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De-laat, Melody, Patterson-Kane, Janet, Pollitt, Christopher, Sillence, Mar-tin, & McGowan, Catherine(2013)Histological and morphometric lesions in the pre-clinical, developmentalphase of insulin-induced laminitis in Standardbred horses.The Veterinary Journal, 195(3), pp. 305-312.

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https://doi.org/10.1016/j.tvjl.2012.07.003

1

Original article

Histological and morphometric lesions in the pre-clinical, developmental phase

of insulin-induced laminitis in Standardbred horses

Melody A. de Laat a*

, Janet C. Patterson-Kane b, Christopher C. Pollitt

a,

Martin N. Sillence c, Catherine M. McGowan

d

a

Australian Equine Laminitis Research Unit, School of Veterinary Science, The

University of Queensland, Gatton, Queensland 4343, Australia. b

School of Veterinary Medicine, College of Medical, Veterinary and Life Sciences,

University of Glasgow, Bearsden Road, Glasgow G61 1QH, Scotland c Faculty of Science and Technology, Queensland University of Technology, Brisbane,

Queensland 4001, Australia. d

Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences,

University of Liverpool, Neston, CH64 7TE, UK

* Corresponding Author: Tel.: +61733650079.

Email address: m.delaat@uq.edu.au (M. A. de Laat)

2

Abstract

Lamellar pathology in experimentally-induced equine laminitis associated

with euglycaemic hyperinsulinaemia is substantial by the acute, clinical phase (~ 48 h

post-induction). However, lamellar pathology of the developmental, pre-clinical phase

requires evaluation. The aim of this study was to analyse lamellar lesions both

qualitatively and quantitatively, 6 h, 12 h and 24 h after the commencement of

hyperinsulinaemia. Histological and histomorphometrical analyses of lamellar

pathology at each time-point included assessment of lamellar length and width,

epidermal cell proliferation and death, basement membrane (BM) pathology and

leucocyte infiltration. Archived lamellar tissue from control horses and those with

acute, insulin-induced laminitis was also assessed for cellular proliferative activity by

counting the number of cells showing positive nuclear immunolabelling for TPX2.

Decreased SEL width and increased evidence of apoptotic SEL epidermal basal (and

suprabasal) cells occurred early in disease progression (6 h). Increased cellular

proliferation in SELs, infiltration of the dermis with small numbers of leucocytes and

BM damage occurred later (24 h, 48 h). Some lesions, such as narrowing of the SELs,

were progressive over this time period (6 - 48 h). Cellular pathology preceded

leucocyte infiltration and BM pathology, indicating that the latter changes may be

secondary or downstream events in hyperinsulinaemic laminitis.

Keywords: Equine; Hyperinsulinaemia; Histology; Epidermal lamellae; TPX2

3

Introduction

Equine laminitis is a complex disease in which the lamellar attachment

between the inner hoof wall and the distal phalanx is compromised. It accounts for up

to 15% of lameness in the equine population (USDA, 2000). Despite improved

methods for diagnosis, management and prevention of the condition (Carter et al.,

2009b; Frank, 2009; Geor and Harris, 2009) specific therapies that target the

underlying pathophysiology of laminitis are limited. The development of efficacious

treatment options requires a deeper understanding of disease pathogenesis.

Endocrinopathic laminitis, associated with pars pituitary intermedia

dysfunction and equine metabolic syndrome, is characterised by hyperinsulinaemia in

both horses and ponies, and this appears to be the key hormonal mechanism

associated with lamellar failure (Field and Jeffcott, 1989; Geor, 2009; McGowan et al.,

2004; Treiber et al., 2006). However, the exact mechanism by which

hyperinsulinaemia causes lamellar pathology is unknown. Research on

endocrinopathic laminitis has been aided by the development of an experimental

model, which induced laminitis with hyperinsulinaemia, using a prolonged

euglycaemic, hyperinsulinaemic clamp (p-EHC), within 72 h in ponies and 48 h in

horses (Asplin et al., 2007; de Laat et al., 2010).

Previous histological assessments of acute, Obel grade 2, insulin-induced

laminitis in horses and ponies treated with a p-EHC have described secondary

epidermal lamellar (SEL) lengthening and narrowing, with increased evidence of

mitotic activity and apoptotic epidermal cells (Asplin et al., 2010; de Laat et al.,

2011b; Obel, 1948). Lesions occurring in the preclinical, developmental phase of this

4

model have not been described. Lamellar inflammation has been reported in the

developmental phase of the black walnut (BWE) and alimentary carbohydrate

overload (ACO) experimental models of laminitis (Black et al., 2006; Faleiros et al.,

2011b; Visser, 2008). However, fewer leucocytes were present in the lamellae of

horses in the clinical phase of insulin-induced (p-EHC) laminitis compared with ACO

laminitis (de Laat et al., 2011b). This suggests that insulin-induced laminitis involves

less overt inflammation of the lamellae than alimentary forms of the disease, and

leucocyte infiltration may not be an early pathogenic lesion.

This study hypothesised that cellular, inflammatory and degradative events

occur in the lamellae prior to the onset of the clinical signs of laminitis, and that these

lesions are not identical to those described in other models of the disease. For the

current study the Standardbred model of p-EHC-induced laminitis (de Laat et al.,

2010) was repeated, with examination of lamellar tissue from earlier time-points,

prior to the onset of clinical signs. Specifically we aimed to analyse microscopic

lamellar lesions both qualitatively and quantitatively, 6 h, 12 h and 24 h after the

commencement of hyperinsulinaemia.

Materials and Methods

Ethical approval for this work was granted by the Animal Ethics Committee of

The University of Queensland (SVS/108/09/RIRDC). The horses were monitored by a

registered veterinarian throughout the study.

Horses

5

Twelve Standardbred geldings (7.3 ± 1.12 years, 440 ± 51.6 kg BW), recently

retired from racing (< 6 weeks), were used in this study. They were in moderate body

condition (body condition score: 4.7/9 (Henneke et al., 1983)) with no phenotypic

indicators (cresty neck score: 0/5 (Carter et al., 2009a)) of insulin resistance (IR).

Routine haematological and biochemical parameters were measured, as indicators of

general health status, before and at the conclusion of the experiment. All horses were

sound on lameness examination and had no evidence of laminitis on clinical and

radiographic inspection of the feet. Horses were randomly allocated to one of three

treatment groups: 6 h (n = 4), 12 h (n = 4) and 24 h (n = 4).

The experiment was conducted as six paired replicates over four weeks. The

subjects were acclimated to the climate-controlled (22.4 ± 0.34 °C) research facility

and fed moderate-quality lucerne hay and chaff for the 48 h preceding the study. Ad

libitum access to water and the same food was allowed during the study. Archived

lamellar samples from horses with acute insulin-induced laminitis (48 h, (n = 4)), and

matched control (n = 4) horses treated with a balanced electrolyte solution for 48 h,

were also used for cell proliferation studies (de Laat et al., 2010). The same control

horse lamellar tissue was used as a reference point for histological examination and

morphometry. The horses in these control and laminitic groups were of similar age,

bodyweight, BCS and breed to the horses in the current study and the experimental

protocols were identical.

Prolonged euglycaemic, hyperinsulinaemic clamp (p-EHC)

All subjects were given an insulin bolus (45 mIU/kg BW; Humulin-R)

followed by a combined insulin (6 mIU/kg BW/min) and glucose (variable rate; 50%

6

dextrose) infusion for 6 h, 12 h or 24 h to maintain euglycaemic hyperinsulinaemia,

using the EHC technique (DeFronzo et al., 1979). Extended-use IV catheters

(MilaCath) were maintained in right and left jugular veins for the administration of

the infusions and blood sampling respectively. Blood samples were collected for

determination of blood glucose (1 mL) and serum insulin (10 mL) concentrations at 0

h, hourly until 8 h, then four-hourly throughout the infusion period. In addition, three

samples were collected 10 min apart, to measure blood glucose and serum insulin

concentrations during a steady state period (< 180 min), which is defined as a 30 min

period when the glucose infusion rate does not require adjustment.

Blood glucose was measured immediately using a handheld glucometer

(Accucheck-Go, Roche) previously calibrated for equine blood against the hexokinase

method (ρc = 0.96) by the authors (de Laat et al., 2010). Serum insulin concentration

was measured using a validated radio-immunoassay (Coat-a-count, Siemens) with

insulin-free serum used as the diluent (McGowan et al., 2008; Tinworth et al., 2009).

Insulin sensitivity (SI) was calculated retrospectively for all horses by measuring the

amount of glucose metabolised (M) and M per unit of insulin (M-to-I ratio), according

to standard protocols (Rijnen and van der Kolk, 2003).

Histology and Immunohistochemistry

At the end of the experiment horses were euthanased and samples of lamellar

tissue were immediately collected from the dorsal mid-section of the hoof wall. The

hoof wall was cut into sagittal blocks with a band-saw, and lamellar specimens (5 mm

x 5 mm) were dissected with a scalpel, rinsed and placed in 10% neutral buffered

formalin for 24 h. Lamellar specimens were processed routinely for histology,

7

embedded in paraffin wax and sectioned at 5 µm. Sections were stained with

haematoxylin and eosin (H & E) and mounted on Superfrost Plus slides (Menzel);

further sections were stained using the periodic acid-Schiff (PAS) method for

visualisation of basement membranes (BM).

Immunohistochemical staining of formalin-fixed, paraffin-embedded sections

from all four feet of the 6 h, 12 h and 24 h groups was performed for leucocyte

identification with a mouse monoclonal macrophage antibody (calprotectin/MAC-387;

Dako) using the EnVision+ System-HRP (Dako). Antigen retrieval was performed

with 0.05% Proteinase K (Dako) for 8 min at room temperature (de Laat et al., 2011b).

Immunolocalisation of proliferating cells was performed in both fore-feet from horses

treated with a p-EHC for 6 h, 12 h, 24 h and 48 h, and control horses, with a mouse

monoclonal antibody to the TPX2 protein (TPX2/ab32795; Abcam) using an

automated stainer and the HRP system (Dako). Antigen retrieval was achieved with a

digital electric pressure cooker (Menarini Antigen Access Unit), in sodium citrate

buffer (pH 6; 125 oC for 1 min 40 s).

Calprotectin is located in the cytoplasm of neutrophils and on the membrane

of monocytes (Striz and Trebichavsky, 2004). TPX2 recognises a nuclear protein

(repp86/p100) associated with cell proliferation in certain tissues, including the

epithelium (Heidebrecht et al., 2003). Unlike the Ki-67 antigen, a more commonly

used immunohistochemical proliferation marker, TPX2 does not immunolocalise cells

in the G1 phase of the cell cycle which avoids over-estimating the number of

proliferating cells (Heidebrecht et al., 1997; Rudolph et al., 1998).

8

The antibodies have been validated for use in horses by western blot (JPK pers

comm., Visser, 2008). Primary antibodies were optimally diluted (MAC-387; 1:400,

TPX2 1:800) in antibody-dilution solution (Dako). Positive control tissues were

lamellae from a horse with ACO laminitis (MAC-387) and human and equine skin

(TPX2). Negative controls used mouse IgG in place of the primary antibody. Sections

were stained (2 x 5 min) with chromagen 3.3’-diaminobenzidine tetrahydrochloride

(DAB, Dako) followed by nuclear staining with Mayer’s haematoxylin (30 s).

Immunoreactivity was analysed in all sections, blinded, by one of the authors

(MAD) using image analysis software (ImagePro). Calprotectin immunoreactivity

was scored using the following scale: 0 = no staining or occasional calprotectin-

positive cell within a vessel lumen; 1 = mild perivascular infiltration of calprotectin-

positive cells into primary dermal lamellar tissue; 2 = moderate, diffuse infiltration of

calprotectin-positive cells throughout dermal connective tissue; 3 = marked tissue

infiltration of calprotectin-positive cells with positive staining of SEL basal and

parabasal cell cytoplasm (de Laat et al., 2011b).

Total TPX2-positive cell counts were made across five consecutive primary

epidermal lamellae (PELs) in both fore-feet. The length of each PEL was measured

and it was divided into abaxial and axial halves. The total number of TPX2-positive

cells in each half of the five PELs was counted to obtain an abaxial, axial and total

value for each front foot. Cell counts from both fore-feet were averaged to obtain a

single value for each horse to account for statistical clustering. A subset (40%) of

tissue sections was measured by a second, blinded author (CCP) to internally validate

the measurement protocol.

9

Histomorphometry

H & E and PAS sections from all four feet of all horses were examined via

light microscopy (Olympus BX-50) by a veterinary pathologist (JPK), who was

blinded to the group of origin. Measurement of lamellar morphometry was performed

blinded, by a different author (MAD), after randomisation of the sections.

Measurements were made on eight, consecutive PELs, using image analysis software

(ImagePro), for each foot. Total (TPELL) and keratinised PEL lengths (KPELL) were

measured. The length of ten SELs was measured separately in the abaxial (SELLB)

and axial (SELLT) halves of each of the eight PELs, for each foot. SEL width (SELW)

of ten consecutive SELs was also measured on both sides (in the mid-section) of all

eight PELs for every horse (n = 20). This measurement protocol has been described

and validated previously (de Laat et al., 2011b).

Statistical analyses

Inter-rater measurement agreement was analysed with Lin’s concordance co-

efficient (ρc) and Bland-Altman’s limits of agreement (LOA) with random variation

assessed with Pearson’s correlation co-efficient (r). An average of forefoot PEL

measurements was compared to averaged hindfeet within each group using

Wilcoxon’s t-test. Average fore-feet measurements, total TPX2 counts and

calprotectin scores were compared between groups using a Kruskal-Wallis ANOVA

on ranks. Multiple pair-wise comparisons were made with Tukey’s test. SELW was

correlated against time with Pearson’s correlation co-efficient (r). TPX2 data was

compared between the abaxial and axial locations for each group with Wilcoxon’s t-

test. All data are presented as mean ± standard error (se) or median (range). Statistical

10

significance was set at P < 0.05. Statistical analyses were performed using R, version

2.7.2.

Results

Prolonged euglycaemic, hyperinsulinaemic clamp (p-EHC)

All subjects remained healthy throughout the experiment with heart rate,

respiratory rate, rectal temperature and haematological and biochemical parameters

remaining unchanged from basal values for each group. Lameness was not detected in

any of the horses. Blood glucose concentrations did not differ between the groups

either before or during the infusion (Table 1). Basal serum insulin concentrations did

not differ between groups. Serum insulin concentrations increased (P < 0.05) from

basal values during the initial 4 h of the infusion and remained elevated (> 800

µIU/mL) for the remainder of the experiment for all groups (Fig. 1). All of the horses

were insulin sensitive (Table 1).

Subjective histological assessment

Lamellar morphology

Compared with control horses, elongation of the SELs, with tapering of the

tips, and a reduction in the angle between the SEL and the PEL axis appeared to be

occurring by 6 h, in treated horses (Fig. 2a, b). The orientation of SEL nuclei had

changed from the long axis being perpendicular to the BM, to parallel (Fig. 3a, b), and

remained in this orientation across all time-points. At 12 h, elongation, tapering and

angulation of the SELs appeared more pronounced, compared with 6 h (Fig. 2c).

SELs appeared further elongated, tapered and angulated at 24 h, and were frequently

11

difficult to distinguish from each other, except at the axial tips of the PELs (Fig. 2d).

The SEL nuclei appeared larger, with enlarged nucleoli, and the epithelial cells more

disorganised. In three of the 24 h horses, there was either slight blebbing of the BM or

more extensive separation from the epidermal cells at SEL tips of a few PEL (axial)

tips (Fig. 3d). In the areas of most marked BM separation, nuclear debris and small

numbers of infiltrating neutrophils were noted within the separated material. Mild

dyskeratosis, with minimally increased keratin, was observed in the SEL and PEL of

some horses at all time-points. Rare keratin pearls were noted in SEL tips of three

laminitic horses at the 6 h time-point; these were regarded to be chronic, background

lesions.

Mitotic figures

Occasional mitotic figures were found in SELs at the axial tips of PELs in

some sections at 6 h (Table 2). A few mitotic figures were present at the 12 h time-

point, and again noted in SELs at or near the axial tips of PELs (Fig. 3c). Mitotic

figures were more frequent again at 24 h than at 6 h or 12 h, but remained localised to

SELs at PEL tips.

Apoptosis

Cells that appeared to be undergoing apoptosis were noted in most sections at

6 h, but varied significantly in number between horses (Table 2). These cells were

identified on the basis of characteristic morphological changes such as rounding,

shrinkage, hypereosinophilia, cytoplasmic budding (formation of apoptotic bodies,

some of which contain nuclear fragments), nuclear chromatin condensation and

fragmentation and phagocytosis by neighbouring epithelial cells (Meyers and

12

McGavin, 2007). There was no evidence of cellular swelling, nuclear changes or

intra-epithelial inflammation that would characterise necrosis. The apoptotic bodies

were located in SELs at any point along the length of the PELs in sections from some

horses, but were concentrated in SELs only at axial tips of the PELs in others (Fig,

3b). Apoptotic cells were again found in SELs at any point along the length of the

PELs in the 12 h group, with clustering in SELs at the axial tips of the PELs noted in

some sections. However, the number of apoptotic bodies or cells appeared decreased

overall, at this time-point, compared to 6 h. Minimal apoptosis occurred in the 24 h

group, often in the same microscopic fields where mitotic figures were observed.

Dermal changes

Myxomatous (fibrillar basophilic) material was seen immediately surrounding

PEL tips and around blood vessels in both the secondary and primary dermal lamellae

(SDL, PDL; Fig. 2b, c). Endothelial cells of all blood vessels were swollen. The

appearance of the dermis was similar at all time-points.

Histomorphometry

Forefoot TPELL and KPELL was increased compared to the hind-foot in the

48 h group, whereas only KPELL differed in the 24 h group. Forefoot TPELL and

KPELL did not differ between groups (Table 3). Neither SELLB nor SELLT was

different between fore- and hind-feet for any group. The length of the forefoot SELs

did not differ between control, 6 h, 12 h or 24 h horses at either location, but there

was a numerical increase in length over time (Table 3). SEL length was only

increased significantly by the acute phase at 48 h (de Laat et al., 2011b). Identical

measurement protocols permitted direct comparison of the data between the two

13

studies. SELs were wider in the fore-feet of the 6 h group compared to the hind feet.

Forefoot SEL width decreased significantly over time (r = -0.92). SELs were wider (P

< 0.05) in the control and 6 h groups (Fig. 4).

Immunohistochemistry

TPX2

Inter-rater variability (Lin’s ρc (lower C.I.) = 0.95 (0.92)) was minimal with

random variation (r = 0.98) largely accounting for disparity. Bland-Altman’s LOA

(C.I.) were 4.4 (-72 - 81) which shows that only minor count differences between

assessors occurred. TPX2-positive nuclei were infrequent in sections from control

horses, and horses treated with the p-EHC for 6 h and 12 h (Fig. 5). However, TPX2

expression was increased (P < 0.05) in both the 24 h and 48 h groups when compared

to all other groups (Fig. 5). TPX2-positive cells were found in SELs along the length

of the PELs and stained cells undergoing mitosis, as well as other cells with no visible

mitotic changes (as expected; Fig. 6). The degree of TPX2 immunoreactivity did not

differ between the abaxial and axial halves of the PEL for any group (Table 2).

Calprotectin

Median (range) calprotectin score did not differ between control horses and

any developmental time-point (Table 2). There was no immunoreactivity to

calprotectin, except for an occasional leucocyte within the vessel lumen, in the hind-

feet of all horses and the fore-feet of the 6 h group (Fig. 6). Only one horse from the

12 h group showed mild immunoreactivity to calprotectin around primary dermal

vessels in the right forefoot, while the remaining feet did not react. In the 24 h group,

14

immunolocalisation of calprotectin was restricted to mild perivascular infiltration of

the PDL tissue in one forefoot of two horses (Fig. 6).

Discussion

The current study is the first to describe the histopathology of the

developmental phase of p-EHC-induced laminitis and provides a temporal analysis of

some key features of the disease. Although significant histomorphometric differences

in SEL length were not found between the developmental time-points examined in

this study, SELs were longer by the onset of clinical laminitis at 48 h (de Laat et al.,

2011b). Failure to detect a significant change in the length of the SELs up to 24 h may

be related to the small sample size or the time-points examined. More marked

increases in SEL length may occur later in the developmental phase, after the 24 h

time-point, but examination of time-points between 24 h and 48 h would be required

to establish this.

Elongation of the SELs may be related to an increase in the number of

epidermal cells. The increased presence of mitotic figures and TPX2

immunolocalisation in SELs at 24 h preceded significant increases in length,

suggesting that increased numbers of epidermal cells contribute to SEL lengthening.

Alternatively, stretching of the existing epidermal cells may cause, or at least

contribute to, early increases in SEL length. Hyperinsulinaemia may alter epidermal

basal cell metabolism which in-turn, may affect the cytoskeleton of SEL cells,

reducing their ability to withstand normal biomechanical stress on the lamellae. Loss

of the normal orientation and ovoid shape of basal cell nuclei may also result from

cytoskeletal compromise. Weakening of basal cells may lead to physical disruption of

15

the basement membrane and further lengthening of the lamellae. Temporal analysis of

epidermal cell size, shape and number in SELs throughout the developmental phase,

and at the onset of the clinical phase would be required to determine whether

stretching or proliferation of cells is predominant. Ultrastructural examination of

lamellar tissue with electron microscopy during the developmental and acute stages of

insulin-induced laminitis would also enhance our appreciation of any cytoskeletal

and/or desmosomal changes that may be occurring.

An interesting finding of the current study was the progressive narrowing of

SELs throughout the developmental phase, with a significant decrease in SEL width

occurring between 6 h and 12 h after the onset of hyperinsulinaemia. Narrowing of

the SELs may be another early indicator of cellular disorganisation and stretching,

occurring in conjunction with SEL elongation. Stretching of the epidermal cells, and

hence SELs, indicates disruption of the normal support for the distal phalanx within

the hoof capsule, which may be a causative factor for the distal displacement of the

distal phalanx that accompanies later stages of the disease.

Insulin has mitogenic potential (Myers and White, 1995) and could be the

primary instigator of the increased cell division occurring in the treated horses.

Elevated numbers of mitotic figures have been reported in alimentary forms of

laminitis, not associated with hyperinsulinaemia (Galey et al., 1991). However, many

different factors can stimulate cellular division and these will not necessarily be

identical in different forms of laminitis. Potential mitogenic factors include cytokines,

growth factors, hormones and a wide variety of cellular stressors. Insulin-like growth

factor-1 (IGF-1) promotes cell survival through an increase in cell proliferation and a

16

reduction in apoptosis (Pavelic et al., 2007). While IGF-1 levels are unlikely to be

increased by hyperinsulinaemia, excessive circulating insulin levels may bind the

IGF-1 receptor and activate the IGF-1 system, favouring cell survival and

proliferation. Increases in inflammatory cytokines have been demonstrated in both

ACO and BWE models of laminitis (Belknap et al., 2007; Leise et al., 2011).

Although their role in hyperinsulinaemic laminitis has not been well defined, pro-

inflammatory cytokines may contribute to increased epidermal cell proliferation.

Evidence of apoptosis was also found to be prevalent following 6 h of

hyperinsulinaemia, but appeared to decrease later in the developmental stages.

Electron microscopic evaluation and immunostaining of the lamellae with an early

marker for apoptosis may support the histological observations made in this study.

However, current immunolabelling methodologies have not been well validated in the

horse and further research into appropriate immunolabels is warranted. The stimulus

for a potential increase in the incidence of apoptosis early in hyperinsulinaemic

laminitis pathology is unknown.

An increase in apoptosis is not unique to insulin-induced laminitis and has

been reported in horses with acute, naturally-acquired laminitis (of unstated cause),

compared with none in normal horses (Faleiros et al., 2004). Hormones, growth

factors, oxidative stress and TNF-α, among other factors, are known inducers of

apoptotic cell death (Avogaro et al., 2010; Kiess and Gallaher, 1998). Although

visceral adipose TNF-α mRNA concentration is similar in both insulin-resistant and

insulin-sensitive horses (Burns et al., 2010) this may not be reflected in the lamellae

and lamellar TNF-α concentration during insulin-induced laminitis development has

17

not been investigated. Current data on the redox status of horses during laminitis does

not support a significant role for reactive oxygen species in disease pathogenesis

(Keen et al., 2004; Treiber et al., 2009), and markers of oxidative stress have not been

found to be increased at any developmental time-point of insulin-induced laminitis

(de Laat et al., 2012). IGF-1 is a known inhibitor of apoptosis and its withdrawal from

a tissue may induce apoptosis (Kiess and Gallaher, 1998). Potentially, apoptosis may

increase secondary to this mechanism during hyperinsulinaemia, if insulin binding of

the IGF-1 receptor reduces tissue IGF-1 concentrations. Furthermore, binding of the

IGF-1 receptor by insulin during hyperinsulinaemia may result in activation of the

IGF system and promotion of cell survival, which could account for the reduction in

apoptosis seen as the study progressed (Chitnis et al., 2008). Studies of the role of

IGF-1 in laminitis are required to better understand its pathogenic potential.

BM separation in SELs at PEL tips was first identified at 24 h, was not present

in all sections and in most cases was not a diffuse or severe change. These findings

suggest that BM separation is a secondary lesion. A lack of extensive BM damage is

also consistent with reports of minimal metalloproteinase activity in the lamellae

during the developmental phase of insulin-induced laminitis (de Laat et al., 2011a).

The calprotectin data supports our previous supposition that the insulin-

induction model is associated with a milder inflammatory response than other

experimental models of laminitis (de Laat et al., 2011b). Less calprotectin

immunoreactivity was seen at 48 h in the insulin-induction model compared with

strong calprotectin expression in the lamellar dermis in the acute and developmental

(18 h and 24 h) phases of ACO laminitis, (de Laat et al., 2011b; Faleiros et al., 2011a;

18

Visser, 2008). The BWE model of laminitis has also been associated with increased

presence of inflammatory cells in lamellar tissue, which begins as early as 1.5 h

following induction (Black et al., 2006; Faleiros et al., 2011b). At the 24 h time-point

of the current study, calprotectin immunolocalisation was limited to being

perivascular, with only small numbers of leucocytes present in 50% of the horses

examined. Neutrophils appear to emigrate to the lamellae after pathology is underway

in hyperinsulinaemic laminitis, which suggests that they respond to pathology, rather

than initiate it. These findings may be important when selecting appropriate treatment

modalities for hyperinsulinaemic laminitis.

Conclusions

Histological evaluation of horses in the pre-clinical phase of insulin-induced

laminitis showed that significant lamellar histopathology occurs prior to the

development of clinical signs at 48 h. Overall, structural changes such as SEL

lengthening and narrowing and some cellular changes, including apoptosis and

nuclear disorientation, occur earlier in disease progression, whereas mitosis and BM

dysadhesion appear to be downstream events. Increased cellular proliferation, cellular

stretching or both, may be associated with significant lengthening of the SELs and

contribute to lamellar dysfunction and lameness. Prevention of exuberant cell

proliferation and differentiation may prove to be a potential avenue for disease

management.

Conflict of interest statement

None of the authors has a financial or personal relationship with other people

or organisations that could inappropriately influence or bias the content of the paper.

19

Acknowledgements

This study was funded by the Rural Industries Research and Development

Corporation, Australia.

20

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24

Table 1. Three groups of horses (n = 4) were treated with a euglycaemic,

hyperinsulinaemic clamp for 6 h, 12 h or 24 h. All horses were insulin sensitive.

Variable 6 h group 12 h group 24 h group

G basal (mM) 5.3 ± 0.32 5.5 ± 0.14 5.6 ± 0.21

G clamp (mM) 4.3 ± 0.19 4.1 ± 0.35 4.4 ± 0.21

I basal (µIU/mL) 10.2 ± 2.95 8.68 ± 1.07 12.0 ± 4.2

I steady state (µIU/mL) 811 ± 132 977 ± 86 747 ± 41

M (mmol/kg/min) 0.02 ± 0.003 0.023 ± 0.004 0.021 ± 0.002

M-to-I ratio (10-6

) 3.3 ± 0.4 3.2 ± 0.7 3.9 ± 0.2

Key: G basal: basal glucose concentration, G clamp: blood glucose concentration during

the clamp, I basal: basal serum insulin, I steady state: serum insulin concentration (I) at

steady state, M: amount of glucose metabolised and a measure of sensitivity of tissues

to exogenous insulin, M-to-I ratio: glucose metabolised per unit of exogenous insulin.

25

Table 2: Median (range) TPX2-positive epidermal cell counts in the abaxial and axial

halves of 5 forefeet PELs and calprotectin score following immunohistochemical

staining. Calprotectin data for control and 48 h horses is from de Laat et al. (2011).

Mitotic figures and apoptotic cell counts/hpf in the lamellae of horses treated with a

prolonged euglycaemic, hyperinsulinaemic clamp for 6 h (n = 4), 12 h (n = 4), 24 h (n

= 4) or 48 h (n = 4) and a control group (CH, n = 4) treated with a balanced electrolyte

solution for 48 h.

Pathology CH 6 h 12 h 24 h 48 h

TPX2

Abaxial 0.5 (0.3 –

1.8)

3.9 (2.4 –

4.3)

1 (0.75 –

1)

63 (39.3

– 98) *

159 (87 –

202) *

Axial 5 (2.5 –

10.5)

6.5 (1 –

12.3)

2.5 (1 –

5)

216 (87 –

454) *

121 (87 –

162) *

Calprotectin

Fore 0 (0 - 0) 0 (0 - 0) 0 (0 - 0) 0 (0 –

0.5)

1.8 (1 –

2) *

Hind 0 (0 - 0) 0 (0 - 0) 0 (0 - 0) 0 (0 - 0) 0 (0 -

0.3)

Mitotic figures/hpf 0 0 0-1 0-5 0-5

Apoptotic cells/hpf 0 0-15 0-5 0-1 0-2

Key: TPX2: Immunohistochemical marker of cellular proliferation, hpf: high power

field (400 X magnification). The asterisks indicate which groups differ (P < 0.05).

26

Table 3: Median (range) length (µm) of the primary (PELs) and secondary epidermal

lamellae (SELs) and width of SELs measured in all four feet of horses treated with a

prolonged euglycaemic, hyperinsulinaemic clamp for 6 h (n = 4), 12 h (n = 4), 24 h (n

= 4) or 48 h (n = 4) and a control group (CH, n = 4) treated with a balanced electrolyte

solution for 48 h. Data for control and 48 h horses is from de Laat et al. (2011).

Parameter CH 6 h 12 h 24 h 48 h

TPELL

Fore 3359 (3008 -

3663)

3049 (2894 –

3418)

3413 (2956 –

3651)

3169 (3100 –

3299) §

3384 (3292 –

3442) §

Hind 2766 (2569 –

3083)

2724 (2655 –

2913)

2953 (2405 –

3417)

2672 (2372 –

2891)

2750 (2631 –

2785)

KPELL

Fore 2895 (2750 –

3180)

2583 (2320 –

2854)

2660 (2533 –

2868)

2626 (2089 –

2717)

2779 (2677 –

2843) §

Hind 2578 (2346 –

2962)

2321 (2250 –

2503)

2527 (2160 –

3666)

2155 (1967 –

2477)

2215 (2195 –

2256)

SELLB

Fore 142 (107 –

173)

176 (149 –

202)

177 (164 –

199)

190 (168 –

219)

226 (213 –

254) *

Hind 130 (117 –

157)

228 (174 –

268)

171 (131 –

326)

135 (120 –

203) 198 (175 - 227)

SELLT

Fore 116 (77 – 155) 169 (149 –

179)

174 (141 –

205)

198 (163 –

218)

270 (187 –

326)

Hind 125 (113 –

130)

172 (148 –

184)

157 (113 –

308)

161 (109 –

211)

216 (201 –

232)

SELW

Fore 25 (21.5 –

28.5) *

25.2 (22.5 –

26.1) *§

19.9 (16.2 -

20.2)

20.2 (19 –

20.7)

12.3 (10.8 –

15)

Hind 23 (20 – 25) 18 (17.9 –

19.5)

21.2 (19.7 –

22.3)

17.7 (15.9 –

18.8)

15.8 (11.5 –

22)

Key: TPELL: total primary epidermal lamellar length, KPELL: keratinised primary

epidermal lamellar length, SELLB: secondary epidermal lamellar length (abaxial end

of the PEL), SELLT: secondary epidermal lamellar length (axial end of the PEL),

SELW: secondary epidermal lamellar width in the mid-section of the PEL. * indicates

forefoot parameters that differ (P < 0.05) between groups. §

indicates if forefeet differ

(P < 0.05) to the hind-feet within each group.

27

Fig. 1. Mean serum insulin concentration (µIU/mL) of three groups of horses (n = 4)

treated with a euglycaemic, hyperinsulinaemic clamp for 24 h (●), 12 h (∆) and 6 h

(▒). Serum insulin increased (P < 0.05) from basal levels over the first 4 h of the

infusion and remained elevated for the remainder of the experiment.

28

Fig. 2. Photomicrographs of the axial ends and entire length (insets) of primary

epidermal lamellae (PELs), with attached secondary epidermal lamellae (SELs). a;

control horse (48 h), b; 6 h time-point. There are changes in the lamellar morphology

relative to the control horse in terms of elongation, narrowing and tapering of the

SELs. Myxomatous (basophilic) material is present around dermal blood vessels. c;

12 h time-point. Subjectively, there is further elongation of SEL. Myxomatous

material (arrow) is still present at this time-point, around now dilated blood vessels in

the dermis. d; 24 h time-point, with similar changes to those noted at 12 h.

Haematoxylin and eosin. Bar = 100 µm.

29

Fig. 3. Photomicrographs of secondary epidermal lamellae (SELs) at the axial tips of

PELs. a; control horse with long axes of the nuclei of basal SEL cells (arrow) oriented

perpendicularly to the basement membrane (BM). b; 6 h time-point, with increased

numbers of apoptotic cells (arrows). Long axes of many nuclei of SEL basal cells are

now oriented parallel to the BM. b inset; two of the apoptotic bodies have been

phagocytosed by epithelial cells, the nuclei of which are slightly compressed (arrows).

c; 12 h time-point, with infrequent mitotic figures (arrow). d; 24 h time-point with

blebbing i.e. slight separation of the BM from tips of the SELs (arrows). The inset

shows one such “bleb”, creating a clear space at the SEL tip; the arrow indicates the

BM. e; 24 h time-point with more advanced, complete separation of the BM from

SEL tips (arrows). a, b, c = Haematoxylin and eosin. Bar = 50 µm. d = Periodic acid-

Schiff (PAS). Bar = 50 μm. e =PAS. Bar = 100 µm.

30

Fig. 4. Secondary epidermal lamellar (SEL) width (µm) in horses treated with a

euglycaemic, hyperinsulinaemic clamp for 6 h (n = 4), 12 h (n = 4), 24 h (n = 4) and

48 h (n = 4) and control horses (CH, n = 4) treated with a balanced electrolyte

solution for 48 h. Median SEL width (―) is shown for each group. SELs were wider

(P < 0.05) in control horses and those treated for 6 h than all other groups. Data for

control and 48 h horses from de Laat et al. (2011).

31

Fig. 5. Immunolocalisation of a cellular proliferative marker, TPX2, was increased (P

< 0.05) in the PELs of horses treated with a euglycaemic, hyperinsulinaemic clamp

for 24 h (n = 4) and 48 h (n = 4), compared to those treated for 12 h (n = 4) and 6 h (n

= 4), and normal control horses (CH; n = 4). The median TPX2-positive cell count (―)

is shown for each group.

32

Fig. 6. Photomicrographs of the axial tips of secondary epidermal lamellae (SEL)

from horses treated with a euglycaemic, hyperinsulinaemic clamp for 6 h (a, b) and 24

h (c, d) stained immunohistochemically with TPX2 (a, c) and calprotectin (b, d).

TPX2 stains cells undergoing mitosis (arrowhead), as well as other cells with no

visible mitotic changes (arrows). Fewer (P < 0.05) proliferating epidermal cells were

seen in horses treated for 6 h (a) compared with 24 h (c). Immunolocalisation of

calprotectin failed to highlight significantly increased numbers of infiltrating

leucocytes after 6 h (b) or 24 h (d) of hyperinsulinaemia.