Akut mezenterik iskemide plazma sitokin düzeylerinin tanısal değeri: deneysel bir çalışma

PDF Dosyası: 

arastirmax_2186_pp_216-221.pdf

Introduction
Acute intestinal ischemia resulting from impaired
blood flow within the mesenteric vascular system is a
catastrophic event, inevitably leading to death if not
diagnosed and treated promptly. Despite the progress in
the art of medicine, diagnosis and treatment of this relatively infrequent disease, still continues to challenge
physicians (1). There is an incidence of 8.6/100.000 in
the population and 1-2 cases per 1000 hospital admissions (2), with reported mortality rates ranging from
44% to 91% depending on etiology and treatment (3).
Although some studies have reported better survival
rates compared to the past decade, there is disagreement
among experts on this issue (4). The improvement,
which still remains to be verified is promising but minimal, has been attributed to advances in supportive
patient care and interventional procedures (2). But early
* Department of General Surgery, Gülhane Military Medical Faculty
** Department of Immunology, Gülhane Military Medical Faculty
***Department of Pathology, Gülhane Military Medical Faculty
Reprint request: Dr. Nazif Zeybek, Department of General Surgery,
Gülhane Military Medical Faculty, Etlik-06018, Ankara
E-mail: nzeybek@hotmail.com
Date submitted: March 05, 2007
Accepted: July 31, 2007
ARAÞTIRMA/ORIGINAL ARTICLE Gülhane Týp Dergisi 2007; 49: 216-221
© Gülhane Askeri Týp Akademisi 2007
Summary
Frequency of mortality in patients with superior mesenteric
artery occlusion is still high due to difficulties in current
diagnosis. The initial symptoms of acute intestinal ischemia
is non-specific and indistinguishable from acute abdomen.
Regardless of the etiology, the time interval between diagnosis and therapy determines the prognosis of acute intestinal ischemia. Our purpose in this study was to compare
serum leukocyte levels and cytokine gradients to evaluate
their prognostic values in the early diagnosis of acute
intestinal ischemia. In this study 80 rats were divided into
4 groups as control, laparatomy, strangulation, and superior mesenteric artery occlusion groups. Serum IL-1β, IL-6
and TNFα and blood leukocyte levels were measured and
the resected intestinal specimens were histopathologically
evaluated for intestinal ischemia and tissue necrosis. The
results showed an increase in cytokine levels in parallel
with tissue ischemia. We believe that, although not specific for intestinal ischemia, serum IL-1β, IL-6 and TNFα levels
may be used as an early indicator of intestinal ischemia if
rapid analysis techniques are developed.
Key words: Cytokine levels, intestinal strangulation,
mesenteric ischemia
Özet
Akut mezenterik iskemide plazma sitokin düzeylerinin
tanýsal deðeri: deneysel bir çalýþma
Süperiyor mezenterik arter oklüzyonlu hastalardaki mortalite oranlarý, mevcut taný yöntemlerindeki zorluða baðlý
olarak hala yüksektir. Akut intestinal iskeminin baþlangýç
bulgularý özgün deðildir ve akut abdomenden ayýrmak zordur. Etiyolojiden baðýmsýz olarak, taný ve tedavi arasýndaki
süre akut intestinal iskeminin prognozunu belirler. Bu çalýþ-
mada amacýmýz serum lökosit düzeyleri ile sitokin düzeylerini karþýlaþtýrarak, bunlarýn akut intestinal iskeminin erken
tanýsýndaki tanýsal önemini deðerlendirmektir. Çalýþmada 80
rat; kontrol, laparatomi, strangülasyon ve süperiyor mezenterik arter oklüzyonu gruplarý olarak 4 gruba ayrýldý. Serum
IL-1β, IL-6, TNFα ve kan lökosit düzeyleri ölçüldü ve rezeke
edilen barsaklar intestinal iskemi ve doku nekrozu açýsýndan
histopatolojik olarak deðerlendirildi. Sonuçlar doku iskemisine paralel olarak sitokin deðerlerinde yükselme gösterdi.
Ýntestinal iskemiye spesifik olmamakla birlikte, serum IL-1β,
IL-6 ve TNFα deðerlerinin, daha hýzlý analiz yöntemleri
geliþtirilirse, intestinal iskeminin erken göstergesi olarak
kullanýlabileceðini düþünüyoruz.
Anahtar kelimeler: Sitokin düzeyleri, intestinal strangülasyon, mezenterik iskemiCilt 49 · Sayý 4 · Gülhane TD IL-1, IL-6 and TNFα in acute mesenteric ischemia · 217
diagnosis is still the most important factor affecting
patient outcome (4). The non-specific nature of symptoms, lack of specific markers and the need for advanced diagnostic procedures such as angiography for accurate diagnosis are the major causes of delay in treatment,
contributing to morbidity and mortality (6). Endogenous substances such as creatinin phosphokinase, alkaline phosphatase, ileal peptide and serum phosphate
levels have been investigated on the search for a possible early marker of intestinal ischemia. Unfortunately
due to lack of specifity and sensitivity, these potential
markers gave positive results usually after the occurrence of irreversible intestinal damage (7-10).
Although the exact mechanism has not been clarified, intestinal ischemia is known to result in gut barrier function failure, initiating systemic immunoinflamatory response (11). Also ischemia has been found to
cause cytokine production by the intestine, Payer's patches and mesenteric lymph nodes (12). Of these cytokines, IL-1β and TNFα have been documented to play
an important role in multiple organ failure in acute
intestinal ischemia (13,14).
This study was designed to investigate if serum IL-
1β, IL-6 and TNFα levels could be used as a potential
indicator of intestinal ischemia.
Material and Methods
Eighty male, 2-month-old Spraque-Dawley strain
rats weighing 200±20 grams were divided into 4 groups consisting of 20 rats in each group. The animals
were provided by the Clinical Sciences Research
Department Division of Experimental Animals. The animals were grouped as follows; Group 1 (Non-operated control group): Only anesthesia was induced and no
surgical procedure was performed, Group 2 (Laparatomy group): Midline incision laparatomy was performed and the abdomen was closed without any further surgical intervention. Group 3 (Segmental ischemia group-Strangulation model): A 10 cm segment
of ileum including the mesentery was ligated 5 cm
proximal to the ileoceacal valve to form a strangulation
ischemia model. Group 4 (Superior mesenteric artery
ligation group): The superior mesenteric artery was ligated 0.5 cm distal to the abdominal aorta following careful dissection of the intestinal mesentery to form total
mesenteric ischemia model.
Anesthesia was induced by Sevoflurane and maintained by Sevoflurane 40% N2O and 60% O2
inhalation.
Blood samples were collected at 5 minutes, 2 hours and
4 hours after operation in all groups to determine the
levels of IL-1β, IL-6 and TNFα. Blood samples at 4
hours were drawn from the heart following thoracotomy.
Relaparatomy was performed at 4 hours post-operative. Ischemic segments in Groups 3 and 4 and corresponding intestinal segments in Groups 1 and 2 were
resected and placed in 10% formalin for histopathological examination. Later on the rats were sacrificed.
Histopathological evaluation was made at the Department of Pathology. The statistical analysis was made
by the Department of Biostatistics using SPSS 10.0
(SPSS Inc. Chicago, IL, USA). Differences between the
groups were evaluated by Friedman and Bonferroni
adjusted Wilcoxon signed ranks tests.
Results
Biochemical and immunological data: The blood leukocyte levels were measured at the Clinical Sciences
Research Department and serum cytokine levels were
measured at the Department of Immunology. The Rat
IL-1β and IL-6 kits were provided from Assay Designs
Inc. AnnArbor Michigan USA and Rat TNFα kits were
provided from CytImmune Sciences Inc. College Park
Maryland USA.
Serum IL-1β levels measured are given in Table I and
Figure 1.
The median values of serum IL-1β levels in Group 1
showed no statistitical significant difference. The
results of IL-1β levels in all other groups showed statistically significant difference (p<0.01).
The values of Group 2 (Laparatomy) showed significant increase by time reaching 206 pg/ml by 4 hours.
Table I. Median serum levels of IL-1β, IL-6 and TNFα by
time
______________________________________________________
Groups Time* IL-1 IL-6 TNF
______________________________________________________
Group 1 (CG) 5. min 26.9 36.6 18.9
2. hr 27.2 35.9 20.0
4. hr 28.5 35.8 20.2
______________________________________________________ p value >0.05 >0.05 >0.05
Group 2 (L) 5. min 26.7 35.9 19.5
2. hr 74.6 92.2 47.6
4. hr 206.3 196.6 175.9
______________________________________________________ p value <0.001 <0.001 <0.001
Group 3 (SIG) 5. min 27.3 35.2 19.5
2. hr 173.6 278.0 180.3
4. hr 300.8 383.5 383.8
______________________________________________________ p value <0.01 <0.01 <0.01
Group 4 (SMIG) 5. min 26.9 36.0 20.9
2. hr 365.8 443.6 435.4
4. hr 708.5 758.2 882.6
p value <0.01 <0.01 <0.01
______________________________________________________
*: Time is duration of ischemia. CG: Control group, L: Laparatomy group, SIG: Strangulation ischemia group, SMIG: Superior
mesenteric ischemia group218 · Aralýk 2007 · Gülhane TD Karaaðaç et al.
Both 2nd hour and 4th hour results were significantly
different from the 5th minute results (p<0.01) and
control group IL-1 levels (p<0.01).
IL-1β levels in Group 3 (Strangulation ischemia)
increased to 173.6 pg/ml by 2nd hour and 300.8 pg/ml
by 4th hour showing a statistically significant chan-ge
from 5 minute and laparatomy group results (p<0.01).
The increase in Group 4 (Superior mesenteric ischemia) was most significant increasing to 365.8 pg/ml by
the 2nd hour and dramatically increasing to 708.5 pg/ml
by the 4th hour. The changes were significantly different from the other groups and 5th minute results
(p<0.01).
The median values of IL-6 levels are shown in Table
I and Figure 2. Changes in the control group were not
found to be significant by time (p>0.05). Fifth minute
values did not show significant difference among all the
groups (p>0.05).
The results of Group 2 showed a significant increase
by time (p<0.01) and the results were also significantly
higher than those of Group 1 (p<0.01).
Serum IL-6 values of Group 3 increased significantly (p<0.01) and was apparently higher than those of
Group 2 (p<0.01).
Serum levels of IL-6 most prominently increased in
Group 4, reaching 758.2 pg/ml at 4th hour, and this
increase was found to be significantly higher than those
of the other groups (p<0.01).
The results of serum TNFα levels are shown in Table
I and Figure 3. The change of TNFα levels in Group 1
was not found to be significant (p>0.05). The difference of 5th minute samples was also not found to be
statistically significant among the groups (p<0.01).
The increase in TNFα levels of Group 2 was significant at 2nd and 4th hour samples (p<0.01) when compared with 5th minute and Group 1 results.
TNFα levels increased significantly by time in Group
3 and was found to be significantly higher by time
(p<0.01) and than the results of Group 1 and 2
(p<0.01).
TNFα levels of Group 4 showed the highest increase. The 2nd hour and 4th hour results were significantly higher than the results of the other groups
(p<0.01) and 5th minute samples (p<0.01).
The standard leukocyte count range of rats was set as
5000-13.000/mm3
as published previously (15). The
median results of leukocyte counts of groups are shown
in Table I and Figure 4.
The median leukocyte counts of Groups 1 and 2 did
800.00
700.00
600.00
500.00
400.00
300.00
200.00
100.00
0.00
Group 1 (CG)
Group 2 (L)
Group 3 (SIG)
Group 4 (SMI)
5 min 2 hr 4 hr
Figure 2. Serum IL-6 levels (pg/ml)
1000.00
900.00
800.00
700.00
600.00
500.00
400.00
300.00
200.00
100.00
0.00
5 min 2 hr 4 hr
Figure 3. Serum TNFα levels (pg/ml)
Group 1 (CG)
Group 2 (L)
Group 3 (SIG)
Group 4 (SMI)
Figure 1. Median serum IL-1β levels (pg/ml)
800.00
700.00
600.00
500.00
400.00
300.00
200.00
100.00
0.00
Group 1 (CG)
Group 2 (L)
Group 3 (SIG)
Group 4 (SMI)
5 min 2 hr 4 hrCilt 49 · Sayý 4 · Gülhane TD IL-1, IL-6 and TNFα in acute mesenteric ischemia · 219
not show significant difference by time (p>0.05). The
results of Group 1 and 2 were not found to be statistically different (p>0.05).
The second hour leukocyte levels of rats in Group 3
showed a statistically significant increase when compared to fifth minute results (p<0.05), the levels increased by the fourth hour but the change was not found
to be significant (p>0.05). However when the results
were compared with Groups 1 and 2, both second hour
(p<0.01) and fourth hour (p<0.05) results were found
to be significantly different.
The leukocyte count results of Group 4 showed statistically significant increase by the second hour
(p<0.05). The change was most apparent by the fourth
hour (p<0.05). These results were higher compared to
Group 3, but the difference was not found to be significant. The change at the 4th hour samples were statistically higher than the 4th hour results of Group 3
(p<0.05).
The intestinal segments were evaluated and graded
on a scale from 1 to 8 for acute intestinal damage as described by Chiu et al. (16). The groups were evaluated
by cross tab variance analysis. The histopathological
abnormality in all rats in Groups 1 and 2 were reported
as grade 0 (Figure 1). Ten rats (50%) in Group 3 developed grade 7 damage (Figure 2) and the remaining 10
rats developed grade 8 damage. When the tissues of
Group 4 were examined 70% (14 rats) of the animals
suffered grade 8 damage while 30% (6 rats) had grade 7
damage. The differences between the groups were
found to be significant (p<0.01) (Figures 1,2,3).
Discussion
Acute mesenteric ischemia is a life threatening emergency causing significant morbidity and mortality if not
diagnosed and treated before irreversible intestinal damage has occurred. Results from studies with limited
number of patients reported a mortality rate of 53% to
93% in this relatively infrequent disease (3). Most of the
survivors of this catastrophic event become total parenteral nutrition dependent and have a reported estimated 5 year survival rate of 18% (17). When diagnosed
prior to irreversible damage, these rates may be significantly reduced. In a recent study by Bingol et al. 24
patients were diagnosed with acute mesenteric ischemia
while hospitalized for cardiac diseases (18). Twelve
patients who were operated within 6 hours survived
while 2 of 9 the patients operated within 12 hours died
and all of the 3 patients operated after 12 hours died.
These numbers point out to the importance of diagnosis on time which is the determinant of patient outcome
for this notorious illness.
Angiography is the gold standard diagnostic technique in evaluation of the cause and diagnosis of acute
intestinal ischemia (19), however it may be time consuming and access to a qualified interventional radiologist
may not be possible in every center. The same is true
with MR angiography and MR spectroscopy capable of
giving excellent view of vasculature, but may be performed in tertiary care centers (20,21). Doppler USG
may be useful in evaluating the mesenteric flow but the
paralytic ileus and the intestinal distention associated
with ischemia may prevent efficient evaluation of flow,
and therefore it may be unreliable in some cases.
In our study we tried to investigate serum IL-1β, IL-
6 and TNFα levels as a cost effective, simple, potential
indicator of intestinal ischemia.
Although the exact mechanism of release is unknown, severe mechanical trauma and thermal damage
have been documented to cause an increase in cytokine
levels and especially in IL-6 (22). IL-1β, TNFα, endotoxins and free oxygen metabolites are some of the factors which may be related to the increment on IL-6 levels (23-26). There are reports on intestinal ischemia
contributing to the intestine becoming a cytokine producing organ. Significant increase in mRNA coding for
immunoregulatory and proinflamatory cytokines have
been demonstrated in the Peyer's patches and mesenteric lymph nodes following intestinal ischemia (11,12).
Intestinal obstruction results with elevation in bacterial content (27), and thereby an increase in IL-6 and
TNFα levels has also been shown following the exposure of enterocytes to E.coli, which may occur following mucosal damage due to ischemia (28). Several
authors have reported increase in plasma cytokine levels and endotoxemia following intestinal ischemia in
their experiment with cat models (29,30). Moore et al.
12000.00
1000.00
800.00
600.00
400.00
200.00
0.00
Group 1 (CG)
Group 2 (L)
Group 3 (SIG)
Group 4 (SMI)
5 min 2 hr 4 hr
Figure 4. Blood leukocyte counts220 · Aralýk 2007 · Gülhane TD Karaaðaç et al.
have also demonstrated the high levels of cytokines in
blood samples drawn from the mesenteric vessels 60-
120 minutes following intestinal ischemia and hypothesized that high cytokine levels and endotoxemia are
early predictors of intestinal ischemia (31).
In our models, we compared intestinal strangulation,
superior mesenteric artery occlusion (SMAO) with laparatomy and anesthesia groups for the levels of IL-1β,
IL-6 and TNFα which began to rise 60 minutes after
the onset of ischemia and reached peak points at 4 hours.
In our study fifth minute levels of IL-1β, IL-6 and
TNFα did not show significant differences in all the
groups. The results in all groups except for anesthesia
(control) group including laparatomy groups showed
an increase at 2 and 4 hours, but the increase was much
more prominent in the strangulation and SMAO groups. The increase was also significant in strangulation
and SMAO groups when compared to laparatomy and
control groups (p<0.01). We also observed higher cytokine levels in fourth hour samples compared to second
hour samples in strangulation and SMAO groups
(p<0.01).
Regardless of the etiology, the ultimate goal for the
operation is to preserve the viability of the intestine. Time is the most important factor determining prognosis.
Once irreversible damage has occurred, regardless of
the operation chosen whether vascular by pass, embolectomy or resection, mortality is high, leaving the patient TPN dependent with only intestinal transplantation
as a hope for a cure (32).
This study has been designed in an effort to find an
easily applicable marker of intestinal ischemia to provide guidance to the hesitating or unsuspecting physician to initiate advanced, invasive and expensive procedures in the diagnosis of this disease before the point
of no return has been reached.
Current tests available for the determination of cytokine levels are still time consuming. The procedures
we have used in our study have taken average 3.5 hours
for the results which are still behind the optimal time
range. We would definitely benefit from the development of faster analysis techniques in this manner. We
believe that serum cytokine levels may be utilized as an
aid for diagnosis if rapid and sensitive ways of measurement become available.