This may be the author’s version of a work that was submitted/acceptedfor publication in the following source:
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.
This file was downloaded from: https://eprints.qut.edu.au/58411/
c© Consult author(s) regarding copyright matters
This work is covered by copyright. Unless the document is being made available under aCreative Commons Licence, you must assume that re-use is limited to personal use andthat permission from the copyright owner must be obtained for all other uses. If the docu-ment is available under a Creative Commons License (or other specified license) then referto the Licence for details of permitted re-use. It is a condition of access that users recog-nise and abide by the legal requirements associated with these rights. If you believe thatthis work infringes copyright please provide details by email to [email protected]
License: Creative Commons: Attribution-Noncommercial-No DerivativeWorks 2.5
Notice: Please note that this document may not be the Version of Record(i.e. published version) of the work. Author manuscript versions (as Sub-mitted for peer review or as Accepted for publication after peer review) canbe identified by an absence of publisher branding and/or typeset appear-ance. If there is any doubt, please refer to the published source.
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: [email protected] (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
References Asplin, K.E., Patterson-Kane, J.C., Sillence, M.N., Pollitt, C.C., McGowan, C.M.,
2010. Histopathology of insulin-induced laminitis in ponies. Equine Veterinary
Journal 42, 700-706.
Asplin, K.E., Sillence, M.N., Pollitt, C.C., McGowan, C.M., 2007. Induction of
laminitis by prolonged hyperinsulinaemia in clinically normal ponies. Veterinary
Journal 174, 530-535.
Avogaro, A., de Kreutzenberg, S.V., Fadini, G.P., 2010. Insulin signaling and life
span. Pflugers Archiv-European Journal of Physiology 459, 301-314.
Belknap, J.K., Giguere, S., Pettigrew, A., Cochran, A.M., Van Eps, A.W., Pollitt,
C.C., 2007. Lamellar pro-inflammatory cytokine expression patterns in laminitis at
the developmental stage and at the onset of lameness: innate vs. adaptive immune
response. Equine Veterinary Journal 39, 42-47.
Black, S.J., Lunn, D.P., Yin, C.L., Hwang, M., Lenz, S.D., Belknap, J.K., 2006.
Leukocyte emigration in the early stages of laminitis. Veterinary Immunology and
Immunopathology 109, 161-166.
Burns, T.A., Geor, R.J., Mudge, M.C., McCutcheon, L.J., Hinchcliff, K.W., Belknap,
J.K., 2010. Proinflammatory Cytokine and Chemokine Gene Expression Profiles in
Subcutaneous and Visceral Adipose Tissue Depots of Insulin-Resistant and Insulin-
Sensitive Light Breed Horses. Journal of Veterinary Internal Medicine 24, 932-939.
Carter, R.A., Geor, R.J., Staniar, W.B., Cubitt, T.A., Harris, P.A., 2009a. Apparent
adiposity assessed by standardised scoring systems and morphometric measurements
in horses and ponies. Veterinary Journal 179, 204-210.
Carter, R.A., Treiber, K.H., Geor, R.J., Douglass, L., Harris, P.A., 2009b. Prediction
of incipient pasture-associated laminitis from hyperinsulinaemia, hyperleptinaemia
and generalised and localised obesity in a cohort of ponies. Equine Veterinary Journal
41, 171-178.
Chitnis, M.M., Yuen, J.S.P., Protheroe, A.S., Pollak, M., Macaulay, V.M., 2008. The
Type 1 Insulin-Like Growth Factor Receptor Pathway. Clinical Cancer Research 14,
6364-6370.
de Laat, M.A., Kyaw-Tanner, M.T., Nourian, A.R., McGowan, C.M., Sillence, M.N.,
Pollitt, C.C., 2011a. The developmental and acute phases of insulin-induced laminitis
involve minimal metalloproteinase activity. Veterinary Immunology and
Immunopathology 140, 275-281.
de Laat, M.A., Kyaw-Tanner, M.T., Sillence, M.N., McGowan, C.M., Pollitt, C.C.,
2012. Advanced glycation endproducts in horses with insulin-induced laminitis.
Veterinary Immunology and Immunopathology 145, 395-401.
21
de Laat, M.A., McGowan, C.M., Sillence, M.N., Pollitt, C.C., 2010. Equine laminitis:
Induced by 48 h hyperinsulinaemia in Standardbred horses. Equine Veterinary Journal
42, 129-135.
de Laat, M.A., van Eps, A.W., McGowan, C.M., Sillence, M.N., Pollitt, C.C., 2011b.
Equine Laminitis: Comparative Histopathology 48 hours after Experimental Induction
with Insulin or Alimentary Oligofructose in Standardbred Horses. Journal of
Comparative Pathology 145, 399-409.
DeFronzo, R.A., Tobin, J.D., Andres, R., 1979. Glucose clamp technique: a method
for quantifying insulin secretion and resistance. American Journal of Physiology 237,
E214-E223.
Faleiros, R.R., Johnson, P.J., Nuovo, G.J., Messer, N.T., Black, S.J., Belknap, J.K.,
2011a. Laminar leukocyte accumulation in horses with carbohydrate overload-
induced laminitis. Journal of Veterinary Internal Medicine 25, 107-115.
Faleiros, R.R., Nuovo, G.J., Flechtner, A.D., Belknap, J.K., 2011b. Presence of
mononuclear cells in normal and affected laminae from the black walnut extract
model of laminitis. Equine Veterinary Journal 43, 45-53.
Faleiros, R.R., Stokes, A.M., Eades, S.C., Kim, D.Y., Paulsen, D.B., Moore, R.M.,
2004. Assessment of apoptosis in epidermal lamellar cells in clinically normal horses
and those with laminitis. American Journal of Veterinary Research 65, 578-585.
Field, J.R., Jeffcott, L.B., 1989. Equine Laminitis - Another Hypothesis for
Pathogenesis. Medical Hypotheses 30, 203-210.
Frank, N., 2009. Equine Metabolic Syndrome. Journal of Equine Veterinary Science
29, 259-267.
Galey, F.D., Whiteley, H.E., Goetz, T.E., Kuenstler, A.R., Davis, C.A., Beasley, V.R.,
1991. Black walnut (Juglans nigra) toxicosis: A model for equine laminitis. Journal of
Comparative Pathology 104, 313-326.
Geor, R.J., 2009. Pasture-Associated Laminitis. Veterinary Clinics of North America-
Equine Practice 25, 39-50.
Geor, R.J., Harris, P., 2009. Dietary Management of Obesity and Insulin Resistance:
Countering Risk for Laminitis. Veterinary Clinics of North America-Equine Practice
25, 51-65.
Heidebrecht, H.J., Adam-Kiages, S., Szczepanowski, M., Pollmann, M., Buck, F.,
Endl, E., Kruse, M.L., Rudolph, P., Parwaresch, R., 2003. repp86: A human protein
associated in the progression of mitosis. Molecular Cancer Research 1, 271-279.
Heidebrecht, H.J., Buck, F., Steinmann, J., Sprenger, R., Wacker, H.H., Parwaresch,
R., 1997. p100: A novel proliferation-associated nuclear protein specifically restricted
to cell cycle phases S, G(2), and M. Blood 90, 226-233.
22
Henneke, D.R., Potter, G.D., Kreider, J.L., Yeates, B.F., 1983. Relationship between
condition score, physical measurements and body-fat percentage in mares. Equine
Veterinary Journal 15, 371-372.
Keen, J.A., McLaren, M., Chandler, K.J., McGorum, B.C., 2004. Biochemical indices
of vascular function, glucose metabolism and oxidative stress in horses with equine
Cushing's disease. Equine Veterinary Journal 36, 226-229.
Kiess, W., Gallaher, B., 1998. Hormonal control of programmed cell death/apoptosis.
European Journal of Endocrinology 138, 482-491.
Leise, B.S., Faleiros, R.R., Watts, M., Johnson, P.J., Black, S.J., Belknap, J.K., 2011.
Laminar inflammatory gene expression in the carbohydrate overload model of equine
laminitis. Equine Veterinary Journal 43, 54-61.
McGowan, C.M., Frost, R., Pfeiffer, D.U., Neiger, R., 2004. Serum insulin
concentrations in horses with equine Cushing's syndrome: Response to a cortisol
inhibitor and prognostic value. Equine Veterinary Journal 36, 295-298.
McGowan, T.W., Geor, R., Evans, H., Sillence, M., Munn, K., McGowan, C.M., 2008.
Comparison of 4 assays for serum insulin analysis in the horse. Journal of Veterinary
Internal Medicine 22, 115.
Meyers, R.K., McGavin, M.D., 2007. Cellular and tissue responses to injury. In:
Zachary, M.D.M.a.J.F. (Ed.), Pathologic Basis of Veterinary Disease. Mosby Elsevier,
St Louis, MO, pp. 16-32.
Myers, M.G., White, M.F., 1995. New frontiers in insulin receptor substrate signaling.
Trends in Endocrinology and Metabolism 6, 209-215.
Obel, N., 1948. Studies on the Histopathology of Acute Laminitis. Dissertation:
Almqvist and Wiksells Boktryckeri A.B., Uppsala, Sweden.
Pavelic, J., Matijevic, T., Knezevic, J., 2007. Biological & physiological aspects of
action of insulin-like growth factor peptide family. Indian Journal of Medical
Research 125, 511-522.
Rijnen, K., van der Kolk, J.H., 2003. Determination of reference range values
indicative of glucose metabolism and insulin resistance by use of glucose clamp
techniques in horses and ponies. American Journal of Veterinary Research 64, 1260-
1264.
Rudolph, P., Knuchel, R., Endl, E., Heidebrecht, H.J., Hofstadter, F., Parwaresch, R.,
1998. The immunohistochemical marker Ki-S2: Cell cycle kinetics and tissue
distribution of a novel proliferation-specific antigen. Modern Pathology 11, 450-456.
Striz, I., Trebichavsky, I., 2004. Calprotectin - a pleiotropic molecule in acute and
chronic inflammation. Physiological Research 53, 245-253.
23
Tinworth, K.D., Wynn, P.C., Harris, P.A., Sillence, M.N., Noble, G.K., 2009.
Optimising the Siemens Coat-A-Count Radioimunnoassay to measure insulin in
equine plasma. In: Proceedings of the Equine Science Society Congress, Colorado.
Treiber, K., Carter, R., Gay, L., Williams, C., Geor, R., 2009. Inflammatory and redox
status of ponies with a history of pasture-associated laminitis. Veterinary Immunology
and Immunopathology 129, 216-220.
Treiber, K.H., Kronfeld, D.S., Geor, R.J., 2006. Insulin resistance in equids: possible
role in laminitis. Journal of Nutrition 136, 2094S-2098S.
USDA, 2000. Lameness and Laminitis in U.S. Horses. In: System, N.A.H.M. (Ed.),
USDA:APHIS:VS,CEAH, Vol. N318.0400, Fort Collins, CO.
Visser, M., 2008. Investigation of proteolysis of the basement membrane during the
development of equine laminitis. In, School of Veterinary Science, Vol. PhD Thesis.
The University of Queensland, Brisbane, p. 275.
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.