Abstract
OBJECTIVE:
It is essential to identify a serological marker of injury in order to study the pathophysiology of intestinal ischemia reperfusion. In this work, we studied the evolution of several serological markers after intestinal ischemia reperfusion injury in rats. The markers of non-specific cell damage were aspartate aminotransferase, alanine aminotransaminase, and lactic dehydrogenase, the markers of inflammation were tumor necrosis factor alpha, interleukin-6, and interleukin-1 beta, and the markers of intestinal mucosal damage were intestinal fatty acid binding protein and D-lactate. We used Chiús classification to grade the histopathological damage.
METHODS:
We studied 35 Wistar rats divided into groups according to reperfusion time. The superior mesenteric artery was clamped for 30 minutes, and blood and biopsies were collected at 1, 3, 6, 12, 24, and 48 hours after reperfusion. We plotted the mean ± standard deviation and compared the baseline and maximum values for each marker using Student’s t-test.
RESULTS:
The maximum values of interleukin-1 beta and lactic dehydrogenase were present before the maximal histopathological damage. The maximum tumor necrosis factor alpha and D-lactate expressions coincided with histopathological damage. Alanine aminotransaminase and aspartate aminotransferase had a maximum expression level that increased following the histopathological damage. The maximum expressions of interluken-6 and intestinal fatty acid binding protein were not significantly different from the Sham treated group.
CONCLUSION:
For the evaluation of injury secondary to acute intestinal ischemia reperfusion with a 30 minute ischemia period, we recommend performing histopathological grading, quantification of D-lactate, which is synthesized by intestinal bacteria and is considered an indicator of mucosal injury, and quantification of tumor necrosis factor alpha as indicators of acute inflammation three hours after reperfusion.
Ischemia/Reperfusion Injury; Intestine; Interleukin-1 Beta; Tumor Necrosis Factor-Alpha; Lactate Dehydrogenase; Chiu Score; D-Lactate
INTRODUCTION
Intestinal ischemia reperfusion (IR), or transient hypoxia followed by
re-oxygenation, is a component of the pathogenesis of all diseases that involve a
reduction or redistribution of bowel blood flow, including shock, neonatal
necrotizing enterocolitis, intestinal transplant, and mesenteric ischemia. As such,
IR can complicate surgical procedures in the elderly and for open-heart surgery
patients (11. Venkateswaran RV, Charman SC, Goddard M, Large SR. Lethal
mesenteric ischaemia after cardiopulmonary bypass: A common complication?
Eur J Cardiothorac Surg. 2002;22(4):534-8,
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mesenteric ischemia after open heart surgery. Angiology. 1998;49(4):267-73,
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-33. Acosta-Merida MA, Marchena-Gomez J, Cruz-Benavides F,
Hernandez-Navarro J, Roque-Castellano C, Rodriguez-Mendez A, et al. Factores
predictivos de necrosis masiva intestinal en la isquemia mesentérica aguda.
Cir Esp. 2007;81(3):144-9,
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). In clinical practice, symptoms are often vague, making a definitive
diagnosis of acute intestinal ischemia notoriously difficult (44. Van der Voort PH. The incomplete puzzle of vasoactive medication
in (abdominal) sepsis. Crit Care Med. 2006;34(5):1565-6,
http://dx.doi.org/10.1097/01.CCM.0000216187.00379.12.
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). When accompanied by clinical signs of peritonitis, this
usually indicates necrosis of the intestinal wall, and the suspicion of intestinal
ischemia is traditionally confirmed by laparotomy (55. Woo K, Mayor K, Kohanzadeh S, Allins AD. Laparotomy for visceral
ischemia and gangrene. Am Surg. 2007;73(10):1006-8.).
In humans, the systematic search for serological markers of intestinal ischemia to
optimize early diagnosis has identified molecules such as D-lactate (66. Garcia J, Smith FR, Cucinell SA. Urinary D-lactate excretion in
infants with necrotizing enterocolitis. J Pediatr. 1984;104(2):268-70,
http://dx.doi.org/10.1016/S0022-3476(84)81010-0.
http://dx.doi.org/10.1016/S0022-3476(84)...
), glutathione S-transferase (GST) (77. Khurana S, Corbally MT, Manning F, Armenise T, Kierce B, Kilty C.
Glutathione S-transferase: a potential new marker of intestinal ischemia.
J Pediatr Surg. 2002;37(11):1543-8,
http://dx.doi.org/10.1053/jpsu.2002.36181.
http://dx.doi.org/10.1053/jpsu.2002.3618...
), and intestinal fatty acid binding protein
(iFABP) (88. Mannoia K, Boskovic DS, Slater L, Plank MS, Angeles DM, Gollin G.
Necrotizing enterocolitis is associated with neonatal intestinal injury.
J Pediatr Surg. 2011;46(1):81-5,
http://dx.doi.org/10.1016/j.jpedsurg.2010.09.069.
http://dx.doi.org/10.1016/j.jpedsurg.201...
,99. Edelson MB, Sonnino RE, Bagwell CE, Lieberman JM, Marks WH,
Rozycki HJ. Plasma intestinal fatty acid binding protein in neonates with
necrotizing enterocolitis: a pilot study. J Pediatr Surg.
1999;34(10):1453-7,
http://dx.doi.org/10.1016/S0022-3468(99)90102-1.
http://dx.doi.org/10.1016/S0022-3468(99)...
). However, a combination of markers, or a “diagnostic
intestinal ischemia panel,” reflecting different aspects of bowel viability
has not been identified (1010. Evennett NJ, Petrov MS, Mittal A, Windsor JA. Systematic review
and pooled estimates for the diagnostic accuracy of serological markers for
intestinal ischemia. World J Surg.
2009;33(7):1374-83.). The temporal
association between IR and serum markers is also not well understood. The
identification of a set of serum markers (SM) that could detect or predict the
outcome of an ischemic insult would be essential for studying the pathophysiology of
IR.
Numerous publications have reported the effects of drugs and therapeutic modalities
on IR injury based on histological damage (1111. Quaedackers JS, Beuk RJ, Bennet L, Charlton A, oude Egbrink MG,
Gunn AJ, et al. An evaluation of methods for grading histologic injury following
ischemia/reperfusion of the emall bowel. Transplantation Proceedings.
2000;32(6):1307-10,
http://dx.doi.org/10.1016/S0041-1345(00)01238-0.
http://dx.doi.org/10.1016/S0041-1345(00)...
,1212. Chiu CJ, McArdle AH, Brown R, Scott HJ, Gurd FN. Intestinal
mucosal lesion in low-flow states. Arch Surg. 1970;101(4):478-83,
http://dx.doi.org/10.1001/archsurg.1970.01340280030009.
http://dx.doi.org/10.1001/archsurg.1970....
) and alterations of serum
levels of molecules that are associated with cell damage, endothelial activation,
inflammation, and oxidative stress (1313. Block T, Nilsson TK, Björck M, Acosta S. Diagnostic
accuracy of plasma biomarkers for intestinal ischaemia. Scand J Clin
Lab Invest. 2008;68(3):242-8,
http://dx.doi.org/10.1080/00365510701646264.
http://dx.doi.org/10.1080/00365510701646...
14. El-Awady SI, El-Nagar M, El-Dakar M, Ragab M, Elnady G.
Bacterial translocation in an experimental intestinal obstruction model.
C-reactive protein reliability?. Acta Cir Bras. 2009;24(2):98-106,
http://dx.doi.org/10.1590/S0102-86502009000200005.
http://dx.doi.org/10.1590/S0102-86502009...
-1515. Kurimoto Y, Kawaharada N, Ito T, Morikawa M, Higami T, Asai Y.
An experimental evaluation of the lactate concentration following mesenteric
ischemia. Surgery Today. 2010;38(10):926-30.). However, the evaluation times are often
fixed or arbitrary, and the temporal dynamics of histological and serological injury
markers remain obscure. Nonetheless, in most studies of experimental IR,
histopathological examination is the gold standard. In 1970, Chiu reported a graded
scale of damage produced by ischemia in the intestinal mucosa based on hematoxylin
and eosin staining (1212. Chiu CJ, McArdle AH, Brown R, Scott HJ, Gurd FN. Intestinal
mucosal lesion in low-flow states. Arch Surg. 1970;101(4):478-83,
http://dx.doi.org/10.1001/archsurg.1970.01340280030009.
http://dx.doi.org/10.1001/archsurg.1970....
). Later, Parks and
Sonnino reported scores, and Park reported a modification to the original Chiu score
(1616. Park PO, Haglung U, Bulkley GB, Fâlt K. The secuence of
development of intestinal tissue injury after strangulation ischemia and
reperfusion. Surgery. 1990;107(5):574-80.). The Chiu and Chiu/Parks
classifications are currently recommended for comparing results reported in the
literature (1111. Quaedackers JS, Beuk RJ, Bennet L, Charlton A, oude Egbrink MG,
Gunn AJ, et al. An evaluation of methods for grading histologic injury following
ischemia/reperfusion of the emall bowel. Transplantation Proceedings.
2000;32(6):1307-10,
http://dx.doi.org/10.1016/S0041-1345(00)01238-0.
http://dx.doi.org/10.1016/S0041-1345(00)...
,1717. Oltean M, Olausson M. The Chiu/Park scale for grading intestinal
ischemia-reperfusion: if it ain`t broke don`t fix it!. Intensive Care
Med. 2010;36(6):1095,
http://dx.doi.org/10.1007/s00134-010-1811-y.
http://dx.doi.org/10.1007/s00134-010-181...
).
In this article, we were interested in identifying combinations of serum and histopathological studies at different time points. Therefore, we assessed the evolution of histopathological injury and serum markers of non-specific cell damage: aspartate aminotransferase (AST), alanine aminotransaminase (ALT), and lactic dehydrogenase (LDH), of acute inflammation: tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and interleukin-1 beta (IL-1), and of intestinal mucosa damage: intestinal fatty acid binding protein (iFABP) and D-lactate during reperfusion.
MATERIAL AND METHODS
Animals procedures
The animal procedures were performed in accordance with the proper use and care of laboratory animals. The experiments were performed on 35 female Wistar rats weighing 200-250 g. The animals were maintained under standard conditions, including stable room temperature, a 12:12-h light/dark cycle, and access to commercial rat pellets and water ad libitum. The ethics committee of our institution approved this study.
Animal model
In brief, after 12 hours of fasting and anesthesia with a 40 mg/kg intraperitoneal injection (IP) of pentobarbital sodium (Anestesal; Pfizer, Mexico City, Mexico), a midline laparotomy was performed, and ischemia was induced by occlusion of the superior mesenteric artery with vascular clamps. An absent pulse was visually verified in the mesenteric vessels with a brown discoloration of the intestine. After 30 minutes of ischemia, blood flow was restored, and the abdominal wall was sutured.
The animals were divided into 7 groups (n = 5) and were named according to the reperfusion time: Sham group or negative control, which underwent surgery without arterial occlusion, and groups with 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, and 48 hours of reperfusion.
Depending on the group, at the end of the reperfusion time, the rats were anesthetized, the sutures were removed, blood samples were obtained from the aorta, and full-bowel wall samples of the ileum, 15 cm from the ileocecal junction, were obtained. The animals were then euthanized by exsanguination.
Morphological examination
Intestinal biopsies were fixed in 10% formalin, processed with conventional
histological techniques, and stained with hematoxylin and eosin. To assess
mucosal injury, the Chiu classification was used (66. Garcia J, Smith FR, Cucinell SA. Urinary D-lactate excretion in
infants with necrotizing enterocolitis. J Pediatr. 1984;104(2):268-70,
http://dx.doi.org/10.1016/S0022-3476(84)81010-0.
http://dx.doi.org/10.1016/S0022-3476(84)...
). The evaluation scale was from 0 to 5, where 0 is a
normal intestinal mucosa, 1 is the development of Gruenhagen subepithelial
spaces, 2 is the extension of the subepithelial space with moderate lifting of
the lamina propria, 3 is the expansion of epithelial lifting with destruction of
the tips of villi, 4 is destroyed villi with exposure of the lamina propria and
dilated capillaries, and 5 is the disintegration of the lamina propria,
hemorrhage, and ulceration.
Serum determinations
After exsanguinating the animal, at least 3 mL of blood was collected and allowed to clot, and the serum was separated by centrifugation and stored at −70°C until further study.
The serum levels of TNF-alpha were determined using a Rat TNF-alpha enzyme-linked immunosorbent assay (ELISA) kit (Peprotech, Rocky Hill, NJ, USA). Serum levels of IL-1 were determined using a Rat IL-1 beta ELISA kit (Peprotech). Serum levels of IL-6 were determined using a Rat IL-6 ELISA kit (Peprotech). The serum levels of iFABP were determined using a Rat iFABP ELISA kit (Biotang, Waltham, MA, USA). The serum levels of D-lactate were determined using a EnzyChrom™ D-Lactate Assay Kit (BioAssay Sytems, Hayward, CA, USA).
The serum levels of AST, ALT and LDH activity were determined by reflectance on a Vitros DT60II System Ortho Clinical Diagnostics by Johnson & Johnson (Rochester, New York, USA).
Statistical analysis
Using SPSS V15 software, we plotted the time variations in the parameters studied, identified the peaks (Pmax) for each parameter and compared them against the baseline (Sham) with t tests for two independent samples. A value of p<0.05 was considered to be statistically significant.
RESULTS
Histopathological changes
The histomorphological damage to the intestinal epithelium according to the Chiu scale was evident in our model at one hour, was maximal at three hours, and returned to baseline levels after 12 hours of reperfusion. The damage was undetectable at 24 and 48 hours (Figure 1).
Temporal evolution of serum marker levels and histological damage at different times of reperfusion. Chiu: histopathological damage scored according to Chiu, LDH: lactic dehydrogenase, IL-1: interleukin-1 beta, AST: aspartate aminotransferase, ALT: alanine aminotransaminase, TNF-a: tumor necrosis factor alpha. *p<0.05 versus Sham.
Non-specific markers of cell damage
Of the non-specific markers of cell damage, LDH peaked at one hour of reperfusion, AST peaked at 6 hours and ALT peaked at 12 hours. The latter two showed a significant elevation at 3 hours (Figure 1).
TNF-alpha, IL-1beta, IL-6, iFABP and D-lactate
The markers whose levels coincided with the peak of injury (Pmax) were TNF-alpha and IL-1beta, which peaked at one hour and remained elevated until 3 hours. No differences were observed at 6 hours compared to the control group. Serum D-lactate did not decrease after reaching its maximum at 3 hours (Figure 1). The levels of IL-6 peaked at 12 hours of reperfusion but did not differ from the controls. The iFABP concentration did not significantly differ from the controls at any time.
Significant differences were documented between the sham group and Pmax in injury scores, non-specific injury markers (LDH, AST and ALT), the inflammatory markers TNF-alpha and IL-1beta, and the specific injury marker D-lactate. There were no significant differences in the concentrations of iFABP or IL-6 (Table 1).
DISCUSSION
The intestinal mucosa is particularly sensitive to IR injury. We observed severe
mucosal injury beginning at 1 hour of perfusion, reaching maximal severity at 3
hours and subsiding at 24 hours. This is consistent with other reports that
morphological intestinal repair begins shortly after 3 hours of reperfusion and is
complete around 24 hours (1818. Chang JX, Chen S, Ma LP, Jiang LY, Chen JW, Chang RM, et al.
Functional and morphological changes of the gut barrier during the restitution
process after hemorrhagic shock. World J Gastroenterol.
2005;11(35):5485-91.
19. Stone WC, Bjorling DE, Southard JH, Galbreath EJ, Lindsay WA.
Evaluation of intestinal villus height in rats after ischemia and reperfusion by
administration of superoxide dismutase, polyethylene glycol-conjugated
superoxide dismutase, and two 21-aminosteroids. Am J Vet Res.
1992;53(11):2153-6.-2020. Illyés G, Hamar J. Sequence of morphological alterations in
a small intestinal ischaemia/reperfusion model of the anesthetized rat.
A light microscopy study. Int J Exp Pathol.
1992;73(2):161-72.). During reperfusion, the cascade of events
that seeks to limit damage and maintain homeostasis involves all the components of
the digestive tract: mucous cells, fibroblasts, neurons, endothelial cells and the
immune system (2121. Fiocchi C. Intestinal inflammation: a complex interplay of
immune and nonimmune cell interactions. Gastrointest Liver Physiol. Am J
Physiol. 1997;273(4 Pt 1):G769-75.). Previous studies have
correlated the duration of ischemia with histological damage to the intestinal
mucosa (2222. Haglund U. Experimental Shock and Gastronintestinal Involmement.
In Jensen SL, editor. Essentials of Esperimental Surgery: Gastroenterology.
1a ed Amsterdam: Harwood academic publishers; 1996. p
33/1-33/10.). At 30 minutes of ischemia,
cellular reserves of oxygen are consumed (1212. Chiu CJ, McArdle AH, Brown R, Scott HJ, Gurd FN. Intestinal
mucosal lesion in low-flow states. Arch Surg. 1970;101(4):478-83,
http://dx.doi.org/10.1001/archsurg.1970.01340280030009.
http://dx.doi.org/10.1001/archsurg.1970....
), and the mucosa loses its ability to accumulate sugars and amino acids
against a concentration gradient (2323. Archer SY, Hodin RA. Intestinal Regeneration and Adaptation
Models. In Wilmore DW, Souba WW, editors. Surgical Research. San Diego: Academic
Press; 2001. p 557-71.).
Histological lesions secondary to ischemia are exacerbated by reperfusion. The
period of time of ischemia, the type of ischemia (arterial, venous, arterial and
venous) (2424. Guzman-de la Garza FJ, Camara-Lemaroy CR, Alargon-Galvan G,
Cordero-Perez P, Muñoz-Espinosa LE, Fernandez-Garza NE. Different patterns
of intestinal response to injury after arterial, venous or arteriovenous
occlusion in rats. World J Gastroenterol. 2009;15(31):
3901-7.), and the time to reperfusion all
determine the severity of injury.
The concentrations of LDH and IL-1 increased before the lesion was identified
histopathologically. LDH is a non-specific marker of cell injury and has been
associated with ischemic events in the intestine (2525. Van Noord D, Mensink PB, de Knegt RJ, Ouwendijk M, Francke, van
Vuuren AJ, et al. Serum markers and instestinal mucosal injury in chronic
gastrointestinal ischemia. Dig Dis Sci. 2011;56(2):506-12,
http://dx.doi.org/10.1007/s10620-010-1303-5.
http://dx.doi.org/10.1007/s10620-010-130...
), while interleukin-1 beta is considered a marker of acute
inflammation, resulting in the activation of macrophages during early stages of
tissue injury. Serum levels of LDH, but not IL-1beta, have been shown to remain
elevated 72 hours after intestinal IR in rats (2626. Hei ZQ, Gan XL, Huang PJ, Wei J, Shen N, Gao WL. Influence of
Ketotifen, Cromolyn Sodium, and Compound 48/80 on the survival rates after
intestinal ischemia reperfusion injury in rats. BMC Gastroenterol. 2008;8:42,
http://dx.doi.org/10.1186/1471-230X-8-42.
http://dx.doi.org/10.1186/1471-230X-8-42...
). Peak serum concentrations of D-lactate and TNF-alpha coincided with
histopathological injury. TNF-alpha, in concordance with previous reports (2424. Guzman-de la Garza FJ, Camara-Lemaroy CR, Alargon-Galvan G,
Cordero-Perez P, Muñoz-Espinosa LE, Fernandez-Garza NE. Different patterns
of intestinal response to injury after arterial, venous or arteriovenous
occlusion in rats. World J Gastroenterol. 2009;15(31):
3901-7.), increased upon injury. Both TNF and IL-1
are essential in the physiopathology of IR and are widely used in studies evaluating
intestinal IR injury (2727. Yamamoto S, Tanabe M, Wakabayashi G, Shimazu M, Matsumoto K,
Kitajima M. The role of tumor necrosis factor-alpha and interleukin-1beta in
ischemia-reperfusion injury of the rat small intestine. J Surg Res.
2001;99(1):134-41, http://dx.doi.org/10.1006/jsre.2001.6106.
http://dx.doi.org/10.1006/jsre.2001.6106...
). Additionally, the
intestine has been identified as a source of TNF during IR-mediated damage (2828. Grotz MR, Deitch EA, Ding J, Xu D, Huang Q, Regel G. Intestinal
cytokine response after gut ischemia. Role of gut barrier failure. Ann Surg.
1999;229(4):478-86,
http://dx.doi.org/10.1097/00000658-199904000-00005.
http://dx.doi.org/10.1097/00000658-19990...
). However, these are non-specific markers of
inflammation and systemic injury, which makes their diagnostic utility
questionable.
Intestinal gut barrier dysfunction is an important aspect of the physiopathology of
IR injury. Intestinal bacteria produce D-lactate, and its presence in serum reflects
a major failure of the mucosal barrier (2525. Van Noord D, Mensink PB, de Knegt RJ, Ouwendijk M, Francke, van
Vuuren AJ, et al. Serum markers and instestinal mucosal injury in chronic
gastrointestinal ischemia. Dig Dis Sci. 2011;56(2):506-12,
http://dx.doi.org/10.1007/s10620-010-1303-5.
http://dx.doi.org/10.1007/s10620-010-130...
).
In a systematic literature review of clinical studies, D-lactate was indeed found to
be one of the most well studied and reliable biomarkers of intestinal ischemia
(1010. Evennett NJ, Petrov MS, Mittal A, Windsor JA. Systematic review
and pooled estimates for the diagnostic accuracy of serological markers for
intestinal ischemia. World J Surg.
2009;33(7):1374-83.). However, in a rat model of gut IR
induced by aortic clamping, D-lactate was not elevated in the serum after 1 hour of
reperfusion following 40 minutes of ischemia, consistent with our results (2929. Collange O, Tamion F, Chanel S, Hue G, Richard V, Thuilliez C,
et al. D-lactate is not a reliable marker of gut ischemia-reperfusion in a rat
model of supraceliac aortic clamping. Crit Care Med. 2006;34(5):1415-9,
http://dx.doi.org/10.1097/01.CCM.0000214517.24064.35.
http://dx.doi.org/10.1097/01.CCM.0000214...
). We did find that D-lactate was elevated at
3 hours, which coincided with severe mucosal injury and, presumably, gut barrier
dysfunction. This suggests that D-lactate sensitivity is time-dependent and
correlates to structural mucosal injury.
Interestingly, D-lactate remained elevated at 48 hours. This could indicate prolonged
functional impairment and bacterial translocation independent of mucosal
morphological integrity. Support for this idea comes from previous studies that
showed elevated D-lactate at 48 hours of reperfusion and correlated to bacteremia
following 1 hour of intestinal ischemia in rats (3030. Wu GH, Wang H, Zhang YW, Wu ZH, Wu ZG. Glutamine supplemented
parenteral nutrition prevents intestinal ischemia- reperfusion injury in rats.
World J Gastroenterol. 2004;10(17):2592-4.). Human studies have confirmed persistent elevations of D-lactate 48
hours after reperfusion during colonic ischemia (3131. Assadian A, Assadian O, Senekowitsch C, Rotter R, Bahrami S,
Fürst W, et al. Plasma D-lactate as a potential early marker for colon
ischaemia after open aortic reconstruction. Eur J Vasc Endovasc Surg.
2006;31(5):470-4, http://dx.doi.org/10.1016/j.ejvs.2005.10.031.
http://dx.doi.org/10.1016/j.ejvs.2005.10...
). The temporal dynamics of tight-junction alterations in the
intestinal mucosa, thought to be a basis for barrier dysfunction after IR (3232. Inoue K, Oyamada M, Mitsufuji S, Okanoue T, Takamatsu T.
Different changes in the expression of multiple kinds of tight-junction proteins
during ischemia-reperfusion injury of the rat ileum. Acta Histochem Cytochem
2006;39(2):35-45.), could partly explain this result.
Additionally, experimental studies in primates have shown that serum D-lactate
levels can predict mortality in models of hemorrhagic shock (3333. Sobhian B, Kröpfl A, Hölzenbein T, Khadem A, Redl H,
Bahrami S. Increased circulating D-lactate levels predict risk of mortality
after hemorrhage and surgical trauma in baboons. Shock. 2012;37(5):473-7,
http://dx.doi.org/10.1097/SHK.0b013e318249cb96.
http://dx.doi.org/10.1097/SHK.0b013e3182...
). This adds to the possible clinical value of D-lactate as a
biomarker in settings of intestinal IR.
2525. Van Noord D, Mensink PB, de Knegt RJ, Ouwendijk M, Francke, van
Vuuren AJ, et al. Serum markers and instestinal mucosal injury in chronic
gastrointestinal ischemia. Dig Dis Sci. 2011;56(2):506-12,
http://dx.doi.org/10.1007/s10620-010-1303-5.
http://dx.doi.org/10.1007/s10620-010-130...
, was unrelated to histopathology during
the acute phase in our study. Rat models of acute intestinal IR have shown
elevations of iFABP early in the course of intestinal mucosal injury (3434. Lieberman JM, Sacchettini J, Marks C, Marks WH. Human intestinal
fatty acid binding protein: report of an assay with studies in normal volunteers
and intestinal ischemia. Surgery. 1997;121(3):335-42,
http://dx.doi.org/10.1016/S0039-6060(97)90363-9.
http://dx.doi.org/10.1016/S0039-6060(97)...
). Clinical studies have documented an
elevation of iFABP as a specific marker for mucosal injury. Although the source of
iFABP is assumed to be the cells of the intestinal mucosa, its utility is based on
the identification of a chronic process of local lesions (88. Mannoia K, Boskovic DS, Slater L, Plank MS, Angeles DM, Gollin G.
Necrotizing enterocolitis is associated with neonatal intestinal injury.
J Pediatr Surg. 2011;46(1):81-5,
http://dx.doi.org/10.1016/j.jpedsurg.2010.09.069.
http://dx.doi.org/10.1016/j.jpedsurg.201...
,99. Edelson MB, Sonnino RE, Bagwell CE, Lieberman JM, Marks WH,
Rozycki HJ. Plasma intestinal fatty acid binding protein in neonates with
necrotizing enterocolitis: a pilot study. J Pediatr Surg.
1999;34(10):1453-7,
http://dx.doi.org/10.1016/S0022-3468(99)90102-1.
http://dx.doi.org/10.1016/S0022-3468(99)...
,2525. Van Noord D, Mensink PB, de Knegt RJ, Ouwendijk M, Francke, van
Vuuren AJ, et al. Serum markers and instestinal mucosal injury in chronic
gastrointestinal ischemia. Dig Dis Sci. 2011;56(2):506-12,
http://dx.doi.org/10.1007/s10620-010-1303-5.
http://dx.doi.org/10.1007/s10620-010-130...
). However, recent clinical studies have
found elevations of iFABP during acute intestinal necrosis (3535. Vermeulen Windsant IC, Hellenthal FA, Derikx JP, Prins MH,
Buurman WA, Jacobs MJ, et al. Circulating Intestinal Fatty Acid-Binding Protein
as an Early Marker of Intestinal Necrosis After Aortic Surgery: A Prospective
Observational Cohort Study. Ann Surg. 2012;255(4):796-803,
http://dx.doi.org/10.1097/SLA.0b013e31824b1e16.
http://dx.doi.org/10.1097/SLA.0b013e3182...
). Not all studies have been consistent: iFABP was not
correlated to intestinal IR at any time point (1, 3 and 6 hours reperfusion) in a
recent study in rabbits (3636. Dundar ZD, Cander B, Gul M, Karabulut KU, Kocak S, Girisgin S,
et al. Serum intestinal fatty acid binding protein and phosphate levels in the
diagnosis of acute intestinal ischemia: an experimental study in rabbits.
J Emerg Med. 2012;42(6):741-7,
http://dx.doi.org/10.1016/j.jemermed.2011.05.051.
http://dx.doi.org/10.1016/j.jemermed.201...
). Together with
our results, this suggests that the role of iFABP as a biomarker for intestinal
ischemia remains unclear.
We identified ALT and AST as molecules that remained elevated even after
histopathological injury had subsided, peaking at 6 and 24 hours post-ischemia. A
previous study showed that AST and LDH elevations occurred early after IR, but no
ALT elevations were evident at 120 minutes of reperfusion (3737. Caglayan F, Caglayan O, Gunel E, Elcuman Y, Cakmak M. Intestinal
ischemia-reperfusion and plasma enzyme levels. Pediatr Surg Int 2002;
18(4):255-7.). This suggested that the origin of AST and LDH was indeed
the intestine and not the liver. In our study, we also found delayed ALT elevations.
During reperfusion, peripheral organs, such as the lung and liver, displayed injury
as a result of polymorphonuclear leukocyte infiltration and the release of
inflammatory mediators into the bloodstream (3838. Simpson R, Alon R, Kobzik L, Valeri CR, Shepro D, Hechtman HB.
Neutrophil and nonneutrophil mediated injury in intestinal ischemia reperfusion.
Ann Surg. 1993;218(4):444-54,
http://dx.doi.org/10.1097/00000658-199310000-00005.
http://dx.doi.org/10.1097/00000658-19931...
). Liver and lung injury indeed occur after intestinal IR, and the
elevation of these enzymes appears to reflect remote organ damage (3939. Cámara-Lemarroy CR, Guzmán-de la Garza FJ,
Alarcón-Galván G, Cordero-Pérez P, Muñoz-Espinosa LE,
Fernández-Garza NE. Effects of thalidomide and pentoxyphylline over local
and remote organ injury after intestinal ischemia/reperfusion. Transplant Proc.
2010;42(5):1624-6,
http://dx.doi.org/10.1016/j.transproceed.2009.12.074.
http://dx.doi.org/10.1016/j.transproceed...
).
The use of fixed time models of intestinal IR injury using serum markers as surrogates for histopathological analysis should be discouraged. Both serum markers and histology are important in grading the severity of IR injury. Additionally, the chosen markers should ideally coincide with mucosal injury. Studying the changes in concentration of these markers during experimentation will allow for a better understanding of the process. When planning a study on IR injury, it is essential to identify the times at which measurements will be carried out, as this could reflect different aspects of the physiopathology.
Our study has several limitations. We only used one ischemia time (30 minutes), making it impossible to directly establish correlations between the serum markers and local or systemic injury. We used a sublethal model of intestinal IR and could not make associations with mortality. We did not directly measure for mucosal barrier permeability, and we did not carry out immunohistochemical or ultrastructural investigations of the injured mucosa. These are areas of future research.
In conclusion, AST, LDH, IL-1 and TNF-alpha are elevated early after reperfusion, and
in the case of TNF-alpha, correlate well with maximal mucosal injury. However, they
are non-specific markers of tissue injury (AST, LDH) or part of the systemic
inflammatory response (IL-1, TNF-alpha). Their utility as diagnostic tools is thus
limited, but they could reflect injury severity. We were surprised not to find
changes in IL-6 levels considering the evidence of its involvement in IR injury
(2626. Hei ZQ, Gan XL, Huang PJ, Wei J, Shen N, Gao WL. Influence of
Ketotifen, Cromolyn Sodium, and Compound 48/80 on the survival rates after
intestinal ischemia reperfusion injury in rats. BMC Gastroenterol. 2008;8:42,
http://dx.doi.org/10.1186/1471-230X-8-42.
http://dx.doi.org/10.1186/1471-230X-8-42...
27. Yamamoto S, Tanabe M, Wakabayashi G, Shimazu M, Matsumoto K,
Kitajima M. The role of tumor necrosis factor-alpha and interleukin-1beta in
ischemia-reperfusion injury of the rat small intestine. J Surg Res.
2001;99(1):134-41, http://dx.doi.org/10.1006/jsre.2001.6106.
http://dx.doi.org/10.1006/jsre.2001.6106...
28. Grotz MR, Deitch EA, Ding J, Xu D, Huang Q, Regel G. Intestinal
cytokine response after gut ischemia. Role of gut barrier failure. Ann Surg.
1999;229(4):478-86,
http://dx.doi.org/10.1097/00000658-199904000-00005.
http://dx.doi.org/10.1097/00000658-19990...
29. Collange O, Tamion F, Chanel S, Hue G, Richard V, Thuilliez C,
et al. D-lactate is not a reliable marker of gut ischemia-reperfusion in a rat
model of supraceliac aortic clamping. Crit Care Med. 2006;34(5):1415-9,
http://dx.doi.org/10.1097/01.CCM.0000214517.24064.35.
http://dx.doi.org/10.1097/01.CCM.0000214...
30. Wu GH, Wang H, Zhang YW, Wu ZH, Wu ZG. Glutamine supplemented
parenteral nutrition prevents intestinal ischemia- reperfusion injury in rats.
World J Gastroenterol. 2004;10(17):2592-4.
31. Assadian A, Assadian O, Senekowitsch C, Rotter R, Bahrami S,
Fürst W, et al. Plasma D-lactate as a potential early marker for colon
ischaemia after open aortic reconstruction. Eur J Vasc Endovasc Surg.
2006;31(5):470-4, http://dx.doi.org/10.1016/j.ejvs.2005.10.031.
http://dx.doi.org/10.1016/j.ejvs.2005.10...
32. Inoue K, Oyamada M, Mitsufuji S, Okanoue T, Takamatsu T.
Different changes in the expression of multiple kinds of tight-junction proteins
during ischemia-reperfusion injury of the rat ileum. Acta Histochem Cytochem
2006;39(2):35-45.
33. Sobhian B, Kröpfl A, Hölzenbein T, Khadem A, Redl H,
Bahrami S. Increased circulating D-lactate levels predict risk of mortality
after hemorrhage and surgical trauma in baboons. Shock. 2012;37(5):473-7,
http://dx.doi.org/10.1097/SHK.0b013e318249cb96.
http://dx.doi.org/10.1097/SHK.0b013e3182...
34. Lieberman JM, Sacchettini J, Marks C, Marks WH. Human intestinal
fatty acid binding protein: report of an assay with studies in normal volunteers
and intestinal ischemia. Surgery. 1997;121(3):335-42,
http://dx.doi.org/10.1016/S0039-6060(97)90363-9.
http://dx.doi.org/10.1016/S0039-6060(97)...
35. Vermeulen Windsant IC, Hellenthal FA, Derikx JP, Prins MH,
Buurman WA, Jacobs MJ, et al. Circulating Intestinal Fatty Acid-Binding Protein
as an Early Marker of Intestinal Necrosis After Aortic Surgery: A Prospective
Observational Cohort Study. Ann Surg. 2012;255(4):796-803,
http://dx.doi.org/10.1097/SLA.0b013e31824b1e16.
http://dx.doi.org/10.1097/SLA.0b013e3182...
36. Dundar ZD, Cander B, Gul M, Karabulut KU, Kocak S, Girisgin S,
et al. Serum intestinal fatty acid binding protein and phosphate levels in the
diagnosis of acute intestinal ischemia: an experimental study in rabbits.
J Emerg Med. 2012;42(6):741-7,
http://dx.doi.org/10.1016/j.jemermed.2011.05.051.
http://dx.doi.org/10.1016/j.jemermed.201...
37. Caglayan F, Caglayan O, Gunel E, Elcuman Y, Cakmak M. Intestinal
ischemia-reperfusion and plasma enzyme levels. Pediatr Surg Int 2002;
18(4):255-7.
38. Simpson R, Alon R, Kobzik L, Valeri CR, Shepro D, Hechtman HB.
Neutrophil and nonneutrophil mediated injury in intestinal ischemia reperfusion.
Ann Surg. 1993;218(4):444-54,
http://dx.doi.org/10.1097/00000658-199310000-00005.
http://dx.doi.org/10.1097/00000658-19931...
39. Cámara-Lemarroy CR, Guzmán-de la Garza FJ,
Alarcón-Galván G, Cordero-Pérez P, Muñoz-Espinosa LE,
Fernández-Garza NE. Effects of thalidomide and pentoxyphylline over local
and remote organ injury after intestinal ischemia/reperfusion. Transplant Proc.
2010;42(5):1624-6,
http://dx.doi.org/10.1016/j.transproceed.2009.12.074.
http://dx.doi.org/10.1016/j.transproceed...
-4040. Braun F, Hosseini M, Wieland E, Sattler B, Müller AR,
Fändrich F, et al. Kinetics and localization of interleukin-2,
interleukin-6, heat shock protein 70, and interferon gamma during
intestinal-rerfusion injury. Transplant Proc. 2004;36(2):267-9,
http://dx.doi.org/10.1016/j.transproceed.2004.01.082.
http://dx.doi.org/10.1016/j.transproceed...
). D-lactate is a more promising clinical biomarker of intestinal
injury and could be used to evaluate functional (barrier) alterations. It coincided
with maximal injury in our study and remained elevated after 48 hours. However,
D-lactate was not elevated in the hyperacute phase of intestinal ischemia (1 hour of
reperfusion). The specific roles of other markers, such as iFABP, coagulation
parameters (4141. Guzman-de la Garza FJ, Camara-Lemaroy CR, Ballesteros-Elizondo
RG, Alarcon-Galvan G, Cordero Perez P, Fernandez- Garza NE. Ketamine and the
Myenteric Plexus in Intestinal Ischemia/Reparfusion Injury. Dig Dis Sci.
2010;55(7):1878-85,
http://dx.doi.org/10.1007/s10620-009-0976-0.
http://dx.doi.org/10.1007/s10620-009-097...
), oxidative stress (4242. Li Ch, Jackson RM. Reactive species mechanisms of cellular
hypoxia-reoxygenation injury. Am J Physiol Cell Physiol.
2001;282(2):C227-41.), and adhesion molecules (4040. Braun F, Hosseini M, Wieland E, Sattler B, Müller AR,
Fändrich F, et al. Kinetics and localization of interleukin-2,
interleukin-6, heat shock protein 70, and interferon gamma during
intestinal-rerfusion injury. Transplant Proc. 2004;36(2):267-9,
http://dx.doi.org/10.1016/j.transproceed.2004.01.082.
http://dx.doi.org/10.1016/j.transproceed...
), requires further investigation.
We would like to thank Gilberto Arevalo Martinez, MVZ, for providing the animals and ensuring their care. We would also like to thank the staff at the Liver Unit, UANL for management of the serum samples. The project was financed by the Department of Physiology of the School of Medicine, the Liver Unit and The Pathology and Cytopathology Service of the “Dr. José Eleuterio González” University Hospital, Universidad Autónoma de Nuevo León. This work was supported by the Programa de Apoyo a la Investigación Científica y Tecnológica of UANL (PAICYT-UANL: SA339-10).
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26Hei ZQ, Gan XL, Huang PJ, Wei J, Shen N, Gao WL. Influence of Ketotifen, Cromolyn Sodium, and Compound 48/80 on the survival rates after intestinal ischemia reperfusion injury in rats. BMC Gastroenterol. 2008;8:42, http://dx.doi.org/10.1186/1471-230X-8-42.
» http://dx.doi.org/10.1186/1471-230X-8-42 -
27Yamamoto S, Tanabe M, Wakabayashi G, Shimazu M, Matsumoto K, Kitajima M. The role of tumor necrosis factor-alpha and interleukin-1beta in ischemia-reperfusion injury of the rat small intestine. J Surg Res. 2001;99(1):134-41, http://dx.doi.org/10.1006/jsre.2001.6106.
» http://dx.doi.org/10.1006/jsre.2001.6106 -
28Grotz MR, Deitch EA, Ding J, Xu D, Huang Q, Regel G. Intestinal cytokine response after gut ischemia. Role of gut barrier failure. Ann Surg. 1999;229(4):478-86, http://dx.doi.org/10.1097/00000658-199904000-00005.
» http://dx.doi.org/10.1097/00000658-199904000-00005 -
29Collange O, Tamion F, Chanel S, Hue G, Richard V, Thuilliez C, et al. D-lactate is not a reliable marker of gut ischemia-reperfusion in a rat model of supraceliac aortic clamping. Crit Care Med. 2006;34(5):1415-9, http://dx.doi.org/10.1097/01.CCM.0000214517.24064.35.
» http://dx.doi.org/10.1097/01.CCM.0000214517.24064.35 -
30Wu GH, Wang H, Zhang YW, Wu ZH, Wu ZG. Glutamine supplemented parenteral nutrition prevents intestinal ischemia- reperfusion injury in rats. World J Gastroenterol. 2004;10(17):2592-4.
-
31Assadian A, Assadian O, Senekowitsch C, Rotter R, Bahrami S, Fürst W, et al. Plasma D-lactate as a potential early marker for colon ischaemia after open aortic reconstruction. Eur J Vasc Endovasc Surg. 2006;31(5):470-4, http://dx.doi.org/10.1016/j.ejvs.2005.10.031.
» http://dx.doi.org/10.1016/j.ejvs.2005.10.031 -
32Inoue K, Oyamada M, Mitsufuji S, Okanoue T, Takamatsu T. Different changes in the expression of multiple kinds of tight-junction proteins during ischemia-reperfusion injury of the rat ileum. Acta Histochem Cytochem 2006;39(2):35-45.
-
33Sobhian B, Kröpfl A, Hölzenbein T, Khadem A, Redl H, Bahrami S. Increased circulating D-lactate levels predict risk of mortality after hemorrhage and surgical trauma in baboons. Shock. 2012;37(5):473-7, http://dx.doi.org/10.1097/SHK.0b013e318249cb96.
» http://dx.doi.org/10.1097/SHK.0b013e318249cb96 -
34Lieberman JM, Sacchettini J, Marks C, Marks WH. Human intestinal fatty acid binding protein: report of an assay with studies in normal volunteers and intestinal ischemia. Surgery. 1997;121(3):335-42, http://dx.doi.org/10.1016/S0039-6060(97)90363-9.
» http://dx.doi.org/10.1016/S0039-6060(97)90363-9 -
35Vermeulen Windsant IC, Hellenthal FA, Derikx JP, Prins MH, Buurman WA, Jacobs MJ, et al. Circulating Intestinal Fatty Acid-Binding Protein as an Early Marker of Intestinal Necrosis After Aortic Surgery: A Prospective Observational Cohort Study. Ann Surg. 2012;255(4):796-803, http://dx.doi.org/10.1097/SLA.0b013e31824b1e16.
» http://dx.doi.org/10.1097/SLA.0b013e31824b1e16 -
36Dundar ZD, Cander B, Gul M, Karabulut KU, Kocak S, Girisgin S, et al. Serum intestinal fatty acid binding protein and phosphate levels in the diagnosis of acute intestinal ischemia: an experimental study in rabbits. J Emerg Med. 2012;42(6):741-7, http://dx.doi.org/10.1016/j.jemermed.2011.05.051.
» http://dx.doi.org/10.1016/j.jemermed.2011.05.051 -
37Caglayan F, Caglayan O, Gunel E, Elcuman Y, Cakmak M. Intestinal ischemia-reperfusion and plasma enzyme levels. Pediatr Surg Int 2002; 18(4):255-7.
-
38Simpson R, Alon R, Kobzik L, Valeri CR, Shepro D, Hechtman HB. Neutrophil and nonneutrophil mediated injury in intestinal ischemia reperfusion. Ann Surg. 1993;218(4):444-54, http://dx.doi.org/10.1097/00000658-199310000-00005.
» http://dx.doi.org/10.1097/00000658-199310000-00005 -
39Cámara-Lemarroy CR, Guzmán-de la Garza FJ, Alarcón-Galván G, Cordero-Pérez P, Muñoz-Espinosa LE, Fernández-Garza NE. Effects of thalidomide and pentoxyphylline over local and remote organ injury after intestinal ischemia/reperfusion. Transplant Proc. 2010;42(5):1624-6, http://dx.doi.org/10.1016/j.transproceed.2009.12.074.
» http://dx.doi.org/10.1016/j.transproceed.2009.12.074 -
40Braun F, Hosseini M, Wieland E, Sattler B, Müller AR, Fändrich F, et al. Kinetics and localization of interleukin-2, interleukin-6, heat shock protein 70, and interferon gamma during intestinal-rerfusion injury. Transplant Proc. 2004;36(2):267-9, http://dx.doi.org/10.1016/j.transproceed.2004.01.082.
» http://dx.doi.org/10.1016/j.transproceed.2004.01.082 -
41Guzman-de la Garza FJ, Camara-Lemaroy CR, Ballesteros-Elizondo RG, Alarcon-Galvan G, Cordero Perez P, Fernandez- Garza NE. Ketamine and the Myenteric Plexus in Intestinal Ischemia/Reparfusion Injury. Dig Dis Sci. 2010;55(7):1878-85, http://dx.doi.org/10.1007/s10620-009-0976-0.
» http://dx.doi.org/10.1007/s10620-009-0976-0 -
42Li Ch, Jackson RM. Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol. 2001;282(2):C227-41.
Publication Dates
-
Publication in this collection
July 2013
History
-
Received
27 Feb 2013 -
Reviewed
18 Mar 2013 -
Accepted
30 Mar 2013