IN VITRO METHOD FOR DIAGNOSING AT EARLY STAGE INTESTINAL ISCHEMIA

Abstract

An in vitro diagnostic method is provided for diagnosing at an early stage an intestinal ischemia in a patient suspected of suffering from intestinal ischemia.

Claims

1. An in vitro diagnostic method for diagnosing at an early stage an intestinal ischemia in a patient suspected of suffering from intestinal ischemia, which comprises: (i) determining in a biological sample from said patient the circulating glucagon-like peptide 2 (GLP-2) level and/or the circulating glucagon-like peptide 1 (GLP-1) level; and (ii) comparing said level with the circulating glucagon-like peptide 2 level and/or the circulating glucagon-like peptide 1 level in a reference sample, wherein the increase of said level in the patient is indicative of said patient suffering from intestinal ischemia.

2. The method according to claim 1, wherein the intestinal ischemia is mesenteric ischemia-reperfusion, especially acute mesenteric ischemia, chronic mesenteric ischemia, or ischemic colitis, or a disease or trouble linked to gut barrier.

3. The method according to claim 1, wherein the biological sample is a blood plasma sample.

4. The method according to claim 1, wherein the level of circulating glucagon-like peptide 2 or circulating glucagon-like peptide 1 is determined by the means of an immunoassay.

5. The method according to claim 4, wherein said immunoassay is ELISA.

6. The method according to claim 1, wherein the reference sample is a biological sample from a healthy subject.

7. Use of a reagent capable of detecting the level of circulating glucagon-like peptide 2 or of circulating glucagon-like peptide 1 for diagnosing in vitro at an early stage an intestinal ischemia in a patient suspected of suffering from intestinal ischemia.

8. The use according to claim 7, wherein the reagent is an antibody directed against glucagon-like peptide 2 or glucagon-like peptide 1.

9. Use of a kit of ELISA containing an antibody directed against glucagon-like peptide 2 or glucagon-like peptide 1 for diagnosing in vitro at an early stage an intestinal ischemia in a patient suspected of suffering from intestinal ischemia.

Description

FIGURES

[0055] FIGS. 1A, 1B and 1C show intestinal injury following acute mesenteric ischemia and reperfusion. Hematoxylin/eosin staining of ileum histological sections after sham treatment (FIG. 1A), 20 minutes of ischemia followed by 15 minutes of reperfusion (FIG. 1B) and 40 minutes of ischemia followed by 15 minutes of reperfusion (FIG. 1C).

[0056] FIGS. 2A and 2B show that intestinal ischemia/reperfusion induces a quick secretion of GLP-1. Ischemia-reperfusion (I/R) of the superior mesenteric artery is applied in a group of 6 mice. Sham-operated mice were used as control. Statistical analysis were performed using an unpaired t test, difference from sham (*) are displayed: *p<0.05, **p<0.01 and ***p<0.001. Values are meanSEM. FIG. 2A: total GLP-1 plasma levels (pM; n=5) after 20 minutes of ischemia followed by 15, 30, 45, 60 and 120 minutes of reperfusion. FIG. 2B: total GLP-1 plasma levels (% of sham; n=5) after 5, 10 and 15 minutes of ischemia followed by 15 min of reperfusion.

[0057] FIG. 3: total GLP-1 and I-FABP plasma levels ( 0/0 of sham, n=6) after a short and a long I/R (20 minutes ischemia followed by 15 minutes or 2 hours reperfusion respectively).

[0058] FIG. 4 shows the comparison of the secretion of inflammation markers after a treatment of I/R (20 min/30 min) in mice of Sham group and of I/R group.

[0059] FIG. 5 shows total GLP-1 arteriovenous differences in patients before and after 45 minutes ischemia and 0, 30 or 120 minutes reperfusion (n=6). All results are expressed as meanSEM.

[0060] FIGS. 6A and 6B show that intestinal ischemia/reperfusion induces a quick secretion of total GLP-2. Ischemia-reperfusion (I/R) of the superior mesenteric artery is applied in a group of 6 mice. Sham-operated mice were used as control. Statistical analysis were performed using an unpaired t test, difference from sham (*) are displayed: *p<0.05, **p<0.01 and ***p<0.001. Values are meanSEM. FIG. 6A: total GLP-2 plasma levels (ng/mL; n=5) after 5, 10 and 15 minutes of ischemia followed by 15 min of reperfusion. FIG. 6B: total GLP-2 plasma levels (ng/mL; n=5) after 20 minutes of ischemia followed by 15, 60 and 180 minutes of reperfusion.

EXAMPLES

[0061] 1. Materials and Methods

[0062] Animals

[0063] WT mice (8-12 weeks old, Charles River) from a homogeneous C57BL6/J background were housed in a controlled environment and fed a standard chow diet (A03 diet; Safe, Augy, France). Animals had free access to water and food. All experiments involving animals were performed in accordance with institutional guidelines and approved by the University of Burgundy's Ethics Committee on the Use of Laboratory Animals (protocol number 5459).

[0064] Animal Model of Intestinal Ischemia/Reperfusion

[0065] Mice were separated into sham-operated groups and ischemia/reperfusion (I/R) groups (n=5/6). They were anesthetized with isoflurane inhalation and placed in a supine position on heating pads to maintain body temperature at 37 C. Midline laparotomy was performed and the superior mesenteric artery (SMA) was isolated. Ischemia was induced by clamping the SMA for 5, 10, 15 or 20 minutes and was followed by 15, 30, 45, 60 or 120 minutes of reperfusion (removal of the clamp). Gut ischemia was confirmed by intestinal color, change and gut reperfusion by the reappearance of pulsation and color. Blood collections were performed to quantify GLP-1, GLP-2 and cytokines. Blood samples were collected in EDTA-coated tubes (BD Vacutainer) from the systemic (retro-orbital or intracardiac puncture) circulation. Plasma was separated by centrifugation at 8000 rpm for 10 minutes at 4 C. Blood and plasma samples were frozen at 20 C. for further analysis. Mice were euthanatized by cervical dislocation and the distal part of the small intestine (ileum) was removed and immediately fixed for histological studies.

[0066] Surgical operation for sham-operated group mice was the same, except that the superior mesenteric artery was not clamped.

[0067] Human Intestinal Ischemia/Reperfusion

[0068] The experimental protocol was performed as previously described (Grootjans et al., 2010). The study was approved by the Medical Ethics Committee of the Maastricht University Medical Center and written informed consent of all patients was obtained. 6 patients with a median age of 66 years (range, 54 to 83 years) undergoing pancreaticoduodenectomy for benign or malignant disease were included in this study. Patients with bile duct obstructive disease were stented before surgery. All patients had normal bile flow at the time of the surgical procedure. During pancreaticoduodenectomy, a variable segment of jejunum is routinely resected in continuity with the head of the pancreas and duodenum as part of the surgical procedure. The terminal 6 cm of this jejunal segment was isolated and subjected to 45 minutes of ischemia by placing two atraumatic vascular clamps over the mesentery. Meanwhile, surgery proceeded as planned. After 45 minutes of ischemia, one third (2 cm) of the isolated ischemic jejunum was resected using a linear cutting stapler. Next, clamps were removed to allow reperfusion, as confirmed by regaining of normal pink color and restoration of gut motility. Another segment of the isolated jejunum (2 cm) was resected similarly after 30 minutes of reperfusion. The last part was resected after 120 minutes of reperfusion. Simultaneously, 2 cm of jejunum, which remained untreated during surgery, was resected, serving as internal control tissue. This segment underwent similar surgical handling as the isolated part of jejunum, but was not exposed to I/R. Arterial blood was sampled before ischemia, immediately on reperfusion, and at 30 and 120 minutes after start of reperfusion. Simultaneous with each respective arterial blood sample, blood was drawn from the venule draining the isolated jejunal segment by direct puncture to assess concentration gradients across the isolated jejunal segment. All blood samples were directly transferred to prechilled EDTA vacuum tubes (Becton Dickinson Diagnostics, Aalst, Belgium) and kept on ice. At the end of the procedure all blood samples were centrifuged at 4000 rpm, 4 C. for 15 minutes to obtain plasma. Plasma was immediately stored in aliquots at 80 C. until analysis.

[0069] Light Microscopy

[0070] The morphologic alterations in the gut were examined by light microscopy (50, 100 and 200). Briefly, tissues from the distal small intestine (ileum) were promptly taken in sham-operated and I/R groups after 20 or 40 minutes of ischemia and 15 minutes of reperfusion. Gut samples were fixed for 48 hours in 10% neutral buffered formalin at room temperature, dehydrated by graded ethanol and embedded in paraffin for histological analysis. Tissue sections (thickness of 5 m) were deparaffinized with xylene, stained with hematoxylin and eosin.

[0071] Biochemical Analysis

[0072] Total GLP-1, GLP-2 and I-FABP concentrations were determined by commercially available ELISA Kits (Millipore, St. Charles, Mo. and Cliniscience) in accordance with manufacturer's protocols.

[0073] Cytokines plasma levels (interleukin (IL)-1, IL-6 and tumor necrosis factor- (TNF-)) were measured by Milliplex MAP 5-Plex Kit using mouse cytokine/chemokine magnetic bead panel (Millipore, Billerica, Mass.) according to the manufacturer's protocol and using a LuminexR apparatus (Bio-Flex 200, Bio-Rad).

[0074] Statistical Analysis

[0075] Numeric data are presented as meanstandard error of mean. Statistical analysis were performed using either the unpaired Sudent's t-test or the nonparametric Mann-Withney U test depending on data distribution's normality. D'Agostino's K.sup.2 test was used to establish whether or not groups of data were normally distributed. A statistical correction was applied when variances were different between groups. A value of P<0.05 was considered statistically significant.

[0076] 2. Results

[0077] 2.1. Rapid GLP-1 Secretion in Mice after Gut Barrier Injury

[0078] The mesenteric ischemia-reperfusion (I/R) was produced in a group of mice. A group of sham-operated mice was used as control. Gut ultrastructure was deeply disorganized after I/R (FIGS. 1A, 1B, 1C). Short times of I/Ft were sufficient to damage intestinal villi compared to sham-operated mice (FIGS. 1A and 1B). Increased damages were observed with increased times of I/R (FIG. 1C). I/R experiments led to a rapid increase of GLP-1 plasma levels (FIGS. 2A and 2B). As shown in FIG. 2A, reperfusion of the mesenteric artery for 2 hours after 20 minutes of ischemia led to a raise of GLP-1 plasma levels. More notably, this increase was significant after only 15 minutes of reperfusion. Shorter times of ischemia (<20 minutes) were still able to induce GLP-1 secretion (FIG. 2B).

[0079] It is revealed that after mesenteric ischemia-reperfusion, GLP-1 secretion precedes I-FABP secretion (FIG. 3) and markers of inflammation, such as IL-1b, IL-6, TNF- (FIG. 4). In contrast to I-FABP, a short I/IR treatment was sufficient to induce a significant plasma GLP-1. Moreover, after mesenteric ischemia-reperfusion, GLP-1 secretion is quantitatively more important than that of I-FABP (FIG. 3).

[0080] These results show that, compared with I-FABP, GLP-1 is more sensitive and can be used as biomarker for diagnosing gut barrier injury at more early stage.

[0081] 2.2. Rapid GLP-1 Secretion in Human after Gut Barrier Injury

[0082] A new model of I/R using human gut tissue (Grootjans et al., 2010) has been used to evaluate if I/R injury in the human gut in vivo was associated with an increase in GLP-1 secretion. Arterio-venous differences in human plasma GLP-1 levels were measured before, after 45 minutes of ischemia and after 30 or 120 minutes of reperfusion. GLP-1 levels were markedly increased after 45 minutes of ischemia and returned to baseline levels after reperfusion (FIG. 5). These results demonstrate that human gut injury is associated with a rapid induction of GLP-1 secretion in vivo.

[0083] 2.3, Rapid GLP-2 Secretion in Mice after Gut Barrier Injury

[0084] GLP-2 secretion level is also measured in mice after gut barrier injury in the experimental conditions similar to that for measuring GLP-1 secretion level, A rapid increase of plasma GLP-2 level is observed after a very short time (15 minutes) of intestinal ischemia (FIG. 6A). An important increase of plasma GLP-2 level is observed after 180 minutes of reperfusion following 20 minutes of ischemia (FIG. 6B).

[0085] These results shows that GLP-2 is also sensitive to gut barrier injury and can be used as a biomarker for diagnosing gut barrier injury at more early stage.