Ultrastructural changes in the equine colonic mucosa after
ischaemia and reperfusion
A. GROSCHE*†, A. J. MORTON, A. S. GRAHAM, L. C. SANCHEZ, A. T. BLIKSLAGER‡, M. M. R. POLYAK†and D. E. FREEMAN
Island Whirl Equine Colic Research Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of
Florida;†Transplant Center, Department of Surgery, College of Medicine, Shands at the University of Florida; and‡Department of Clinical
Sciences, North Carolina State University, Raleigh, USA.
Keywords: horse; colon; TEM; tight junctions; autophagy; phagocytosis; immune cells
Reason for performing study: Ultrastructural changes in the
epithelium can provide information on early changes in
barrier properties, repair and inflammation in equine colon
after ischaemia and reperfusion (I/R).
Objectives: To describe the morphology and ultrastructure of
the epithelium in equine large colonic mucosa after I/R, and
the response of inflammatory cells to injury.
Methods: Ischaemia was induced for 1 h followed by 4 h of
reperfusion in a 40 cm segment of the pelvic flexure in 6
horses. Mucosal biopsies before and afterischaemia, and after
1, 2 and 4 h of reperfusion were fixed in glutaraldehyde/
paraformaldehyde and osmium tetroxide, and embedded in
epon. Morphological and ultrastructural changes were
evaluated in toluidine blue-stained semithin sections by light
microscopy and in thin sections stained with uranyl acetate/
lead citrate by transmission electron microscopy.
Results: Ischaemia caused swelling of epithelial cells and their
organelles, opening of tight junctions, detachment from the
basement membrane, early apoptosis and single cell necrosis.
Autophagy was a prominent feature in epithelial cells after
ischaemia. Reperfusion was characterised by apoptosis,
epithelial regeneration and restoration of apical cell junctions.
Phagocytic-like vacuoles containing cellular debris and
bacteria were evident in epithelial cells after reperfusion.
Paracellular and subepithelial clefts formed, accompanied
by infiltration of neutrophils, lymphocytes and eosinophils
into the epithelium. Subepithelial macrophages and luminal
neutrophils had increased phagocytic activity.
Conclusions: Ischaemia caused ultrastructural damage to the
colonic epithelium, but epithelial cells recovered during
Potential relevance: Transmission electron microscopy can
demonstrate subtle ultrastructural damage to epithelial cells
and evidence of recovery after I/R in equine colon.
Strangulation obstruction of the large colon is the most devastating
form of colic in horses. Rapid ischaemic degeneration of the
colonic epithelium facilitates translocation of bacterial toxins
through the damaged epithelial barrier resulting in endotoxic
shock and possible death (Snyder et al. 1989; Gibson and Steel
1999). Although prompt restoration of blood flow by surgical
correction of the volvulus is essential to prevent irreparable
damage, reperfusion can exacerbate epithelial damage (Meschter
et al. 1991; Moore et al. 1995). However, the importance of colonic
ischaemia-reperfusion (I/R) injury in horses is not fully understood
(Rowe and White 2002).
Intestinal I/R injury can be identified early by increase in
capillary and epithelial permeability (Snyder et al. 1992; Darien
et al. 1995). First histological signs of colonic mucosal damage
are characterised by lifting of small clusters of epithelial cells, their
detachment from the basement membrane and subsequently death
by apoptosis or necrosis (Meschter et al. 1991; Snyder et al. 1992).
Although the colonic epithelium is completely denuded after 4 h
of low-flow ischaemia, intracellular degenerative processes and
abnormalities of the cell structure are apparent long before
epithelial detachment occurs (Snyder et al. 1992). One hour of
experimentally induced ischaemia in the equine colon caused
minor morphological alterations but severe epithelial barrier
failure, characterised by decreased transepithelial resistance
(Graham et al. 2011). However, this measurement of barrier
integrity returned to normal after 4 h of reperfusion, without any
apparent morphological explanation based on light microscopy
(LM; Graham et al. 2011). Possible degenerative processes and
ultrastructural abnormalities of the epithelial barrier not seen by
routine LM might be responsible for loss of the barrier function.
The few published studies on colonic I/R injury in horses at the
cellular level (Meschter et al. 1991; Wilson et al. 1994; Darien
et al. 1995; Dabareiner et al. 2001) have not demonstrated early
ultrastructural changes of the colonic epithelium in horses that
could affect its barrier function during I/R (Graham et al. 2011).
The purpose of the present study was to illustrate alterations
of the equine colonic epithelium after 1 h of ischaemia
and during 4 h of reperfusion, and to describe ultrastructural
abnormalities demonstrated by transmission electron microscopy
(TEM) and morphological changes on semithin sections evaluated
by LM. The hypothesis was that early ischaemic injury results in
distinct but reversible ultrastructural alterations of epithelial cells
that play a role in barrier dysfunction and recovery.
*Corresponding author email: firstname.lastname@example.org
[Paper received for publication 14.01.11; Accepted 12.03.11]
© 2011 EVJ Ltd
8 EQUINE VETERINARY JOURNAL
Equine vet. J. (2011) 43 (Suppl. 39) 8-15
Materials and methods
Six horses used in this study were of mixed breeds with a mean age
of 16 years and a mean bodyweight of 548 kg. They were donated
for research purposes and they were free of gastrointestinal
diseases determined by physical, clinicopathological (white blood
cell count and differentiation, total protein and albumin) and faecal
examinations. The study was performed with approval and under
guidelines of the Institutional Animal Care and Use Committee of
the University of Florida. Horses were fed grass hay (2% of their
bwt/day) and water was provided ad libitum. Horses were adapted
to their diet and environment for at least one week before the study.
A 14 gauge, 13.3 cm Teflon catheter was inserted into the left
jugular vein for administration of anaesthetic drugs and isotonic
fluids. Horses were placed under general anaesthesia according
to the following protocol: xylazine (1.0 mg/kg bwt i.v.) to provide
sedation, and then general anaesthesia was induced with diazepam
(0.1 mg/kg bwt i.v.) to effect followed by ketamine (2.2 mg/kg bwt
i.v.) as a bolus injection. General anaesthesia was maintained with
isoflurane (1–3%) in 100% oxygen. Horses were mechanically
ventilated at 6 breaths/min. Isotonic polyionic fluids were infused
i.v. continuously at 2.5–5 ml/kg bwt/h. Mean arterial blood
pressure was monitored through a 20 gauge, 5.1 cm Teflon catheter
in the facial artery and was maintained at or above 60 mmHg.
electrocardiography, blood gas analysis, capnography and pulse
Horses were positioned in dorsal recumbency and prepared for
an aseptic ventral midline celiotomy. The large colon was
exteriorised and placed on a plastic drape on the ventral abdomen.
To induce ischaemia, a 40 cm segment of colon at the pelvic flexure
was subjected to transmural compression by intestinal clamps
at each end of the selected segment, and combined venous and
arterial occlusion was achieved with umbilical tape ligatures.
After induction of ischaemia, the colon, colonic vasculature and
associated mesentery were surgically divided at the pelvic flexure
so that two 20 cm segments of colon (dorsal and ventral) did
not communicate. To accomplish this, the colon was transected
and sutured at each end over an intestinal clamp in Parker-Kerr
fashion with 2–0 polydioxanone, and this layer was oversewn in a
continuous Cushing fashion to completely close the transected
bowel. The blind ends so created were lavaged with warm sterile
saline and placed in the abdomen during periods between biopsies.
After the colon was replaced in the abdomen, the abdominal
incision was closed temporarily with towel clamps. After 1 h of
ischaemia, the colon was re-exteriorised and one of the two
20 cm ischaemic segments and a contiguous 5–7 cm of control
(nonischaemic) colon were resected for histological evaluations
and in vitro experiments, alternating ventral and dorsal segments
between horses (Graham et al. 2011). The transected end created
by removal of these segments was closed by Parker-Kerr technique
as described above.At the same time, the clamps and ligatures were
removed from the remaining segment of colon and it was replaced
in the abdomen to allow resumption of blood flow for 4 h of
reperfusion under general anaesthesia. Small mucosal biopsies
(1–2 cm2) were taken before (control) and after ischaemia (1hI),
and after 1 (1hR), 2 (2hR) and 4 h of reperfusion (4hR). After
the reperfused tissues were sampled, the horses were humanely
subjected to euthanasia with an overdose of sodium pentobarbital
(88 mg/kg bwt i.v.) while under anaesthesia. The same investigator
performed all surgeries and sampling (D.E.F.).
In each horse, four 2 mm punched mucosal biopsies of control
tissues, and tissues after 1hI, 1hR, 2hR and 4hR were fixed in 2.5%
glutaraldehyde and paraformaldehyde at 4°C overnight, washed
in 0.1 mol/l cacodylate buffer, and placed in 1% aqueous osmium
tetroxide for 1 h at room temperature. After washing in 0.1 mol/l
cacodylate buffer and deionised water, fixed samples were
dehydrated in graded concentrations of acetone, and embedded
in epon. Tissues were cut into semithin sections (500 nm) with
an ultramicrotome equipped with a glass knife. Sections were
mounted on glass slides, stained with toluidine blue and examined
by LM. Tissue blocks were further trimmed to a size of 0.5 ¥
0.5 mm at the area of interest for thin sectioning. Thin sections
(70 nm) were cut on the same microtome equipped with a diamond
knife and mounted on Formvar-coated copper mesh grids. The
grids were stained with 2% uranyl acetate and lead citrate and
examined with the Hitachi H-7000 or Zeiss EM10A TEM at
magnifications varying from 2500–80,000.
Additionally, samples were fixed in 10% neutral buffered
formalin for 36 h, subsequently embedded in paraffin, and cut into
5 mm sections. After deparaffinising and rehydration, slides were
stained with periodic acid Schiff (PAS) in a routine manner to
characterise basement membranes.
Adescriptive evaluation of morphological changes assessed by
LM was performed, and compared with ultrastructural alterations
of epithelial cells evaluated by TEM. Furthermore the reaction of
subepithelial immune cells during I/R was characterised by TEM.
In toluidine blue-stained semithin sections, minor pathohistological
alterations of the colonic epithelium were evident after 1hI
(Fig 1b) compared to controls (Figs 1a,d). Epithelial injury was
characterised by cell oedema, microvilli disintegration, apoptosis,
subepithelial fluid accumulation and detachment of epithelial
cells from the vacuolated basement membrane (Figs 1b,e). Lamina
propria oedema, accumulation of necrotic debris, and swollen
or necrotic immune cells (lymphocytes, eosinophils, mast cells)
were detectable in the subepithelial space after 1hI (Fig 1b).
Cytoplasmic granules in subepithelial mast cells and eosinophils
were also reduced.
Further deterioration of epithelial injury was not evident
after reperfusion (Figs 1c,f). The colonic epithelium responded to
reperfusion with dilation of the paracellular space, and infiltration
of neutrophils, lymphocytes and eosinophils (Fig 1c). After 1hR,
repair of the epithelial layer could be detected as a covering of
small epithelial defects by interconnections between detached
epithelial cells or between membrane extensions from intact
adjacent neighbouring cells (Fig 1c). Numerous macrophages with
large phagocytic vacuoles, neutrophils and mast cells were located
in the subepithelial lamina propria after 4hR.
© 2011 EVJ Ltd
A. Grosche et al.9
Ischaemia: After ischaemia, most of the epithelial cells were
shorter and dilated (Fig 1h) compared to controls (Fig 1g).
Microvilli were reduced in size and number, and their core
appeared less electron-dense (Fig 2a). The surface coat was
diminished or disappeared partially, and goblet cells were
rarefied and their granules reduced. The apical part of
terminal tight junctions (TJ) was partly disrupted or dilated
After ischaemia, the less electron-dense cytoplasm of epithelial
cells appeared vacuolated and contained a reduced number of
mitochondria, rough endoplasmic reticulum (rER) and golgi
complexes, all of which were swollen (Figs 1f and 2a,c,d). The
mitochondrial matrix was lucent, and the christae were dilated
and disrupted (Fig 2d). Most of the nuclei appeared rounded and
enlarged. Their less electron-dense plasma, and the condensation
and margination of nuclear chromatin indicated early apototic
features (Figs 1h, 2f). Large numbers of autophagosomes evident
in the cytoplasm after 1hI contained damaged cell organelles,
lytic cytoplasm and lysosomes (Fig 2c, e). Only single epithelial
cells appeared necrotic, as characterised by cytoplasmic lucency
and vacuolisation, and severely dilated and lytic nuclei and cell
Fig 1: Epithelium and subepithelial lamina propria of the equine colon after ischaemia and reperfusion: a, d, g: control; b, e, h: 1 h of ischaemia;
c, f, i: 4 h of reperfusion; toluidine blue (a–c, ¥400), periodic acid Schiff (d–f, ¥400), TEM (g–h).
© 2011 EVJ Ltd
10 Mucosal ultrastructure after colonic I/R
After ischaemia, large subepithelial vacuoles separated
Consequently, small groups of epithelial cells detached from the
distorted basement membrane to form subepithelial clefts.
Detached epithelial cells, however, remained connected to each
other by their apical cellular junctions. Numerous lymphocytes,
phagocytic active neutrophils and eosinophils infiltrated the
subepithelial clefts and migrated through the paracellular space
towards the intestinal lumen.
The subepithelial lamina propria contained large vacuoles.
were located in the subepithelium. Subepithelial mast cells,
lymphocytes and eosinophils were swollen and necrotic (Fig 2f).
decreased intracellular granules and damage to the plasma
membrane. Numerous neutrophils were attached to subepithelial
venules, and they migrated into the lamina propria (Fig 2f).
Reperfusion: Within 4 h of reperfusion, the ultrastructural
damage to epithelial cells did not progress. A prominent feature
during reperfusion was an enlargement of the paracellular and
subepithelial space over time that further separated detached
2 µm 2 µm
2 µm 10 µm
Fig 2: Epithelial cells and subepithelial structures after 1 h of ischaemia: TEM. a) Apical part of 2 epithelial cells: shortened degenerated microvilli,
cytoplasmic lucency and decreased cytoplasmic granules; b) apical junction complex between 2 epithelial cells with disrupted tight junctions (arrowhead;
arrow: adherens junction); c) vacuolated cytoplasm, degenerated cell organelles (endoplasmic reticulum, golgi apparatus, lysosomes, mitochondria) and
autophagosomes; d) degenerated mitochondria (lower epithelial cell); e) autophagosome; f) apoptotic epithelial cells (nuclear chromatin margination),
subepithelial vacuoles, disrupted basement membrane, vacuolated subepithelial lamina propria, degranulated mast cells.
© 2011 EVJ Ltd
A. Grosche et al. 11
epithelial cells from the basement membrane (Figs 1i, 3a). Within
4hR, there was no evidence that these detached epithelial cells
reattached. Instead these cells became shorter and appeared to
adhere to each other at the luminal surface by membrane extensions
and intact apical cell junctions, thus preserving coverage of large
underlying clefts (Figs 1i and 3a,d). Subepithelial clefts and
paracellular spaces were infiltrated with intact and apoptotic
neutrophils (Fig 3a), lymphocytes and eosinophils. Numerous
close proximity to the epithelial surface and contained vacuoles
with phagocytosed bacteria and necrotic debris. Some apoptotic
cells and apoptotic bodies were evident within the epithelium
Epithelial cell nuclei appeared shrunken, irregularly lobulated
and partly pyknotic, and contained large nucleoli and electron-
dense chromatin (Fig 3e). Although reduced in their number,
mitochondria, rER and golgi complexes looked normal (Fig 3e). In
addition to autophagosomes, large membrane-bound vacuoles
containing necrotic debris, bacteria and apoptotic bodies were
observed in the cytoplasm of epithelial cells, possible evidence that
they became phagocytic during reperfusion (Figs 3b,c). Numerous
apoptotic cells, phagocytic active macrophages and neutrophils,
and mast cells and lymphocytes were located in the subepithelial
lamina propria (Fig 3f).
Although 1hI of the equine colon resulted in minor mucosal
injury on LM, examination of the mucosa by TEM demonstrated
ultrastructural alterations in individual epithelial cells in response
to the short ischaemic period. Examination of toluidine blue-
stained semithin sections by LM revealed distinct morphological
alterations of the epithelium. Damage to microvilli, dilated
paracellular spaces, subepithelial cleft formation and single cell
necrosis evident on TEM after ischaemia could cause cellular
dysfunction and disruption of the intestinal barrier. Reperfusion
1 µm 5 µm
Fig 3: Epithelial cells and subepithelial structures after reperfusion: TEM. a) Increased intercellular and subepithelial spaces with infiltrated neutrophil
(2hR). Apical part of epithelial cells remains connected to each other; b) apical part of epithelial cells containing numerous phagocytic vacuoles with
necrotic cell organelles, lytic plasma, lysosomes and apoptotic bodies (4hR); c) phagocytic vacuoles (4hR); d) apical junction complex between 2 epithelial
cells with intact tight junction (arrow head; arrow: adherens junction; 2hR); e) pyknotic, lobulated epithelial cell nuclei, apoptotic nucleus, and intact
mitochondria (2hR); f) subepithelial lamina propria with phagocytic active macrophage, and lymphocytes and mast cells (4hR).
© 2011 EVJ Ltd
12 Mucosal ultrastructure after colonic I/R
of the ischaemic injured mucosa for 4 h was sufficient to allow
the damaged epithelium to recover epithelial barrier function
(Graham et al. 2011). Whereas the naturally occurring lesion in
clinical cases is an ischaemia of variable duration and intensity,
the lesion induced in this study may represent a milder disease
because of the short duration of ischaemia and because the
subjects were on 100% oxygen. However, the type of injury
inflicted was designed to be reversible and capture the gross
and microscopic elements that are typical of colonic ischaemia
(Snyder et al. 1989).
Semithin sections stained with toluidine blue can demonstrate
specific morphological changes of the colonic epithelium by
LM after I/R. Histomorphometric and morphological studies on
paraffin-embedded colonic mucosal biopsies have shown that
short periods of ischaemia cause minor but significant changes
characterised by detachment of epithelial cells from adjacent
cells and basement membranes, oedema formation, haemorrhage
and accumulation of necrotic debris in the lamina propria
(Meschter et al. 1991; Darien et al. 1995; Graham et al. 2011).
These changes could be demonstrated in the results of the present
study. Additional changes demonstrated by semithin sections
included disrupted microvillar integrity, dilated paracellular spaces,
vacuolisation of the basement membrane, single cell necrosis, early
apoptosis and epithelial repair.
Epithelial alterations did not progress during reperfusion and
epithelial repair started at 1hR in the present study. Although
epithelial alterations after 1hI were minor in the present study,
metabolic and ultrastructural changes of single epithelial cells can
cause epithelial barrier dysfunction (McAnulty et al. 1997; Graham
et al. 2011). Sun et al. (1998) calculated a strong positive
correlation between short ischaemic times of 20 and 40 min, and
disruption of epithelial barrier permeability in rats, consistent with
our recent findings of epithelial barrier failure after 1hI (Graham
et al. 2011). However, 4hR resulted in full recovery of the barrier
function in the equine colon (Graham et al. 2011).
Ischaemia: Acascade of cellular enzymatic and metabolic changes
in the epithelium during hypoxia leads to reversible ultrastructural
alterations(Snyderet al.1992;McAnultyet al.1997)characterised
by swelling and vacuolisation of epithelial cells, dilation of cell
organelles and structural changes of the nucleus consistent with
features of early apoptosis (Labat-Moleur et al. 1998). In addition
to hypoxia, activation or necrosis of subepithelial mast cells,
neutrophils and eosinophils, as demonstrated in our study, could
also play a potential role in ischaemic mucosal injury by releasing
toxic and inflammatory mediators (Wardlaw 1996; Boros et al.
1999; Gayle et al. 2000).
One of the key findings in the present study was the
disintegration or dilation of TJ between epithelial cells after
paracellular permeability and, therefore they play a major role in
maintaining the epithelial barrier. Additionally, the lateral
intercellular space is also thought to contribute mechanically to the
transepithelial resistance (Madara 1998; Blikslager et al. 2007).
Thus, separation of epithelial cells from their neighbouring cells
by intercellular fluid accumulation and expanded TJ could explain
epithelial barrier failure after ischaemia (Graham et al. 2011).
A prominent change in colonocytes after ischaemia in the
present study was autophagy, a homeostatic process that removes
damaged or surplus organelles, supplies nutrients and energy,
eliminates intracellular pathogens and toxic proteins, and delivers
endogenous antigens for presentation (Levine and Deretic 2007;
Levine and Kroemer 2008). Amino acids or fatty acids recovered
through autophagy may be used forATPproduction, and misfolded
proteins and damaged mitochondria may be removed under
hypoxic conditions (Sadoshima 2008). Alternatively, a marked
upregulation of autophagy and accompanying upregulation of
lysosomal enzymes can cause self-digestion and eventual cell death
(Sadoshima 2008). Although autophagy in epithelial cells might
have caused single cell death after ischaemia in the present study,
it might also favour epithelial cell survival during hypoxia
(Sadoshima 2008), as evident by epithelial repair and functional
recovery within 4hR (Graham et al. 2011).
Reperfusion: Reperfusion of colonic tissues did not exacerbate
epithelial cell damage in the present study, and ischaemic injured
epithelial cells appeared to restore the epithelial lining during
reperfusion by reattaching to adjacent cells (Fig 4). Rapid self-
sealing by epithelial cells usually begins within 15 min after injury,
and allows neighbouring cells to reestablish cell to cell contacts and
restore epithelial integrity (Wilson and Gibson 1997; Mammen and
Matthews 2003; Blikslager et al. 2007; Fig 4). However, epithelial
Fig 4: Schematic model of epithelial cell injury after 1 h of ischaemia, and
epithelial recovery after 4 h of reperfusion: ischaemia causes some single-
cell necrosis (middle cell) but the majority of cells (remainder in ischaemia
panel) undergo some degree of degeneration, nuclear chromatin
margination, detachment from the basement membrane and disruption of
terminal tight junctions. Paracellular and subepithelial clefts formed,
accompanied by infiltration of neutrophils and lymphocytes during
reperfusion. Enterocytes and apical junction complexes recover during
reperfusion, and single-cell defects (caused by loss of the middle cell in this
example) are closed by development of apical cell-to-cell connections
between intact neighbouring cells.
© 2011 EVJ Ltd
A. Grosche et al. 13
morphology and ultrastructure did not appear completely normal
after 4hR. Although the final fate of damaged epithelial cells
cannot be established conclusively from this study, our findings
are consistent with previous descriptions of a rapid recovery
process and are within timeframes previously determined for
restitution (Wilson and Gibson 1997; Mammen and Matthews
2003; Blikslager et al. 2007). Final repair of the ischaemic injured
epithelium starts later and involves proliferation and re-
epithelialisation (Blikslager et al. 2007). Our observation of
closure of TJ or sealing of membrane extensions between surviving
neighbouring cells could explain functional recovery in the same
tissues in Ussing chambers after 4hR (Graham et al. 2011).
Although a larger epithelial defect requires restitution by migration
of surviving cells in the periphery of the injury, the injury induced
in our model appeared predominantly to involve recovery of the
epithelial lining by reattachment between remaining cells in the
zone of epithelial damage (Fig 4).
Many factors that control epithelial repair are released by
epithelial cells themselves, or they are produced by mucosal
immune cells. Among these immune cells, neutrophils are thought
to play a key role in tissue injury and repair (Serhan and Savill
2005; Nathan 2006). Despite the presence of neutrophils within the
intercellular space andTJ after 4hR, the colon had improved barrier
at this time, as determined by transepithelial resistance and
transmucosal mannitol flux (Graham et al. 2011). Accumulation
and transepithelial migration of neutrophils persist in ischaemic
injured colonic mucosa for at least 18 h after ischaemia injury
without impairment of epithelial barrier integrity (Grosche et al.
2008; Matyjaszek et al. 2009). This is contrary to what has been
demonstrated in porcine ileum (Gayle et al. 2002). It is possible
that activated neutrophils that are recruited to the site of colonic
injury (Grosche et al. 2008) secrete anti-inflammatory and pro-
resolution factors that also promote repair (Nathan 2006; Serhan
et al. 2008). Apoptosis of neutrophils, and their clearance
by inflammatory macrophages, is also an essential step in
inflammation reduction and initiation of repair (Savill et al. 2002).
Thus, neutrophils could play a potential role in resolution of
inflammation and promoting tissue repair during reperfusion of the
ischaemic colonic mucosa in horses.
The results of the current study also indicated that epithelial
cells displayed phagocyticactivity
intracellular phagocytic vacuoles. The role of this process is not
clear. Phagocytosis of foreign material could provide more
nutrients and energyduring
microenvironment for regulation of innate and adaptive immune
responses, and possibly initiate repair (Artis 2008). Because
epithelial cells can phagocytose adjacent cells, apoptotic cells and
bacteria (Monks et al. 2005; Neal et al. 2006), they could control
the inflammatory response and minimise injury after I/R in
Results of the present study indicate that 1hI causes structural
alterations of the equine colonic epithelium. Initially, mucosal
injury occurs at the cellular level, and leads to epithelial barrier
failure. However, epithelial cells can survive short-term hypoxia
and recover during 4hR. It was evident that the cells remaining in
the zone of injury had re-established connections with adjacent
cells, despite their abnormal appearance. This is also consistent
with our finding that the same tissues had re-established functional
integrity within this timeframe, based on their performance in
Ussing chambers. (Graham et al. 2011). Phagocytosis of apoptotic
and necrotic cells could minimise inflammation and assist
epithelial repair during reperfusion. The results also indicate that
repair can proceed in the presence of mucosal neutrophil activity
during I/R in the equine colon.
Conflicts of interest
No conflicts of interest have been declared.
Source of funding
The study was funded in part by the American College of
Veterinary Surgeons and the Deedie Wrigley-Hancock Fellowship.
We thank the service team of Dr Byuong-Ho Kang from the
Bioimaging and Electron Microscopy Lab at the Interdisciplinary
Center for Biotechnology Research, University of Florida,
especially Karen Kelley, for her outstanding technical and
scientific support with transmission electron microscopy.
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