Pathological and clinicopathological studies on ... (1-26).pdfEgypt. J. Comp. Path. & Clinic. Path....

Egypt. J. Comp. Path. & Clinic. Path. Vol. 21 No. 2 (April) 2008; 1- 26

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Pathological and clinicopathological studies on reperfusion of ischemic

intestine

By A. H. Osman*; M.M. Bashandy**; Noha M.Said**; W.S. Elghoul***

*Department of Pathology; ** Department of Clinical Pathology; *** Department of Surgery, Faculty of Veterinary Medicine Cairo University

SUMMARY The present work was carried on dogs as experimental model to assess the

histological and clinicopathological changes that associated with ischemia-reperfusion injury of intestine.

Serum biochemical changes with correlation to histopathological alterations as a result of induction of ischemia-reperfusion were clarified.

A total 15 healthy male mongrel dogs of weighing 9 to 12 kg and ageing one to five years old were allocated into 3 groups according to the pathway of induction of intestinal ischemia.

Blood samples were taken from dogs before and after induction of ischemia-reperfusion injury for determination serum biochemical and blood coagulation changes. A significant hypercoagulability in all groups at the end of ischemic and reperfusion times were recorded. Serum biochemical changes associated with IRI in intestine revealed a significant increase in (ALT, AST, ALP, LDH and AMYL), enzymes activities with ischemic and reperfusion times, when compared with their activity at zero-time. Blood urea, creatinine and glucose determination showed significant increase in their concentration during ischemia and reperfusion times. Also C-reactive protein revealed a significant increase in their titer with all groups at the end of ischemic time and with reperfusion time.

Ischemia/ reperfusion lesions of intestinal region revealed congestion, hemorrhages and cyanosis associated with damage of intestinal epithelium and serosal layer with different grades. The histological findings demonstrated severe destruction of intestinal segment after reperfusion in comparison with ischemia. On the other hand, ischemia/ reperfusion were reflected on hepatic and renal parenchyma with different grades according to ischemic pathway. Keywords: Ischemia/ reperfusion – Colon - Coagulation changes- Serum biochemical changes- Gross Pathology – Histopathology.

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Prof. Dr. Mahmoud Samy Ahmed Professor of Pathology, Fac. Vet. Med., Cairo Univ.

Prof. Dr. Rawhia M. Emran Professor of Pathology, Animal Health Research Institute, Dokki


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INTRODUCTION

schemia means failure of blood supply or inadequate blood flow

to an area of tissues (Guido and Isabelle, 1996). It is a dual defect of oxygen deficit and carbon dio-xide excess (Benjaminute et al., 1993) and it is a state existing when an organ or tissue has it's arterial perfusion lowered relativ-ely to it's metabolic need (Neville, 2000). It occurs when blood flow to an organ or tissue has ceased "complete ischemia" or is abnorm-ally low "partial ischemia" (Roder-ick and Keith, 1992).

The chief causes of arterial local ischemia are usually, (1)-occlusion of the arterial supply, (2)- narrowing of arterial lumen, or (3)- mechanical external pressure upon an artery (Ritchie, 1990).

Reperfusion injury is tissue injury that occurs following re-establishment of circulation after an ischemic event (Rustin et al., 1994). Reperfusion is the treatment of choice to save viable tissue foll-owing acute ischemia of a vascular territory, but reperfusion of ische-mic tissue is associated with local and systemic leukocytic activation and trafficking "specially neutroph-il" (Danielle et al., 2005).

Reperfusion injury occurs aft-er restoration of blood flow subse-quent to an episode of ischemia

(Rustin et al., 1994). Reperfusion of ischemic tissues can lead to sev-eral complications that may worsen the ischemic lesion and produce sy-stemic alterations and a life threate-ning situation (Marcelo and Wins-ton, 2005). Reperfusion injury of ischemic tissue represents an acute inflammatory response that can cause significant morbidity and mortality (Ming Zhang et al., 2004).

Bowel ischemia is a common complex disorder with various pri-mary causes, clinical presentation, high mortality rate and it account for at least 3% of human deaths in USA. In horses ischemic gastroint-estinal disease is the most common cause of death; especially acute mesenteric thrombosis carries high morbidity and mortality rate so any delay in diagnosis or treatment ag-gravates the patients out come (Hung et al., 1999). Acute mesent-eric ischemia is life threatening vascular emergency, due to inadeq-uate tissue perfusion so it requires early diagnosis, intervention to ad-equately restore mesenteric blood flow and prevent bowel necrosis and patient death (Upendra et al., 2005).

One of the most common cau-

ses of intestinal ischemia are mech-anical obstruction which occur ma-inly due to, hernial strangulation, volvulus "twisting of intestinal

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segment" and intussusception. In the latter form excessive peristaltic contraction drives the affected seg-ment of bowel immediately distal lies in a sleeve like manner over it and some of the mesentery is inclu-ded in the portion of bowel that is pushed forward and the resulting local edema lead to local ischemia (Neville, 2000).

Intestinal ischemia reperfusi-on injury may lead to intestinal barrier dysfunction, resulting in ba-cterial translocation, which can le-ad to adult respiratory distress syn-drome and sepsis. The most impor-tant side effect of ischemia reperf-usion injury are multiple organ dysfunction, which may lead to death (Upendra et al., 2005).

The purpose of this study was to determine the degree intestinal damage during ischemia and reper-fusion as well as liver and kidney tissue reaction. Coagulation chang-es, serum biochemical and C- reactive protein alterations were to be clarified. MATERIALS AND METHODS Animals

his study was done on fifteen healthy male mongrel dogs,

weighing 9 to 12 kg and ageing one to five years old. The dogs were housed in a separate kennels, and kept under observation for 14 days prior to the day of operation.

Experimental design:- A total of 15 male dogs were

subjected to induction of intestinal ischemia and reperfusion. The animals were classified into three groups each one contained five animals for induction of (I/RI) through three different pathways.

Surgical procedures:- Anesthesia: dogs were premedicat-ed with 0.04 mg/Kg atropine sulph-ate (S/C) and 0.1-0.5 mg/Kg diazp-am (I/M), induction of anesthesia was performed by injection of sodium thiopental 2.5 %solution. Group (1): Cranial mesenteric

artery (CMA) occlusion Five dogs were subjected to 2

hours of CMA occlusion and 2 ho-urs of reperfusion. After ischemic period the plastic clips were rem-oved and reperfusion allowed for 2 hours.

Group (2):Segmental ligation

Five dogs were subjected to 2 hours of segmental ligation and 2 hours reperfusion “ligation release-ed” .A 30-cm of jejunal segment; a 30-cm of colon segment and their arteries and veins were identified and isolated. Each segment was ligated by using a plastic clips placed at each end of and their arteries and veins were identified and isolated.

Group (3): Intraluminal distention

and decompression

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Five dogs were subjected to 2 hours of intraluminal distention and 2 hours of decompression. A 30 cm of jejunal segment was occluded by using a plastic clips placed at each end. Sterile Ringer's lactated solution was infused into the lumen to induce an intraluminal distention. After ischemic period the plastic clips were removed and reperfusion allowed. Sampling A- Blood samples:-

Five blood samples were coll-ected from abdominal aorta from each animal before operation and one & two hours from induction of ischemia as well as perfusion. The blood samples were divided into two parts. The first part was antico-agulated by sod. citrate solution 3.8% (1.8 ml blood +0.2 ml sod. citrate solution), then centrifuged at 2000 rpm for ten minutes for plas-ma separation and was used for pe-rforming the coagulation paramet-ers. The second one collected in plain centrifuge tube and was allo-wed to clot, then centrifuged at 2000 rpm for ten minutes for serum separation.

I-Coagulation studies: a) Prothrombin time (PT):- measu-

rement the time of fibrin clot formation is according to Moll and Ortel (1997).

b) Activated partial thromboplastin time (APTT):- measurement the

time of fibrin clot formation is according to Poller and Thom-son, (1992).

II-Serum studies: 1. Alanine and aspartate amino

transferases (ALT and AST) : Colorimetric determination of

ALT and AST activities was perf-ormed according to Reitman and Frankel (1957). 2. Alkaline phosphatase (ALP):

Colorimetric determination of alkaline phosphatase activity was done according to Tietz (1986).

3. Lactate dehydrogenase (LDH): The principle of the test was

depending on the idea that the LDH specifically catalyzes the oxi-dation of lactate to pyruvate with subsequent reduction of NAD to NADH. The rate at which NADH forms is proportional to LDH activ-ity. The method described determ-ines NADH absorbance increase per minute at 340 nm (Kachmar and Moss, 1976).

4. Amylase (AMYL): Colorimetric determination of

amylase activity was done accord-ing to Marshall (1980).

5. Blood urea: Blood urea was measured cal-

orimetrically at wave length 578 nm was done according to (Searcy, et al., 1967).

6. Serum creatinine: Serum creatinine was determi-

ned according to Henery (1968).


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7. Blood glucose: Colorimetric determination of

glucose was determined according to Howanitz (1984) 8. C-reactive protein (CRP):

CRP was determined semi-quantitatively by rapid latex agg-lutination test according to Fischel (1967). III. Statistical analysis of data:

All numerical data were stati-stically evaluated for the mean and standard error for each group. The significance of the results was dete-rmined by conducting the least sig-nificance difference between diff-erent times outlined by Sendecor and Cochran (1989). IV. Histopathological examination:

Tissue specimens were taken from mucosa and serosa of colon segment at the end of ischemic ti-me and at the end of reperfusion. Liver and kidney specimens were taken at the end of reperfusion time. All specimens were fixed in 10% neutral buffer formalin. The fixed specimens were then trimm-ed, washed and dehydrated in asce-nding grades of alcohol, cleared in xylene, embedded in paraffin, sect-ioned at 4–6 µ thickness and stai-ned with haematoxylene and eosin Bancroft (1994). Histological, gra-ding for epithelial damage of intes-tinal mucosa was assessed as desc-ribed by Park et al. (1991), table (1). Hepatic injury was estimated using an ordinal scale modified from Camargo et al. (1997) table (2).

Table (1) Park's histological grading of intestinal epithelial damage:

Grade Description

0 Normal mucosa

1 Subepithelial space

2 Extended Subepithelial space

3 Epithelial lifting along villous side

4 Denuded villi

5 Loss of villous tissue

6 Crypt layer infarction

7 Transmucosal infarction

8 Transmural infarction


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Table (2): Histological grading of liver injury

Grade Description 0 No apparent injury on light microscopic examination I Hepatocytes swelling II Cytoplasmic vacuolization, nuclear pyknosis, apoptosis III Focal necrosis IV Massive necrosis of whole hepatic cords & hemorrhages

RESULTS

I- Coagulation changes: esults of the coagulation study are illustrated in table (3).

Intestinal groups showed signific-ant prolongation in PT and PTT

time at the end of ischemic and reperfusion times, associated with a significant increase in INR ratio at the end of ischemic time.

Table (3): *Values of coagulation changes during intestinal I/R in different groups

Group Times PT (seconds)

PTT (seconds)

INR Ratio

Zero-time 12.67 ± 0.85a 19.00 ± 1.08a 1.11 ± 0.01a 1 hr ischemia 30.50 ± 0.20b 30.00 ± 0.41b 2.93 ± 0.02b 2 hr ischemia 60.00 ± 0.00c 60.00 ± 0.00c 6.65 ± 0.06c

1 hr reperfusion 60.00 ± 0.00c 60.00 ± 0.00c 7.10 ± 0.04e

Cranial Mesenteric

Artery occlusion (CMA) 2 hr reperfusion 60.00 ± 0.00c 60.00 ± 0.00c 7.77 ± 0.13f

Zero-time 11.75 ± 0.63a 21.33 ± 0.62a 1.03 ± 0.04a 1 hr ischemia 15.33 ± 0.47b 26.33 ± 0.85b 1.39 ± 0.0b 2 hr ischemia 20.00 ± 0.82c 60.00 ± 0.00c 1.89 ± 0.10c

1 hr reperfusion 60.00 ± 0.00d 60.00 ± 0.00c 5.58 ± 0.05d

Segmental ligation

(SL) 2 hr reperfusion 60.00 ± 0.00d 60.00 ± 0.00c 7.63 ± 0.15e

Zero-time 12.67 ± 0.47a 12.33 ± 0.24a 1.11 ± 0.04a 1 hr ischemia 15.67 ± 0.62b 14.67 ± 0.47b 1.42 ± 0.07ab 2 hr ischemia 18.00 ± 0.41c 18.67 ± 0.47c 1.64 ± 0.03b

1 hr reperfusion 60.00 ± 0.00d 60.00 ± 0.00d 5.65 ± 0.18c

Dilatation Decompression

(DD) 2 hr reperfusion 60.00 ± 0.00d 60.00 ± 0.00d 7.39 ± 0.20d

*Values represent mean ± S.E. Mean with different alphabetical letters are significantly difference

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II-Biochemical Parameters:- Statistical analysis of bioch-

emical parameters of canine model subjected to intestinal ischemia reperfusion are illustrated in table (4).

Assay of serum enzymes (ALT, AST, ALP, LDH and AMYL,) in intestinal ischemic gro-ups gave a significant increase in their activities with ischemic and reperfusion times, when compared with those of corresponding at zero- time.

The results of blood urea, cr-eatinine and glucose showed sign-ificant increase in their concent-ration during ischemia and reperf-usion times table (5).


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Table (4): *Changes in enzymatic activities during intestinal I/R in different groups

Group Times ALT (iu/L)

AST (iu/L)

ALP (iu/L)

LDH (iu/L)

Amylase (iu/L)

Zero-time 13.25 ± 2.29a 18.75 ± 1.38a 28.80 ± 1.76a 119.67 ± 3.27a 512.00 ± 9.34a 1 hr ischemia 18.25 ± 1.78a 22.50 ± 1.55a 35.95 ± 1.25b 176.93 ± 4.59b 597.25 ± 7.34b 2 hr ischemia 24.50 ± 1.44b 30.75 ± 1.65b 37.65 ± 0.92b 206.45 ± 5.90c 676.00 ± 5.40c

1 hr reperfusion 30.75 ± 0.63c 42.00 ± 2.83c 37.68 ± 0.93b 230.00 ± 2.45d 682.00 ± 11.97c

Cranial Mesenteric

Artery occlusion (CMA) 2 hr reperfusion 35.25 ± 1.75c 63.67 ± 3.27d 40.68 ± 0.78c 290.00 ± 3.67e 696.67 ± 7.32c

Zero-time 13.25 ± 1.75a 17.00 ± 2.12a 26.98 ± 0.96a 114.00 ± 4.92a 542.00 ± 13.18a 1 hr ischemia 19.25 ± 1.44b 27.50 ± 1.94b 30.73 ± 0.79b 157.45 ± 5.84b 609.50 ± 12.37b 2 hr ischemia 27.00 ± 2.39c 31.00 ± 2.68b 32.85 ± 1.90b 210.25 ± 5.82c 631.67 ± 6.98b

1 hr reperfusion 29.25 ± 0.48c 39.75 ± 2.95c 32.13 ± 0.72b 237.00 ± 5.16d 628.00 ± 11.52b

Segmental Ligation

(SL) 2 hr reperfusion 36.50 ± 1.55d 62.50 ± 3.47d 32.77 ± 1.50b 289.67 ± 2.87e 642.25 ± 11.54b

Zero-time 9.50 ± 0.87a 16.00 ± 1.96a 26.40 ± 1.34a 109.67 ± 2.72a 530.75 ± 5.48a 1 hr ischemia 16.00 ± 1.22b 21.00 ± 2.20a 29.13 ± 0.88a 166.00 ± 5.12b 599.00 ± 11.84b 2 hr ischemia 21.50 ± 1.32c 29.00 ± 2.04b 31.23 ± 0.79b 215.00 ± 2.68c 670.00 ± 9.39c

1 hr reperfusion 35.00 ± 1.63d 31.67 ± 2.05b 32.10 ± 1.14b 255.00 ± 2.04d 677.50 ± 6.12c


(DD) 2 hr reperfusion 36.50 ± 2.25d 46.33 ± 1.84c 34.83 ± 1.19c 286.50 ± 4.57e 660.50 ± 14.08c

*Values represent mean ± S.E. Mean with different alphabetical letters are significantly difference.


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Table (5): *Values of urea, creatinine & glucose change intestinal I/R

Group Times Urea (mg/dl)

Creatinine (mg/dl)

Glucose (mg/dl)

Zero-time 14.03 ± 1.16a 0.67 ± 0.03a 59.63 ± 5.08a

1 hr ischemia 25.75 ± 0.10b 0.70 ± 0.09a 79.27 ± 2.46a

2 hr ischemia 28.70 ± 2.48b 0.75 ± 0.07b 173.95 ± 5.74b

1 hr reperfusion 45.45 ± 0.59c 0.90 ± 0.04c 184.00 ± 9.80b

Cranial

Mesenteric

Artery

occlusion

(CMA) 2 hr reperfusion 44.90 ± 0.20c 1.08 ± 0.02d 197.50 ± 8.78b

Zero-time 17.75 ± 1.03a 0.57 ± 0.03a 83.40 ± 2.90a

1 hr ischemia 29.16 ± 1.68b 0.60 ± 0.04a 116.80 ± 2.94b

2 hr ischemia 29.16 ± 1.68b 0.72 ± 0.03b 119.50 ± 1.43b

1 hr reperfusion 29.33 ± 1.22b 0.91 ± 0.03c 165.00 ± 2.86c

Segmental

ligation

(SL)

2 hr reperfusion 36.67 ± 1.81c 0.96 ± 0.02d 182.93 ± 8.56c

Zero-time 15.73 ± 1.21a 0.70 ± 0.04a 67.67 ± 3.70a

1 hr ischemia 20.50 ± 0.41b 0.72 ± 0.03ab 77.80 ± 1.73a

2 hr ischemia 24.78 ± 0.95c 0.74 ± 0.03b 135.00 ± 5.72b

1 hr reperfusion 32.00 ± 1.63d 0.83 ± 0.04c 183.60 ± 0.99b

Dilatation

decompression

(DD)

2 hr reperfusion 33.67 ± 1.25d 0.86 ± 0.04c 187.83 ± 8.70b

*Values represent mean ± S.E.

Mean with different alphabetical letters are significantly difference. III-C-reactive protein changes:-

A significant increase in C - reactive protein titer noted in all intestinal groups at the end of

ischemic time and with reperfusion time table (6).


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Table (6): *Value of CRP changes during intestinal I/R

Group Times CRP (mg/µl) Zero-time 6.00 ± 0.00a

1 hr ischemia 7.50 ± 1.50b 2 hr ischemia 9.00 ± 1.70c

1 hr reperfusion 15.00 ± 1.70d

Cranial Mesenteric

Artery Occlusion

(CMA) 2 hr reperfusion 21.00 ± 1.70e Zero-time 6.00 ± 0.00a

1 hr ischemia 6.00 ± 0.00a 2 hr ischemia 7.50 ± 1.50b

1 hr reperfusion 10.50 ± 1.50c

Segmental Ligation

(SL) 2 hr reperfusion 15.00 ± 1.73d

Zero-time 6.00 ± 0.00a 1 hr ischemia 10.50 ± 1.50b 2 hr ischemia 13.50 ± 1.50c

1 hr reperfusion 15.00 ± 1.22d


(DD) 2 hr reperfusion 19.50 ± 1.50e

*Values represent mean ± S.E. Mean with different alphabetical letters are significantly difference. IV-Pathological results: A-Cranial mesenteric artery

occlusion: The gross lesions were recor-

ded after one hour from cranial mesenteric artery occlusion reveal-ed congestion of mesenteric blood vessels and oedema of intestinal wall. On the other hand, severe int-estinal congestion and hemorrhages from mesenteric borders and vess-els were demonstrated after 2 hours from occlusion with cyanosis of in-testinal wall (Plate, A-1). Histolog-ically, cranial mesenteric artery oc-clusion revealed subepithelial oed-

ema and shrinkage of intestinal villi grade (2). The lamina propria of colon segment showed hemorrh-ages and leukocytic infiltration ma-inly neutrophils, macrophages and lymphocytes (Plate, A-2). Sloughi-ng of serosal mesothelial lining of colon with intact basement membr-ane was noticed. The subepithelial connective tissue showed oedema with inflammatory cells infiltration mainly neutrophils. Focal hemorrh-agic areas were also seen (Plate, A-3).

Hemorrhages, swelling and cyanosis of colon were noticed


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after 1 hour from reperfusion. Mes-enteric hemorrhages and cyanosis were remarked after 2 hours from reperfusion (Plate, A-4). Histologi-cal section of colon revealed sube-pithelial oedema with atrophy of intestinal villi grade (2). The lami-na propria showed severe hemorrh-ages with leukocytic infiltration mainly neutrophils and macropha-ges (Plate, A-5). Serosal mesothel-ial lining was desquamated with intact basement membrane. The su-bepithelial connective tissue revea-led congestion, oedema, hemorrha-ges and inflammatory cells infiltr-ation mainly neutrophils and macr-ophages (Plate, A-6).

The pathological alterations in case of reperfusion of colon app-eared more severe in comparison with ischemic stage.

Liver after two hours of repe-rfusion showed perihepatitis chara-cterized by capsular and subcaps-ular infiltration with neutrophils and macrophages. The hepatocytes showed swelling, granular cytopla-sm and pyknotic nuclei grade (II). Some hepatocytes revealed intracy-toplasmic fat globules of variable size with peripheral eccentric nucl-ei (Plate, A-7).

Kidney after two hours of re-perfusion showed shrinkage of glo-merular tuft and widening of Bow-man's space. The proximal convol-uted tubules showed swelling of its

epithelial lining with intraluminal albuminus cast (Plate.A-8).

B-Segmental ligation: Hemorrhages and congestion

appeared from the mesenteric bord-ers and vessels of the ligated colon-ic segments after one hour of ligati-on. Colonic segments showed mo-derate congestion and hemorrhage with increase of its thickness after two hours from ligation (Plate, B-1). Histologically, the colonic muc-osa showed subepithelial oedema extended along villous side and atrophy of some intestinal villi grade (3). The lamina propria rev-ealed inactive intestinal glands and leukocytic infiltration mainly neut-rophils and macrophages (Plate, B-2). Colonic serosa after 2 hours of segmental ligation revealed desqu-amation of mesothelial lining with intact basement membrane. The su-bepithelial connective tissue show-ed oedema, congestion of blood ca-pillaries and inflammatory cells in-filtration mainly neutrophils (Plate, B-3).

One hour after release of lig-ation colon segment revealed hem-orrhages, congestion and cyanosis of colonic segment in comparison with non ligated segments. Severe congestion and hemorrhages were observed after 2 hours from ligat-ion released (Plate, B-4). Reperfus-ion of colon segment after 2 hours was associated with damage of colonic mucosa characterized by


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transmucosal infarction accompa-nied with severe hemorrhages and leukocytic infiltration grade (7). The lamina propria showed necro-sis of intestinal gland, hemorrhages and leukocytic infiltration (Plate, B-5). Colonic serosa showed slou-ghing of mesothelial lining. Oede-ma of subepithelial connective tiss-ue with marked dilation of lymph vessels as well as inflammatory ce-lls infiltration mainly neutrophils, macrophages and lymphocytes we-re observed (Plate, B-6).

Liver showed subcapsular le-ukocytic infiltration mainly neutro-phils and macrophages which exte-nded to the hepatic parenchyma. The hepatic lobules showed conge-stion of its sinusoids with few mononuclear cells infiltration. Sw-elling of hepatic cells with central pyknotic nuclei were seen. Some hepatocytes showed focal area of coagulative necrosis characterized by loss of cell details with presence of architecture outline grade (III) (Plate, B-7).

Kidney showed hypercellular-ity of glomerular tufts that included proliferation of endothelial and me-sangial cells with leukocytic infiltr-ation. The proximal and distal con-voluted tubules showed swelling of their epithelial lining with intralu-minal albuminus droplets (Plate, B-8).

C- Segmental dilatation / decom-pression:

Mild congestion was observ-ed after one hour from dilatation. Congestion of colon segment was increased after two hours from dilatation, with an engorgement of colonic arteries with blood (Plate, C-1). Histologically, colonic muco-sa showed sloughing of its epitheli-al lining and loss of villous tissue grade (4). The lamina propria was infiltrated with neutrophils and ma-crophages (Plate, C-2). Colon sero-sa after two hours of dilatation dis-played sloughing of some mesoth-elial cells. Oedema and leukocytic infiltration of subepithelial connec-tive tissue were observed. Moreov-er the inflammatory reaction was extended to the muscular layer wh-ich characterized by focal aggrega-tion of inflammatory cells inbetwe-en the muscle bundles mainly neut-rophils and macrophages, massive leukocytic infiltration of both sero-sa and muscular layers by numer-ous numbers of neutrophils were noticed (Plate, C-3).

One hour after decompressi-on, colon segment became more congested and hemorrhagic with severe engorgement of blood vess-els with blood in comparison with colon segment during dilatation. Hemorrhage and congestion was increased after two hours from decompression (Plate, C-4). Colon-ic mucosa demonstrated loss of int-estinal villi and oedema of subep-


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ithelial connective tissue grade (5). The lamina propria showed atrophy of intestinal gland with massive hemorrhages (Plate, C-5). Decomp-ression of colon segment after two hours was accompanied with desq-uamation of mesothelial lining with intact basement membrane. The su-bepithelial connective tissue show-ed oedema which extended to muscular layer. Inflammatory cells were infiltrated both serosa and muscular layers which consisted of neutrophils and macrophages (Pla-te, C-6).

Liver showed congestion of both central veins and sinusoids after two hours of colon segment decompression. Inflammatory cells were infiltrated the sinusoids and

also around the central veins which consisted mainly of neutrophils and macrophages. Hepatic lobules sho-wed disorganization of hepatic cor-ds, nuclear pyknosis and apoptosis of hepatocytes grade (II) (Plate, C-7).

Kidney revealed obvious his-tological reaction especially the co-rtical region. The glomerular tufts were dilated and occupied most of Bowman's capsule which also con-tained free red blood cells. The ren-al tubules showed necrobiotic cha-nges especially the proximal con-voluted tubules (Plate, C-8).


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Plate. (A) Fig.(1)Colon showing congestion and hemorrhages from mesenteric vessels were demonstrated after 2

hours from occlusion. Fig.(2)Intestinal villi of colon revealed grade (2) and the lamina propria showed hemorrhages and

leukocytic infiltration after 2 hours from occlusion (H&E X200). Fig.(3)Colon showing sloughing of serosal mesothelial lining oedema and inflammatory cells

infiltration subepithelial connective tissue after 2 hours from occlusion (H&E X200). Fig.(4)Colon showing mesenteric hemorrhages and cyanosis were remarked after 2 hours from

reperfusion. Fig.(5)Intestinal villi revealed grade (2) and the lamina propria showed severe hemorrhages with

leukocytic infiltration after 2 hours from reperfusion (H&E X200). Fig.(6)Colon showing desquamation of serosal mesothelial lining with congestion, oedema,

hemorrhages and inflammatory cells infiltration subepithelial connective tissue (H&E X200). Fig.(7)Hepatic lobule showing perihepatitis and hepatocytes revealed grade (II) (H&E X200). Fig.(8)Kidney showing shrinkage of glomerular tuft and swelling of tubular epithelial lining with

intraluminal albuminus cast (H&E X200).


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Plate. (B) Fig.(1)Colonic segments showing moderate congestion and hemorrhage after two hours from ligation Fig.(2)Colonic mucosa showing subepithelial oedema extended along villous side grade (3) (H&E

X200). Fig.(3)Colonic serosa after 2 hours of segmental ligation revealed desquamation of mesothelial lining

with oedema, congestion and inflammatory cells infiltration of subepithelial connective tissue (H&E X200).

Fig.(4)Reperfusion of colon segment after 2 hours showing hemorrhages, congestion and cyanosis. Fig.(5)Reperfusion of colon segment after 2 hours showing transmucosal infarction grade (7) (H&E

X200). Fig.(6)Colon showing desquamation serosal mesothelial lining and the subepithelial connective tissue

revealed congestion, oedema, hemorrhages and inflammatory cells infiltration (H&E X200). Fig.(7)Hepatic lobules showing perihepatitis and hemorrhage; hepatocytes revealed grade (III) (H&E

X200). Fig.(8)Kidney showing hypercellularity of glomerular tufts and swelling of tubular epithelial lining

with intraluminal albuminus cast (H&E X200).


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Plate.(C) Fig.(1)Colon segment after two hours from dilatation showing engorgement of colonic arteries with

blood Fig.(2)Colonic mucosa showing sloughing of its epithelial lining and loss of villous tissue grade (4)

(H&E X200). Fig.(3)Colon serosa after two hours of dilatation displayed sloughing of mesothelial cells, oedema and

leukocytic infiltration of subepithelial connective tissue (H&E X200). Fig.(4)Colon showing hemorrhage and congestion after two hours from decompression (H&E X200). Fig.(5) Colonic mucosa demonstrating grade (5), the lamina propria showing atrophy of intestinal

gland with massive hemorrhages after two hours from decompression (H&E X200). Fig.(6) Decompression after two hours of colon segment after showing desquamation of mesothelial

lining, oedema and inflammatory cells infiltration of subepithelial connective tissue extended to muscular layer (H&E X200).

Fig.(7) Hepatic lobule showing disorganization of hepatic cords, nuclear pyknosis and apoptosis of hepatocytes grade (II) (H&E X200).

Fig.(8)Kidney showing dilatation of glomerular tufts with red blood cells with necrobiotic changes renal tubules (H&E X200).


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DISCUSSION imely diagnosis and surgical intervention for ischemia are

challenging clinical problems (Wil-liams, 1998). The restoration of bl-ood flow is the treatment of choice to save viable tissue following ac-ute ischemia of a vascular territory. Nerveless, reperfusion of ischemic tissues can be accompanied by sig-nificant local, remote and systemic inflammatory events that may limit the beneficial effects of blood flow restoration (Danielle et al., 2005).

The current study was planed to demonstrate the gross and histo-pathological changes following isc-hemia-reperfusion injury as well as some serum biochemical constitue-nts.

Ischemia-reperfusion injuries was promoted the regional product-ion of inflammatory mediators, ex-pression of cell adhesion molecules on endothelial and immune cell surfaces and increase the precoagu-latory properties of vascular endo-thelial cells (Schwarz et al., 1999). The increase in precoagulatory pro-perties was in agreement with the result of the present work, as it ob-vious from results that prothrombin time (PT), activated partially thro-mboplastine time (PTT) and INR ratio was significant prolonged aft-er induction of ischemia in all gro-ups in comparison with value befo-re induction of ischemia. In addit-ion, more prolongation occurs after reperfusion. The same results were

noticed by (Monreal et al., 2000). Imaz et al., (2002) reported that hypercoagulation may be attributed to the release of endogenous medi-ators such as platelet activation fac-tor in inflammatory disorders. Oth-er studies claimed hypercoagulab-iltity to absorption of endotoxin from intestine (Weiss and Rashid, 1998).

Measurement of serum bioch-emical markers may be useful in management and diagnosis of isch-emia-reperfusion injury. Assay of serum ALT and AST in our exper-iment showed a significant increase in its activity and may be due to tissue injury and inflammatory me-diators which are associated with ischemia-reperfusion injury. It is well known that ALT enzyme is nearly specific for hepatocellular injury in dogs and increased serum levels parallel the magnitude of he-patocellular injury in acute cases. AST enzymes occur in most cells; however, it is useful in evaluating hepatocellular injury (Kaneko, 1997).

Reports of Upendra et al.,

(2005), were in a agreement with our results as they found that eleva-tion in ALT and AST values in ischemia reperfusion mainly due to chemical mediators that cause cell injury and leakage of enzymes. This result was confirmed by the histopathological changes observed in liver after ischemia reperfusion

T


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injury, liver cells showed degener-ative changes associated with con-gestion, mononuclear cells infiltra-tion and some hepatocytes showed coagulative necrosis.

In the present work serum ALP showed a significant increase with intestinal ischemia-reperfu-sion, this could be attributed to tissue injury associated with ische-mia and reperfusion (Wollin et al., 1981).

Serum ALP is found primarily in intestine, kidney, liver and bone; moreover kidney and intestine have the greatest activity per gram of tissue (Kaneko et al., 1997). This was in close agreement with the findings of higher ALP activities were associated with greater intes-tinal damage and renal infarction (Hoover et al., 1988). So increase in ALP increase probability of sur-gery and worse prognosis (Saulez et al., 2004).

LDH results of the present ex-periment recorded a significant inc-rease in its activity with intestinal and renal ischemia-reperfusion. LDH activity is present in all the cells of the body predominantly in cytoplasm of the cell. Even small mass of damaged tissues causes leakage of enzymes and increasing its level in serum significantly (Kaneko et al., 1997). Such results were in agreement with that report-ed by Thompson (1990). As he fo-und that LDH were more likely to

be elevated during intestinal ische-mia. Furthermore, Hoover et al., (1988), observed that LDH was significantly increased during renal ischemia-reperfusion. This increase may be contributing to tissue brea-kdown that occur during ischemia-reperfusion injury (Uday Kumar et al., 2003).

It is well known that α-amyl-ase enzyme found in several tissues of dogs including intestine and kid-ney (Mocharla et al., 1990). More-over the specificity of serum amyl-ase increases as a clinical marker of exocrine pancreas (Corazza et al., 1994). The present study recor-ded a significant increase in amyla-se activity during intestinal ischem-ic time and continued with reperfu-sion, this was in agree with that recorded by Yang et al., (2004). This finding might be attributed to that formation of free radicals due to ischemia and reperfusion causi-ng pancreatic injury resulting in escape of these enzymes to the cir-culation.

The obtained data of serum urea and creatinine in canine model subjected to intestinal ischemia-reperfusion injury showed a signi-ficant increase. These results were in agreement with Yang et al., (2004), who found that urea conce-ntration increase significantly in rat after intestinal ischemia-reperfus-ion. The increase in urea concentr-ation may be contributing to perfu-


19

sion defect that occur as a result of dehydration. Hypovolemia occur in this study mainly due to surgical intervention and hemorrhage from mesentery during ischemia-reperf-usion. Dogs don't tolerate hypovol-emia which may lead to developm-ent of acute renal failure. Develop-ment of azotemia associated with increase in creatinine concentration noted in the present study may be due to dehydration which lead to decrease in glomerular filtration ra-te or primary acute renal failure which developed from perfusion defect (Balint et al., 1975). Histop-athological changes observed in re-nal tissue after ischemia-reperfu-sion as congestion and inflammat-ory cells infiltration mainly neutro-phils support the changes in renal function tests.

Glucose estimation revealed significant hyperglycemia which appeared during ischemia and after reperfusion in all groups. This may be due to the effect of anesthetic drugs used during experiment; Ilkiw (2002) reported that dogs are capable of increasing cortisol lev-els in response to surgical stimula-tion. So that hyperglycemia occurs in response to increase in the cor-tisol level.

CRP is considered the most diagnostically important of the acu-te phase protein in humans (Maur-ey, 1985). Moreover, Caspi et al., (1987) indicates that it may be equ-

ally valuable in dogs. This interest in generated the potential for use of acute phase proteins to provide an early and reliable signal to the clin-ician of the presence of any infla-mmatory disease. The results of CRP in the present study indicated that CRP was significantly increa-sing in all groups. This result was in agreement with experimentally induction of inflammation in horse showed significant increase in CRP (Imaz et al., 2002).

The increase in CRP in all gr-oups may be contributing to a defe-nsive mechanism against further injury that occur during ischemia and continued after reperfusion (Kaneko et al., 1997). CRP level increase with the severity of bacter-ial translocation in acute intestinal obstruction (Cevikel et al., 2004).

Intestinal ischemic segment

appeared thick, dark red to purple in color and edematous. The mu-cosa and neighboring mesentery become congested and hemorrh-agic. During reperfusion, the bowel wall thickened progressively and become more congested hemorrh-agic and blackened in color there were sever congestion, edema and hemorrhage of the mesentery. The-se findings were coincided with th-at reported by (Carol et al., 1991).

Acute intestinal ischemia was accompanied by massive infarction of the mucosa and inner muscular


20

layer. The bowel wall becomes co-ngested, edematous and the mucosa undergoes necrosis and ulceration. Bleeding into the lumen is a cons-equence of the pervious changes. The lesions are characteristically patchy and may affect both the small intestine and colon (Vinay et al., 1997).

The ascending colon is more susceptible to seromuscular layer damage (Robin et al., 2001). Expe-rimental pathological study on hor-ses subjected to ischemia for 70 minute and followed by 60 minute reperfusion revealed that after 70 minute of jejunal ischemia there was serosal capillary congestion but minimal edema or leukocyte in-filtration. The mesothelial cell lay-er was either partially or complete-ly absent. After reperfusion the me-sothelial cell loss, serosal edema, erythrocyte and leukocyte infiltrat-ion were significantly increased. These findings agreed with that re-ported by Robin et al. (2001).

Other experiment on rats fou-nd that after ligation of mesenteric artery for one hour total villous lo-ss with some crypts, normal stroma and patent vessels were noted (Up-endra et al., 2005). While (Byron, 2001) found that after ligation of mesenteric artery for one hour and followed by one hour reperfusion in rats, the jejunal serosal and mu-scle layers loss it's mesothelial cell

layer, serosal edema and increase neutrophils numbers.

After distention and decompr-ession of the colon in rat, the mes-othelial cell layer was completely absence with serosal edema and in-creased serosal neutrophils. The hi-stological changes associated with clinical cases of colonic infarction include epithelial sloughing, cong-estion, hemorrhage, edema and ne-crosis of mucosa and edema and hemorrhage were observed in sub-mucosa (Meschter et al., 1986). These findings were similar to that observed in our study in dogs.

Horse subjected to intestinal intraluminal distention /decompres-sion for 120 minute recorded a mesothelial cell loss, moderate ser-osal edema, lymphatic dilution, and erythrocyte infiltration. After deco-mpression, serosal edema, hemorr-hage, and leukocyte infiltration inc-reased (Robin et al., 2001). In oth-er study performed in foals distent-ion of the jejunum for 2 hour follo-wed by decompression resulted in serosal edema and cellular infiltrat-ion (Lundin et al., 1989). In simil-ar study intraluminal distention for 2 hour followed by 60 minute of decompression revealed mucosal neutrophilic infiltration (Dabarein-er et al., 1993).

The changes that observed in small bowel are more dramatic than that observed in large bowel


21

over the same period of ischemia (Jubb and Kennedy, 2000). The abnormalities observed in the isch-emic or reperfusion of colonic ser-osal layer was partially mesothelial cell loss (Robin et al., 2001). Col-on suffer from thromboembolism disease showed epithelial sloughi-ng, ulceration, necrosis and hemo-rrhage of the under lying lamina propria, and inflammatory cell infi-ltration. These findings were agre-ed with that observed by Barclay et al. (1980). On the other side, the epithelial sloughing was similar to that observed in spontaneous or ex-perimentally induced intestinal isc-hemia in several species of animals and man (Hagluns et al., 1980).

Intestinal ischemia reperfusi-on injury may lead to intestinal ba-rrier dysfunction, resulting in bact-erial translocation, which can lead to adult respiratory distress syndro-me and sepsis. The most important side effect of ischemia reperfusion injury is multiple organ dysfuncti-ons, which may lead to death (Yang et al., 2004 and Upendra et al., 2005).

Conclusion: Coagulation values revealed

hypercoagulability is characteristic for ischemia - reperfusion injury. Also values of serum enzymes showed significant increase in their activities was associated with isch-emia and continued even after rep-erfusion. A marked increase in C-

reactive protein levels was highly associated with bacterial translocat-ion and multiple organ dysfunct-ions that associated with ischemia reperfusion injury. Histopathologi-cal findings, clarified the ischemic picture of colon by different path-ways as well as demonstrated that reperfusion lesions which were more severe in comparison with ischemia.

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