Equine Colic: III. Intestinal Response to Injury

1. Introduction

The pathophysiologic events that take place during an acute abdominal crisis may include bowel distention, bowel ischemia, tissue reperfusion, necrosis, inflammation, apoptosis, and changes in bacterial flora. The responses to these events include changes in intestinal motility, water and electrolyte secretion absorption, vascular permeability, inflammatory cell activation, and, ultimately, tissue structure. Colic (the clinical signs caused by abdominal pain) is initiated by stimulation of nervous reflexes and formation of chemical mediators that cause an increase in heart rate, venous pooling, fluid sequestration, and alterations in tissue perfusion and oxygenation. Understanding the clinical signs created by these physiologic alterations helps the veterinarian determine the type of disease and its severity.

2. Normal Function

The equine gastrointestinal tract is designed to digest and absorb carbohydrate and protein in the small intestine and to absorb volatile fatty acids produced by digestion of cellulose in the cecum and large colon [1]. Since the majority of the horse’s energy is derived from volatile fatty acids formed in the large intestine, a primary function of the stomach and small intestine is to hydrate the ingesta and move it into the cecum and large colon for fatty acid production. This entails large fluid shifts from the extracellular fluid space into the intestine in the proximal gastrointestinal tract and net resorption of fluid in the small intestine, cecum, and colon (Fig. 1).[1,2].

The secreted fluid is isotonic and contains mostly Na+ and Cl- or HCO3- [1]. Water follows Na+ into the intestinal lumen and is partially resorbed in the cecum and large colon with volatile fatty acids. The result is an enterosystemic cycle, in which water is removed from extracellular fluid space and secreted predominantly into the proximal gastrointestinal tract where it is used to liquefy and buffer the ingesta being delivered to the cecum and colon. The water is then returned to the extracellular space by absorption in the small intestine, cecum, and large colon. This water exchange is equivalent to 1.5 X the animal’s total extracellular fluid volume in a 24-hour period [2].

The digestive processes of the equine stomach and small intestine are similar to those of other monogastric animals. Water absorption is linked to glucose absorption, and dipeptides, tripeptides, and amino acids are predominately absorbed in the small intestine. Fat is digested to micelles for absorption in the small intestine. The cecum and colon function like the forestomach of the ruminant. These organs normally contain a variety of microorganisms, including gram-negative and gram-positive bacteria. The breakdown of cellulose to volatile fatty acids (butyrate, propionate, and acetate) can fluctuate with feeding [1,3]. Active absorption of these fatty acids enhances absorption of sodium and water. If horses are fed a highly soluble carbohydrate twice daily, rapid intraluminal production of fatty acids lowers the luminal pH. The resulting increase in osmolarity causes a shift from absorption to secretion of fluid, which reduces the plasma volume [4]. The systemic response is an increase in plasma renin and angiotensin concentrations [1]. This shift is due to the osmotic pressure from the fatty acids and lactate concentrations within the intestinal lumen and secretion of HCO3- to buffer the increased acidity [1,5]. The increase in fatty acids shifts water secretion to absorption as it follows fatty acid absorption into the systemic circulation producing relative dehydration of the ingesta [5]. If horses are fed at 2-hour intervals, plasma volume and renin angiotensin response remain unchanged [4].

Figure 1. An illustration of the enterosystemic circulation (in gallons). A fluid volume equivalent to 1.5 X the horse’s extracellular fluid volume is secreted and nearly all reabsorbed in 24 hours. Stomach, ST; small intestine, SI; cecum, CE; large colon, LC; small colon, SC.

Figure 2. The ingesta from the right dorsal colon in horses on an all hay diet has more water than the ingesta from horses fed hay and grain. Also, ingesta from horses fed hay and grain has less course material, less water, and obvious gas formation [6].

When a horse’s diet is changed from strictly forage to a diet with both forage and concentrate, there is an increase in dry matter content of ingesta in the right dorsal colon and evidence of increased gas production within 48 hours (Fig. 2 and 3) [6].

The decrease in water content is likely due to a decrease in fiber content and an increase in soluble carbohydrate consumption, which also increases gas production. Significant shifts in colon water content were not observed in this experimental model, however there were significant increases in rectal temperature and heart rate in horses fed grain and hay compared to hay alone. Serum electrolytes were significantly different between horses on an all-hay diet and those on a hay and grain diet, although no values were abnormal [6]. This suggests a systemic response to changes in a horse’s diet, particularly when adding soluble carbohydrate in the form of grain.

Intestinal motility that moves ingesta and fluid aborally (distally) is cyclic and controlled by numerous factors within the intestine. The musculature of the duodenum, cecum, and pelvic flexure responds to distention with ingesta by starting a peristaltic wave. In the small intestine, rhythmic slow waves, or migrating myoelectrical complexes, migrate aborally in the intestine and initiate motor events and are measured as spiking activity that is classified as none, intermittent, or regular [7]. These electrical spikes initiate muscular activity, some of which is transmitted aborally along the intestine. Distention increases this spike activity anywhere within the intestinal tract by activating peristalsis oral (proximal) to the distention and inhibiting peristalsis aborally. A pacemaker in the colon’s pelvic flexure may start the electrical activity with a regular basal cycle that is increased with eating, increased fiber content, and distention [7-10].

The cecum and colon produce haustral motility to mix and retain ingesta. Pacemakers, identified by initiation of motility and neuron density in the pelvic flexure and right dorsal colon, produce spike potentials, which create both propulsive and retropulsive motility. Spike burst potentials may be short and produce local mixing contractions or may be long and initiate the oral (proximal) and aboral (distal) movement of ingesta [10]. These movements are increased by distention and by reflexes from other intestinal activities, including filling of the stomach, the gastrocolic reflex, and filling of the ileum, the enterocolic reflex [9,11]. The normal time required for fluid to travel from the stomach to the cecum is 30 - 180 minutes. Total transit from the stomach to anus is as short as 12 hours for fluids and as long as 7 - 10 days for large particulate matter such as plant stems [2,7,12]. Larger particulate matter is retained in the large colon for longer periods by mixing retropulsive movements that allow completion of cellulose digestion [7,12].

Figure 3. Graph of colon water content during a 48-hour monitoring period. Horses’ diets were all hay changing from all hay to hay and grain, or hay and grain during the monitoring period. The water content of the right dorsal colon decreased within 48 hours of switching from a hay to a hay/concentrate diet [6].

Intestinal blockage alters intestinal motility, distends the intestine, initiates secretion of fluid into the intestinal lumen, and eventually causes mucosal damage. Ischemia due to strangulation of mesenteric vessels rapidly results in cellular damage throughout the bowel wall and especially within the mucosa and serosa. The diseases that produce these effects are categorized as obstruction (simple obstruction), strangulation obstruction, (hemorrhagic strangulation obstruction and ischemic strangulation obstruction), and non-strangulation infarction, which in the horse is synonymous with thromboembolic colic.

3. Intestinal Inflammation: General Concepts

Bowel inflammation results from a complex system of responses to one or more stimuli. Though macrophages and endothelial cells are known to respond to ischemia or sepsis in the horse, work in other species demonstrates release of inflammatory mediators from numerous cell types including mucosal cells, endothelial cells, fibrocytes, myocytes, mesothelial cells, and neurons [13]. Tissue ground substance, such as mucopolysaccarides and glycoaminoglycans, can also initiate an inflammatory response by transmitting signals between immunocytes, such as macrophages or histocytes, and afferent neurons. Theoretically, all cells in the intestine can act as effector cells by producing cytokines that act on other cells or by responding individually to the insult.

Cytokines, growth factors, and adhesion molecules initiate an inflammatory response. This response can delay cell apoptosis resulting in delayed removal of mucosal cells and neutrophils or early death of immunocytes [14]. Cytokines such as interleukin-1 (IL-1β), tumor necrosis factor (TNF-α), platelet activating factor (PAF) complement (C5a), interferon (INF-γ), and histamine are all reported to be involved in inciting intestinal inflammation [15]. Although describing all of the effects and interactions of the inflammatory cytokines and eicosanoids is beyond the scope of these proceedings, it is apparent these substances can stimulate and inhibit the inflammatory reaction by directing cell communication and cell response to injury. As a result, the disease process should be viewed as a sequence of altered cell functions, which are integrated and designed to protect the intestine from permanent injury [16].

Intestinal inflammation is initiated by the response of mucosal cells, endothelial cells, neurons, fibroblasts, mast cells, eosinophils, neutrophils, and macrophages. Rather than responding independently, these cells likely all respond to the initial insult, each with their inherent cytokine production or cell activation, which stimulates or suppresses cell responses. The sequence likely proceeds from an initial stimulus such as ischemia, which causes release of cytokines from macrophages and lymphocytes. Subsequent involvement of the vascular endothelium or enteric neurons occurs via recruitment by cytokine stimulation, enzyme activation, or direct neural stimulation [17].

Endothelial cells are key to the neutrophil’s response to ischemia. Cytokines from macrophages, direct insult from ischemia, or neuropeptides stimulate endothelial cells to release cytokines and chemotactic factors that attract neutrophils and eosinophils. Subsequently, the expression of adhesion molecules enable neutrophils to adhere to and migrate through the endothelium into the interstitium. Afferent neurons detecting cytokine increases initiate neuropeptide, cytokine, or eicosanoid release from the efferent neurons, resulting in activation of numerous cells including mast cells, eosinophils, PMNs, macrophages, fibroblasts, muscle cells, and lymphocytes (Fig. 4) [18-20].

Fibrocytes, responding to the initial cytokine messages, subsequently release cytokines and growth factors such as IL-1α, IL-1β, TNF-α, transforming growth factor, and platelet derived growth factor. Metalloproteinases released in response to inflammatory cytokines alter the basement membranes and collagen in the different tissues, allowing migration of inflammatory cells to the extracellular space and stimulating other cells to release chemotactants. Muscle cells, once thought to be neutral in the inflammatory cycle, appear to be able to release cytokines, thereby participating in the inflammatory reaction [13,22]. Bowel dysfunction after surgical manipulation has been linked to neutrophil infiltration as a result of increased adhesion molecule production in muscle [23].

Cells participating in the inflammatory response can suppress the function of effector cells. Each cell appears to communicate with other cells locally to cause or suppress the inflammatory response. Production of nitric oxide by endothelial cells can reduce neutrophil adhesion, while increased release of growth factor by fibroblasts and the vascular endothelium speeds healing of the mucosa [24]. The response to receptor activation may only take seconds to minutes to up-regulate cytokine production. In some cases, the lack of suppression, perhaps due to chronic or overwhelming stimulation or severe damage to cells, allows amplification of inflammation and permanent cell damage. Because the cytokines can stimulate a response in other organs, local inflammation creates a systemic response which, when overwhelming, causes the Systemic Inflammatory Response Syndrome (SIRS) [14,25].

The role of certain cells in the overall process of intestinal inflammation is better understood than others. Reperfusion, cytokines, and complement initiate endothelial cell changes. Subsequent production of cytokines and eicosanoids by endothelial cells attracts neutrophils and macrophages. Endothelial cells also stimulate the inflammatory response by altering capillary permeability, promoting neutrophil adhesion, and physically altering blood flow. The interaction between the endothelial cells and neutrophils or eosinophils is facilitated by PAF, leukotrienes (LTB4), and adhesion molecules produced by endothelial cells and PMNs [26]. Neutrophil migration into affected tissues subsequently causes severe damage, including damage to cells and tissue ground substance, which further stimulates the inflammatory response.

Figure 4. Neuropeptides released after stimulation of afferent neurons react with neuropeptide receptors on immunocytes with subsequent production and release of cytokines. The inflammatory response is both local and systemic as cytokines circulate, activating cells in multiple organs [21]. Reprinted from Payan DG, The role of Neuropeptides in Inflammation, Chapter in Basic Principles and Correlates, 2nd ed, Edited by J.J. Gallin, L.M. Goldstein, and R. Snyderman, Raven Press, Ltd. New York, 1992.

Although all intestinal cells can be involved in the inflammatory process, the nervous system is integral in the inflammatory response. Release of potassium, adenosine triphosphate (ATP), bradykinin, and prostaglandin E2 stimulate afferent neurons [27]. Neuropeptides are released from efferent neurons in response to afferent signals from the products of cell injury including cytokines, eicosanoids, and histamine [28]. Substance P, neurokinin, calcitonin generelated protein, and vasoactive peptide are all increased in inflamed tissues, suggesting that they act as messengers to histiocytes or immunocytes (Fig. 4) [21].

Inflammatory responses by mast cells, PMNs, T-cells (cytokine release), B-cells, macrophages, fibroblasts, and muscle cells are due, in part, to neuropeptide stimulation. The coordination of this response is not totally understood, but neuropeptides can serve as pro-inflammatory mediators or suppressors. This system allows for immediate response of cells to a stimulus and can likely cause persistent inflammatory reactions in response to previously inflamed tissue [13].

Local inflammation of the intestine causes change in other organs, especially in the lung [29,30]. Circulating cytokines and activated neutrophils rapidly initiate a pulmonary inflammatory response after intestinal inflammation. This response is well known in experimental models and man but has not been reported during intestinal disease in the horse. There is evidence that the lung acts as a shock organ during endotoxemia in horses, and there is clinical evidence that this injury is responsible for clinical signs seen in horses with colic [31]. Other organs are likely affected, creating signs of multiple organ involvement [14].

4. Ischemia

Ischemia is a deficiency of blood flow in tissue or in an organ. After 5 minutes of total intestinal ischemia in dogs breathing room air, mitochondrial swelling and disruption of cristae is detectable by electron microscopy. Cytoplasmic and membrane changes within intestinal epithelial cells occur in the first 30 minutes due to activation of phospholipases, cytokine production, and initiating of the arachidonic metabolite cascade. If ischemia persists, cell degradation continues due to failure of the membrane ion pumps, allowing calcium to move into the cytoplasm [32]. Calcium accumulation within the cell activates proteases with resultant cell membrane damage and nuclear clumping. Calcium uptake in the mitochondria is increased, which inhibits oxidative phosphorylation [32].

Microscopic changes become evident at 30 minutes, when the mucosal epithelial cells and serosal mesothelial cells separate from their basement membranes [33,34]. Initially, this appears to be a mechanical separation caused by water movement from the vasculature into the subepithelial or submesothelial space. Activated metalloproteinases may also be involved in altering the basement membrane. The space, created by the initial separation at the tip of the small intestinal villus, is named Grunehagan’s space (Fig. 5).

If ischemia continues, cell damage progresses and the mucosal cells progressively slough from the lamina propria toward the intestinal crypts (Figure 5). The change is similar, but somewhat slower, in the colon, with epithelial cells sloughing from the surface of the mucosa.

The serosa reacts in a similar fashion with mesothelial cells lifting off the basement membrane before there is visible cell membrane or cytoplasmic change (Fig. 6).

Other than vascular congestion, there is relatively minimal change in the architecture of the supporting tissues beneath the mucosa or the serosa for the first 60 minutes of total ischemia. After 180 minutes of ischemia, the lamina propria and mucosal vascular tuft is necrotic and becomes homogeneous with lack of nuclear definition and cell structure, leaving no chance for regeneration [33].

Differences in the experimental methods used to study ischemic injury in the horse and other animals have resulted in reporting of different rates of mucosal degeneration. Animals anesthetized with inhalants in oxygen logically have differences in the time required to create ischemic injury, most likely because of higher tissue oxygen concentrations at the beginning of the experiment [33,35,36]. Specific cells, such as muscle, appear more resistant to ischemia depending on their intracellular energy reserves. There are also differences in the response to low-flow ischemia versus total arteriovenous obstruction. Nevertheless, lesions caused by ischemia will often progress after reperfusion, with the degree of injury dependent on the length of ischemia and the inherent ability for cell recovery and healing.

Figure 5. (A) Transmission electron photomicrograph of the attachment of enterocytes to the basement membrane adjacent to a capillary in a villus from a horse with jejunal distention. Fluid has accumulated around the cell attachment on the basement membrane. (B-D) Photomicrographs (H&E) of the initial separation of the mucosal cells at the tip of the villus. Fluid collects in the space between the cells and the basement membrane creating "Grunehagan’s space" (arrows) (B). The mucosal cells which are still adhered together slough away from the capillary tuft (C) and eventually expose the necrotic villus tip (D). Figs. 5C and 5D are reprinted from White NA et al. Mucosal alterations in experimentally induced small intestinal strangulation obstruction in ponies. AJVR 1980;41:193-198.

Figure 5. (continued).

5. Reperfusion

Reactive hyperemia is the initial response when blood flow to ischemic tissue is first restored [37]. Reperfusion hyperemia has been observed in clinical cases and documented with blood flow measurements and thermography in the small intestine (Fig.7) [38,39]. In the large colon, this reactive hyperemia is equally distributed from the mucosa to the seromuscular layer [26,40].

Reperfusion injury is initiated within hypoxic cells where a pivotal reaction occurs during delivery of oxygen to the ischemic tissues. Oxidation reduction reactions within the cell result in the release of free electrons from the cytochrome proteins in the mitochondria and create free oxygen radicals when an unpaired electron (extra electron) moves to an outer electron orbit of the oxygen atom [41]. Oxygen-free radicals are produced during normal cellular metabolism, but several endogenous antioxidants such as superoxide dismutase, catalase, and glutathione peroxidase are present in cells to protect against oxidation. Non-enzymatic molecules, which can absorb oxygen-free radicals, include alpha tocopherol, ascorbate, and beta-carotene [42]. After ischemia, reperfusion initiates rapid generation of oxygen-free radicals such as the superoxide-free radicals (O2-•), which overwhelm the protective antioxidants allowing the free radicals and their metabolites to cause cell injury [26,41].

The most frequently reported intracellular reaction, which occurs during reperfusion, involves activation of xanthine oxidase, an enzyme present in the cytosol of most animal cells. Most of the tissue xanthine oxidase is retained as xanthine dehydrogenase. During ischemia, decreased ATP concentrations result in accumulation of hypoxanthine. As calcium accumulates in the cells due to membrane pump failure during ischemia, calpain stimulates conversion of xanthine dehydrogenase to xanthine oxidase. The excess hypoxanthine which accumulates during ischemia is catalyzed by xanthine oxidase (Fig. 8) [43].

During normal cell metabolism, superoxide dismutase converts superoxide radicals into hydrogen peroxide (H2O2), which is then degraded to water (H2O). After ischemia, excess free radical production allows formation of hydroxyl radicals (OH-•) via the iron dependent Haber-Weis reaction [26]. Hydroxyl radicals damage cell membranes causing stimulation of cytokine production and, potentially, cell death. Although xanthine dehydrogenase is present in the small intestine, there is little of this enzyme or xanthine oxidase in the large colon of ponies, suggesting that production of alternative reactive oxygen is from leukocytes or aldehyde oxidase [44].

Figure 6. Transmission electron photomicrographs of the jejunal serosa.The normal mesothelial cell (A) is attached to a basement membrane and has microvilli and pores for equilibration of water in abdominal fluid. After 30 minutes of ischemia, the mesothelial cells are lifting off the basement membrane (B) partially due to water accumulation beneath the cells.

Endothelial cells are the primary initiators of reperfusion injury [16] . Endothelial cells are metabolically active cells, which have specialized roles in the regulation of blood pressure, vascular permeability, vascular tone, inflammatory cell adhesion, coagulation, and platelet aggregation [45]. The endothelium synthesizes and releases several chemical mediators which regulate vasomotor tone. When activated or injured by lipid peroxidation, endothelial cells swell, causing disruption of the tight cell junctions and increasing capillary permeability. Fluid, protein, and erythrocytes leak into the interstitium. The subsequent increase of interstitial pressure causes capillary collapse. Endothelial cell swelling and extramural capillary pressure subsequently decrease blood flow, resulting in a "no-reflow phenomena" which creates a new ischemic event in the affected tissue (Fig. 9) [46-48].

Figure 7. Laser Doppler images measuring red blood cell flow in the equine jejunum (yellow and red indicate the greatest serosal blood flow, while blue indicates no blood flow). The initial blood flow at approximately 30 ml/min/kg (A) is significantly decreased during low flow ischemia at 2 ml/min/kg (B). During release of the arterial clamp, blood flow rapidly increases to twice normal flow (C) and subsequently decreases below normal after 2 - 3 hours of reperfusion.

Figure 8. Production of superoxide radicals in the cytoplasm of an endothelial cell. As energy stores are utilized during ischemia, adenosine accumulation leads to production of hypoxanthine. Calcium accumulation in the cell due to failure of the ion pumps activates calpain, which catalyzes the production of xanthine oxidase. When increased concentrations of oxygen are presented to the cell during reperfusion, xanthine oxidase converts hypoxanthine to uric acid and a superoxide radical (O2-•)26 Reprinted from Moore RM, Muir WW, Granger DN. Mechanisms of gastrointestinal ischemia-reperfusion injury and potential therapeutic interventions: A review and its implications in the horse. J Vet Int Med 1995; 9(3):115-132.

When stimulated by oxygen radicals or platelets, endothelial cells generate cytokines which attract and activate neutrophils during reperfusion injury [16]. Studies in feline intestine demonstrated curtailment of neutrophil infiltration by pretreatment with allopurinol(inhibits xanthine oxidase) [49], superoxide dismutase [49], catalase (catalyzes conversion of H2O2 to H2O), and deferoxamine (inhibits conversion of O2-• to OH-•) [50]. Cytokines such as leukotriene B4, complement C5a, and platelet activating factor promote chemotaxis, as well as adherence and extravasation of the leukocytes, during reperfusion [51,52]. Neutrophil migration into the interstitium from the capillaries or venules is preceded by adhesions to the endothelium (Fig. 10). Neutrophil adhesion occurs via an interaction between the neutrophil adhesion receptor complex (CD18), the intracellular adhesion molecule-1 (ICAM-1), and the endothelial leukocyte adhesion molecule-1 (ELAM-1). Chemotactic agents, such as LTB4, complement C5a, or PAF, increase the activity of the CD18 receptor complex, and cytokines can also stimulate the endothelial cell resulting in expression of ICAM-1 and ELAM-1 [53].

Elastase, which is released from activated neutrophils, degrades the intercellular barriers between the endothelial cells, allowing migration of neutrophils from the microvasculature into the interstitium (Fig. 10) [54]. When the neutrophils adhere to and migrate through the endothelium, they further injure the endothelium and increase vessel permeability (Fig. 11).

Neutrophil migration and accumulation initiate a second phase of reperfusion injury. Activated neutrophils degranulate and release free radicals, proteases, and hypochlorous acid, adding to the vascular and interstitial tissue injury (collagen and ground substance) during reperfusion [55]. Neutrophils also produce damaging superoxide free radicals via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, which catalyzes the reduction of oxygen to the superoxide anion and hydrogen peroxide. The production of these oxidative compounds by neutrophils is called the "respiratory burst" and is the main mechanism by which the neutrophils contribute to reperfusion injury and phagocytize and, subsequently, destroy microorganisms (Fig. 12) [56]. NADPH catalyzes the enzymatic conversion of hydrogen peroxidase and chloride ions to hypochlorous acid, a potent oxidant [57]. Hypochlorous acid rapidly reacts with other compounds to form other potent oxidants, such as the lipophilic Nchloramines [58,59]. Hypochlorous acid can also cause tissue damage by inactivating alpha-anti-proteases and activating gelatinase and collagenases and is believed to kill bacteria via halogenation, deamination, and decarboxylation of microbial proteins and nucleic acids [59]. These same mechanisms are thought to be involved in the tissue injury and may explain the ability of the hypochlorous acid to disrupt cell membranes and affect permeability of the mucosa [60].

Neutrophils infiltrate the mucosa and serosa of equine small intestine during reperfusion (Fig. 13) [34,61,62]. Increased numbers of neutrophils in the serosa and increased vascular permeability during acute reperfusion are associated with serosal inflammation after 48 hours and scarring 10 days later, suggesting that the changes during the acute phase of reperfusion are linked to subsequent complications [63]. Neutrophils increase throughout the depth of the mucosa in the large colon of horses during low-flow ischemia, with a subsequent increase during reperfusion [40].

Figure 9. Transmission electron photomicrographs of jejunal serosal venules. The normal vessel is thin walled and contains red blood cells (A). After ischemia and reperfusion, the vessel collapses from fluid pressure in the interstitium, and neutrophils plug the lumen as they adhere to the endothelial surface effectively decreasing blood flow. 9A reprinted from Dabareiner RM, et al. Effects of Carolina rinse solution, dimethyl sulfoxide, and 21 aminosteroid, U-74389G, on microvascular permeability and morphology of the equine jejunum after low-flow ischemia and reperfusion, AJVR 2005; 66:525-536. 9B reprinted from Dabareiner RM, et al. Effects of intraluminal distention and decompression on microvascular permeability and hemodynamics of the equine jejunum. AJVR 2001; 62:225-236.

Figure 10. Transmission electron photomicrograph of a jejunal serosal venule. Neutrophils roll along the vessel wall and then adhere to the vessel wall prior to migration into the interstitium.

Figure 11. Transmission electron microphotograph of a jejunal serosal venule with multiple RBC in the lumen. A neutrophil is migrating between endothelial cells into the perivascular tissue during reperfusion of the serosa.

Figure 12. Transmission electron photomicrograph of the surface of the jejunal serosa during reperfusion. Neutrophils migrate from blood vessels into the interstitium where they release lysosomes, which disrupt collagen and ground substance and subsequently cause an inflammatory reaction.

Figure 13. Neutrophils and mononuclear cells (arrows) migrate between enterocytes after ischemia and reperfusion of the small intestine. The response was likely triggered by the response of the endothelial cells and enterocytes to ischemia and then to the chemical events initiated by reperfusion. Reprinted from Dabareiner RM, et al. Effects of Carolina rinse solution, dimethyl sulfoxide, and 21 aminosteroid, U-74389G, on microvascular permeability and morphology of the equine jejunum after low-flow ischemia and reperfusion. AJVR 2005; 66:525-536.

A recent study evaluating the effect of depleting leukocytes with a filter in an extracorporal circuit revealed that the filter did not have a profound effect on attenuating the reperfusion injury to the equine small intestine [64]. This suggests that other resident cells or neutrophils, rather than circulating neutrophils, can mediate reperfusion injury.65

Proteases, enzymes which cleave peptide bonds and which can have damaging effects on cells, also contribute to reperfusion injury. Granulocytes contain at least three serine proteases (elastase, neutral proteases, and chymotrypsin-like proteases such as cathepsin G), which are released during phagocytosis [26]. These serine proteases catalyze the degradation of proteins or polypepetides. Lysosomes, small intracellular organelles found in the cytoplasm of most cells, are also a source of proteases that degrade or digest cellular debris (Figure 12). During normal physiologic conditions, endogenous protease inhibitors protect tissues from these enzymes, but, during reperfusion, excess protease activity appears to be responsible for tissue injury as indicated by decreased mucosal injury when protease inhibitors are administered in conjunction with reperfusion of feline intestine [66]. Granulocytes and lysosomes are likely to be the most important sources of proteases in the large intestine and may contribute to mucosal cell injury [26].

Though intestinal ischemia reperfusion injury has been largely attributed to cell death, research also suggests apoptosis, or programmed cell death, is caused by ischemia and reperfusion [67]. Whereas cellular necrosis is initiated by lack of oxygen, apoptosis results from activation and transcription of specific genes and affects individual cells of a highly specific cell type. Apoptosis starts by alteration of mitochondrial permeability (mitochondrial permeability transition) and subsequent activation of caspase 9 and 3 during reperfusion [68]. When the mitochondrial permeability transition or caspase activation is blocked, apoptosis is prevented. Apoptotic cells in the mucosa, circular and longitudinal muscle, myenteric plexus, and serosa are significantly increased in clinical cases of equine intestinal strangulation and simple obstruction (Fig. 14) [69]. This could be clinically important because apoptosis may affect the integrity of the mucosal barrier and may be a stimulus for additional inflammation during reperfusion [70-72].

Occasional apoptotic epithelial cells in the normal intestine do not cause alterations in the intestinal barrier integrity, whereas epithelial and endothelial barrier permeability increases with increased intestinal mucosal apoptosis [72]. In naturally occurring colic cases, apoptosis was evident in distended and ischemic intestine. Intestine distant to the primary lesion was often affected, supporting the concept of circulating cytokines or inflammatory mediators stimulating apoptosis (Fig. 15) [69].

Figure 14. Apoptosis stain (brown staining cells) of large colon using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL method). In normal intestine, very few or no apoptotic cells are found using the TUNEL method (A). In horses with intestinal distention or strangulation, apoptosis was evident in mucosal cells, myocytes in circular and longitudinal muscle (B), in glial cells and in neurons in the myenteric plexus (C). 14C reprinted from Rowe EL, et al. Detection of apoptotic cells in intestines from horses with and without gastrointestinal tract disease. AJVR 2003; 64:982-988.

6. Intestinal Obstruction

In the small intestine, obstruction of the intestinal lumen is usually due to intraluminal blockage from a dehydrated food mass or extraluminal pressure from adhesions, thickening of the intestinal wall, or infection. Adynamic ileus may recur after surgery or may be associated with thromboembolism and cause functional obstruction due to lack of intestinal movement. Adynamic ileus is associated with peritonitis, ischemic intestinal insults, anesthesia, endoparasitism, and electrolyte imbalances [7,73-77]. Obstruction of the cecum is due to tympany, impaction (dehydrated ingesta), dysfunction (hydrated ingesta), or intussusception. In the large colon or small colon, impaction by dehydrated ingesta, concretions, or sand most often cause blockage.

Figure 15. Graph of the number of apoptoic cells in obstructed and strangulated intestine, both from the primary lesion and distant to the lesion. There was a significant (*) increase in the number of apoptotic cells in intestine oral or aboral to strangulated intestine. Although the clinical effect of apoptosis in intestine is not known, the marked increase in apoptosis in muscle and nerves suggests this may affect motility and increase the amount of inflammation, potentially causing clinical complications such as ileus or adhesions.

The immediate response to intestinal obstruction is increased motility of the segment of bowel oral (proximal) to the blockage due to distention and relaxation of the intestine aboral (distal) to the blockage [10,78,79]. The muscular activity of the intestine is increased around the obstruction, increasing intraluminal pressure in this segment of intestine. Distention of the intestine by the obstruction or fluid or gas oral to the obstruction stretches the wall of the intestine and combines with the reflex muscular spasm to initiate colic [9].

Obstruction of the Small Intestine

When the small intestine is obstructed, the enterosystemic circulation of fluid is blocked so that fluid from saliva, stomach, bile, pancreas, and small intestinal secretion is prevented from passing to the large intestine to be reabsorbed. Because normal secretion of fluid continues, the small intestine becomes distended oral (proximal) to the obstruction. When the luminal pressure becomes elevated, the tissue pressure compresses the capillaries and reduces venous drainage. Blood flow to the intestine is decreased as the capacitance of the vascular system changes (Fig. 16) [80].

Because of increased capillary hydrostatic pressure, water moves into the lymphatic system, the intestinal lumen, or through the serosa [81]. The increased intraluminal hydrostatic pressure created by enhanced secretion of fluid initiates cyclic increases in secretion by continuing to increase the intraluminal pressure (Fig. 17) [82-84].

Figure 16. Scanning electron photomicrographs of latex injections of serosa. Increased intraluminal pressure (18 cm H2O) collapses the small veins and capacitance vessels (A) compared to the normal filling of capillaries and vessels in non-distended intestine [80]. 16A and B reprinted from Dabareiner RM, et al. Evaluation of the microcirculation of the equine small intestine after intraluminal distention and subsequent decompression. AJVR 1993; 54:1673-1682.

Because the secreted fluid is isotonic, there is minimal acute change in serum electrolyte values with acute distention. The consequences of intestinal distention are dehydration from third space sequestration of secreted fluid, mucosal injury, abdominal pain, and increased movement of protein across the serosa into the abdominal cavity [85]. The distention eventually inhibits motility in the affected segment of intestine, as well as other portions of the intestinal tract [86]. The clinical signs that result are colic, increased heart rate due to pain and decreased circulatory fluid volume, reduced borborygmi, gastric and intestinal fluid sequestration (gastric reflux), and increased protein concentration in peritoneal fluid. An example of this type of disease process is ileal impaction (Fig. 18).

If the intraluminal pressure is acutely decreased, such as occurs with surgical decompression, intestinal hyperemia and reperfusion injury occur. All layers of the intestine are affected, but the serosa appears to be more susceptible to the inflammatory response created by reperfusion [87]. The edema already present from increased vascular leakage is increased [88].

Figure 17. (A) Blood flow to the small intestinal villus courses through a single arteriole to the villous tip. The blood is returned via venules while interstitial fluid balance is maintained by fluid drainage through the lymphatics. (B) Increased intraluminal pressure increases the interstitial pressure which collapses veins and increases capillary back pressure, which causes the capillaries to leak fluid and protein into the tissue, the bowel lumen, and the peritoneal cavity [85].

Endothelial cells are damaged, initiating cytokine production and chemotaxis of inflammatory cells. Neutrophils migrate into the serosa and cause further inflammation (Figure 12) [34]. After the initial hyperemia from reperfusion, blood flow in serosal vasculature is decreased. This "no reflow phenomena" is caused by swollen endothelial cells, neutrophils, and platelets plugging capillaries, and by the collapse of capillaries from increased interstitial fluid (Fig. 19) [80]. Post-distention reperfusion injury can cause ileus and secondary fluid secretion leading to bowel distention. This can cause colic, gastric reflux, and an inflammatory response reflected by an increase in protein and neutrophils in the peritoneal fluid. If the inflammation is severe enough, the response can continue for several days, causing fibrin deposition within and on the surface of the serosa. The long-term result is scarring and adhesion formation of the intestine [87].

Figure 18. (A) Distended small intestine due to an ileal obstruction. The increased intraluminal pressure causes thin wall vessel collapse, which increases transcapillary pressure thereby forcing fluid into the interstitium, peritoneal cavity, and the bowel lumen. The increased fluid secretion in the bowel creates a cyclic increase in pressure with more secretion. (B) Photomicrograph of a small intestinal villus during small intestinal distention. Excess fluid accumulates in the lymphatics (L). Neutrophils (arrows) and macrophages invade the vasculature and the tissue in response to the endothelial damage caused by the ischemia [b].

Figure 19. Photomicrograph of small intestine serosa from a foal. The normal serosa is made up of a single mesothelial cell layer on a layer of fibrous tissue (violet colored tissue) made predominantly of collagen (A). After 2 hours of distention at 18 cm of water pressure, the serosa is edematous with infiltration of RBC and WBC and dilated lacteals. The edema and poor perfusion (no reflow phenomena) in the capillaries increases during reperfusion.

Obstruction of the Cecum and Large Colon

Obstruction of the cecum or large colon often blocks passage of ingesta but allows intraluminal gas to move aborally. Exceptions to this include extremely dehydrated ingesta, sand impactions, enteroliths, or displacements of the colon with entrapment. Initially, obstruction causes an increase in reflex muscular contractions with increased frequency causing intraluminal pressure increases [8]. Pain is intermittent and is associated with intestinal contractions or marked distention.

Cecal obstruction is usually caused by dehydrated ingesta or by cecal dysfunction resulting in marked distention by hydrated ingesta. When distention reaches a critical level, fluid leaks into the interstitium, as demonstrated in Figure 20.

If the distention and increased pressure is not relieved, blood leaks from the capillaries as part of the ischemic injury. This involves all layers of the cecal wall and creates an inflammatory response which can be amplified by reperfusion when cecal distention is relieved (Fig. 21).

During obstruction of the large colon, fluid entering from the small intestine continues to be absorbed in the cecum and segments of colon oral to the obstruction. The process slowly produces systemic dehydration due to lack of water intake and also results in intermittent pain with reduction of borborygmi. Clinical signs seen with this type of obstruction include intermittent colic (usually mild to moderate), a slight increase in heart rate, mild dehydration, reduced borborygmi, and peritoneal fluid total protein concentrations ranging from normal to increased, depending on the duration of the obstruction. The cecum and colon appear to be more compliant than the small intestine, with higher intraluminal pressure required to cause ischemic and reperfusion injury.

Examples of disorders associated with obstruction of the large colon include large colon impaction, entrapment of the large colon in the renosplenic space, and right dorsal colon displacement. In most instances, damage after distention of the large colon does not appear to be permanent. However, recurrence of colic after large colon impaction or displacements appears to be more common than expected in the normal horse population [89,90]. Colon specimens from horses with obstructions that persisted for more than 24 hours had significantly decreased numbers of neurons in the myenteric plexus at the pelvic flexure (Fig. 22). It is not known if this change was present before the obstruction, thus acting as a predisposing factor, or if the neuronal dropout was caused by the obstruction. Similar to laboratory animals with congenital loss of myenteric plexus neurons, the longitudinal layer of the affected bowel was hypertrophied compared with normal horses [91]. This change suggests that the loss of neurons did not allow inhibition of contractions, thus leading to muscle hypertrophy similar to that observed in horses with cecal hypertrophy associated with chronic cecal impaction [91].

Figure 20. Photomicrographs of the normal cecum (A) and a cecum which was distended (B) with hydrated ingesta in a horse diagnosed with cecal dysfunction (impaction). The submucosa in the distended cecum (B) is filled with fluid due to leakage of the capillaries as a result of increased intraluminal pressure. The edema will resolve after the pressure is relieved and inflammatory response is controlled.

Figure 21. (A) Distended cecum with hyperemia, petechial hemorrhages, and wall thickening secondary to accumulation of fluid ingesta. (B) Photomicrograph of the cecal musculature with hemorrhage and infiltration with neutrophils. The injury represented by the cellular infiltrate is sufficient to cause ileus and could result in necrosis depending on the response to reperfusion.

Figure 22. Neuron numbers (per cm) in the pelvic flexure (PF) and right dorsal colon (RD) were significantly decreased (* indicating values with P < 0.05) compared to normal horses after chronic obstruction, or in the pelvic flexure, left dorsal colon (LD), and transverse colon (TC) after large colon volvulus. Red bars, normal colon; blue bars, less than 24 hours of obstruction; green bars, more than 24 hours obstruction; yellow bars, large colon volvulus [91].

When large colon obstruction is complete and gas no longer escapes past the obstruction, rapid distention occurs in the proximal segments of the bowel. In many instances, this may include the entire colon and cecum. Severe stretching of the intestinal wall produces severe pain, and intestinal motility ceases due to intestinointestinal reflex [9]. Increased intra-abdominal pressure also reduces diaphragmatic movement and decreases tidal volume. The increase in intra-abdominal and intra-thoracic pressures reduces venous return to the heart, causing reduced cardiac output, and reduced arterial oxygenation, which can lead to severe shock and death. These advanced clinical signs include rapid heart rate, pale cyanotic mucous membranes, bloat, no borborygmi, and severe unrelenting pain.

Adynamic ileus can occur in the small or large bowel and produce signs similar to those of mechanical obstruction with subsequent bowel distention. Adynamic ileus in the stomach and/or small intestine can lead to signs identical to those of small intestinal obstruction, including gastric distention from fluid sequestration and eventual rupture of the stomach [73]. Grain overload with volatile fatty acid production can cause stasis in both the stomach and small intestine. Chronic distention of the intestine secondary to a mechanical obstruction can stretch the musculature and injure the mucosa, causing adynamic ileus. This same sequence of events can occur in the cecum and large colon due to rapid distention of these segments of intestine with gas. The gas cannot escape as motility is decreased both from the by-products of rapid fermentation and massive distention of the bowel. This can lead to severe tympany with clinical signs similar to those observed with complete obstruction of the large bowel.

Figure 23. Photomicrographs (H&E stain) of small intestine subjected to 60 minutes of strangulation. Venous strangulation causes accumulation of blood in the interstitium (A) while arteriovenous strangulation causes ischemic injury with mucosal cell separation without blood accumulation (B). The severity of the injury in either type of strangulation is dependent on the duration of ischemia.

Figure 24. (A) Transmission electron photomicrograph of a normal small intestinal villus. (B) Photomicrograph of a small intestinal villus after 60 minutes of arteriovenous ischemia and subsequent reperfusion. The villous tuft is exposed with sloughing of the mucosal cells, creating a grade III injury. This is also observed in the scanning electron photomicrograph demonstrating the exposed capillaries of the villus (C). 24C reprinted from White NA, et al. Mucosal alterations in experimentally induced small intestinal strangulation obstruction in ponies. AJVR 1980; 41:193-198.

Reduced colonic motility also occurs in the large colon secondary to administration of atropine, alpha-2 receptor stimulation with drugs such as xylazine or detomidine, or opiate receptor agonists [7,92]. The stasis following administration of xylazine or detomidine is normally short-lived and does not cause a clinical problem even when treating intestinal obstruction [92]. Atropine or low serum calcium concentrationsa can cause long periods of bowel stasis with accumulation of both gas and fluid, creating the same problems seen with an intraluminal obstruction or extraluminal compression.

Ileus may also occur due to sympathetic stimulation in response to pain or physical or ischemic trauma to the bowel. This form of functional obstruction is normally temporary and often not complete. Clinically this results in a lack of borborygmi on auscultation. Although mixing sounds from the colon may be present, long progressive sounds are absent until long spike burst activity returns.

Although intestinal distention may appear benign, particularly when it is easily corrected, it is important to remember that intestinal distention leads to inflammation in the bowel wall. When severe enough, this distention can decrease motility and eventually lead to adhesions [87,88].

7. Strangulation Obstruction

Strangulation obstruction of intestine is a combination of luminal obstruction and mural ischemia [85]. Strangulation obstructs the blood supply by vascular constriction and is usually due to intestinal torsion-volvulus or incarceration of the small intestine in a variety of intra-abdominal sites, including the epiploic foramen, inguinal ring, or rents in the mesentery. Two types of strangulation have been defined, venous and arteriovenous [35,93]. With less than complete constriction, venous return is impeded at the site of strangulation without a loss of arterial flow. This causes accumulation of blood in the interstitium of the intestinal layers, with most of the blood accumulating in the submucosa and lamina propria (Fig. 23) [35].

Arteriovenous constriction can occur acutely or may follow venous constriction (Figure 23). Both types of circulatory interruption result in the development of lesions in the intestine. Although both forms of strangulation cause ischemia, engorgement of the intestinal wall causes the bowel to appear more devitalized than may be detected with a microscopic examination, so that the intestine may not be as severely injured as that which occurs with arteriovenous strangulation for the same amount of time [93]. Because of the cascade of events which occur during intestinal strangulation, the injury and resulting clinical signs of strangulation obstruction are caused by the combined effects of ischemia, reperfusion injury, and endotoxemia.

Ischemia causes similar lesions in the large and small intestine. The lack of perfusion reduces oxygen delivery, which affects the mucosal cells first due to their high metabolic activity [33]. The cells start to separate from the basement membrane because of the formation of extracellular edema at their attachment to the capillary tuft of the lamina propria. Thus, the cells slough before development of severe intracellular damage of the mucosal cells [94]. Sloughing of the epithelial cells of the villi occurs in the small intestine and from the surface and into the crypts of the cecum and colon [33,36]. The slough is graded from 0 (normal) to 5 (total slough) with necrosis of the crypt cells (Fig. 24) [33,36,95,96].

The loss of cells is sequential starting at the villous tip and progressing to the crypts. At the same time, the mesothelium of the serosa also sloughs, and the fibrous tissue layer becomes disrupted and edematous. Ischemic changes continue as progressive necrosis unless blood flow resumes. As the pH in the tissue drops, enzymatic processes cease and the cells remain static with a brief period of preservation of cell membrane and cytosolic contents. When the cell’s source of energy is exhausted, cell membrane degradation occurs, followed by cell death.

Vascular damage initiates leakage of protein and erythrocytes out of the vascular space into the bowel lumen and peritoneal fluid [23]. Loss of the mucosal barrier allows bacteria and endotoxin to escape into the peritoneal cavity causing increased neutrophil numbers in peritoneal fluid. With severe necrosis, bacteria are present in the fluid or within the neutrophils, and the fluid itself becomes serosanguineous.

Initially, strangulation obstruction causes clinical signs similar to those of simple obstruction. Clinical signs that separate strangulating lesions from simple obstruction relate to the rapid bowel distention, disruption of the mucosal barrier, and absorption of bacteria and endotoxin from the intestine into the circulation. Strangulating lesions often cause severe abdominal pain from stretching of the mesentery, necrosis of the intestine, and endotoxin release with subsequent cytokine and prostaglandin production [97-105]. Loss of mucosal epithelium exposes the lamina propria and its capillary system to the contents of the bowel lumen. Bacteria and endotoxins invade the tissue, pass through to the peritoneal cavity, and eventually enter the bloodstream. These events initiate shock by movement of fluid from the extracellular and intracellular compartments into the intestinal lumen, and by the systemic response to endotoxemia [106].

Figure 25. (A) Photomicrograph (H&E stain) of small intestine strangulated in an incarceration. (B) Photomicrograph of bowel located proximal to the primary lesion. This bowel (B) was distended and had evidence of mucosal injury, even though grossly it appeared pink and viable.

Figure 26. Photomicrograph (H&E stain) of the fascia between the circular and longitudinal muscle. Neutrophils migrate into all layers of the intestine during reperfusion. Neutrophil infiltration into the muscle and myenteric plexus likely affects intestinal motility and function.

Figure 27. Photomicrograph of a villus with mucosal cells covering the capillary tuft within 48 hours of a grade-3 injury in the jejunum. The initial healing relies on villous contraction and migration of the surviving enterocytes to cover the villus. The result is a flattened villus, which is eventually restored to its normal height with enterocyte proliferation.

Figure 28. Table of peritoneal exudate cell (PEC) activity (cell stimulation was in vitro with bacterial endotoxin) after abdominal surgery in rabbits. PMNs increased immediately, while macrophages increased after 3 days. Superoxide activity was highest immediately after surgery. Plasminogen activator inhibitor was increased while plasminogen activator was decreased during the first three days after surgery. Interleukin increases which are linked to increases in plasminogen activator occurred after five days (*, not applicable) [125]. Reprinted from Rogers KE, diZerega GS. Function of peritoneal exudate cells after abdominal surgery. J Invest Surg 1993; 6:9-23.

The intestine proximal and distal to the primary site of strangulation frequently has evidence of injury [96]. This may be due to a response to cytokine release such as tumor necrosis factor which is released into the systemic circulation [107,108]. Additionally, poor perfusion resulting from hypovolemia and endotoxemia may create poor perfusion to multiple organs including the intestine. Apoptosis is stimulated in bowel distant from the primary lesion (Figure 15) [69]. Although not as severe as the injury caused by a strangulating lesion, the reduced flow also creates ischemia and subsequent intestinal injury. This is predominantly a mucosal lesion, with mucosal cell loss and edema of the lamina propria (Fig. 25). This secondary bowel injury is potentially due to endotoxin or reperfusion injury making affected bowel prone to ileus, fluid secretion, and eventual infarction unless the shock sequence can be reversed.

Figure 29. Neutrophil accumulation in the serosa after 2 hours of ischemia or 2 hours of distention followed by 3 hours of reperfusion and decompression, respectively. Neutrophils were significantly increased (P<0.05) after low flow ischemia (blue bar) or distention (green bar) compared to control intestine or intestine treated with Carolina rinse (red bars) [38,126].

Figure 30. (A) Photomicrograph of jejunal serosa from a foal 10 days after 60 minutes of ischemia. The arrows indicate the original level of the serosa before injury. No mesothelial regeneration is evident on the new serosal surface, leaving this intestine prone to adhesion formation. (B) Transmission electron photomicrograph of jejunal serosa 10 days after ischemia. Fibroblasts are proliferating and producing excess collagen within the newly added serosal layer.

Reperfusion Injury

Reperfusion injury can be initiated by several mechanisms with a resulting inflammatory response and progressive injury, which is more severe than created by ischemia alone [66]. Reperfusion injury occurs after partial or low flow ischemia as well as after complete ischemia, unless severe cell injury does not allow the cells to respond to reperfusion. In the horse, experimental ischemia and reperfusion have documented reperfusion injury in both small intestine and the large colon [33,34,47,81,87,88,109-114]. It appears that neutrophils are responsible for most of the damage associated with reperfusion injury. As they migrate into the injured tissue, they cause more damage by releasing proteinaces, oxygen radicals, and hypochlorous acid. Tissue damage is progressive, including progressive mucosal epithelial cell slough, collagen disruption, edema, and neutrophil infiltration of the submucosa and muscular layers and serosa (Fig. 26) [49,112,115-117].

Figure 31. Adhesions of the small intestine resulting from a small intestinal volvulus corrected at surgery 6 months earlier. The scar formation is due to serosal inflammation after ischemia and subsequent reperfusion with an excessive fibrous tissue response, which makes permanent adhesions, as well as circumferential constriction and mesenteric shortening which compromise the mesenteric blood supply. Reprinted from White NA, Pathophysiology of obstruction, strangulation, strangulation/obstruction schema, Chapter in Equine Medicine and Surgery Ed. by Colahan P, Merritt A, Moore J, Mayhew I, American Veterinary Publications, Santa Barbara, 1999; 590-602.

Reperfusion injury of the intestine is usually evident after surgery but is thought to occur after any condition which causes decreased blood flow to the intestine. During surgery, the hyperemic response may prevent the surgeon from recognizing bowel undergoing reperfusion injury. Progressive degeneration of intestine due to reperfusion injury usually occurs 24 - -48 hours after surgery and causes colic and/or depression, ileus, increased heart rate, discolored mucous membranes, and alterations in the peritoneal fluid. Although increases in neutrophils and protein concentration is expected after abdominal exploration, cell degeneration and indications of sepsis often accompany bowel injured by reperfusion or continued distention.

When trying to relate all of the clinical signs that evolve from endotoxemia and bowel inflammation or necrosis, the overlap of many reactions makes delineating specific causes and effects difficult. The signs of shock and pain from the intestinal lesion are amplified by an adrenergic response causing sweating, tacky mucous membranes, poor membrane refill, tachycardia, and ileus.

The severity of the signs observed during shock and reperfusion are dependent on the amount of intestine involved. Large colon torsion or small intestinal volvulus that involves a large segment of bowel induces more rapid and severe systemic changes than conditions involving only small segments of intestine. Conditions such as intussusceptions or some inguinal hernias, in which the strangulated bowel is isolated from the abdomen, do not produce the same acute signs of shock until endotoxins or cytokines move into the systemic circulation. Other than pain from stretching of the mesentery, these conditions may cause few systemic signs or biochemical changes or may have a slower onset [118].

9. Intestinal Healing and Adhesions

Mucosal and serosal regeneration can be a rapid and a potentially totally reparative process that follows any intestinal injury. The mucosa can heal as long as viable enterocytes are present to migrate from the crypt or villus over the lamina propria. During the loss of epithelial cells, the villus is shortened by contraction and loss of the capillary tuft. The villus is covered by epithelium within 12 - 24 hours by restitution of the mucosal barrier (Fig. 27). Restitution allows the epithelial cells remaining after the injury to cover the villus. This part of the repair is modulated by several factors including contraction of the villus, which is dependent on the nervous system and somewhat controlled by prostaglandins [119]. Growth factors, such as transforming growth factor and epidermal growth factor, ground substance (collagen, mucopolysaccarides, glycoaminoglycan), and enzymes, play an important role by stimulating cell migration along the villus [120-122]. Prostaglandins help stimulate muscle contraction within the villus. Surviving cells develop lamellipodia which move along the basement membrane as the cell migrates along the villus. Ultimately, tight junctions between the epithelial cells must be restored to maintain an effective mucosal barrier against bacteria and endotoxin. Because PGE2 is involved in tight junction closure, treatment with non-selective cyclooxygenase (COX) inhibitors delays recovery of the barrier function allowing molecules to move between the cells [123]. The lack of tight junction closure may allow the movement of bacteria and endotoxin through the new mucosal lining resulting in the systemic response during the healing process. Currently, a COX2 inhibitor, which does not delay healing but provides analgesia and decreases inflammation, has not been identified or tested for horses.

If there is lack of blood supply or excessive necrosis or inflammation, intestinal healing is delayed, leaving the exposed lamina propria open to bacteria and endotoxin which can migrate into the peritoneum and vascular system.

Surviving precursor cells form new enterocytes which migrate up the villus. This allows villus elongation, and eventually new cells replace the original enterocytes which slough into the lumen. Enteral nutrition appears to stimulate this proliferation of cells, and glutamine is an essential nutrient for this reparative process in the small intestine [124]. Within 10 days, the villus can regenerate to normal length, and the intestine can return to its normal absorptive capacity.

Serosal responses to ischemia and reperfusion include edema, deposition of fibrin, and infiltration of neutrophils and macrophages. A study of cell kinetics after experimental intestinal anastomosis or abdominal surgery in rabbits measured numbers of leukocytes and concentrations of IL-1, TNFα, plasminogen activator, and plasminogen inhibitor in peritoneal fluid [125]. After surgery, the highest concentration of neutrophils occurred on day 1, whereas macrophages peaked on days 3 and 28. TNFα concentrations peaked on days 3 and 14. Superoxide production was highest from neutrophils on day 4 and highest from macrophages on days 1 and 4. Thromboxane, PGE2, and prostacyclin peaked and maintained concentrations from days 5 - 10. Plasminogen activator concentration was lower than normal from days 1 - 5 and peaked on day 14 and maintained high concentrations until day 28. Plasminogen inhibitor concentration was highest on days 1 and 10. The cyclic response of these cytokines, specifically IL-1 from macrophages, stimulates plasminogen activator to breakdown fibrin and reduce adhesion formation (Fig. 28). Though similar research examining cytokine and plasminogen activator concentrations in abdominal fluid has not been completed in horses, the response is likely similar to this experiment.

The response is nullified if the inflammatory process causes excess fibrin production and inhibition of fibrin breakdown. The reaction within the serosa appears to predict the eventual amount of scar or adhesion formation. The initial response to ischemia includes significant increases in neutrophil numbers, compared to non-ischemic intestine or intestine treated with NSAIDS or multimodal solutions, such as Carolina rinse, aimed at inhibiting the initial reperfusion injury cascade (Fig. 29) [38,62,126].

Subsequent to the serosal inflammatory response, fibroblast proliferation heals the serosa by adding a new layer of collagen to the original boundary of the basement membrane. Fibroblasts migrate into the fibrin deposited on the surface of the serosa (Fig. 30). This inflammatory response inhibits the restoration of the serosal mesothelium so there is no protective (lubricating) surface during this stage of healing. When adjacent intestine or abdominal wall is equally inflamed, the adhesion of these structures can become permanent as the fibrous tissue matures.

The final scar formation results in adhesions, restrictive mesenteric shortening, and scar contracture which cause narrowing of the intestinal lumen (Fig. 31) [127]. The fibrous response is particularly evident in foals under 3 months of age [87]. Several weeks after an apparent successful surgery, these horses have recurrent obstruction which causes colic and, in some horses, distended small intestine with gastric reflux. The adhesions will often require surgery to by-pass or remove the affected bowel.

Footnotes

[a] White NA. Unpublished data, 1976
[b] The Equine Acute Abdomen, Teton New Media, in press.