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Keywords:

  • horse;
  • colon;
  • TEM;
  • tight junctions;
  • autophagy;
  • phagocytosis;
  • immune cells

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Source of funding
  9. Acknowledgements
  10. References

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 after ischaemia, 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 reperfusion.

Potential relevance: Transmission electron microscopy can demonstrate subtle ultrastructural damage to epithelial cells and evidence of recovery after I/R in equine colon.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Source of funding
  9. Acknowledgements
  10. References

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.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Source of funding
  9. Acknowledgements
  10. References

Animals

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.

Experimental procedures

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. Other monitoring tools used during anaesthesia included electrocardiography, blood gas analysis, capnography and pulse oximetry.

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.).

Sample preparation

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 µm sections. After deparaffinising and rehydration, slides were stained with periodic acid Schiff (PAS) in a routine manner to characterise basement membranes.

A descriptive 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.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Source of funding
  9. Acknowledgements
  10. References

Morphological changes

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.

image

Figure 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).

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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.

Ultrastructural changes

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 (Figs 2a,b).

image

Figure 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.

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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 organelles.

After ischaemia, large subepithelial vacuoles separated epithelial cells from the basement membrane (Fig 2f). 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. Enlarged macrophages containing phagocytic vacuoles and granules were located in the subepithelium. Subepithelial mast cells, lymphocytes and eosinophils were swollen and necrotic (Fig 2f). Their ultrastructure was characterised by cytoplasmic vacuolisation, 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 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 neutrophils migrated into the intestinal lumen. They were located in 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 (Fig 3b,e).

image

Figure 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).

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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).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Source of funding
  9. Acknowledgements
  10. References

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 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).

Morphological changes

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).

Ultrastructural changes

Ischaemia: A cascade of cellular enzymatic and metabolic changes in the epithelium during hypoxia leads to reversible ultrastructural alterations (Snyder et al. 1992; McAnulty et 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 ischaemia. Tight junctions are principally responsible for regulating 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 for ATP production, 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 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).

image

Figure 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.

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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 and TJ after 4hR, the colon had improved barrier function 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 phagocytic activity as demonstrated by intracellular phagocytic vacuoles. The role of this process is not clear. Phagocytosis of foreign material could provide more nutrients and energy during reperfusion or sample the 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 horses.

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.

Source of funding

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Source of funding
  9. Acknowledgements
  10. References

The study was funded in part by the American College of Veterinary Surgeons and the Deedie Wrigley-Hancock Fellowship.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Source of funding
  9. Acknowledgements
  10. References

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.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Source of funding
  9. Acknowledgements
  10. References