DETECTION OF APOPTOTIC CELLS IN INTESTINE FROM HORSES WITH AND WITHOUT

GASTROINTESTINAL DISEASE by

Emma Lucy Rowe BSc.BVMS (Hons.) Thesis submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Master of Science In Veterinary Medical Science APPROVED: ___________________________________ Nathaniel A. White II, Chairman ____________________________ _____________________________ Virginia Buechner-Maxwell John L. Robertson April, 2003 Leesburg, Virginia Key words: horse, intestine, gastrointestinal disease, apoptosis Copyright 2003, Emma Lucy Rowe

DETECTION OF APOPTOTIS CELLS IN INTESTINE FROM HORSES WITH AND WITHOUT GASTROINTESTINAL DISEASE by Emma L. Rowe BSc.BVMS (Hons.) Committee Chairman: Nathaniel A. White II, DVM, MS Diplomate American College of Veterinary Surgeons Theodora Ayer Randolph Professor of Surgery Veterinary Medical Sciences (ABSTRACT) A study was performed to identify apoptotic cells in the equine intestine and to determine

if the occurrence of apoptosis is affected by gastrointestinal disease and tissue layer of intestine.

Samples of intestine were collected from 38 horses that underwent surgery or were humanely

destroyed for small or large bowel obstruction, strangulation or distension. Samples were also

taken from 9 horses which were humanely euthanized for reasons other than gastrointestinal

disease or systemic disease. Specimens were collected at surgery from intestine involved in the

primary lesion, distant to the primary lesion, or at necropsy from several sites including the

primary lesion. Tissues were fixed, serially sectioned and stained with hematoxylin and eosin

(H&E) and for apoptosis by the terminal deoxynucleotidyl transferase mediated dUTP nick end

labeling (TUNEL) technique. The number of apoptotic cells per high power field were counted

in the mucosa, circular muscle, longitudinal muscle and serosa for each sample of intestine.

Apoptotic staining nuclei were seen in all layers of intestine. An increased number of apoptotic

cells were found in the circular muscle of the intestine from horses with simple obstruction.

Intestine distant from the primary strangulating lesion had higher numbers of apoptotic cells than

intestine distant from a simple obstruction lesion or intestine taken at the site of a strangulating

or simple obstructive lesion. Intestine from horses with obstructing or strangulating lesions in

the small intestine and large colon has increased numbers of apoptotic cells. Further

investigation is required to determine whether increased apoptosis affects intestinal function.

iv

GRANT INFORMATION

This research was funded by the Patricia Bonsall Stuart Equine Research Award

v

DEDICATED TO

Christopher and Anne Rowe

My mother and father who have provided never-ending support and friendship throughout my

life and who gave me the opportunity to pursue my love of veterinary science.

vi

ACKNOWLEDGEMENTS

Nathaniel A. White- my research advisor and mentor, for his support, patience and enthusiasm

not only involving this project but in all aspects of my training.

Virginia Buechner-Maxwell- for her enthusiasm, guidance, knowledge and friendship.

Daniel L. Ward- who performed the statistical analysis of data for his skills, knowledge and

patience.

John L. Roberston- for his knowledge and insight into the pathology of gastrointestinal disease.

Patsy Dillon-Long- who provided my technical support in the laboratory.

vii

TABLE OF CONTENTS

1. LIST OF FIGURES…………………………………………………………ix

2. LIST OF TABLES…………………………………………………….........xiii

3. LIST OF ABBREVIATIONS………………………………………………xiv

4. INTRODUCTION…………………………………………………….........1

5. LITERATURE REVIEW

A. Gastrointestinal Disease in the equine

1. Importance………………………………………….3

2. Pathophysiology of gastrointestinal disease………..4

3. Ischemia……………………………………….........9

4. Reperfusion…………………………………………11

5. Intestinal Distension………………………………..18

6. Inflammation of the peritoneum..…………………..20

B. APOPTOSIS

1. Apoptosis-programmed cell death..…………………..21

2. Immunohistochemical staining to detect apoptosis…...27

3. Apoptosis and the intestine……………………………29

4. Apoptosis and ischemia/reperfusion injury…………...33

5. Role of nitric oxide in apoptosis………………………35

6. Apoptosis and inflammation...…………………………37

7. Apoptosis and non-steroidal anti- inflammatory drugs...38

C. LITERATURE CITED…………………………………………………41

viii

6. DETECTION OF APOPTOTIC CELLS IN HORSES WITH AND WITHOUT

GASTROINTESTINAL DISEASE………………………………………………….66

A. Abstract…………………………………………………….67

B. Introduction………………………………………………...68

C. Materials and methods……………………………………..69

D. Results……………………………………………………..72

E. Discussion………………………………………………….74

F. References………………………………………………….81

G. List of figures………………………………………………86

7. CONCLUSION..…………………………………………………………………98

8. VITA……………………………………………………………………………..99

ix

LIST OF FIGURES Page

Figure legends: 88

Figure 1: Photomicrograph of the mucosa in the small intestine from

a horse with gastrointestinal disease. All dark staining nuclei represent

apoptotic cells. Healthy or necrotic cells did not stain. Diaminobenzidine

(DAB) stain and methyl green counterstain; bar equals 50µ.

Figure 2: Photomicrograph of the mucosa in the small intestine 88

from a horse in the control group. No apoptotic nuclei are present.

Diaminobenzidine (DAB) stain and methyl green counter stain;

bar equals 50µ.

Figure 3: Photomicrograph of the circular muscle in the small 89

intestine from a horse with gastrointestinal disease. All dark

staining nuclei represent apoptotic cells. Healthy or necrotic cells

did not stain. Diaminobenzidine (DAB) stain and methyl green

counterstain; bar equals 50µ.

Figure 4: Photomicrograph of the circular muscle in the small 89

intestine from a horse in the control group. No apoptotic nuclei

are present. Diaminobenzidine (DAB) stain and methyl green counter

stain; bar equals 50µ.

x

Page

Figure 5: Photomicrograph of a myenteric plexus in the large colon. 90

All dark staining nuclei represent apoptotic cells. Healthy cells or

necrotic cells did not stain. Neurons, glial cells and Schwann cells

were apoptotic in this myenteric plexus. Diaminobenzidine (DAB) stain

and methyl green counterstain; bar equals 50µ.

Figure 6: Photomicrograph of the serosa from the large colon. 90

All dark staining nuclei are apoptotic mesothelial cells on the

surface of the serosa. Healthy cells or necrotic cells did not stain.

Diaminobenzidine (DAB) stain and methyl green counterstain;

bar equals 50µ.

Figure 7: Graph of the mean numbers of apoptotic cells in the intestine 91

from horses in all 4 categories- simple obstruction, strangulation,

musculoskeletal, control. A significant increase in apoptotic cells

was present in the simple obstruction category (P< 0.0088) which

is indicated by the star (*). Columns represent the geometric mean

number of apoptotic cells while error bars represent the 95% confidence

intervals.

xi

Page

Figure 8: Graph of the mean number of apoptotic cells in intestine 92

taken from the primary lesion or at a distant site in horses with simple

obstruction and strangulation. A significant increase in apoptotic cells

was present at a distant site to a strangulation lesion (p< 0.0186)

which is indicated by the star (*). Columns represent the geometric

mean number of apoptotic cells while error bars represent the 95%

confidence intervals.

Figure 9: Graph of the mean number of apoptotic cells in the four 93

tissue layers from horse with simple obstruction or strangulation.

Columns represent the geometric mean number of apoptotic cells

while error bars represent the 95% confidence intervals.

Figure 10: Photomicrograph of thymus from mouse not treated with 94

dexamethasone. All dark staining nuclei are apoptotic thymic cells.

Healthy cells or necrotic cells did not stain. Diaminobenzidine (DAB)

stain and methyl green counterstain; bar equals 50µ.

Figure 11: Photomicrograph of thymus from a mouse treated with 94

Dexamethasone. All dark staining nuclei are apoptotic thymic cells.

Healthy cells or necrotic cells did not stain. Diaminobenzidine (DAB)

stain and methyl green counterstain; bar equals 50µ.

xii

Page

Figure 12: Photomicrographs of serial sections of cir cular muscle from 95

the jejunum of a horse with a strangulating lesion. The one on the left

is stained with Diaminobenzidine (DAB) and methyl green counterstain

and the one on the right with hematoxylin and eosin (H&E).

xiii

LIST OF TABLES

Page

Table 1: The ANOVA for split-plot with colic, intestine, and tissue as factors. 96

Results of Type 3 Tests of Fixed Effects.

Table 2: The ANOVA for split-plot with colic, intestine, and tissue as factors. 96

Results of The Mixed Procedure Tests of Effect Slices.

Table 3: The ANOVA for split-plot with affected (code for intestine at primary 97

site of the lesion compared with intestine at a distant site), colic, intestine, and

tissue as factors. Results of the Mixed Procedure Tests of Effect Slices.

xiv

LIST OF ABBREVIATIONS

ATP = adenosine triphosphate

O2-. = superoxide free radicals

OH-. = hydroxyl radical

NADPH = nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system

TNF-a = tumor necrosis factor alpha

IL-1ß = interleukin-1-beta

IL-6 = interleukin-6

Ca++ = calcium ion

FADD = fas associated death domain

TNFR1 = tumor necrosis factor receptor

TRADD = TNFR1 associated death domain

RIP = receptor interacting protein

Apaf-1 = apoptosis protease activating factor

ADP = adenosine diphosphate

DNA = deoxyribonucleic acid

PT = mitochondrial permeability transition

NO = nitric oxide

H202 = hydrogen peroxide

iNOS = inducible NO synthase

TUNEL = terminal deoxynucleotide transferase mediated dUTP nick end labeling

3-OH = 3-hydroxy

PI = propidium iodide

xv

LPS = lipopolysaccharide

IGF-1 = insulin like growth factor

EMAP-11 = endothelial monocyte-activating polypeptide II

Hg2+ = mercury ion

NSAID = non-steroidal anti- inflammatory drugs

PG = prostaglandin

PGE2 = prostaglandin E2

ISNT = in situ nick translation

H & E = hematoxylin and eosin

PBS = phosphate buffered saline

TdT = terminal deoxynucleotidyl transferase

DAB = diaminobenzidine

1

INTRODUCTION

Gastrointestinal disease is the cause of high morbidity and mortality in the horse.

Experimental evidence suggests that one reason for morbidity and mortality in horses with

obstructive diseases of the gastrointestinal tract is reperfusion injury. 1,2 In the horse

experimental ischemia has been shown to cause mucosal and serosal damage, which is

exacerbated during reperfusion 2,3. These studies have been performed with and without

treatments directed at interrupting the inflammatory pathways associated with reperfusion injury

and have yielded conflicting results. 4 5 Intestinal ischemia-reperfusion injury has been largely

attributed to cellular necrosis. Apoptosis, a distinct form of cell death, has recently been

recognized as having an important role in both ischemia and reperfusion injury. 6,7 Apoptosis is

a natural physiologic mechanism to allow death of unwanted cells with minimal tissue

inflammation. Apoptosis has an important role in embryogenesis, tissue homeostasis,

lymphocyte development and function and tumor regression. 8

In the gastrointestinal tract apoptosis can be associated with the maintenance of

homeostasis and shedding of cells from the villous tips in the small intestine, or with

pathological states such as the development of intestinal adenocarcinomas, the pathogenesis of

inflammatory bowel disease and radiation induced gut damage. 9 The apoptotic response appears

to be activated by membrane changes in the mitochondria with subsequent transcription of

specific genes which affect individual cells of a specific cell type 10. Apoptosis has been

observed following experimental ischemia-reperfusion injury to the brain, heart, adrenals, liver,

and kidneys of other species. 7 Apoptosis has found to be important an important source of

inflammation in the kidney 11,12 and muscle injury 6. In human medicine non-steroidal anti-

2

inflammatory drugs have been found to induce apoptosis in the colon with sufficient magnitude

to disrupt the epithelial barrier causing inflammation. 13

Apoptosis is thus part of the homeostatic processes of the body but if it is present in

excess in certain cell types or if it is delayed in inflammatory cells, this form of cell death can

create inflammation. This inflammation has been documented with increased apoptosis in

ischemia reperfusion injury of the muscle and kidney and therefore may be important in

ischemia-reperfusion injury in the equine intestine. To date experimental and clinical

investigations into therapeutic intervention in reperfusion injury have been at time conflicting

and unrewarding. Apoptosis can be controlled and thus may have important therapeutic

interventions in certain disease states including ischemia reperfusion. To our knowledge

apoptosis has not been investigated following ischemia-reperfusion of the equine intestine.

Apoptosis can be examined using the TUNEL ( terminal deoxynucleotide transferase

mediated dUTP nick end labeling ) technique. This technique distinguishes apoptotic from

necrotic cells by specifically detecting DNA cleavage associated with apoptosis. This technique

has been shown to be effective in the detection of apoptosis in the formalin fixed samples of

human large intestine. 14

The objectives of this study were to 1) identify apoptosis in the equine intestine using the

TUNEL method, 2) to identify the type(s) of cells undergoing apoptosis, 3) determine whether

the occurrence of apoptotic cells was affected by gastroint estinal disease and tissue layer of the

intestine. We hypothesized that equine intestine directly or indirectly affected by naturally

occurring simple or strangulation obstruction would have increased numbers of apoptotic cells.

3

LITERATURE REVIEW

GASTROINTESTINAL DISEASE IN THE EQUINE IMPORTANCE

Acute abdominal pain (colic) in the horses is one of the most frequent problems

encountered by veterinarians in large animal practice 15 and these diseases causing colic have a

great economic impact due to the high case fatality rate 16. The annual national incidence of

colic has been estimated to be 4.2 colic events/100 horses per year with a case fatality rate 11%.

The annual cost of colic in the United States has been estimated to be $115,300,000 16.

Strangulating obstruction of the small intestine and large colon is a frequent cause of this acute

abdominal disease requiring surgery. 17-19 In a study with 2,385 horses referred to 14 veterinary

teaching hospitals with signs of abdominal pain, 18.4% had strangulating intestinal obstruction,

with overall mortality of 79.9% 19. Recent reports have documented improved survival with

surgery for strangulation of the large and small intestine. However, intestinal strangulation still

has the highest case fatality of diseases that cause colic, and it is associated with the highest rate

of post-operative complications. In a recent study the long-term survival rates of horses

recovered from anesthesia after surgery for a small intestinal lesion at 7 months and greater than

12 months following surgery, were 75% and 68% respectively. 20 Original reports suggested

survival rates for horses with a strangulating obstructing lesion of the large colon are lower than

for small intestinal strangulation with recovery rates expected between 21-42% after surgery.

21,22 However, recent short term survival rates of 83% and 93% have been reported for survival

after surgery for large colon volvulus. 23,24

4

PATHOPHYSIOLOGY OF GASTROINTESTINAL DISEASE

Any interference of the aboral flow of ingesta through the gastrointestinal tract has the

potential to cause bowel distention and severe pain. 25 There are many conditions which can

result in an acute abdomen and these can be grouped into categories: 1. primary gastrointestinal

tympany, 2. simple obstruction, 3. strangulation obstruction, 4. nonstrangulating infarction, and

5. inflammatory (enteritis and peritonitis). Primary gastrointestinal tympany is the result of

accumulation of gas within the lumen of the intestine or stomach. 25 The gas accumulation

causes distention and abdominal pain due to excessive stretch of enteric nerves. 25

Simple obstruction occurs commonly in both the small and large intestine. It occurs

when there is occlusion of the intestinal lumen by intraluminal obturation, an intramural mass, or

by external compression. 25 Simple obstruction of the small intestine can be caused by lesions

within the bowel wall that constrict the lumen, obstruction of the lumen by an intraluminal food

mass or foreign body, or by external compression by a mass such as an adhesion, abscess or

neoplasm. 26, 27, 28, 29 Failure of the small intestinal musculature to contract which may occur

with a physical blockage, surgical manipulation of the bowel, or which may be secondary to

peritonitis will also result in an obstruction. 30, 31,32 Functional obstructions may also be

produced by spastic contractions which can occur during spasmodic colic. 33

When small intestinal occlusion then prevents the aboral movement of ingesta, fluid and

gas, there is subsequent increase in osmolality of the intestinal content and water is then drawn

into the lumen from the mucosal vasculature. 34 If the distention worsens the increased pressure

compromises venous drainage and the mucosa will become edematous and congested. If the

obstruction is present for a prolonged period of time blood is shunted away from the mucosa and

serosa resulting in ischemia. 25, 35

5

The two most common cause of simple obstruction in the large colon are impactions and

nonstrangulating displacements. 26,33 Large colon impactions cause approximately 30% of all

colics diagnosed at referral centers. 36, 37 Large colon impactions will often occur at sites where

the lumen diameter is narrowed, for example at the pelvic flexure or just proximal to the

transverse colon in the right dorsal colon. 38 The mechanism of these impactions is not

completely understood, but poor quality feed, old age, debilitation, poor dentition, parasites,

overeating, inadequate water intake, motility disorders, and limited exercise have all been

reported as possible causes of this condition. 38 Impactions develop slowly over time (several

days to weeks) unless there is an enterolith or foreign body causing an acute obstruction. There

is progressive distention of the large colon proximal to the obstruction, but in the early stages

some ingesta and gas can still pass the mass. 38 If the obstruction becomes complete, ingesta

and gas accumulate more rapidly and marked distention occurs oral to the obstruction. The

distention may become so great that it exerts pressure against the diaphragm and vena cava

which results in impaired pulmonary function and venous return. This will ultimately cause

hypovolemic and hypoxemic shock. 38 Impactions also occur in the cecum. Prolonged

distention of the cecum or large colon may impair mucosal perfusion resulting in the mucosa

becoming devitalized and eventually rupturing, which is usually fatal. 38 Total blockages of the

large intestinal lumen with an enterolith or foreign body are usually located at the pelvic flexure

or transverse colon. Stretching of the mass over the bowel wall will cause pain and impairment

of venous drainage. If there is severe ischemia the mucosa will degenerate from pressure

necrosis. 38 The left ventral and dorsal colons are freely movable in the abdomen and can

become displaced or twisted. The cause of the abnormal positioning is not known but

predisposing factors may include the horse rolling from abdominal pain, motility changes with

6

stasis and hypermotility of segments of the large colon, and excessive and rapid gas production

with rotation of the colon due to lifting of the gas-filled segments. 38 The large colon may also

rotate 900 or 1800 without causing vascular occlusion but obstructing the passage of gas and

ingesta to cause a simple obstruction. 39 Lymphatic drainage distal to a non-strangulating

displacement results in edema of the bowel wall and mesentery eventually leading to intestinal

compromise. 39

Strangulating obstruction of the intestines involves simultaneous vascular occlusion and

luminal obstruction. 40 This type of obstruction can be the result of incarceration of bowel in an

intra-abdominal (mesenteric rent, epiploic foramen) or extra-abdominal (scrotum, diaphragm)

location or from the twisting of the mesentery associated with volvulus. 40 Strangulating

obstruction of the colon is usually the result of torsion along its long axis. The rotation has to be

greater than 1800 to produce venous occlusion and more than 2700 to obstruct arterial flow. 33

There are two types strangulating obstruction; hemorrhagic strangulating obstruction and

ischemic strangulating obstruction. In the horse most cases of strangulating obstruction are

hemorrhagic. 40 During hemorrhagic strangulating obstruction there is venous occlusion with

continued arterial flow resulting in congestion, hemorrhage, and edema of the bowel wall. 3

With the venous occlusion there is an increase in the hydrostatic pressure and the microvascular

permeability also increases in response to inflammatory mediators. 41 The other type of

strangulation, ischemic strangulation involves simultaneous arterial and venous occlusion. 3 In

this case there is no increase in hydrostatic pressure because the arterial supply is also constricted

and there is less hemorrhage and edema compared with the hemorrhagic form. During this form

of ischemia the serosa becomes blanched and cyanotic. 41

7

During hemorrhagic or ischemic strangulating obstruction in the small intestine fluid

sequesters in the subepithelial space causing the epithelium to loosen from the underlying

basement membrane at the villus tip. 42 The epithelial cells slough in sheets with the cells

attached to each other. This separation continues along the villus into the crypts. Loss of the

entire villus epithelium occurs after 3 hours of experimentally induced total arteriovenous

occlusion. 40 After 4-5 hours of occlusion there is complete necrosis of the mucosal epithelium

extending to the base of the crypts. 42 The degeneration progresses so that after 6-7 hours

necrosis has extended beyond the muscular layers. 40

Complete ischemia of the large colon induces cellular necrosis of groups of surface

epithelial cells which loosen from their basilar attachments and from the neighboring cells. 43

Compared to the small intestinal mucosa, cell degeneration becomes irreversible before epithelial

sloughing occurs. 44 Arteriovenous occlusion and transmural compression causing complete

ischemia for a period of 3-4 hours results in irreversible damage to the mucosa of the large colon.

43 Sloughing of 97% of the surface epithelium and at least 50% of the glandular epithelium is

associated with death in the naturally acquired large colon volvulus. 22

A nonstrangulating infarction results from reduced blood flow to a segment of bowel

caused by an intravascular occlusion. 45-47 In the horse the occlusion is usually due to a thrombus

or embolus and occurs most frequently in the ileocecocolic branch of the cranial mesenteric

artery. 25 Damage and thrombosis of these vessels is caused by migration of the Strongylus

vulgaris larvae. 33,48,49 Reduced blood flow and tissue perfusion result from verminous

thrombosis which leads to focal or multifocal infarction of the ileum, cecum or colon. Emboli

can break from the thrombus and can lodge in the peripheral branches of the ileocecocolic artery.

39,50 In some cases the emboli or thrombi are small and the resulting ischemia is only transient as

8

the circulation is re-established by collateral circulation. 27, 51 However, when the ischemic lesion

is extensive and cannot be reached by the collateral circulation an infarct develops, and the

affected intestinal segment becomes necrotic. 25

Inflammatory conditions of the bowel or enteritis can be caused by bacterial or viral

infections, or may result from parasites, trauma (surgical) or toxins. The mucosa is the most

susceptible layer of the intestine to this type of injury as it is the most metabolically active. 25 In

the small intestine inflammation extending to the submucosa and into the muscularis causes ileus

with the accompanying intestinal distention and gastric reflux. 52 Conversely in the large colon

any increase in water delivered by the small intestine, or any alteration in the absorptive

capabilities of their mucosa due to enteritis, results in the accumulation of large amounts of water

in the lumen and subsequent diarrhea. 53 Very large quantities of water and electrolytes may be

lost from the body when oral or aboral loss occurs, resulting in dehydration and hypovolemia.

Endotoxemia and/or septicemia may also occur when damage to the mucosa is severe

enough to allow bacteria and endotoxin to pass through the lamina propria into the circulating

blood. The development of the endotoxemia is enhanced when the endotoxin and bacteria leaks

into the peritoneal cavity through the bowel wall. 25 Inflammation of the peritoneum is usually

caused by ischemia or bacterial contamination through part of the gastrointestinal tract which is

devitalized, perforated or ruptured. Surgical procedures that involve entry into the abdomen or

accidental perforation of the abdominal wall may also cause peritonitis. 54 Extension of enteric

infections into the peritoneal cavity is a common cause of peritonitis in the horse where as blood-

borne infections are rare. 54

9

ISCHEMIA

Ischemia is a deficiency in blood flow to an organ or tissue. 55 A functional constric tion

or mechanical obstruction of blood vessels supplying intestines can lead to ischemia causing

inadequate tissue perfusion and oxygenation. 5 Distention of the intestines also causes ischemia-

this occurs in obstruction of the small intestine, resulting in severe distention proximal to the

obstruction, or distention of the cecum due to dysfunction. As the intraluminal pressure

increases, the capillaries and venules are compressed and the subsequent decrease in perfusion

and increased accumulation of interstitial fluid causes further vascular compromise. 2

Cell viability is dependant on the maintenance of an adequate blood flow for the delivery

of oxygen and the removal of waste products. 5 Cells have the capability to increase oxygen

extraction if there is a substantial decrease in blood flow and they continue to function with

energy reserves. 5 However, if the blood flow is decreased below the amount necessary to

maintain cellular viability or if the cellular metabolic rate and oxygen consumption increase, the

cell metabolism is compromised and there is change in both cellular structure and function. 5

Different cells in the intestine have different metabolic demands and susceptibility to ischemia.

As an example mucosal epithelial cells of the intestine are highly energy dependant and if there

is a reduction in blood supply and decreased oxygenation of the tissues, rapid injury and death

occur. 5

When ischemia is continuous, the cell metabolism goes through several alterations which

eventually lead to cell necrosis. In the cell, oxygen is consumed in the mitochondria where it is

reduced to water by the electron transport chain 56,57 which is coupled with the synthesis of

adenosine triphosphate (ATP). When the oxygen supply to a tissue decreases, both cellular

oxygen stores and stored energy are decreased 5 initiating anaerobic glycolysis. Anaerobic

10

gycolysis results in an accumulation of lactic acid within the cell causing an intracellular

acidosis, which inhibits the ATP production. 58 The synthesis of ATP is relatively inefficient

during anaerobic glycolysis and can result in increases in lactate and proton concentrations in the

bloodstream which have been associated with a poor prognosis in horses with ischemia-

reperfusion injury of the large colon. 59 The cell membrane and cytosol remain intact until the

energy dependant pumps, which maintain normal ionic balance within the cell, fail causing an

influx of calcium into the cell. 57 The accumulation of calcium within the cell activates proteases

which cause cell membrane damage and nuclear clumping. Calcium uptake within the

mitochondria is increased and this inhibits oxidative phosphorylation. 60 As the cell membranes

deteriorate intracellular homeostasis fails, resulting in swelling of the cell and the leakage of

cellular elements such as enzymes and electrolytes into the interstitium. 5 If the ischemia

continues hydrogen ions increase and the cellular proteins and structures are permanently altered.

57 Eventually proteases, lipases, and other degradative enzymes initiate autolytic destruction of

organelles resulting in irreversible injury and death.

Cell structure may change within 5 minutes of ischemia, with changes in the

mitochondria including swelling and disorganization of the cristae. 61 These mitochondrial

changes occur before the cytoplasmic and membrane changes which occur in the first 30 minutes

by activation of phospholipases, cytokine production and accumulation of arachidonic acid.

Microscopic changes are evident following 30 minutes of ischemia when the mucosal epithelial

cells and serosal mesothelial cells separate from their basement membranes creating a space at

the tip of the villus in the small intestine called Grunehagan’s space. 42 The separation appears to

be caused by water accumulation in the subepithelial space. Alterations in the basement are

11

caused by activation of metalloproteinases. 55 Concurrent with the sloughing of the mucosal cells

toward the crypts, cell degeneration progresses in the capillary tuft and the lamina propria.

The change in the large colon is similar, but the sloughing of epithelial cells off the

surface of the mucosa is slower as it proceeds to the crypts. The serosa also reacts with the

mesothelial cells lifting off the basement membrane before there is visible cell membrane or

cytoplasmic change. 55 There is minimal change in the architecture of the supporting tissues in

the mucosa or the serosa for the first 60 minutes of total ischemia. 55 After 180 minutes of

ischemia the lamina propria and mucosal vascular tuft has lost its architecture and the tissue is

necrotic with lack of definition and cell structure. 55

REPERFUSION

If oxygen and energy return to the tissue just prior to membrane and organelle failure the

cells can still survive. However if the blood flow returns when the cells are still viable, a cascade

of events is set in motion by the delivery of oxygen to the previously ischemic tissue. 5 62 This

resuscitation can be destructive and the resultant injury is called reperfusion injury. Reperfusion

injury relies on renewed oxygen in the tissue with participation of endothelial cells and afferent

receptors to create the inflammatory response which ensues. 55 If cells have been damaged the

reperfusion injury can result in inflammation, apoptosis and necrosis, thereby delaying healing.

Reperfusion injury is observed more commonly following partial or low flow ischemia 63 but can

be seen following complete ischemia unless severe cell injury does not allow the cells to respond

to reperfusion. 63, 64

Initially during reperfusion there is a reactive hyperemia due to increased bloodflow. 65-68

The response of the blood vessels’ smooth muscle to these substances depends on the particular

agent, the smooth muscle condition and whether there is an intact or functional endothelium. 69

12

The bloodflow and tissue perfusion is regulated by the endothelium which alters the smooth

muscle tone by releasing a balance of endothelium derived vasoconstrictors and vasodilators. 70

Vasodilatory mechanisms include the stimulation of the muscarinic receptors on intact

endothelial cells by acetylcholine which results in the release of nitric oxide from the

endothelium which causes vasodilation. 71 The endothelial cells synthesize nitric oxide from L-

arginine via NO synthase and this synthesis can be blocked by NO synthase inhibitors. 72-74

Prostacyclin is released from the endothelium and contributes a little to the endothelium

dependent relaxation in blood vessels and also inhibits platelet aggregation. 75 The endothelium

also releases vasoconstrictive substances including endothelin and eicosanoids. 76 During

physiologic and pathologic conditions the large colon can release other agents that cause arterial

constriction including angiotensin, histamine, serotonin and norepinephrine. Serotonin and

histamine stimulate smooth muscle contraction via the release of endothelium-derived substances

such as endothelin and therefore require a functional endothelium to work. 77

Damage to the endothelium of the arteries and veins has been thought to result in loss of

endothelium derived vasodilatory agents such as nitric oxide and thus an increased sensitivity to

vasoconstrictive agents. The contractile force in the vessels of the large colon are the strongest

with intact endothelium, less strong with a nitric oxide synthase inhibitor (Nw-Nitro-L-Arginine

methyl esther, L-NAME) and the least strong with a denuded endothelium. Contractile agents

such as thromboxane and vasopressin usually cause a minor response in the endothelium when

the vessel is damaged but their effect is enhanced if the endothelium has been damaged. 78 If the

vessels of the large colon are damaged endothelium derived contractile agents may be lost which

may contribute to the decreased colonic bloodflow and the pooling of blood in the venous

circulation. 77

13

The hyperemic response during immediate reperfusion in the large colon is equally

distributed from the mucosa to the seromuscular layer. 79,80 Mechanical or biochemical response

to reperfusion exacerbates the mucosal edema, hemorrhage and necrosis. 81 Enteric

neuropeptides, which increases during reperfusion of the large colon including calcitonin gene-

related peptide and substance P may be responsible for the vascular smooth muscle relaxation. 82

At the cellular level reperfusion initiates immediate formation of oxygen metabolites,

which occurs immediately on reperfusion. 83 Free radicals have an unpaired electron in their

outer orbit and are formed during chain reactions when electrons are released as part of

oxidation-reduction reactions within. 84 When there is a decrease in blood flow severe enough to

cause cellular hypoxia, the oxidative phosphorylation is uncoupled and there is a decrease in

ATP. 84 The electrons formed during the uncoupled reactions leak from the mitochondria and

react with oxygen when blood flow returns during reperfusion. There is a rapid generation of

oxygen free radicals during reperfusion, such as the superoxide free radicals (02-). 84, 5 Oxygen

free radicals are produced during normal cellular metabolism and can damage cells, but anti-

oxidants normally present in cells protect against injury. 85 The endogenous anti-oxidants are

both enzymatic (superoxide dismutase, catalase and glutathione peroxidase) and nonenzymatic

(alpha tocopherol, ascorbate and betacarotene). 85 These protective anti-oxidants are

overwhelmed during reperfusion injury allowing the free radicals and their metabolites to cause

injury to the cells. 5

Free radicals are also produced from intracellular reactions- the most frequently reported

reaction involves xanthine oxidase, an enzyme present in the cytosol of most animal cells.

Xanthine oxidase is retained as xanthine dehydrogenase which is linked to nicotinamide

dinucleotide. During ischemia the ATP decreases causing the cell to metabolize existing stores

14

which results in the accumulation of hypoxanthine. As the Na+K+ATPase pump fails calcium

accumulates within the cell and the high intracellular calcium concentration and calpain converts

xanthine dehydrogenase to xanthine oxidase. 86,87 The xanthine oxidase catalyzes the conversion

of hypoxanthine to xanthine which when oxygen is present results in superoxide production. 5

The superoxide radical is normally converted into hydrogen peroxide by endogenous superoxide

dismutase, which is then further degraded to water. 85 However, during reperfusion, the

endogenous mechanisms are overwhelmed and hydroxyl radicals (OH-) are formed through the

iron-dependant Haber-Weis reaction. Hydroxyl radicals are active against cell membranes and

cause further perturbations in cell metabolism. 5 The importance of the xanthine oxidase in

postischemic injury has been demonstrated with pretreatment of animals with superoxide

dismutase or allopurinol. 88-90 Detectable levels of xanthine oxidase/xanthine dehydrogenase

have been measured in the horses following one hour of small intestinal ischemia causing

conversion of xanthine dehydrogenase to xanthine oxidase. 91 However, Vatistas et al. did not

find conversion of this enzyme system during reperfusion after low-flow ischemia. 92 The large

colon of horses or other species has low levels of xanthine oxidase and increased levels of

aldehyde oxidase another enzyme which generates free radicals. 93 The cells in the lamina

propria of the feline large colon have been shown to have xanthine oxidase activity 94 and the

subepithelial cells can also produce free radicals. 95 Oxygen free radicals damage the tissue by

causing lipid peroxidation of cell membranes and intracellular organelles with subsequent

production of phospholipid mediators due to the activation of phospholipaseA2. 5

Malondialdehyde and conjugated dienes, which are byproducts of cell membrane lipid

peroxidation, have been detected in intestinal reperfusion injury in cats, rats and dogs. 96, 97,98

15

One of the primary initiators of reperfusion is the endothelial cell. 55 Endothelial cells are

metabolically active and they have specialized functions including the regulation of blood

pressure, vascular permeability, vascular tone, inflammatory cell adhesion, coagulation and

platelet aggregation. 99 During reperfusion injury when the endothelial cells are activated or

injured they swell, and there is disruption of the tight cell junctions which results in extravasation

of erythrocytes and neutrophils. 1 Increased permeability allows fluid and protein move into the

interstitium causing an increased interstitial pressure with capillary collapse. 1, 55 The vascular

collapse and endothelial swelling decreases the blood flow to the tissue causing further ischemia.

When stimulated by oxygen radicals or platelets, endothelial cells generate cytokines, which

attract and activate neutrophils during reperfusion injury. 55, 5 Studies in the feline intestine

have demonstrated that the neutrophil infiltration during reperfusion can be prevented with

pretreatment with allopurinol (inhibits xanthine oxidase) 100, superoxide dismutase (promotes the

dismutation of 02- to H2O2) 100, catalase (catalyzes conversion of H2O2 to H2O) 101 and

deferoxamine (inhibits conversion of 02- to OH-) 101. Neutrophil migration and accumulation in

the interstitium can cause a second phase of reperfusion injury with a decrease in reduced

glutathione and superoxide dismutase in the feline intestine. 100 Pretreatment with allopurinol or

administration of superoxide dismutase prevents the neutrophil influx and the decrease in

reduced glutathione levels 100 which suggests a relationship between the oxygen free radicals and

neutrophil migration as a cause of mucosal damage. This neutrophil response is seen in the

intestine affected by ischemia and also in the bowel adjacent or at a distant site to it. 102,103

When neutrophils are activated they degranulate and release free radicals, proteases and

hypochlorous acid which cause the vascular and mucosal injury seen in reperfusion injury. 5,94,96

Neutrophils also produce damaging superoxide free radicals via the nicotinamide adenine

16

dinucleotide phosphate (NADPH) oxidase system 85,87, which catalyzes the reduction of oxygen

to the superoxide anion and hydrogen peroxide. The production of these oxidative compounds is

the main mechanism by which the neutrophils phagocytize and subsequently destroy micro-

organisms and this process is called the “respiratory burst”. 104

White blood cells contain an enzyme called myeloperoxidase which is inactive in the

dormant neutrophil. 87 Various infectious or inflammatory conditions trigger the priming

response which activates neutrophils 105 and subsequent release of myeloperoxidase into

extracellular fluids 95. A significant increase in myeloperoxidase activity in the large colon has

been demonstrated during reperfusion following a period of ischemia. 106 Myeloperoxidase

catalyzes the enzymatic conversion of hydrogen peroxides and choride ions to hypochlorous

acid. 104 Hypochlorous acid is a potent oxidant and reacts with other compounds to form other

potent oxidants, such as the lipophilic N-chloramines. 95,107 Hypochlorous acid can cause tissue

damage by inactivating alpha-antiproteases and activating gelatinase and collagenases 95 and has

the ability to disrupt cell membranes 95,108. When applied in vitro to the equine right ventral

colon hypochlorous acid can affect the permeability of the mucosa and can cause cellular

damage 109 which suggests that neutrophils cause significant damage to the large colon during

reperfusion injury. Neutrophils have been shown to increase in the large colon lamina propria

and mucosa during low-flow ischemia with a subsequent increase during reperfusion. 110

Neutrophils increase significantly in the serosa of small intestine during reperfusion an event

associated with subsequent scarring. In a recent study the role of the neutrophil seemed less

clear when a filter in an extracorporeal circuit, which depleted leukocytes, failed to attenuate

reperfusion injury in the small intestine. 111 It is speculated that resident rather than circulatory

17

neutrophils mediate reperfusion injury 94 and/or that the production of free radicals alone may be

as important as a cause of tissue damage in the small intestine.

Proteases are enzymes that cleave peptide bonds and they contribute to reperfusion injury

by causing tissue damage. Granulocytes contain at least three serine proteases including

elastase, neutral proteases, and chymotrypsin- like proteases (cathepsin G), which are release

during phagocytosis. 5 These serine proteases catalyze the degradation of proteins or

polypeptides 112. For example elastase released from activated neutrophils, degrades the

intracellular barriers between the endothelial cells which allows the migration of the neutrophils

from the microvasculature to the interstitium. 5 Lysosomes are also a source of proteases and are

small intracellular organisms found in the cytoplasm of most cells. They degrade or digest

cellular debris. 112 Normally endogenous inhibitors protect against this type of injury 112 and

mucosal injury during reperfusion has been prevented experimentally with protease inhibitors in

the feline intestine 113. Granulocytes and lysosomes appear to be the most important source of

proteases in the large intestine. 5 In addition to these sources the pancreas is also a very

important protease source in the small intestine 112. The pancreatic endoproteases can maintain

some enzymatic activity when they bind to the intestinal mucosa and thus may cause injury by

proteolytic destruction of the mucosal cells. 5

Intestinal ischemia-reperfusion injury has been largely attributed to cellular necrosis,

however recent research indicates another form of cell death known as apoptosis may be

involved. Cellular necrosis is characterized by changes resulting from lack of oxygen, where as

apoptosis results from an intracellular signaling which initiates the transcription of specific

genes. 10 Cellular necrosis normally affects groups of cells or entire organs, while apoptosis

affects individual cells of a highly specific cell type. 10 Apoptosis has been recognized in several

18

species as having an important role in ischemia-reperfusion injury. Apoptosis has been observed

following ischemia-reperfusion injury to the brain, heart, adrenals, liver and kidneys in

experimental studies 7 and has been recognized in several species as having a role in

exacerbating reperfusion injury.

INTESTINAL DISTENTION

Distended intestine often appears to have a normal color and motility after

decompression, however the intestine has sustained damage which may not be evident grossly.

During intestinal distention the intraluminal pressure increases causing collapse of the veins and

capillaries, even when the blood pressure is normal. It has been demonstrated in clinical disease

when the small intestinal pressure is increased at 18cm of water there is a decrease in mesenteric

blood flow to the intestine by 50% resulting in low flow ischemia. 114 There is an increase in

capillary back pressure but the arterial pressure is maintained which alters the Starling forces 114

within the intestinal wall and subsequent secretion of fluid from the vasculature. Water and

protein escape into the interstitium causing submucosal and serosal edema. 55 At the higher

intraluminal pressures there is more secretion than absorption of water and thus the intraluminal

pressure increases further after the bowel compliance has reached its limit. 115 Eventually fluid

and protein leak into the peritoneal cavity and if the pressure is maintained the intestinal

compliance allows blood flow to gradually increase. 55 The serosa and submucosal edema

progresses, mucosal and serosal lymphatics dilate and red blood cells and white blood cells

migrate into the serosa and submucosa. 55 However in clinical cases with simple obstruction of

the small intestine or colon there is minimal mucosal injury. 55

If the bowel is decompressed there is a hyperemic response as is seen after low flow or

total ischemia. 55 The blood flow can initially double but then will decrease below normal. 114

19

The mucosa is relatively resistant to short term distention, however in the serosa the edema

causes capillary closure and increased vascular permeability. 55 Neutrophils migrate into the

serosa and cause destruction of collagen and ground substance. 103, 116, 117 The intestinal smooth

muscle is also affected by edema and neutrophil migration into the fascial planes around the

myenteric plexi. 55 The bowel can heal after this type of insult, however the serosa is frequently

thickened by fibrous tissue with the possibility of adhesion formation and permanent mesenteric

constriction. 116,118 It is rare that prolonged distention causes bowel necrosis. Bowel necrosis is

more common when focal distention is caused by foreign bodies or impactions. 55

The small intestine is more susceptible to distention injury compared to the large colon. 55

Ileus is common in horses with previously distended small intestine and the severity can be

predicted by the bowel pressure measured at surgery. 119,120 Clinical measurement of the

intraluminal pressure of the large colon during obstruction indicates that the colon can withstand

at least twice the distention pressure with no adverse effects. 119,120 The correlation of increased

intraluminal pressures and survival indicates that intestinal distention is an important cause of the

injury that is seen with those conditions of the acute abdomen causing distention, in addition to

the peritoneal changes and failure of the cardiovascular system. 35,114,116,120,121

Intestinal distention also effects the enteric nervous system. Impaction of the colon or

cecum resulting in chronic obstruction has been associated with a significant decrease in the

number of neurons in the myenteric plexi but with similar numbers of myenteric plexi as normal

horses. 122,123 The change in neuron number was associated with increased thickness of the

longitudinal muscle in the pelvic flexure or both circular and longitudinal muscle hypertrophy in

the cecum. 123 This may be similar to the pseudo-obstruction in humans and to the experimental

denervation of intestinal segments in rats. 55 The lack of nervous inhibition has been

20

hypothesized to allow constant and uncoordinated muscular contractions which result in the

hypertrophy and eventually the poor transit of ingesta. 55 Affected myenteric plexuses have also

been shown to have an increase in the number of glial cells. 55 This may be an inflammatory

response by the enteric nervous system to the intestinal distention, or the inflammation and

decrease in neurons may be responsible for the colon dysfunction in the affected horses due to

colon obstruction.

INFLAMMATION OF THE PERITONEUM

The serosal layer of the intestine has the important function of maintaining a lubricated barrier

at the bowel surface which is vital for normal intestinal motility. 55 The serosa is made up of a

single layer of mesothelial cells that attaches to a basement membrane which is adjacent to an

elastic layer. Some of the mesothelial cells are short with channels linking the peritoneal surface

to the serosa and others have long microvilli which help trap fluid on the surface of the

peritoneum, thus maintaining lubrication. The mesothelial cells react to circulating or

intraperitoneal lipopolysaccharide, infection and surgery by releasing TNF-a, Il-1ß, Il-6 and

macrophage inflammatory protein 124 resulting in the attraction and migration of neutrophils into

the serosal connective tissue. 55 During an ischemic process the mesothelium is rapidly lost with

serosal swelling and edema. With reperfusion the serosal vascular permeability increases and

white blood cells migrate through capillaries or venules and infiltrate into the serosa connective

tissue layer. 55 Neutrophils accumulate at the basement membrane around vessels and within

lymphatics and fibrin accumulates within the serosa and at the surface. The neutrophils release

lysozymes with apparent disruption of the collagen and the denuded serosal surface becomes

covered with a fibrin clot. 55 There is a massive accumulation of cells, predominately

neutrophils, in the serosa and at the surface within 24-48 hours. 55 The larger the inflammatory

21

reaction within the serosa and on the sur face, the more fibroplasia which delays the resurfacing

of the mesothelium and increases the chance of scarring and adhesion formation. The severity of

the adhesions has been experimentally correlated with increasing concentrations of TNF-a in the

peritoneal fluid and TNF- a antibodies have been shown to decrease adhesion formation. 125,126

Following small intestinal ischemia there is an initial hyperemia in most of the bowel but

actually a reduction of perfusion to the serosa. 55 The same process occurs during bowel

distention and is greater when the distention is relieved following decompression. There is

immediately edema formation in distended bowel which hypothetically increases serosal

pressure. 55 The increased serosal pressure exerts extravascular pressure and closes capillaries

and venules. 114,127 This process continues after decompression and a reduction in the distention

and thus during reperfusion there is ischemic injury, serosal endothelial cell swelling, and

capillary plugging. 55 This may explain adhesions involving bowel which was distended and was

proximal to an obstruction or strangulating lesion but not actually involved in the primary

ischemic lesion. 55 Adhesion formation due to septic peritonitis also occurs in response to the

massive inflammatory response in the serosa with neutrophil migration and fibrin deposition in

and on the serosa. 128 In some cases the inflammatory response is so great that it is possible that

lysozymes prevent adhesions by breaking down the adhesions. 55

APOPTOSIS

APOPTOSIS-PROGRAMMED CELL DEATH

Cellular death can occur in two biochemically and morphologically distinct ways-

cellular necrosis and apoptosis. Cellular necrosis refers to the light microscopic changes

resulting from enzymatic degradation of the nucleus and cytoplasm and is characterized by

22

biomechanical and morphologic alterations resulting from anoxia. 10 Necrosis typically occurs in

response to acute cellular dysfunction and is a passive and destructive process which results in

the release of intracellular contents into the extracellular environment. There is a subsequent

activation of a marked inflammatory response which exacerbates the condition. 129 Apoptosis is,

in contrast, a highly regulated process characterized by cell shrinkage. Cells’ contents are

subsequently packaged into apoptotic bodies that are subsequently engulfed by macrophages

thereby prevent ing an inflammatory response. 129 Apoptosis results from activation and

transcription of specific genes and affects individual cells of a highly specific cell type in

contrast to cellular necrosis in which there are large numbers of all cell types affected in tissue or

entire organs. 10 There is evidence that protein molecules, which are involved in the induction of

apoptosis, are constitutively expressed in mammalian cells and are normally inactivated by

antiapoptotic proteins. 10 These protein molecules are synthesized in the same cell in response to

certain survival signals including growth factors, extracellular matrix or fluid or a variety of

hormones. 10 Loss of these protective factors10 or activation of the harmful proteins induced by

genotoxic stimuli 130 can initiate apoptotic pathways which can ultimately result in the demise of

the cell. 131

Apoptosis is characterized by cellular shrinking, condensation and margination of the

chromatin and ruffling of the plasma membrane which is called budding. 137 The cell eventually

becomes divided into the apoptotic bodies which consist of cell organelles and/or nuclear

material surrounded by an intact plasma membrane. 137 These apoptotic cells are mostly

engulfed by neighboring cells, particularly macrophages. 132 The macrophage recognizes the

apoptotic cell fragments by their expression of phosphatidylserin on the outside of the plasma

membrane. 138 Other mechanisms of phagocytosis include expression vitronectin receptors or

23

stimulation by certain carbohydrates. 139 140 Apoptotic cells are not always recognized by

phagocytes and then they may undergo secondary141 or apoptotic necrosis142 Necrosis can be

thought of as the end stage of any cell death process. 142

Apoptosis is a physiological cell death that has a critical role in the development and

tissue homeostasis. 132, 8, 133 Apoptosis eliminates superfluous or potentially harmful cells. 134 It

is crucial for an organism to remove apoptotic cells before they become secondarily necrotic and

release intracellular contents, causing tissue damage or an inflammatory reaction. 135

Macrophages are the key cells in this process, using a number of receptors to recognize surface

changes appearing as cells die. 135 In addition to removing potentially inflammatory and

immunogenic cell debris, macrophages also inhibit inflammation by engulfing apoptotic cells

through the release of transforming growth factor ß and other inflammatory mediators. 135

Therefore as apoptosis is part of normal cellular turnover, the clearance by macrophages and

other phagocytes plays a critical role in tissue homeostasis. 135 Macrophages have also been

found to induce apoptosis. 135 Apoptotic cell engulfment has an important role in various

diseases such as cancer, virus- induced pathologic conditions, autoimmune diseases, and other

inflammatory states. 135

Apoptosis is involved with embryogenesis, tissue homeostasis, lymphocyte development

and function, and tumor regression, resulting from DNA damage induced by various factors. 8

Apoptosis is an energy-dependant, active form of cell death, which not only occurs normally

during development and in the immune system but also in response to injury. 132 Normal cells

will typically die by undergoing apoptosis or by entering a state of irreversible growth arrest,

termed replicative senescence, at the end of the lifespan. 129 The regulation of apoptosis has a

direct influence on the ability of a cell to survive cellular insult from both external and internal

24

sources and thus the genes involved in apoptosis influence cellular longevity. 129 A variety of

molecules have the ability to regulate apoptosis. Inhibitory molecules include heat shock

proteins, anti-apoptotic members of the Bcl2 family and anti-oxidant molecules. Molecules

which can promote apoptosis include pro-apoptotic members of the Bcl2 family and p53.

Almost every tissue can undergo apoptosis when provided with an appropriate stimulus and thus

many apoptotic genes are present in all tissues. However, the exact compositions of the

apoptosis regulators vary in tissues and this variation contributes to different responses of

different cells and tissues to different apoptotic stimuli. 136

Mitochondria play a central role in both apoptosis and necrosis. 143 Mitochondria provide

a key amplification step in the apoptotic pathway by releasing apoptogenic proteins in the

cytosol of the cells. 144 Mitochondria have a multiprotein complex formed at the contact site

between the inner and outer mitochondria membranes which is called the mitochondria

permeability transition (PT) pore. 145 The PT pore is involved in the regulation of matrix Ca++,

voltage, pH, transmembrane potential and volume and functions as a Ca++, voltage, pH and

redox-gated channel with several levels of conductance and little or no ion selectively. 145

Certain proteins in the cell, when activated, move from the cytoplasm or nucleus to the

mitochondrial membranes, where they interact with mitochondrial receptors such as hsp70,

BclXL, PTPC, Bax and Bcl2. 131 When these proteins are translocated to the mitochondrial

membranes they promote permeabilization of the membranes with the consequent release of

caspases or nuclease activators from the mitochondrial intermembrane space. 131 One of the

most critical proteins in this process is cytochrome c which in the presence of ATP and dATP,

forms a complex with apoptosis activating factor 1 and procaspase 9. 143 This induces cleavage

of procaspase 9 resulting in the release of active caspase 9 which then cleaves and activates

25

procaspase 3. 143 Active caspase 3 then induces proteolytic cleavage of a range of target

proteins. 143 These target proteins then cause changes in the cytosol, nucleus and plasma

membrane that are the characteristic of apoptosis. 146-148, 149

There are three major mechanisms through which apoptosis can be induced by the

activation of caspases. These include a receptor- ligand binding with activation of caspase-8; a

mitochondrial mechanism with activation of caspase-9; a process involving the endoplasmic

reticulum with activation of caspase-12. 150 The first mechanism involves the binding of a ligand

on a specific receptor which is located on the cell membrane. This binding activates the

conversion of procaspases to caspases. Two well known ligands are the Fas ligand, which has

Fas as the receptor, and TNF-a which binds to TNFR1. 137 Both of these receptors contain an

analogous cytoplasmic domain called the ‘death domain’, which is responsible for the signal

transduction in apoptosis. 151 There are two molecules which are linked to the death domain- the

Fas associated death domain (FADD) for Fas 152, and the TNFR1 associated death domain

(TRADD) for TNFR1 153. TRADD binds to FADD and thus both receptors use FADD to

transducer the death signal. 137 Procaspase 8 binds with its death domain to FADD and becomes

activated to caspase 8. 137 Activated caspase 8 results in the conversion of procaspase-3 into the

effector caspase 3 which seems essential for apoptosis. 154 TNF- alpha can induce apoptosis by

the binding of receptor interacting protein (RIP) to the TRADD which leads to the activation of

caspase 2. 155 There is also a highly specialized form of Fas mediated apoptosis caused by

cytotoxic T lymphocytes. 137 Other receptor- ligand mediated forms of apoptosis are known but

their exact mechanisms are not yet understood. 137 The second mechanism involves the

mitochondria. Certain cytotoxic agents such as nitrogen monoxide and radiation can cause

apoptosis which involves the mitochondrial protein cytochrome c. 156, 157 Cytochrome c is

26

localized on the outside of the inner mitochondrial membrane and in the intermembrane space 158

and has an important function in the intracellular electron transport chain for the production of

ATP 137. Cytochrome c is released into the cytosol during apoptosis and then it binds to the

apoptosis protease activating factor (Apaf-1). The cytochrome c and the Apaf-1 form a complex

together with dATP which then activates procaspase 9 to caspase 9. 148 This results in the

activation of procaspase-3 into caspase-3 which leads to the known characteristic morphological

consequences of apoptosis. 137 Most agree that the release of cytochrome c into the cytosol is

regulated by the proteins coded by the genes of the Bcl2 family. 137 Bcl2 and BclxL are both

apoptosis inhibitors 137 and Bid and Bax are two pro-apoptotic proteins 144. The proteins of the

Bcl2 family are mainly localized in the outer mitochondrial membrane 137 and the ratio of Bax

transcripts to the bcl2/bcl-xL transcripts will determine whether cytochrome c is release or not.

The mechanism of release of cytochrome c by Bax is not understood as yet. Some authors

believe that these proteins regulate the mitochondrial permeability via the PT pore. 159 The

opening of this channel results in the influx of ions which results in swelling of the

mitochondrion and breaks in the outer membrane while the inner membrane remains intact due

to its larger surface. 137 Cytochrome c then breaks through the outer membrane and enters the

cytosol. 159 This ion influx explains the decrease in transmembrane potential of the inner

mitochondrial membrane which has been documented in apoptotic cells. 160 Recent studies

suggest that the mitochondrial mechanism and the receptor- ligand mechanism are not completely

independent. 137 Caspase 8 can activate the protein Bid. Activation of Bid leads to the release

of cytochrome c and activation of caspase 9. 161 Another recent study showed that the outer

membrane permeabilization by Bcl2 family proteins does not require the mitochondrial matrix,

the inner mitochondrial membrane or other proteins. 162 Bid, or its BH3-domain peptide,

27

activated monomeric Bax to produce membrane openings that allowed passage of very large (2

megadalton) dextran molecules which explains the translocation of large mitochondrial proteins

during apoptosis. 162 This process requires cardiolipin and is inhibited by antiapoptotic BclxL. A

third mechanism in the apoptotic process has also been demonstrated in transgenic mice. Agents

that stress the endoplasmic reticulum such as tunicamycin and thapsigargin cause much

apoptosis in embryonic fibroblasts of normal mice but not caspase-12-/- knock-out mice. 150

This process appears to be independent of both former pathways described above.

In vascular smooth muscle cells it has been shown that cytochrome c activates K+

channels before inducing apoptosis. In vascular smooth muscle cells, K+ is the dominant cation

and Cl- is the dominant anion in the cytoplasm. Cytoplasmic K+ at a normal concentration

(~140mM) suppresses apoptosis by inhibiting caspase activation. 163 In a cell- free system with

only isolated nuclei, a decrease in K+ from 140-180mM caused a 1.6-fold increase of apoptosis

induced by the apoptosis-inducing factor. 164 Efflux of K+ through K+ channels, therefore, plays

an important role in initiating the apoptotic volume decrease and apoptosis. The cytochrome c-

mediated decrease in cytoplasmic K+ resulting from opened Kv channels, in addition to the

volume decrease which occurs during apoptosis, may contribute to induction of DNA

fragmentation and apoptosis by reducing the inhibitory effect of cytoplasmic K+ on the

cytoplasmic caspases and the internucleosomal DNA cleavage nuclease. 163 It is possible that

apoptosis inducers such as staurosporine, may also open K+ and Cl- channels through cyt-c

independent mechanisms. 165

IMMUNOHISTOCHEMICAL STAINING TO DETECT APOPTOSIS

In many of the pathways that are initiated in apoptosis, permeabilization of the

mitochondrial membrane is a critical event that results in release of various molecules from the

28

mitochondrial intermembrane space. 131 These molecules include procaspases (enzymes),

cytochrome c (a caspase activator), Smac/Diablo ( a caspase coactivator) and an apoptosis-

inducing factor. 131 The apoptosis inducing factor activates the nucleases that cleave the DNA

into small fragments. 131 The DNA strand breaks that result from the endonuclease(s) activation

can be labeled in situ, in individual fixed, permeabilized cells or tissues in sections, by the

terminal deoxynucleotide transferase mediated dUTP nick end labeling (TUNEL) technique

187,188. In this technique, the 3’-OH termini which are generated as a result of the DNA

fragmentation during apoptosis, are labeled with modified nucleotides by terminyl

deoxynucleotide transferase. 189 This enzyme selectively detects apoptotic rather than necrotic

cells and is more specific than DNA polymerase. 189 The new ends that are generated upon DNA

fragmentation are typically localized in the morphologically identifiable nuclei and apoptotic

bodies.

There is a high correlation between TUNEL staining and apoptosis. 189 190 When

compared to another technique, the ISNT (in situ nick translation), the TUNEL technique was

more sensitive. 191 A major advantage of the TUNEL technique is the ability to reveal early

DNA breaks during apoptosis which occur before histological changes characteristic of apoptosis

can be detected. The TUNEL technique is very successful in detecting apoptotic lymphocytes

and neutrophils in the lamina propria of fixed human intestinal samples. 14 Dexamethasone is

known to induce apoptosis in the murine thymus 192, 193,194 ApopTag® Peroxidase kits are used

to successfully stain apoptotic rat thymus lymphocytes treated in vitro with dexamethasone.

195,187

A potential concern of detecting apoptosis in formalin fixed tissue was that of random

DNA damage with long term fixation, resulting in nonspecific labeling. This effect has been

29

investigated in rat testicular tissue. Results indicated that formaldehyde is able to replace

protons in the purine and pyrimidine bases but does not react with sugar hydroxyls or phosphate

ester groups in nucleic acids. 196 It is possible that with prolonged fixation any single-stranded

regions of DNA, or ‘free ends’ are locked up and they are thus inaccessible to the polymerase. 196

Any damage resulting from fixation or cytopathological mechanisms such as necrosis would thus

not be detectable with this technique. 196 Therefore the test has a risk of a false negative result

rather than a false positive result.

APOPTOSIS AND THE INTESTINE

Apoptosis has been suggested to play a complementary and opposite role to mitosis in the

regulation of animal cell populations. 132 In the gastrointestinal tract apoptosis is important in

physiologic cell renewal. 197,198 Apoptosis in the gastrointestinal tract is associated with the

shedding of cells from the villous tips in the small intestine, and with the development of

intestinal adenocarcinomas, the pathogenesis of inflammatory bowel disease, and radiation

induced gut damage. 9 The apoptotic targeting of damaged stem cell populations, early response

for apoptotic removal of DNA-damaged cells, and early repair of DNA-damaged cells are

considered major indicators of differences in levels of tumorigenesis in the intestine. 199

Enterocyte apoptosis is increased in patients with untreated celiac disease which results in the

formation of villous atrophy. 200 Thus apoptosis plays an important role in the maintenance of

normal gastrointestinal homeostasis and in the induction of gastrointestinal disease. 201 202

Endothelial and epithelial barrier functions protect against potential pathogenic invasion.

9 An intact intestinal barrier prevents invasion of potential pathogens 203 and macromolecules

which are not normally permeable through the barrier may pass to variable degrees under

pathologic conditions. 204 The number of apoptotic cells in the colon in human patients with

30

diseases such as untreated inflammatory bowel disease, non-steroidal anti- inflammatory drug-

related colitis, and active celiac disease. 13, 200 In one study a model of intestinal epithelial

apoptosis was induced by injecting doxyrubicin into the peritoneum of rats. When the

endothelial and epithelial permeability and the amount of apoptotic cells were measured there

was a significant increase in gut water content, albumin flux, and bidirectional clearance of

albumin with a significantly higher apoptotic index. 9 This data indicates that occasional

apoptotic cells which appear in normal intestine may not cause alterations in intestinal barrier

integrity, whereas increased intestinal apoptosis causes increased endothelial and epithelial cell

permeability.

Both the glycolytic and protein synthetic pathways may play a crucial role in the

formation of mucosal endothelial apoptosis and dysfunction. 9 Chemotherapeutic agents can

induce apoptosis in the intestinal epithelium and cause an increase in barrier permeability. 205, 9

An increase in barrier permeability enables pathogenic bacteria and/or their toxins to pass

through the intestinal barrier which can result in gut-origin sepsis and other complications. 9 The

inflammatory response associated with the non-steroidal anti- inflammatory induced apoptosis in

the human colon indicates that apoptosis achieves sufficient magnitude to disrupt the epithelial

barrier and cause inflammation. 13

Morphological and biochemical evidence suggests that glutamine deprivation induces

apoptosis in rat small intestinal epithelial cells. 206 Glutamine is rapidly absorbed and

metabolized by enterocytes from intraluminal or arterial sources 207 and is the most abundant

amino acid in the blood 208. In human patients treated with sustained parenteral nutrition which

does not contain glutamine, have decreased mucosal weight and DNA and protein content. 209

Gut atrophy induced by glutamine starvation is caused in part by an increased rate of apoptosis in

31

intestinal epithelial cells which affects the homeostatic balance between cell proliferation and

cell death. Glutamine prevents apoptosis in the gut by supplying an essential energy source to

maintain cellular adenoside triphosphate (ATP) concentrations. 206 Administration of

antioxidants such as dimethyl sulfoxide prevents glutamine deprivation-induced apoptosis in

HuH-7 cells which may indicating that in some cells glutamine prevents apoptosis by

maintaining glutathione levels rather than ATP. 206

Recently apoptosis has been found to be a major mode of cell death in the destruction of

rat small intestinal epithelial cells induced by ischemia and ischemia/reperfusion injury. 210 The

intestinal mucosa is one of the most sensitive tissues to ischemia/reperfusion injury. 210

Apoptosis has been characterized in rat jejunum and ileum following occlusion of the superior

mesenteric artery for 15 or 60 minutes followed by a period of reperfusion. 211 Apoptosis does

not occur in the duodenum as blood flow is not decreased after occlusion of the artery. 211 The

percentage of fragmented DNA increases during ischemia and is maximal after 1hour of

reperfusion. 211 The magnitude of DNA fragmentation is directly proportional to the duration of

the ischemic insult with a significantly higher percentage of fragmented DNA after 60 minutes of

ischemia compared with 15 minutes of ischemia. 211 Because maximal DNA fragmentation

occurrs at 1 hr postreperfusion followed by a return to baseline values by 6hours, induction of

intestinal apoptosis and mucosal recovery appear are rapid processes. 211 There may be a time-

dependant increase in apoptosis-promoting factors during ischemia and early phases of

reperfusion which rapidly decreased with prolonged reperfusion. 211 There may also be a

simultaneous induction of inhibitors of apoptosis as well as promoters of tissue repair during the

later phase of reperfusion. 212,213 The exact mechanism for the induction of apoptosis is not

known. However studies have shown that free radicals can cause apoptosis 212,213 which is

32

consistent with the increase in free radical production during ischemia/reperfusion injury. 212,213

Certain authors have reported alterations in cell adhesion molecules or substratum along the

crypt-villous axis, and used this to support the idea of cell loss by shedding. 215,210 However, the

regulation of intestinal cell death and proliferation is highly complex and is most likely

controlled by a variety of factors. 214 It has been proposed that altered expression of ß1 integrins

along the crypt-villous axis facilitates cell loss. 215 Recently the interaction between extracellular

matrix materials and cells has been proposed as a critical role in causing apoptosis, with the cell

death occurring when the contacts are disrupted; a phenomenon is known as anoikis. 216

Affected epithelial cells lose their contact with the villous stroma indicating apoptosis is induced

by ischemia and ischemia/reperfusion could represent anoikis, however in this study not all the

detached cells demonstrated positive staining with the TUNEL method and some cells had

evidence of necrotic cell death when examined by DNA binding fluorochromes PI (propidium

iodide). 210

The intestine is susceptible to stress during surgery and surgical trauma induces oxidative

stress and damage through increased production of reactive oxygen species. 217 Surgical stress

increases intestinal permeability and induces bacterial translocation in the systemic circulation.

218 There are profound metabolic changes, organ dysfunction, and immunosuppression during

gastrointestinal surgery which form part of the surgical stress response to trauma. 219 The

intestine is highly susceptible even to surgery at remote locations. Mild handling of the intestine

following laparotomy caused oxidative stress in the enterocyte, accompanied by functional

alterations in the intestine. 220 Furthermore during surgical stress there is increased activity of

xanthine oxidase and increased oxygen free radicals which activate proteases in the enterocyte

mitochondria. 221 This protease activation was not seen in xanthine oxidase deficient animals.

33

Increased mitochondrial protease activity may be responsible for the induction of the

mitochondrial permeability transition and mitochondrial dysfunction seen during surgical stress.

221 Surgical stress also activates cytosolic proteases, which are modulated by thiol redox status

and are maximally activated by 60 min after surgical stress and return to normal by 24hours.

APOPTOSIS AND ISCHEMIA/REPERFUSION INJURY

Pro-apoptotic stimuli will stimulate cells to undergo apoptosis. However intracellular

ATP concentration is a determining factor in the reactions causing apoptosis. Decreased ATP

levels will favor necrosis over apoptosis and an increased ATP provided by glycolytic substrates

has the opposite effect. 166 The concept that the mitochondria can trigger apoptosis when

caspases are activated and necrosis when such proteases fail to act is compatible with the finding

that many drugs induce necrosis at high doses and apoptosis at lower doses. 145 Induction of the

mitochondrial PT pore in a smooth protracted fashion allows for the activation and action of

proteases before ATP depletion causes cell death. Alternatively when the PT pore is induced in

a massive, rapid fashion, heavily compromising cellular metabolism then necrosis will occur

before apoptogenic proteases come into action. 145 Several apoptosis- inducing agents such as

pro-oxidants and nitric oxide can act on the critical cysteine residues of caspases provoking their

inactivation. 167 Low doses of these compounds induce the PT pore and cause apoptosis, where

as high doses induce the PT pore but inhibit caspases causing necrosis. Therefore the intensity

of the PT-inducing stimulus may determine which among the two major consequences of PT will

dominate over the other resulting in either a bioenergetic catastrophe culminating in cytolysis, or

the activation/action of apoptogenic caspases. 145

The susceptibility to either apoptosis or necrosis is also influenced by the ratio between

glycolytic and respiratory ATP generation, which is differentially affected by the disruption of

34

mitochondrial function. This explains why lymphocytes in which glycolytic ATP generation

dominates tend to undergo apoptosis, whereas neurons in which oxidative ATP generation

dominates tend to undergo necrosis in response to stress. 145 The existence of an alternative

ATP source to oxidative phosphorylation may be a critical factor allowing cells to progress to

apoptosis. 168 For example if the onset of the mitochondrial permeability transition occurs in a

subpopulation of mitochondria as would occur during the early stages of TNFa-induced

apoptosis in hepatocytes, then the remaining functional mitochondria may produce sufficient

ATP to permit apoptosis to proceed. 168 In ischemic tissue pH decreases rapidly due to anerobic

glycolysis and the hydrolysis of ATP. During reperfusion normal pH is restored. While the

naturally occurring acidosis is protective against loss of cell viability during ischemia, the

restoration of a normal pH during reperfusion can accelerate cell death. A pH below 7 inhibits

conductance through the PT pore of the mitochondria so it is inhibited during ischemia.

However an increasing pH has been shown to trigger the onset of the mitochondrial permeability

transition which depolarizes mitochondria with uncoupler-stimulated mitochondrial ATPase

activation. 169 170 If the rise in pH is prevented after reperfusion the mitochondrial permeability

transition is blocked and there is improved ATP recovery and inhibition of cell death. 171

Cyclosporin A has also been shown to prevent cell death by blocking the mitochondrial

permeability transition without blocking recovery of pH. 171 Because acidosis and cyclosporin A

both act to block onset of the mitochondrial permeability transition, accelerated ATP hydrolysis

caused by onset of the mitochondrial permeability transition is likely to be the basis for

continued ATP depletion and subsequent cell death. 171

35

ROLE OF NITRIC OXIDE IN APOPTOSIS

Nitric oxide (NO) is an important molecule with diverse roles including the modulation

of apoptosis. Nitric oxide can either induce or inhibit apoptosis depending on the cell type and

coexisting metabolic or experimental conditions. Nitric oxide can inhibit caspases by S-

nitrosylation, by inhibiting Fas induced apoptosis, or by increasing anti-apoptotic proteins such

as Bcl2. 172 Some studies indicate that NO may be an important protective molecule against

ischemia/reperfusion injury, 173,174 while other studies demonstrate that NO is involved in the

breakdown of the intestinal mucosa after an ischemia/reperfusion insult. 175 Further tissue NO

generation increases during ischemia/reperfusion of the rat small intestine when adenosine is

added to the preservation solution and decreases when the adenosine receptors are blocked with

theophylline, even in the presence of exogenous xanthine. 176 The adenosine administration

increases NO generation that is concomitant with an increase in caspase-3- like activity and DNA

fragmentation. 176 These findings indicated that adenosine is related to NO production in

ischemia/reperfusion injury and there is a direct role of NO in the activation of apoptosis during

the ischemia/reperfusion of the rat small intestine. 176 This finding contraindicates the NO

inhibition of apoptosis previously found in rat gastric mucosa cells177, but is in agreement with

previous results demonstrating that NO is required for the activation of apoptosis173,178. DNA

fragmentation and morphologically recognizable apoptotic injury in small intestinal tissue

occurrs with the addition of exogenous xanthine which increases caspase-3-like activity. 176 The

protective effects of blocking adenosine function by theophylline are reversed by further addition

of xanthine. 176 The administration of NO donors to the xanthine-treated group induces the same

effect on apoptotic parameters as that produced by the administration of theophylline. This

indicates a NO-independent effect of xanthine on apoptosis which involves reactive oxygen

36

species. 176 The role of xanthine in the development of apoptosis during ischemia/reperfusion is

supported by the production of apoptotic tissue injury with the addition of exogenous xanthine

even when adenosine receptors are blocked.

The effect of NO on mitochondria has been examined in other studies. Short-term

exposure to low levels of NO results in a completely reversible inhibition of respiration (at

cytochrome oxidase), but longer-term exposure to high levels of NO, or exposure to NO in the

presence of reactive oxygen species, leads to irreversible inhibition of respiration. 179 The

conversion from the reversible to the irreversible phase of inhibition is mediated by the following

factors a) accumulating inhibition by peroxynitrite or NO, b) increased reactive-oxygen-species

production by mitochondria, c) increased H2O2 levels due to inhibition of catalase, d) reaction of

H2O2 and NO with superoxide dismutase to produce peroxynitrite, and e) secondary effects of

cytochrome oxidase inhibition, for example glutamate release from nerve terminals. 179 Nitric

oxide has specific interactions with mitochondria at the level of both cytochrome c oxidase and

complex III. 180 One study demonstrated NO restoration of the mitochondrial PT pore at high

release rates in excess of those to be encountered in vivo, even in a pathologic situation with

maximal stimulation of inducible NO synthase. 180 This study identified inhibition of

mitochondrial PT pore opening as a novel site of action for NO signaling in apoptosis. 180 The

mechanism involves the depolarization of the mitochondrial membrane and inhibition of calcium

accumulation and favors partial inhibition of mitochondria. 180

Nitric oxide can induce necrotic or apoptotic cell death. 181 Nitric oxide- induced necrosis

in the absence of glucose is due to the inhibition of mitochondrial respiration and subsequent

ATP depletion. 181 However when glucose, nitric oxide donors and mitochondrial inhibitors are

available, apoptosis is induced. This demonstrates that necrosis versus apoptosis induced by

37

either NO or mitochondrial inhibitors depends critically on the glycolytic capacity of the cell. 181

Nitric oxide- induced apoptosis has been shown to be mediated by caspase activation and

specifically the caspase-3 and caspase-3-processing-like proteases. 181 Nitric oxide also induces

cell death in several other cell types including cardiac myocytes and smooth muscle cells. 182,183

Inducible NO synthase (iNOS) expression and NO production is associated with an increase in

caspase-3 activity and apoptotic cells death in adult cardiac fibroblasts. 184 The cardiac fibroblast

apoptosis in response to IL-1ß is attenuated by the caspase-3 inhibitor. 184 In the smooth muscle

cells and cardiac myocytes , the proapoptotic effect of cytokines is usually mediated by the

induction of iNOS protein which only occurs in pathological conditions when there are high

levels of NO production. 185, 182,183 Nitric oxide as discussed before can have a proapoptotic or

antiapoptotic effects- low levels of NO generated by endothelial NOS may have a protective

effect, whereas the higher levels produced by iNOS may exert a proapoptotic effect. 184

APOPTOSIS AND INFLAMMATION

Apoptosis has been shown to be an important source of inflammation in the kidney which

is an important documentation of the fact that apoptosis is not only a benign process causing

minimal tissue damage. Apoptosis of glomerular endothelial cells may contribute to the

development of glomerulosclerosis during severe forms of glomerulonephritis. 11 Tumor

necrosis factor-a and lipopolysaccaride (LPS) induce apoptosis of bovine glomerular endothelial

cells which is characterized by early mitochondrial cytochrome c release, mitochondrial

permeability transition, Bak protein upregulation, BclXL protein downregulation and caspase-3

activation. 222 Co-treatment of bovine glomerular endothelial cells with 10nM dexamethasone

and TNF- a or LPS has been demonstrated to block approximately 90% of apoptotic cell death.

222 Using a murine model of ischemia and reperfusion Daeman demonstrated administration of

38

the anti-apoptotic agents IGF-1 and ZVAD-fmk (a caspase inactivator) prevented the early onset

of not only renal apoptosis and also inflammation and tissue injury. 12 When these agents were

added after the onset of apoptosis the effects were completely attenuated. 12 The presence of

apoptosis in this study was directly correlated with posttranslational processing of the endothelial

monocyte-activating polypeptideII (EMAP-II) which could explain apoptosis- induced influx and

sequestration of leukocytes in the reperfused kidney. 12 These results indicate that apoptosis is a

crucial event that initiate the inflammation observed in ischemia/reperfusion injury of the bovine

kidney and subsequent tissue injury. The potent nephrotoxin Hg2+ at cellular concentrations

does not compromise cellular energy production, yet impairs activation of NF-kB. This

increases the sensitivity of the kidney cells to the apoptosis- inducing effects of other toxicants

and infectious agents to which kidney cells are otherwise resistant. 223 Apoptosis is thought to

play an important role in the pathogenesis of renal failure associated with toxicant injury to

tubular epithelial cells. 224,225

Recently muscle injury (measured by creatinine kinase) caused by aortic clamping was

reduced with a pancaspase inhibitor. 6 In this study the muscle injury resulting from 90-120

minutes of ischemia was reduced by the pancaspase inhibitor z-VAD. 6 The pancaspase inhibitor

leads to a blockade of apoptosis which reduced inflammation and subsequent injury. 6 Thus in

this study apoptosis was associated with inflammation in the muscle which could be reduced

with a caspase inhibitor.

APOPTOSIS AND NON-STEROIDAL ANTI-INFLAMMATORY DRUGS Nonsteroidal anti- inflammatory drugs (NSAIDs) are associated with

gastrointestinal inflammatory damage and aggravation of gut inflammatory conditions in human

medicine and veterinary science. In human medicine NSAIDs have been shown to exert a

39

protective effect against colon cancer due to an increased colon cell apoptosis. 226 NSAIDs have

also been shown to modulate the release of colony stimulating factors in some cells. In one

study reduced secretion of granulocyte-colony stimulating factor and macrophage-colony

stimulating factor and increased HT-29 cell apoptosis was observed with the highest

concentrations of nonselective NSAIDs. 226 NSAID administration in rats induces systemic

release of TNF-a and drives gastric epithelial cells to apoptosis activating the pro-apoptotic

cascade of caspases. 227 In response neutrophils are recruited into the gastric microcirculation

through a process which involves activation of the adhesion molecules. 227 In the gastric mucosa

in vivo PGE2 is one of the most important protective factors and NSAID’s are thought to damage

the gastric mucosa by inhibiting cyclooxygenase and decreasing the amount of PGs at the level

of the gastric mucosa. It has been demonstrated that PGE2 shows cytoprotective effects on

cultured gastric mucosal cells in relation to apoptosis but not with respect to necrosis. 228 PGE2

has also been shown to inhibit spontaneous apoptosis in vitro. 229 It appears that the target of

PGE2 as an inhibitor of apoptosis is a protein such as caspase 3 which is common to both

apoptotic pathways. 228 The antiapoptotic effect of PGE2 in human colon cancer cells is due to

an increase in the expression of Bcl2 230 and therefore PGE2 may inhibit the cytochrome c-

dependent activation of caspase-9 through an increase in the expression of Bcl2 to inhibit gastric

irritant-induced apoptosis. 228 An increase in apoptotic bodies in equine right dorsal colon tissue

incubated with an NSAID compared with controls has been recently documented. 231 Possible

causes for this increased apoptosis in the presence of NSAID could be reactive oxygen

metabolites and proteases in addition to inhibition of prostaglandin synthesis. 231 In this study it

was postulated that even after extensive cell loss through apoptosis barrier function may be

preserved by rearrangement of tight junctions and desmosomes and by flattening and spreading

40

neighboring cells over the defect. 232 The inflammatory response associated with NSAID-

induced apoptosis in human colon suggests that apoptosis can achieve sufficient magnitude to

disrupt the epithelial barrier and cause inflammation so it is reasonable to suggest that the same

pathology may occur in the equine intestine and that intestinal function could be altered. 13

41

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Detection of apoptotic cells in intestine from horses with and

without gastrointestinal disease.

EMMA L. ROWE† BSc. BVMS(Hons.), NATHANIEL A.WHITE† DVM. DACVS, VIRGINIA

BUECHNER-MAXWELLƒDVM, DACVIM., JOHN L. ROBERTSON?VMD, PhD, AND

DANIEL L. WARDz MS.

Marion duPont Scott Equine Medical Center† (Leesburg, Virginia), the Department of Large

Animal Clinical Sciencesƒ, the Department of Pathobiology?, and the Office of Research and

Graduate Studiesz, Virginia-Maryland College of Veterinary Medicine, Virginia Tech,

Blacksburg, Virginia.

This research was funded by the Patricia Bonsall Stuart Equine Research Award.

Submitted to: American Journa l of Veterinary Research

67

Abstract

Objective : To identify apoptosis in the equine intestine and determine if occurrence of apoptosis

is affected by gastrointestinal disease and tissue layer of intestine.

Animals : Samples of intestine were collected from 38 horses that underwent surgery or were

humanely euthanized for small or large bowel obstruction, strangulation or distension. Samples

were also taken from 9 horses which were humanely euthanized for reasons other than

gastrointestinal disease or systemic disease.

Procedure: Specimens were collected at surgery from intestine involved in the primary lesion,

distant to the primary lesion, or at necropsy from several sites including the primary lesion.

Tissues were fixed, serially sectioned and stained with hematoxylin and eosin (H&E) and for

apoptosis by the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling

(TUNEL) technique. The number of apoptotic cells per high power field was counted in the

mucosa, circular muscle, longitudinal muscle and serosa.

Results: Apoptotic staining nuclei were seen in all layers of intestine. An increased number of

apoptotic cells were found in the circular muscle of the intestine from horses with simple

obstruction. Intestine distant from the primary strangulating lesion had higher numbers of

apoptotic cells than intestine distant from a simple obstruction lesion or intestine taken at the site

of a strangulating or simple obstructive lesion.

Conclusion: Intestine from horses with obstructing or strangulating lesions in the small intestine

and large colon has increased numbers of apoptotic cells, possibly due to ischemic cell injury and

subsequent inflammation. Further investigation is required to determine whether increased

apoptosis affects intestinal function.

68

Introduction

Gastrointestinal disease is the cause of high morbidity and mortality in the horse.

Strangulating obstruction of the small intestine and large colon is a frequent cause of the acute

abdominal disease requiring surgery.1-3 Even with surgical intervention, the overall prognosis

for long-term survival of horses with a strangulating lesion of the small intestine or large

intestine is guarded, with long-term survival rates of 68% 4 and 24% 5. Experimental evidence

suggests that one reason for morbidity and mortality in horses with obstructive diseases is

reperfusion injury.6,7 Mucosal and serosal damage which occurs during ischemia is exacerbated

during reperfusion.7,8 To date, intestinal ischemia-reperfusion injury has been largely attributed

to cellular necrosis, although apoptosis has recently been recognized as having an important role

in both ischemia and reperfusion injury.9-10

Apoptosis, a distinct form of cell death, is a natural physiologic mechanism for the

removal of cells which are not needed, with minimal tissue inflammation. Apoptosis has an

important role in embryogenesis, tissue homeostasis, lymphocyte development and function and

tumor regression.11

Apoptotic cells have been observed following experimental ischemia-reperfusion injury

to the brain, heart, adrenals, liver, and kidneys of other species.9 In the gastrointestinal tract,

apoptosis can be associated with the maintenance of homeostasis and shedding of cells from the

villous tips in the small intestine, or with pathological states such as intestinal adenocarcinomas,

inflammatory bowel disease and radiation induced gut damage.12 Apoptotic cells can be

detected using the terminal deoxynucleotide transferase mediated dUTP nick end labeling

(TUNEL) technique. This technique distinguishes apoptotic from necrotic cells by specifically

detecting DNA cleavage associated with nuclear change during apoptosis. This technique has

69

been shown to be effective in the detection of apoptosis in the formalin fixed samples of human

large intestine. 13

The objectives of this study were to 1)determine if apoptosis could be identified in the

equine intestine using the TUNEL method, 2)to identify the type(s) of cells undergoing apoptosis

and, 3)to determine whether the occurrence of apoptotic cells was affected by gastrointestinal

disease and tissue layer of the intestine. We hypothesized that equine intestine directly or

indirectly affected by naturally occurring simple or strangulating obstruction would have

increased numbers of apoptotic cells.

Materials and Methods

Samples of intestine from thirty eight horses (gastrointestinal disease group) that

underwent abdominal surgery or were humanely euthanized for small or large bowel

strangulation, obstruction or distension were collected. Intestine from 5 horses (musculoskeletal

group) which were humanely euthanized, due to acute or chronic orthopedic conditions including

capital physis fracture (1), chronic bilateral degenerative suspensory desmitis (1), neoplasia of

the right mandible (1), cervical vertebral malformation (1), and comminuted second phalangeal

fracture (1) was sampled for comparison. Samples of small intestine, large colon, cecum and

small colon were also obtained from 4 normal horses (control group) which had no clinical signs

of gastrointestinal disease, had not received systemic medication for at least 6 months, and were

donated for other reasons including behavioral problems (2), osteochondrosis of the left

tarsocrural joint and behavioral problems (1), or cutaneous sarcoids (1).

Intestinal specimens were collected at surgery or at necropsy and consisted of one sample

per designated region. Samples collected at surgery were full thickness biopsies from resected

70

bowel, from the enterotomy site in the small intestine or large colon or from the edge of bowel

segments that were incorporated in an anastomosis. When possible samples were taken at the

primary lesion and at sites distant to the lesion. Samples collected at necropsy were collected

immediately after death (within 5 minutes post lethal injection with concentrated pentobarbital)

from the primary lesion and at various sites proximal and distal to the lesion. For the nine horses

with no gastrointestinal disease, samples (one specimen from each site) were taken from the

duodenum, mid jejunum ileum, cecum, large colon and small colon. All samples were

immediately placed in 10% neutral buffered formalin, fixed for a minimum of 48 hours,

dehydrated, and then embedded in paraffin. Serial sections of each specimen were cut and

stained with hematoxylin and eosin (H&E) and with the TUNEL technique for apoptotic cells.

Apoptotic cells were detected by in situ DNA end labeling (TUNEL method) 13 using the

Apoptag® In Situ Apoptosis Detection Kit (Intergen Co., Purchase, NY). All reagents used were

contained in the Apoptag® kit unless otherwise specified. Unstained tissue intestinal sections

were deparaffinized and transferred to phosphate buffered saline (PBS) for 5 minutes. The tissue

sections were pretreated with 20µg/ml Proteinase K ( Sigma, St. Louis, MO ), washed in dH20,

quenched with hydrogen peroxide (3.0%), covered in 75µg/ml equilibration buffer and then

incubated with 55µg/ml terminal deoxynucleotidyl transferase (TdT enzyme) for 3 hours in a

humidified chamber. The reaction was terminated with stop wash buffer and the tissue sections

were incubated with 65µg/ml anti-digoxigenin conjugate for 30 minutes in a humidified chamber

and then washed with PBS. The tissue sections were stained with 75µg/ml diaminobenzidine

(DAB), washed and then counterstained with methyl green and mounted.

As a detection control for the TUNEL technique, sections of thymus were taken from

untreated (negative control) and mice treated with dexamethasone (positive control mice).

71

Dexamethasone is known to induce apoptosis in the thymic lymphocytes. 14-16 Thymus samples

were stained simultaneously with intestinal samples to ensure the staining technique was

effective and that apoptotic cells were being detected. Stained slides were examined in a blinded

manner and scored for the presence or absence of apoptotic cells (see below). Hematoxylin and

eosin stained sections were reviewed to identify the specific cell types undergoing apoptosis in

the various locations by comparison with TUNEL stained slides.

In each specimen the apoptotic cells located in the mucosa, circular muscle, longitudinal

muscle and serosa were counted in three high power fields at 400X magnification. The mean

number of apoptotic cells in each tissue layer was calculated for all samples. Samples were

assigned to one of four diagnostic categories for statistical analysis- simple obstructed,

strangulated, musculoskeletal and control. The data for the samples was log transformed to

stabilize the variances. The data for the samples was back transformed for presentation. The

MIXED procedure of the SAS system (ver. 8.2, SAS Institute Inc., Cary, NC 27513) was used to

perform a split plot analysis of variance to test for effects of colic, intestine and tissue layer and

their interactions. Gastrointestinal disease (colic) was the whole plot factor, type of intestine

(small intestine or large intestine) was the subplot factor and tissue layer (mucosa, circular

muscle, longitudinal muscle and serosa) was the subplot factor in the analysis of variance.

Significant interactions were further investigated using the SLICE option to test for simple main

effects. Simple contrasts were used to compare means with simple main effects. Small colon

and cecum samples were not included in the analysis, because the number of these horses

recovered from anesthesia did not include a representative number of these intestine types across

all categories (including simple obstructed, strangulated, musculoskeletal and control categories).

72

Results

Samples were collected from 43 clinical cases and from four horses donated to the

Veterinary Teaching Hospital. One intestinal sample was taken in 22 horses, and a sample from

more than one location in the intestinal tract was taken from 25 of the horses. Of 106 samples

collected, 50 were from the small intestine, 38 from the large intestine, 10 from the cecum, 4

from the small colon and 4 from the stomach. The numbers of intestinal sections examined in

the disease categories are as follows: simple obstruction (14), peritonitis (4), strangulation

obstruction (23), distant to strangulation (different intestinal segment) (12), distant to

strangulation (same intestinal segment) (6), no gastrointestinal disease (chronic musculoskeletal)

(8), distant simple obstructed (other intestinal segment) (2), adjacent to strangulated lesion (4),

inflammatory (same intestinal segment) (1), no gastrointestinal disease (other) (6), control (no

systemic medication) (24).

Control slides for the TUNEL procedure reacted properly and the procedure was

considered to provide valid and reproducible staining of equine intestinal samples. Apoptotic

cells were evident in all 4 layers of intestine, except in five horses with no clinical evidence of

gastrointestinal disease (one in the musculoskeletal category and four in the control category).

One slide was unreadable due to the very poor condition of the devitalized intestine. Apoptotic

nuclei were observed in both the capillaries and connective tissue of the lamina propria,

predominately at the tip of intestinal villi, and in the mucosal epithelial cells (Figures 1 & 2).

Apoptotic nuclei were observed in circular (Figures 3 & 4) and longitudinal muscle. Apoptotic

nuclei were also identified in the connective tissue of fascial planes between groups of muscle

fibers. Schwann cells, glial cells or activated satellite cells and neurons were also apoptotic in

five of the samples of intestine in horses with gastrointestinal disease(Figure 5); however the

73

number of affected cells in the ganglia was not counted. Apoptosis was rare in the serosa but did

occur in the endothelial cells in the small vessels and in the mesothelial cells. (Figure 6)

Apoptotic neutrophils in the serosa and other regions of intestine were uncommon. In the

musculoskeletal group no apoptotic cells were identified in one horse with a left hind second

phalanx fracture, but apoptotic cells were identified in the other four horses of this group. The

horse with severe degenerative suspensory desmitis had generalized apoptosis; the horse with a

chronic capital physis fracture had apoptosis only in the cecum; the horse with cervical vertebral

malformation had apoptosis predominately in the large colon; whereas and one with a tumor on

the mandible had apoptosis predominately in the small intestine. Very small numbers of

apoptotic cells (no more than 10% of the number of apoptotic cells present in the horses with

gastrointestinal disease) were identified in the samples from the four control horses.

Horses with gastrointestinal disease (colic) had a significantly greater number of

apoptotic cells (P<0.007) when compared with the horses with musculoskeletal disease and the

control horses. There was a statistically significant difference in the number of apoptotic cells in

the different tissue layers (P<0.004) over all the categories. There was a significant three way

interaction between colic, tissue and intestine was found (P<0.003). The test for simple main

effects revealed a significantly higher number of apoptotic cells in the circular muscle of the

intestine in horses with simple obstruction compared to the other tissue layers in the

strangulation obstruction, musculoskeletal and control categories (P<0.009). (Figure 7) There

was also a significantly higher number of apoptotic cells in the large colon compared with the

small intestine for all categories (P<0.001). A significantly increased number of apoptotic cells

were found in the intestine at a distant site to the primary lesion in the horses with strangulation

obstruction compared to those with simple obstruction and compared to the intestine taken at the

74

primary strangulating obstruction lesion or simple obstruction lesion. (P< 0.019)(Figure 8) A

trend for a higher number of apoptotic cells in the circular muscle compared to the other tissue

layers was found in the horses with a simple obstruction or strangulating lesion of the intestine.

(Figure 9)

Discussion:

Cellular death occurs in two biochemically and morphologically distinct ways; cellular

necrosis and apoptosis. Apoptosis results from activation and transcription of specific genes.

In many of the pathways that are initiated in apoptosis, permeabilization of the mitochondrial

membrane is a critical event that results in release of various molecules from the mitochondrial

intermembrane space.17 These molecules include procaspases (enzymes), cytochrome c (a

caspase activator), Smac/Diablo (a caspase co activator) and an apoptosis- inducing factor.17 The

apoptosis inducing factor activates the nucleases that cleave the DNA into small fragments.17

The DNA strand breaks that result from the endonuclease(s) activation can be labeled in situ, in

individual fixed, permeabilized cells or tissues in sections, by the terminal deoxynucleotide

transferase mediated dUTP nick end labeling (TUNEL) technique.18,19 In this technique, the 3’-

OH termini that are generated as a result of the DNA fragmentation during apoptosis are labeled

with modified nucleotides by terminyl deoxynucleotide transferase. 20 This enzyme selectively

detects apoptotic rather than necrotic cells and is more specific than DNA polymerase.20 There

is a high correlation between TUNEL staining and apoptosis.20,21 The TUNEL technique had

greater sensitivity when it was compared to the in situ nick translation (ISNT).22 A major

advantage of the TUNEL technique is the ability to reveal early DNA breaks during apoptosis

which occur before the light microscopic changes characteristic of apoptosis. The TUNEL

75

technique using the same kit as was used in this study has been very successful in detecting

apoptosis of lymphocytes and neutrophils in the lamina propria of fixed human intestinal

samples.13

A potential concern of detecting apoptosis in formalin fixed tissue was that of random

DNA damage with long term fixation, resulting in nonspecific labeling. This effect has been

investigated in rat testicular tissue. Results indicated that formaldehyde is able to replace

protons in the purine and pyrimidine bases but does not react with sugar hydroxyls or phosphate

ester groups in nucleic acids.23 It is possible that with prolonged fixation any single-stranded

regions of DNA, or ‘free ends’ are locked up and they are inaccessible to the polymerase.23 Any

damage resulting from fixation or cytopathological mechanisms such as necrosis would not be

detectable with this technique.23 Therefore there is the risk of a false negative result rather than a

false positive result. Apoptotic cells may have been missed in this study, but the positively

stained cells are unlikely to be the result of any mechanism other than apoptosis.

There were significantly higher numbers of apoptotic cells in the circular muscle from

the intestine in horses with simple obstructive gastrointestinal disease compared to the control

and musculoskeletal groups. The difference in the pathophysiology of simple obstruction and

strangulation may explain increased apoptosis found in intestine during simple obstruction. The

mitochondrial PT pore can initiate apoptosis or necrosis, and the induction of the pore in a

sequential fashion, allows for the activation and action of proteases which will stimulate

apoptosis before ATP depletion results in cell death.24 However when the pore is activated

rapidly, as in ischemia, the cellular metabolism may be severely compromised and necrosis will

occur before the apoptosis- inducing proteases are activated.24 Caspase activation and the

intracellular ATP content are critical factors which determine whether a cell will undergo

76

necrosis or apoptosis.24 ATP is required for activation of cytochrome c which induces

apoptosis.24 If ATP is decreased, thereby inhibiting caspases which activate the apoptotic

process, cells will undergo necrosis.24 If glycolytic substrates can provide an alternative source

of ATP, then apoptosis is likely to occur. Though speculative, intestinal hypoxia during

strangulation may cause this intracellular mechanism to induce more cells to undergo necrosis,

whereas in the case of simple obstruction, inflammatory mediators may induce apoptosis when

there is sufficient ATP. This is not an all or nothing event as renal apoptosis after ischemia is

induced by hypoxia 25 and ATP depletion 26.

Apoptosis is a physiologic mechanism that allows cells to be eliminated without

associated tissue inflammation. In the intestine apoptosis plays a role in the maintenance of

normal gastrointestinal homeostasis 27 and apoptotic cells are ingested by phagocytic cells or are

shed into the lumen 28. While apoptosis occurring as part of the intestine’s normal function

should not cause inflammation, excessive apoptosis has been shown experimentally to cause

inflammation. For example in the rat, occasional apoptosis in the intestine does not cause an

alteration in the intestinal barrier integrity, however when intraperitoneal doxyrubicin is

administered there is an increase in mucosal apoptosis and a concomitant increase in epithelial

and endothelial permeability.12 Apoptosis is considered an important source of inflammation in

the kidney.29 During severe forms of glomerulonephritis apoptosis of the glomerular endothelial

cells may contribute to the development of glomerulosclerosis.30 In a murine model

administration of antiapoptotic agents at the time of reperfusion of the kidney following ischemia

prevented the early onset of apoptosis, inflammation and tissue injury.10 Glutamine deprivation

induced apoptosis in the rat intestinal epithelial cells and this subsequent disruption in

homeostatic balance between cell proliferation and cell death was proposed to contribute to gut

77

atrophy.31 The significant increase in numbers of apoptotic cells in the circular muscle of

intestine in horses with simple obstruction, and in intestine distant to a primary strangulating

obstruction lesion, suggests the apoptosis may be associated with intestinal inflammation.

It was interesting that there were significantly higher numbers of apoptotic cells in the

circular muscle of the intestine taken distant to the site of a strangulating lesion compared to that

taken distant to a simple obstruction lesion or at the site of either type of lesion. As an example

high numbers of apoptotic cells were present in the large colon of horses with a strangulating

obstruction of the small intestine. Increases in apoptotic cells at distant sites may be a result of

systemic inflammatory mediators resulting from the inflammation during ischemia and

reperfusion. Many mediators such as cytokines, growth factors, chemotactic peptides, hypoxia,

and acidosis which can activate leukocytes and can initiate cell apoptosis.32 For example in

human patients following elective surgery the interleukin 6 (IL-6) was notably increased at 24

hours after surgery and at this concentration inhibited neutrophil apoptosis. This effect was

attenuated with the addition of recombinant human interleukin 10 (IL-10). The authors from this

study concluded that imbalances of pro- inflammatory and anti- inflammatory cytokine release

affected the apoptotic capacity of the plasma.33 Ischemia/reperfusion following occlusion of the

superior mesenteric artery in the rat small intestine has been shown to induce apoptosis in the rat

jejunum and ileum.34 The exact mechanism of ischemia/reperfusion induced apoptosis in this rat

small intestine was not explained as the regulation of intestinal cell death and proliferation is

highly complex and is likely to be controlled by a variety of factors.35 Since free radicals have

been shown to cause apoptosis 36,37, it is reasonable to hypothesize that free radicals produced

during reperfusion in the equine intestine may stimulate apoptosis.34

78

Another possible explanation of the significantly increased apoptotic cells distant to a

strangulating lesion in intestine from horses undergoing surgery may be the effect of surgical

manipulation of the intestine. The intestine is known to be susceptible to stress at surgery and

studies have demonstrated that surgical trauma induces oxidative stress and damage through

enhanced production of reactive oxygen species.38 Surgical stress induced in the rat intestine

(induced by opening the abdominal wall and handling the intestine as done during exploratory

laparotomy) was shown to induce and activate protease activity in the villus and crypt cells.39

Both the mitochondrial and cytosolic proteases were activated in this study and free radicals

generated by xanthine oxidase were proposed as possible mediators of protease activation after

surgical stress in the intestine.39 The increased protease activity observed in the mitochondria

may be responsible for the mitochondrial permeability transition and mitochondrial dysfunction

leading to apoptosis.39 The intestine is highly susceptible to surgical manipulation at remote

locations and mild handling of intestine following laparotomy has been shown to cause oxidative

stress in the enterocyte and functional alterations in the intestine.40

There was trend for there to be higher numbers of apoptotic cells in the circular muscle of

the intestine from horses with either a simple obstructive or strangulating lesion. If the apoptosis

is associated with an increased inflammatory response in the muscle, it could affect the

contractility of the muscle. Contractility of equine intestine has been reduced experimentally by

inhibition of the circular muscle with nitric oxide in both jejunum41 and ventral colon. Recently

muscle injury (measured by creatinine kinase) resulting from 90-120 minutes of ischemia was

reduced by the pancaspase inhibitor z-VAD.10 This suggests tha t apoptosis which is blocked by

pancaspase inhibitor, is responsible for or associated with muscle inflammation and subsequent

injury.10 A better understanding of the relationship of intestinal muscle cell apoptosis and

79

intestinal inflammation may help development of new therapy to attenuate apoptosis in intestinal

muscle.

The locations of the different types of apoptotic cells in the intestine were recorded;

however the number of cells in the specific regions of the four intestinal layers was not

quantitated. Apoptotic cells were observed in the mucosa and large numbers were located within

the connective tissue of the lamina propria at the tip of the intestinal villi. Apoptosis has been

proposed to be associated with the shedding of the cells from the villus tips in the small intestine

12 and has been induced in the mucosa of the ileum and jejunum of rats following occlusion of

the superior mesenteric artery.34 In these rats apoptotic cells were most evident at the villus

tips.34 The apoptosis, which, increased during the ischemic period in the rat small intestine, was

maximal after one hour of reperfusion and then significantly decreased after only 6 hours

indicating, it was a transient event.34 Apoptosis was also observed in the muscle fibers of the

longitudinal muscle and within the fascial planes between the groups of muscle fibers. Further

investigation is required to determine whether this apoptosis in the muscle is detrimental to

motility.

Apoptosis of schwann cells, glial cells or activated satellite cells and neurons in the

myenteric plexus also suggests a possible relationship of apoptosis and bowel function. Since

the myenteric plexus is important for functional motility and for other gastrointestinal functions

such as secretion, apoptosis of cells in the enteric nervous system may alter intestinal function.42

Few apoptotic cells were observed in the serosa even though ischemia and reperfusion,

and the intraluminal distention and decompression, have been reported to cause severe

morphologic changes in the equine jejunal serosa.7 Both mesothelial cells and endothelial cells

were apoptotic in the serosa but appeared to be few in number compared to other layers. In this

80

study both the musculoskeletal and gastrointestinal disease groups of horses had large numbers

of apoptotic cells compared with the control group. Three of the five horses in the

musculoskeletal group had been treated with phenylbutazone for greater than or equal to 14 days

prior to euthanasia. Because phenylbutazone has been shown to cause an increase in apoptosis in

vitro in the equine right dorsal colon 43, the apoptosis present in this musculoskeletal group of

horses may have been related to the systemic inflammation or medication. The horses with

gastrointestinal disease in this study all received flunixin meglumine at variable times prior to

sample collection. Though it may be possible for flunixin meglumine to induce apoptosis in the

intestine of treated horses, this does not explain the differences in the number of apoptotic cells

observed between simple obstructed and strangulated intestine or those differences observed

cells from the primary lesion to those at a distant site. Further investigation is required to

determine the effect of non steroidal anti- inflammatory drugs on apoptosis in the equine

intestine.

In summary the TUNEL technique is an effective method for detecting apoptotic cells in

the equine intestine. The large numbers of apoptotic cells in the intestine of horses with

gastrointestinal disease may be important in the initiation of inflammation and subsequent

intestinal injury. The significant increase in apoptotic cells in intestine distant to strangulating

lesions may be very important clinically in understanding the post-operative response of horses

and possible complications following diseases causing ischemia. Specifically the increased

apoptosis in circular muscle and enteric nervous tissue in intestine which is not removed at

surgery may be important in post-operative bowel function. However further investigation is

required to understand the apoptotic events during gastrointestinal injury and to determine if

81

apoptosis is related to changes in intestinal function and inflammation seen with ischemia and

reperfusion.

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86

Figure legends:

Figure 1: Photomicrograph of the mucosa in the small intestine from a horse with

gastrointestinal disease. All dark staining nuclei represent apoptotic cells. Healthy or necrotic

cells did not stain. Diaminobenzidine (DAB) stain and methyl green counterstain; bar equals

50µ.

Figure 2: Photomicrograph of the mucosa in the small intestine from a horse in the control

group. No apoptotic nuclei are present. Diaminobenzidine (DAB) stain and methyl green

counter stain; bar equals 50µ.

Figure 3: Photomicrograph of the circular muscle in the small intestine from a horse with

gastrointestinal disease. All dark staining nuclei represent apoptotic cells. Healthy or necrotic

cells did not stain. Diaminobenzidine (DAB) stain and methyl green counterstain; bar equals

50µ.

Figure 4: Photomicrograph of the circular muscle in the small intestine from a horse in the

control group. No apoptotic nuclei are present. Diaminobenzidine (DAB) stain and methyl

green counter stain; bar equals 50µ.

Figure 5: Photomicrograph of a myenteric plexus in the large colon. All dark staining nuclei

represent apoptotic cells. Healthy cells or necrotic cells did not stain. Neurons, glial cells and

Schwann cells were apoptotic in this myenteric plexus. Diaminobenzidine (DAB) stain and

methyl green counterstain; bar equals 50µ.

87

Figure 6: Photomicrograph of the serosa from the large colon. All dark staining nuclei are

apoptotic mesothelial cells on the surface of the serosa. Healthy cells or necrotic cells did not

stain. Diaminobenzidine (DAB) stain and methyl green counterstain; bar equals 50µ.

Figure 7: Graph of the mean numbers of apoptotic cells in the intestine from horses in all 4

categories- simple obstruction, strangulation, musculoskeletal, control. A significant increase in

apoptotic cells was present in the simple obstruction category (P< 0.0088) which is indicated by

the star (*). Columns represent the geometric mean number of apoptotic cells while error bars

represent the 95% confidence intervals.

Figure 8: Graph of the mean number of apoptotic cells in intestine taken from the primary lesion

or at a distant site in horses with simple obstruction and strangulation. A significant increase in

apoptotic cells was present at a distant site to a strangulation lesion (p< 0.0186) which is

indicated by the star (*). Columns represent the geometric mean number of apoptotic cells while

error bars represent the 95% confidence intervals.

Figure 9: Graph of the mean number of apoptotic cells in the four tissue layers from horse with

simple obstruction or strangulation. Columns represent the geometric mean number of apoptotic

cells while error bars represent the 95% confidence intervals.

88

FIGURES Figure 1: Jejunal mucosa with apoptotic cells from horse with GI disease

Figure 2: Jejunal mucosa with no apoptosis from control horse

89

Figure 3: Jejunal circular muscle with apoptotic cells from horse with GI disease

Figure 4: Jejunal circular muscle with no apoptosis from control horse

90

Figure 5: Apoptotic cells in myenteric plexus from horse with GI disease

Figure 6: Apoptotic cells in serosa from horse with GI disease

91

Figure 7: Graph of number of apoptotic cells in intestine from horses in the 4 categories of disease.

0

5

10

15

20

25

30

35

Simple obstructed Strangulated Musculoskeletal Control

Disease Category

Mea

n N

umbe

r of

Apo

ptot

ic C

ells

*

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Figure 8: Graph of number of apoptotic cells in intestine taken from the primary site and distant to the site of strangulating and simple obstructive lesions.

0

5

10

15

20

25

30

35

40

45

Distant site Lesion site

Site of sample collection

Mea

n n

um

ber

of

apo

pto

tic

cells

Simple obstructionStrangulation

*

93

Figure 9: Graph of number of apoptotic cells in the 4 layers of intestine over all disease categories.

0

5

10

15

20

25

30

35

40

45

Mucosa Circular Muscle LongitudinalMuscle

Serosa

Tissue

Mea

n n

um

ber

of

apo

pto

tic

cells

94

Figure 10: Thymus stained for apoptotic cells from mouse not treated with dexamethasone

Figure 11: Thymus stained for apoptotic cells from mouse treated with dexamethasone

95

Figure 12: Jejunal circular muscle stained for apoptosis with DAB on the left and with H&E on the right to allow identification of apoptotic nuclei.

96

TABLES Table 1: The ANOVA for split-plot with colic, intestine, and tissue as factors.

Results of Type 3 Tests of Fixed Effects.

Effect Num

DF Den DF

F Value Pr>F

Colic 3 48 4.59 0.0066 Tissue 3 160 4.66 0.0037 Colic*Tissue*Intestine 9 160 2.89 0.0034 Table 2: The ANOVA for split-plot with colic, intestine, and tissue as factors.

Results of The Mixed Procedure Tests of Effect Slices.

Effect Colic Tissue Intestine Num

DF Den DF

F Value

Pr>F

Colic*Tissue*Intestine Simple obstructed

Mucosa 1 160 2.39 0.1241

Colic*Tissue*Intestine Simple obstructed

Circular muscle

1 160 7.04 0.0088

Colic*Tissue*Intestine Simple obstructed

Longitudinal muscle

1 160 2.47 0.1180

Colic*Tissue*Intestine Simple obstructed

Serosa 1 160 3.24 0.0740

Tissue*Intestine Small intestine

3 160 1.06 0.3683

Tissue*Intestine Large intestine

3 160 5.99 0.0007

97

Table 3: The ANOVA for split-plot with affected (code for intestine at primary

site of the lesion compared with intestine at a distant site), colic, intestine, and

tissue as factors. Results of the Mixed Procedure Tests of Effect Slices. Effect At primary lesion

or distant site Num DF

Den DF

F Value Pr>F

Colic*affected Distant site 1 48 5.93 0.0186 Colic*affected Primary site 1 48 1.97 0.1671

98

CONCLUSION

Apoptotic cells were detected in the equine intestine using the TUNEL method. This

method was successful in detecting the apoptotic nuclei which stained a distinctive brown color.

Horses with gastrointestinal disease had a greater number of apoptotic cells compared to horses

with no gastrointestinal disease. Horses with simple obstructive disease had a higher number of

apoptotic cells in the circula r muscle compared to the other tissue layers in horses with

strangulation or no gastrointestinal disease. There was also a higher number of apoptotic cells in

the large colon compared to the small intestine for all the samples. There was a trend for a

higher number of apoptotic cells in the circular muscle of intestine over all. This finding of

increased apoptosis in the presence of disease may be of clinical importance as apoptosis has

been shown to be associated with inflammation and tissue injury in other species both in the

gastrointestinal system and other body systems.

There were a significantly higher number of apoptotic cells found in intestine distant to

the primary lesion in horses with strangulating obstruction compared to those with simple

obstruction and compared to the intestine taken at the primary strangulating obstruction lesion or

simple obstruction lesion. This indicated that intestine which is not directly affected by a lesion

has increased apoptosis which may be the result of systemic inflammatory mediators or free

radical production. This is may be important clinically as the intestine which remains following

a resection anastomosis for a strangulating lesion has increased apoptosis based on this finding.

Further investigation is required to determine if increased apoptosis causes inflammation and

tissue injury and affects intestinal function. As apoptosis can be controlled at the mitochondrial

level there may be potential therapeutic implications for the future. Apoptosis and the associated

inflammation has been inhibited in human smooth muscle and bovine and murine kidney.

99

VITA

Name : Emma Lucy Rowe

Date of Birth: 02/20/75

Tertiary education: Murdoch University School of Veterinary Studies 1992-1997

Bachelor of Science 1995

Bachelor of Veterinary Medicine and Surgery (Hons.) 1997