolism during the development of laminitis in the oligofructose model. Equine Vet J. 2016;48(2):246–252. doi:10.1111/evj.12417
- 19.↑
Stokes SM, Bertin FR, Stefanovski D, et al. The effect of continuous digital hypothermia on lamellar energy metabolism and perfusion during laminitis development in two experimental models. Equine Vet J. 2020;52(4):585–592. doi:10.1111/evj.13215
- 20.↑
Stokes SM, Bertin FR, Stefanovski D, et al. Lamellar energy metabolism and perfusion in the euglycaemic hyperinsulinaemic clamp model of equine laminitis. Equine Vet J. 2020;52(4):577–584. doi:10.1111/evj.13224
- 21.↑
Catunda APN, Alves GES, Paes Leme FO, et al. Apoptosis in epithelial cells and its correlation with leukocyte accumulation in lamellar tissue from horses subjected to experimental sepsis-associated laminitis. Res Vet Sci. 2021;136:318–323. doi:10.1016/j.rvsc.2021.03.009
- 22.↑
Janke LJ, Ward JM, Vogel P. Classification, scoring, and quantification of cell death in tissue sections. Vet Pathol. 2019;56(1):33–38. doi:10.1177/0300985818800026
- 23.↑
Gupta D, Singla R, Mazzeo AT, et al. Detection of metabolic pattern following decompressive craniectomy in severe traumatic brain injury: a microdialysis study. Brain Inj. 2017;31(12):1660–1666. doi:10.1080/02699052.2017.1370553
- 24.↑
Pawlak EA, Geor RJ, Watts MR, Black SJ, Johnson PJ, Belknap JK. Regulation of hypoxia-inducible factor-1α and related genes in equine digital lamellae and in cultured keratinocytes. Equine Vet J. 2014;46(2):203–209. doi:10.1111/evj.12092
- 25.↑
Burns TA, Watts MR, Belknap JK, van Eps AW. Digital lamellar inflammatory signaling in an experimental model of equine preferential weight bearing. J Vet Intern Med. 2023;37(2):681–688. doi:10.1111/jvim.16662
- 26.↑
Anderton H, Alqudah S. Cell death in skin function, inflammation, and disease. Biochem J. 2022;479(15):1621–1651. doi:10.1042/BCJ20210606
