Animal Models of Ischemia-Reperfusion Induced Intestinal Injury: Progress and Promise For Translational Research

Articles in PresS. Am J Physiol Gastrointest Liver Physiol (November 20, 2014). doi:10.1152/ajpgi.00112.
2013
1 Animal models of ischemia-reperfusion induced intestinal injury: progress and promise
2 for translational research
4 Liara M. Gonzalez,1 Adam J. Moeser,2 Anthony T. Blikslager1
5 Center for Comparative Medicine and Translational Research, 1Department of Clinical Sciences,
2
6 Department of Population Health and Pathobiology, North Carolina State University, Raleigh,
7 NC, USA
9 Correspondence:
10 Anthony T. Blikslager
11 College of Veterinary Medicine
12 1060 William Moore Drive
13 Raleigh, NC 27607
14 Telephone: 919-513-7725
15 Fax: 919-513-6336
16 Email: Anthony_Blikslager@ncsu.edu
17
18 Running Title: Animal Models of Ischemic Injury
19
20 Abstract
21 Research in the field of ischemia-reperfusion injury continues to be plagued by the inability to
22 translate research findings to clinically useful therapies. This may in part relate to the complexity
23 of disease processes that result in intestinal ischemia, but may also result from inappropriate
24 research model selection. Research animal models have been integral to the study of ischemia-
25 reperfusion induced intestinal injury. However, the clinical conditions that compromise intestinal
26 blood flow in clinical patients ranges widely from primary intestinal disease to processes
Copyright © 2014 by the American Physiological Society.

27 secondary to distant organ failure and generalized systemic disease. Thus, models that closely
28 resemble human pathology in clinical conditions as disparate as volvulus, shock, and
29 necrotizing enterocolitis are likely to give the greatest opportunity to understand mechanisms of
30 ischemia that may ultimately translate to patient care. Furthermore, conditions that result in
31 varying levels of ischemia may be further complicated by the reperfusion of blood to tissues
32 that, in some cases, further exacerbates injury. This review assesses animal models of
33 ischemia-reperfusion injury as well as the knowledge that has been derived from each in order
34 to aid selection of appropriate research models. In addition, a discussion of the future of
35 intestinal ischemia-reperfusion research is provided to place some context on the areas likely to
36 provide the greatest benefit from continued research of ischemia-reperfusion injury.
37
38 Introduction
39 The survival rate for human and veterinary patients with ischemic intestinal injury is generally
40 less than 50% (17, 69, 172, 183) because tissue hypoxia, inflammation and cell infiltration result
41 in loss of the mucosal barrier. This barrier is primarily composed of a single layer of columnar
42 epithelial cells bound together by interepithelial junctions, which collectively prevents the
43 translocation of bacteria and associated toxins into the systemic circulation (140). Intestinal
44 ischemia is associated with a broad range of clinical conditions including neonatal necrotizing
45 enterocolitis (125), acute mesenteric ischemia (AMI) (183), volvulus (155), trauma (41),
46 cardiopulmonary disease (151), hemorrhagic shock (41, 53) and intestinal transplant rejection
47 (112) (Figure 1). Ischemic injury may be coupled with subsequent reperfusion of tissue that is
48 known to exacerbate injury in some of these disease processes (112). Since intestinal ischemia
49 is rarely preventable, most research in the field has focused on advancing techniques for early
50 detection of ischemia and the development of novel therapeutic approaches that target the post-
51 ischemic insult (the reperfusion period). Injury attributed to reperfusion is thought to be primarily
52 attributable to reactive oxygen metabolites, associated with activation of oxidant-producing

53 mucosal enzymes, release of lipid chemoattractants from injured cellular membranes, and
54 subsequent infiltration of neutrophils (Figure 2). Therefore, proposed therapeutic interventions to
55 protect against reperfusion injury have targeted inhibition of oxidant injury and of neutrophil
56 activation (144, 157). However, due to the relative failure of clinical therapy directed at
57 reperfusion injury following early studies, the importance of targeting reperfusion injury in cases
58 of intestinal ischemic injury has been brought into question (105). The most recent clinical
59 perspectives on the efficacy of these treatments are derived from studies of tissues more
60 commonly affected by reperfusion, such as the myocardium following infarction (7). These too
61 have failed to demonstrate the clinical utility of these therapies. Encouragingly, recent studies
62 have recognized the modulation of cell death pathways as a novel target for therapeutic
63 intervention (87, 181). Mechanisms of cell death mediated by necrosis and apoptosis have been
64 shown to further exacerbate injury induced by ischemia and reperfusion. In fact, insufficient
65 clearance of these dying cells has been shown to lead to increased inflammation and impaired
66 tissue repair (181). As a result, there has been a shift of emphasis toward modulation of
67 pathways of cell death (87, 181) and regenerative processes (113, 161). However, initial
68 consideration of the animal model to select for study is of paramount importance, given the
69 highly variable clinical presentation of diseases that involve ischemia. Animal models have been
70 indispensable to the study of mechanisms of ischemia and reperfusion injury (22, 39, 134-137),
71 but each model has distinct advantages and disadvantages so that none of these models can
72 perfectly recapitulate the natural onset and progression of human disease (Table 1 and 2). The
73 aim of this review is to provide an in depth assessment of key animal models used for the study
74 of ischemia-reperfusion injury, with the goal of allowing investigators to understand and select
75 from available models for translational research. In addition, the future of intestinal regenerative
76 medicine in light of recent advances will be discussed.
77 Mechanisms of Ischemia
78 Ischemia is the reduction or complete occlusion of blood flow to a target organ that results in a
79 state of tissue oxygen deprivation. In the intestine, overt obstruction of the blood supply can
80 result from mesenteric vascular occlusion derived from thrombus formation or emboli secondary
81 to cardiopulmonary disease (143, 166). Compromise of normal blood flow to the intestine can
82 also be associated with severe intestinal distension and mechanical obstruction as occurs in
83 cases of intestinal strangulation associated with hernia, volvulus and intussusception (1, 183).
84 Additionally, ischemic injury is thought to contribute to the pathogenesis of neonatal necrotizing
85 enterocolitis (NEC), the most common life-threatening gastrointestinal emergency in neonatal
86 patients (69, 125, 127). Other major sources of intestinal ischemic injury result from diseases
87 that reduce systemic blood flow such as cardiopulmonary diseases and shock states (53, 151).
88 Finally, the interruption of blood flow is an important component of the process of intestinal
89 transplantation that inevitably results in a period of ischemia (106, 112) (Figure 1).
90 Decreases in blood flow compromise the oxygen supply required for normal cellular function
91 and results in both cellular damage and death. The intestinal epithelium has a particularly high
92 energy demand, largely as a result of basolateral Na+-K+ATPase used to drive secretion and
93 absorption, and is therefore particularly sensitive to reductions in blood flow. The process of
94 epithelial cell loss that results from ischemic injury is well documented in many research animal
95 species (39, 135, 137, 147). Cells closest to the intestinal lumen on the villus tips in the small
96 intestine and inter-crypt surface epithelium in the colon initially lose their attachment to the
97 basement membrane with progressive loss of cells extending toward the crypt base as the
98 duration of ischemia increases (39, 135) (Figure 3). Interestingly, the cells closest to the luminal
99 surface normally function in a state of ‘physiologic hypoxia’ due to an oxygen gradient that is
100 derived from the mucosal vascular architecture (146, 171). Although there are species
101 differences in the anatomy of the mucosal vasculature (Figure 4), a general construct of counter
102 current exchange exists whereby oxygen diffuses from the arterial blood supply that ascends
103 the mucosa toward the lumen and venous drainage flowing in the opposite direction,
104 progressively decreasing the arterial concentration of oxygen as the vessels near the luminal
105 surface (3, 9). This creates a steep oxygen gradient that makes the villus tips hypoxic (33, 159).
106 A similarly decreasing gradient of oxygen from the crypt base to the luminal surface exists within
107 the colon (171). Therefore, further decreases in oxygen concentrations, as occurs with intestinal
108 ischemia, impact the cells at the villus tips and colonic surface epithelium first. This initially
109 creates a distinct lesion identified histopathologically, called Gruenhagen’s space (16, 39)
110 (Figure 3). Originally, this space was solely attributed to the accumulation of cytoplasmic fluid
111 from ischemically damaged cells (39). More recent studies using in vitro techniques and a novel
112 in vivo human study have shown that contraction of the myofibroblasts within the lamina propria
113 also contribute to the creation of this initial defect (46, 141). In fact, the creation of the space
114 was shown to occur within 30 minutes of complete ischemia and reseal by 60 minutes if the
115 blood supply was subsequently returned (46). This demonstrates the remarkable capacity of the
116 intestine to repair rapidly in cases where vascular compromise can be reversed. However, in
117 most cases the duration of ischemia is likely to be lengthier and the tight junctions that serve to
118 anchor the cells together break down, resulting in cell loss into the intestinal lumen. Epithelial
119 cell separation from the basement membrane and complete cell loss then continue and
120 progress toward the crypt base in a time-dependent manner (39). It has been specifically
121 demonstrated in a porcine model of jejunal mesenteric vascular occlusion that by 60 minutes,
122 epithelium is lost from the upper third of the villus and by 120 minutes there is near complete
123 loss of villus epithelium (22). Any exposure of the basement membrane causes marked
124 compromise of barrier function, allowing luminal bacteria and toxins to gain access to the
125 underlying vasculature within the lamina propria (75, 111, 140). The sequelae are septicemia
126 and multiple organ dysfunction, which are responsible for the high morbidity and mortality rates
127 associated with severe intestinal ischemic injury (86, 143, 166, 183).
128 Models of Ischemia
129 Complete Vascular Occlusion
130 Complete ischemia by temporary vascular occlusion (using atraumatic vascular clamps) or
131 permanent vascular occlusion (using ligation) of the superior mesenteric artery (SMA –
132 technically the cranial mesenteric artery in animals) in rodent models is currently the most
133 commonly used method of inducing intestinal ischemic injury. The SMA is approached through
134 a midline incision and occluded for varying lengths of time (87). In most of these studies,
135 heparin is injected intravenously to prevent thrombus formation within the SMA which enables
136 the circulation to be re-established once the atraumatic vascular clamps are removed for
137 measures of reperfusion injury. However, an early study by Megison et. al. noted a highly
138 variable degree of injury and often high mortality of rats in studies in which occlusion of the SMA
139 was performed (116). That study noted that SMA occlusion alone was unreliable in creating
140 consistent and reproducible injury, whereas SMA occlusion combined with ligation of collateral
141 arcades created a greater degree of injury and a higher but consistent level of mortality in the
142 animals. These findings lent support to a similar study performed in cats in which SMA
143 occlusion reduced blood flow to the mucosa an average of 35% in the proximal duodenum, 61%
144 in the distal duodenum, 71% in the jejunum and ileum, and 63% in the proximal colon. No
145 reduction of blood flow was identified in the mid and distal colon (142). Thus, the effect of SMA
146 occlusion is highly dependent on the intestinal segment being evaluated. These findings may be
147 complicated further by the fact that different segments of the intestine may be more or less
148 sensitive to ischemic injury. For example, differences in resistance to ischemic injury have been
149 identified between the jejunum, ileum and large colon with studies indicating a progressive
150 increase in resistance to ischemic injury in aboral segments (37, 107, 147). Furthermore, a
151 porcine study of small bowel transplantation demonstrated microscopic injury in the ileum only
152 after 5 hours of ischemic injury compared to 1.5 hours in the jejunum (35). Little reperfusion
153 injury was noted in either segment. It should be noted, however, that this study only evaluated
154 injury histologically.
155 Unfortunately, many studies of ischemic injury either assess injury in a single segment of
156 intestine, many times the ileum, or do not specify which segment is being evaluated (Table 3).
157 Additionally, few studies have focused on the colon despite the fact that colonic ischemia is a
158 more common clinical entity in people than small intestinal ischemia (1, 51, 73, 122). The colon
159 does, in fact, respond differently to insult. For example, a study compared regional differences
160 in susceptibility to injury between the small intestine and colon and demonstrated that the colon
161 was more resistant to ischemia in rats (107).
162 A more recent technique used to achieve complete irreversible ischemia was established in a
163 porcine model by embolization of the SMA. The SMA was accessed either percutaneously (96)
164 or via endovascular catheterization (2, 25, 148) and a solution of either buthyl-2-cyanoacrylate
165 (96) or polyvinyl alcohol particles and gel foam (2, 25, 148) were injected intravascularly. These
166 models are particularly useful for imaging studies aimed at improving diagnosis in patients
167 suspected of having AMI. Using this approach, the abdominal contents are not exposed or
168 manipulated in creating the vascular obstruction. Since bowel manipulation alone creates
169 inflammation that otherwise would not be present in clinical cases of AMI, minimally invasive
170 approaches to the SMA may be optimal for studies modeling AMI.
171 The critical assessment of the type of ischemia model with regard to studies of AMI cannot be
172 overstated. Acute mesenteric ischemia is a complex of diseases that can be classified based on
173 whether the mechanism of obstruction is occlusive or nonocclusive. Occlusive intestinal
174 ischemia is further subdivided into acute or chronic and whether arteries or veins are affected.
175 Nonocclusive mesenteric ischemia is a disease process that is more poorly understood but
176 occurs with patent mesenteric arteries and is associated with disease processes such as
177 congestive heart failure, aortic insufficiency, renal or hepatic disease and patients following
178 cardiac surgery (172). Therefore, other approaches to create ischemic injury such as the low-
179 flow model likely better recapitulate this form of injury.
180 Low-Flow Ischemia
181 Most of the original low-flow ischemia studies were performed in cats (Table 3).This model likely
182 best recreates the type of intestinal injury that would be expected following hemorrhagic shock
183 or other events that dramatically decreases the volume of blood delivered to an intestinal
184 segment. In the low-flow ischemia model, blood flow is reduced to 20% of baseline levels (~25-
185 35 mmHg). Using this method, the abdomen is approached through a midline incision and a
186 segment of ileum is typically isolated. An arterial circuit is then established between the superior
187 mesenteric and femoral arteries and pressure cannulas are used within the vessels in order to
188 measure vascular pressure. Reduced blood pressure within the superior mesenteric artery (25-
189 30 mm Hg) is achieved using an adjustable vascular clamp. The majority of injury associated
190 with this model has been attributed to reperfusion as a result of increases in xanthine oxidase
191 and neutrophil activation, making the ischemic event more of a priming mechanism for
192 subsequent injury. This may or may not adequately represent clinical scenarios, in which injury
193 caused by ischemia may be so severe that reperfusion injury is of limited significance. Again,
194 careful selection of models depending upon the human condition being studied is critical.
195 Segmental Mesenteric Vascular Occlusion
196 An additional model of ischemia is segmental mesenteric vascular occlusion, in which the blood
197 flow within a discrete loop of intestine is interrupted by clamping the local mesenteric vascular
198 supply and cross-clamping the bowel. Within a single animal, multiple loops can be subjected to
199 varying lengths of ischemia with or without reperfusion, thereby titrating the degree of injury
200 within each loop. This model can be readily performed in large animals, such as the pig, and
201 rodents (73, 74), and has the advantage of providing multiple treatment groups and controls
202 within a single animal. However, this model of strangulating obstruction relies on complete
203 occlusion of the arterial and venous blood supply, whereas clinically the venous circulation is
204 commonly compromised prior to the arterial blood supply due to differences in vessel wall
205 thickness and compliance (135). This results in continued arterial supply of oxygenated blood
206 for a variable period of time, until the tissue becomes so congested in the absence of venous
207 drainage that the arterial vascular supply ultimately collapses. This complex form of ischemic
208 injury includes elements of ischemia as well as excessive interstitial pressure, which together
209 likely affect the level of epithelial sloughing.
210 Perhaps the most interesting and significant application of the mesenteric vascular occlusion
211 model of intestinal ischemia has been its application in human subjects (46, 47). In these
212 studies, a normal segment of jejunum from patients undergoing Roux-en-Y or similar intestinal
213 reconstruction were isolated and subjected to 30-45 minutes of mesenteric vascular occlusion,
214 with varying lengths of reperfusion, prior to removal from the patient (46, 47, 75, 115). The
215 limitation of these studies is that severe ischemic damage as applies to most patients suffering
216 from ischemia-reperfusion injury cannot be induced in these clinical studies.
217 With regard to each of these models of ischemia, the research animal species used may
218 contribute significantly to the outcome. As previously noted, variations of the mucosal vascular
219 supply, particularly in the small intestine, exist between species (Figure 4). The pig and human
220 villus microvascular architecture are very similar, arborizing at the tip of the villus into a fountain-
221 like pattern and converging into one or two venules located beside the centrally located arteriole
222 (9, 33). In contrast, in the rat, the flattened leaf-shaped villi are supplied by a single arteriole that
223 passes unbranched from the base to the tip of the villus where it bifurcates into a capillary
224 network, a so-called “netted bag” pattern (38). The arterioles begin to converge and form two
225 venules as the vasculature approaches the base of the villus (33, 38). In the mouse, two
226 arterioles supply oxygenated blood to the villus, and then divide into a capillary network that
227 rejoins to form a single venule at the tip of the villus. The venule then travels down the center
228 toward the base of the villus (34). These differences may impact the physiological function of
229 the intestinal mucosa between species and potentially their susceptibility or response to injury.
230 Multiple in vivo and in vitro intestinal permeability studies have demonstrated significant
231 differences between man and rodents and greater correlation between humans and pigs (15,
232 45, 126). In particular, Bijlsma et. al. hypothesize that the differences in permeability between
233 rodents and humans is due to the interspecies variation in villus blood vessel architecture
234 resulting in varying countercurrent exchange efficiency that directly impacts villus epithelial cell
235 function (15).
236 Mechanisms of Reperfusion Injury
237
238 The concept of progressive injury that occurs following the restoration of normal vascular
239 circulation to an ischemically compromised region seems counter-intuitive. After all, re-
240 establishing blood flow is ultimately critical to rescue and maintain cell function. However,
241 ischemia creates an environment primed for the production of injurious metabolites, the influx of
242 inflammatory cells and epithelial cell apoptosis and necrosis. Early studies of reperfusion in cats
243 and rodents attributed the exacerbation of injury during this period to the production of reactive
244 oxygen metabolites that directly contribute to injury as well generation of neutrophils
245 chemoattractants (26, 48, 65, 66, 68, 70, 77, 78, 82, 98, 99, 101, 128-130, 136-138, 153, 164).
246 During ischemic conditions, as tissues continue to utilize adenosine triphosphate as a source of
247 energy, the metabolic byproduct, hypoxanthine, accumulates. In addition, in this oxygen
248 deprived environment, xanthine dehydrogenase which would normally function to metabolize
249 hypoxanthine is converted to xanthine oxidase by locally produced tissue proteases (130).
250 When tissue perfusion is re-established, xanthine oxidase utilizes the now freely available and
251 abundant oxygen as an electron acceptor as it metabolizes hypoxanthine, producing superoxide
252 (66-68, 70) (Figure 2). The direct injury induced by oxygen free radicals combined with their role
253 in the activation and chemoattraction of neutrophils creates the severe tissue injury attributed to
254 reperfusion.
255 Neutrophils inflict tissue injury by a multitude of proposed mechanisms that include occlusion
256 and increased permeability of the microvasculature (44, 83), release of reactive oxygen
257 metabolites (48, 130, 165), cytotoxic enzyme release (186) and mechanical injury induced by
258 migration (58). The vast majority of the studies used to determine these findings used a feline
259 low-flow ischemia model. Upon activation, neutrophils release the contents of their granules that
260 include potent proteases such as elastase, myeloperoxidase, protease-3 and
261 metalloproteinases as well as reactive oxygen species including H2O2 and hypochlorous acid
262 (108, 153, 186). These compounds are bactericidal and aid in neutrophil migration as
263 connective tissues are lysed, but concomitantly cause extensive collateral damage to
264 surrounding matrix proteins and nearby cells. Additional injury is created by the mechanical
265 damage induced by their migration across the epithelial monolayer which has been shown to
266 increase paracellular permeability (58). The detrimental role of neutrophil accumulation on
267 epithelial barrier function post ischemia was supported by findings in a porcine model of
268 complete mesenteric vascular occlusion (58) in which pre-treatment of animals with anti
269 CD11/CD18 monoclonal antibody, targeting neutrophil adhesion, significantly reduced
270 neutrophil infiltration and improved measures of epithelial barrier function (58).
271 Model Dependent Reperfusion Injury
272 The significance of reperfusion to intestinal injury following an ischemic event has been and
273 continues to be controversial. This is partly due to the fact that the degree of reperfusion injury
274 appears to depend on the type of ischemia (complete versus incomplete vascular occlusion)
275 and duration of ischemic injury as well as the research animal species and segment of affected
276 intestine.
277 Complete Vascular Occlusion
278 Currently, the complete vascular occlusion method for induction of ischemic injury in rodents is
279 the most commonly used approach for the study of ischemia-reperfusion injury (Table 5 and 6).
280 Rodents are advantageous to use because of their relatively low cost, ease of maintenance,
281 and rapid reproduction rate. In recent ischemia-reperfusion research, mouse models have been
282 used to determine genes associated with oxidative stress (12) as well as to provide evidence of
283 increased epithelial proliferation and barrier function in response to endogenous factors such as
284 keratinocyte growth factor administration (30). Furthermore, mice are highly amenable to
285 genetic manipulation. Attenuation of reperfusion injury in mice genetically modified to
286 overexpress superoxide dismutase has provided further proof of the deleterious role of reactive
287 oxygen metabolites in reperfusion (48). Additionally, other transgenic mouse models have been
288 used to demonstrate the protective role of mu opioid receptor signaling (61), and the detrimental
289 effects of cytokines such as IL-17A (104) in ischemia-reperfusion injury. A large number of
290 studies have also utilized rat models. Aside from transgenic murine models, the role of a host of
291 factors has been successfully investigated in rodents, highlighting the beneficial effects of
292 epidermal growth factor (11, 59), heparin-binding epidermal growth factor (36, 50, 114), anti-
293 apoptotic signaling (86) and xanthine oxidase blockade (157) to prevent injury or improve
294 healing. Other studies using rats have shown that atenolol (31), a beta-blocker and L-arginine
295 (62), a substrate of nitric oxide biosynthesis both attenuate enteric nervous system dysfunction
296 that occurs as a result of ischemia-reperfusion injury. However, multiple studies have
297 demonstrated that the degree of injury attributed to reperfusion following mesenteric vascular
298 occlusion is variable. The degree of reperfusion injury appears to depend on the duration of
299 preceding ischemia and the segment of intestine (107, 135). Evidence of reperfusion injury has
300 been shown to occur following intervals between 30 to 60 minutes of ischemia but not when the
301 duration of ischemia is shorter or longer (107, 135). Additionally, the colon appears to be more
302 resistant to reperfusion injury compared to the small intestine and susceptibility to injury along
303 the length of the small intestine is variable with the ileum appearing to be more resistant to
304 reperfusion injury than the jejunum. The reason for these differences remains poorly understood
305 (35, 37, 107), although the lack of oxidant-producing enzymes in the colon may be one reason
306 that injury differs from the small intestine (76, 120, 121).
307 Low-Flow Ischemia
308 As noted above, in models utilizing the low-flow method of ischemia, the majority of tissue
309 damage occurs during reperfusion. In vivo feline studies have shown a direct correlation
310 between elevated xanthine oxidase levels and tissue damage as well as microvascular injury
311 (65, 68, 164, 184). However, important differences exist between species that influence their
312 susceptibility to reperfusion injury. The robust reperfusion injury identified in cats and rodents as
313 a result of mucosal xanthine oxidase-induced oxidant release is not found in other species (22).
314 In fact, humans and pigs lack mucosal xanthine oxidase at birth, and levels remain low even
315 into adulthood making the clinical relevance of the feline and rodent studies questionable (14).
316 Furthermore, no definitive clinical utility has been identified for xanthine oxidase inhibitors in
317 patients with intestinal ischemia-reperfusion injury (105, 132). Perhaps with the development of
318 the novel human model of ischemia-reperfusion injury (47) the controversy regarding the
319 contribution of xanthine oxidase to reperfusion injury will be resolved.
320 Another important species difference is the role of neutrophils in the induction and perpetuation
321 of reperfusion injury. The resident mucosal population of neutrophils, as opposed to neutrophils
322 recruited from circulation during onset of injury, appears to play a significant role in reperfusion
323 injury in rodents and cats (48, 101, 130, 153). In a low-flow cat model, acute versus chronic pre-
324 treatment with a CD18-specific monoclonal antibody enabled the investigators to assess the
325 role of resident mucosal neutrophils, with chronic administration of the antibody depleting
326 mucosal neutrophil reserves. These studies indicated that it was the resident neutrophils, rather
327 than those infiltrating from the microvasculature, that were responsible for the majority of the
328 reperfusion injury documented (101, 130, 153). However, humans have a relatively small
329 population of resident neutrophils (22), a finding that was also found to be the case in horses.
330 Interestingly, equine ischemia studies have shown very limited evidence of reperfusion injury
331 (21, 120). Together, differences in oxidant enzyme expression and neutrophil populations likely
332 affect the reliability of basic studies to translate findings to the treatment of humans with
333 ischemia-reperfusion injury.
334 Segmental Mesenteric Vascular Occlusion
335 Most of the segmental mesenteric vascular occlusion studies have been performed in pigs
336 (Table 7) although other species have been used (Tables 3, 4 and 6). This is likely because the
337 larger size of the pig facilitates temporary occlusion of smaller vessels, such as those found
338 within the mesentery, without compromising the integrity of the vessels, thereby allowing
339 reperfusion. There are other advantages to using the porcine model for the study of reperfusion
340 injury. Pigs share a similar pattern of xanthine oxidase expression as humans (22), whereas in
341 rodents, enzyme expression is 4-5 fold higher (14, 22). Additionally, pigs like humans have a
342 relatively small population of resident neutrophils unlike rodents and cats (22).
343 The larger size of pigs is also advantageous for models requiring surgical manipulation, such as
344 Thiry-Vella loops in which an isolated cannulated segment of intestine can be subject to
345 ischemic injury (19), or where research involves tissue transplantation (182) or testing advanced
346 diagnostic imaging (28). Similar surgical manipulations in rodents have been fraught with
347 complications (6).

348 Although evidence supports the use of pigs to more closely recapitulate human intestine,
349 obvious morphological differences exist which may influence the use of pigs for the study of
350 reperfusion injury (10, 45, 92, 149). In many mammalian species, including humans, the small
351 intestinal epithelium is composed of stem cells that reside within the crypt base, and four post-
352 mitotic cell types: absorptive enterocytes, goblet cells, enteroendocrine cells, and Paneth cells.
353 However, the existence of Paneth cells in pigs remains controversial and there is currently no
354 definitive evidence to prove or disprove the existence of a similarly functioning cell type (63,
355 123, 124). Paneth cells are unique in that they reside adjacent to the intestinal stem cells within
356 the small intestinal crypt base, and are thought to migrate toward the crypt base following
357 complete maturation as compared to other maturing cell lineages that migrate toward the lumen.
358 Paneth cells are thought to play an integral role in modulating stem cell self-renewal or
359 differentiation especially during times of insult (40, 57, 152), making the study of regenerative
360 events following ischemic injury potentially more complex in porcine models. However, there is
361 recent evidence that Paneth cells may actually contribute to the injury induced by ischemia-
362 reperfusion injury (72, 93, 104). Work in rodent models attribute injury within the gut, systemic
363 inflammation and remote organ injury following intestinal ischemia-reperfusion injury to Paneth
364 cell degranulation. In fact, it appears that Paneth cell depletion may attenuate ischemia-
365 reperfusion injury (104).
366 Knowledge regarding the advantages and disadvantages to each model is critical to appropriate
367 experimental design and data interpretation.
368 Future Directions
369 New Insight into Mechanisms of Cell Death
370 Recent insight into the process of cell death by apoptosis and necrosis in both rodent and
371 human models of intestinal ischemia-reperfusion injury have begun to shed light into the role of
372 these pathways in the process of injury and subsequent repair (32, 46, 115, 156, 181). An
373 important component to the combined injury attributed to ischemia and reperfusion is the direct
374 impact on the intestinal epithelial cells. Controlled cell death is critical to the maintenance of
375 normal barrier function and serves to maintain epithelial continuity along the luminal surface
376 while expelling dying cells. Under homeostatic conditions, cells mature as they gradually
377 migrate along the crypt-villus axis. As they near the luminal surface a normal process of
378 desquamation occurs that results in epithelial cell shedding. Briefly, this process is thought to be
379 driven by an interaction between the extracellular matrix and integrins and cadherins, the
380 transmembrane proteins that anchor cells to the basement membrane, and which signal cell
381 survival through pathways including focal adhesion kinase (p125fak), phosphatidylinositol 3’-
382 kinase (PI-K)/Akt and mitogen-activated protein kinases (ERK, JNK, p38 MAPK) (49, 54, 55, 84,
383 174). Changes in these surface proteins and signaling pathways occur as cells approach the
384 villus tip that signal a normal process of cell shedding into the intestinal lumen (29). During
385 ischemia and reperfusion, cell injury results in death by both apoptotic and necrotic processes
386 (75, 87, 109). Sustained tissue injury appears to be associated with inflammation that is
387 perpetuated by the presence of excessive apoptotic and necrotic cells mediated by the
388 complement system (79, 162, 178, 185). However, the exact pathophysiologic mechanisms that
389 occur are complex and incompletely understood (181). Nonetheless, inhibition of apoptosis as
390 well as the facilitated clearance of apoptotic cells appears to reduce bacterial translocation and
391 promote repair my minimizing prolonged inflammation (46, 86, 115, 181). Based on research
392 dedicated to understanding ischemia-reperfusion injury, it seems that the attenuation or
393 modulation of the inflammatory response would be critical to future therapeutic approaches
394 aimed at reducing tissue destruction. This is discussed in a few recent reviews; one that
395 addresses the contribution of oxidative stress to the pathogenesis of gastrointestinal disease
396 (13) and another that summarizes the therapeutic potential for neutrophil elastase inhibitors to
397 decrease inflammatory tissue injury (81). These reviews note that despite the large amount of
398 preclinical research dedicated to these particular approaches to modulation of inflammation, the
399 challenges to translate findings to clinical application remain. However, compared to the field of
400 gastrointestinal research, more current and novel therapeutic strategies appear to be available
401 for ischemic cardiovascular disease (81, 139). Nevertheless, little progress has been made in
402 improving clinical outcome in either field. Therefore a better understanding of reparative
403 processes may provide an alternative means to improve care.
404 Advances in Understanding Epithelial Renewal
405 Recent advances in the field of intestinal stem cell biology and regenerative medicine have
406 improved our understanding of epithelial renewal (145). The recent discovery of biomarkers to
407 clearly distinguish intestinal stem cell populations has contributed to the rapid advancement of
408 the field (8, 64, 167, 175, 176). Interestingly, rodent models of intestinal ischemia have shown
409 architectural preservation of the intestinal epithelial stem cells (56, 88, 134, 173). Additionally, the
410 ability of the intestinal epithelial stem cell compartment to expand after intestinal resection, radiation
411 injury, and doxorubicin treatment has been demonstrated in rodent models (42, 43, 85, 177).
412 Together this makes the intestinal epithelial stem cells a promising therapeutic target to hasten
413 mucosal epithelial regeneration following ischemia-reperfusion injury. It is our hope that this new
414 knowledge and advanced technology will facilitate targeted therapies to improve treatment and
415 outcome of this devastating disease.
416 Summary
417 Ischemia and reperfusion contribute to intestinal injury in a multitude of clinical
418 gastroenterological conditions. Despite this, advances in the field have remained modest. This
419 review aimed to shed light on the most commonly used animal models for the study of ischemia
420 and reperfusion to aid in interpretation of study results and perhaps influence model selection in
421 the future. For example, if investigation of reperfusion injury were of particular interest, a feline
422 model of ischemia would be helpful; whereas strangulating intestinal ischemia would be more
423 appropriately studied using a porcine mesenteric vascular occlusion model. Appropriate
424 modeling of disease states or model selection are arguably components of the failure to
425 translate bench top discoveries to clinical application.
426 Figure Legend
427 Figure 1. Clinical conditions that cause ischemic intestinal injury
428 Figure 2. Mechanism of ischemia-reperfusion injury
429 Figure 3. Epithelial loss associated with ischemic intestinal injury in porcine jejunum. As the
430 duration of ischemia increases there is progressive loss of epithelium that begins at the villus tip
431 and continues toward the crypt base. Asterisk indicates Gruenhagen’s space. Scale Bar 50 µm.
432 Figure 4. Species differences in villus microvascular architecture
433
434
435
436
437
438
439
440
441
Table 1. Comparison of Models of Ischemia
Complete Vascular Occlusion Low-Flow Segmental
SMA Ligation SMA Embolization Vascular
Occlusion
Advantages • Models • Models occlusive • Models • Models
occlusive causes of ischemia nonocclusiv occlusive
causes of • Intestinal tract not e causes of causes of
ischemia exposed or ischemia ischemia
• Ease of surgical manipulated (2, 25, • Improved • Creation of
model creation 96, 148) model of multiple loops
• Ideal for diagnostic reperfusion of varying
imaging studies injury duration of
ischemia within
a single animal
• Applied in
human
subjects (46,
47, 73-75, 115)
Disadvantages • Poor model of • Poor model of • Increase • Both arterial
chronic ischemia chronic ischemia difficulty of and venous
• Only arterial • Only arterial supply surgery supply ligated;
supply occluded occluded • Minimal most clinical
• Unreliable to • Requires advanced ischemic cases of
create surgical approach injury strangulating
consistent injury • Likely requires ischemia
(116) large animal venous supply
• High mortality • Ischemia not obstructed first
rate (116) reversible
• No reduction of prohibiting study of
blood flow to reperfusion
distal colon
(142)
• Different
segments of
intestine vary in
resistance to
ischemic injury
(37, 107, 107,
147, 147)
442
443
444
Table 2. Comparison of Large and Small Animal Models of Digestive Disease

Advantages Disadvantages
Large • Select models closely approximate • High cost
Animal human disease • More difficult to manage
(Porcine, • Approximate human size (pig) • Special housing facilities
Dog, • Possess highly developed central and • Few transgenic models available
Cat) peripheral nervous system • Time consuming
• Ease of surgical manipulation • Pathophysiological differences,
specifically with regard to
reperfusion injury (cat)
• Morphological differences in
mucosal epithelial cell populations
compared to humans
Small • Low cost • Pathophysiological differences,

Animal • Ease of maintenance specifically with regard to
(Rodent) • Ease of genetic manipulation reperfusion injury
• Rapid reproduction rate • Absence of clinical signs of human
• Number of available and well disease in some models
established models • Technically difficult to create
surgery models due to size
• Mortality rate
• Genome is ~14% smaller than
humans
445
446
447
448
449
450
451
452
453
454
455 Table 3. Experimental details of in vivo ischemia-reperfusion models in cats
Type of Experimental IR Segment of Intestine Reference

Injury
SMA Complete Occlusion Ileum (95)
SMA Complete Occlusion Jejunum + Ileum (52)
Mesenteric vascular Small intestine segment not (80)
occlusion specific
Low-flow SMA occlusion Ileum (67, 70, 82, 83, 90, 97, 100,
101, 128, 129, 136-138, 153,
164, 165, 180, 186, 187)
Low-flow SMA occlusion Jejunum + Ileum (98)
Low-flow SMA occlusion Jejunum (130)
Low-flow SMA occlusion Small intestine does not (91, 99, 184)
specify segment
456 SMA, Superior Mesenteric Artery
457
458
459
460
461
462
463
464
465 Table 4. Experimental details of in vivo ischemia-reperfusion models in dogs

Injury
SMA occlusion Ileum (5, 94, 163)
Mesenteric vascular Jejunum (27, 71)
occlusion
466
467 SMA, superior mesenteric artery
468
469 Table 5. Experimental details of in vivo ischemia-reperfusion models in mice

Injury
SMA occlusion Jejunum (12, 181)
SMA occlusion Jejunum + Ileum (185)
SMA occlusion Colon (144)
SMA occlusion* Jejunum + Ileum (104)
SMA occlusion* Jejunum (103)
SMA occlusion* Small intestine Segment not (48, 162)
specified
SMA occlusion + collateral Ileum (61)
vessel ligation*
470
471 SMA, superior mesenteric artery; Asterisk indicates transgenic animal use.
472
473
474
475 Table 6. Experimental details of in vivo ischemia-reperfusion models in rats

Injury
Mesenteric Vascular Proximal colon (73, 74)
Occlusion
SMA occlusion Proximal colon (107, 122, 179)
SMA occlusion Jejunum + Ileum (11, 107, 114, 157)
SMA occlusion Jejunum (30, 31, 60, 62, 86, 88, 131)
SMA occlusion Small intestine does not (59, 181)
specify segment
SMA + jejunal and colic Jejunum (87, 116)
artery occlusion
SMA + jejunal and colic Ileum (173)
artery occlusion
476
477 SMA, superior mesenteric artery
478
479
480
481
482
483
484 Table 7. Experimental details of in vivo ischemia-reperfusion models in pigs

Injury
SMV Occlusion Jejunum, ileum, colon (170)
Mesenteric Vascular Ileum (4, 18-20, 22-24, 28, 58, 89,
Occlusion 96, 102, 110, 117-119, 133,
154, 160, 170)
Mesenteric Vascular Jejunum + Ileum (35)
Occlusion
SMA Embolism Jejunum + Ileum (148)
SMA Embolism Small intestine + colon (96)
SMA Embolism Small Intestine segment not (2, 25)
specified
Mesenteric Vascular Jejunum (46, 47, 75, 115)
Occlusion
Mesenteric Vascular Proximal Colon (73, 74)
Occlusion
485
486 SMV, superior mesenteric vein; SMA, superior mesenteric artery
487 Table 8. Experimental details of in vivo ischemia-reperfusion models in humans

Injury
Mesenteric Vascular Jejunum (46, 47, 75, 115)
Occlusion
Mesenteric Vascular Proximal Colon (73, 74)
Occlusion
488
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Animal Models of Ischemia-Reperfusion Induced Intestinal Injury: Progress and Promise For Translational Research

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Articles in PresS. Am J Physiol Gastrointest Liver Physiol (November 20, 2014). doi:10.1152/ajpgi.00112.

1 Animal models of ischemia-reperfusion induced intestinal injury: progress and promise

2 for translational research

4 Liara M. Gonzalez,1 Adam J. Moeser,2 Anthony T. Blikslager1

11 College of Veterinary Medicine

12 1060 William Moore Drive

18 Running Title: Animal Models of Ischemic Injury

21 Research in the field of ischemia-reperfusion injury continues to be plagued by the inability to

Copyright © 2014 by the American Physiological Society.

28 resemble human pathology in clinical conditions as disparate as volvulus, shock, and

34 to aid selection of appropriate research models. In addition, a discussion of the future of

36 provide the greatest benefit from continued research of ischemia-reperfusion injury.

52 attributable to reactive oxygen metabolites, associated with activation of oxidant-producing

54 subsequent infiltration of neutrophils (Figure 2). Therefore, proposed therapeutic interventions to

76 medicine in light of recent advances will be discussed.

84 Additionally, ischemic injury is thought to contribute to the pathogenesis of neonatal necrotizing

85 enterocolitis (NEC), the most common life-threatening gastrointestinal emergency in neonatal

129 Complete Vascular Occlusion

154 injury histologically.

161 was more resistant to ischemia in rats (107).

173 whether the mechanism of obstruction is occlusive or nonocclusive. Occlusive intestinal

179 flow model likely better recapitulate this form of injury.

180 Low-Flow Ischemia

195 Segmental Mesenteric Vascular Occlusion

209 likely affect the level of epithelial sloughing.

216 from ischemia-reperfusion injury cannot be induced in these clinical studies.

235 function (15).

236 Mechanisms of Reperfusion Injury

260 include potent proteases such as elastase, myeloperoxidase, protease-3 and

269 CD11/CD18 monoclonal antibody, targeting neutrophil adhesion, significantly reduced

271 Model Dependent Reperfusion Injury

277 Complete Vascular Occlusion

285 genetic manipulation. Attenuation of reperfusion injury in mice genetically modified to

307 Low-Flow Ischemia

319 contribution of xanthine oxidase to reperfusion injury will be resolved.

333 ischemia-reperfusion injury.

334 Segmental Mesenteric Vascular Occlusion

347 complications (6).

365 reperfusion injury (104).

367 experimental design and data interpretation.

368 Future Directions

369 New Insight into Mechanisms of Cell Death

392 dedicated to understanding ischemia-reperfusion injury, it seems that the attenuation or

403 processes may provide an alternative means to improve care.

404 Advances in Understanding Epithelial Renewal

415 outcome of this devastating disease.

417 Ischemia and reperfusion contribute to intestinal injury in a multitude of clinical

425 translate bench top discoveries to clinical application.

426 Figure Legend

427 Figure 1. Clinical conditions that cause ischemic intestinal injury

428 Figure 2. Mechanism of ischemia-reperfusion injury

432 Figure 4. Species differences in villus microvascular architecture

Table 2. Comparison of Large and Small Animal Models of Digestive Disease

Small • Low cost • Pathophysiological differences,

Type of Experimental IR Segment of Intestine Reference

SMA Complete Occlusion Jejunum + Ileum (52)

Mesenteric vascular Small intestine segment not (80)

101, 128, 129, 136-138, 153,

164, 165, 180, 186, 187)

Low-flow SMA occlusion Jejunum + Ileum (98)

Low-flow SMA occlusion Jejunum (130)

456 SMA, Superior Mesenteric Artery

Type of Experimental IR Segment of Intestine Reference