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Rapid reversal of human intestinal ischemia-reperfusion induced damage by shedding of injured enterocytes and reepithelialisation.

Derikx JP, Matthijsen RA, de Bruïne AP, van Bijnen AA, Heineman E, van Dam RM, Dejong CH, Buurman WA - PLoS ONE (2008)

Bottom Line: Although intestinal IR has been studied extensively in animals, results remain inconclusive and data on human intestinal IR are scarce.It reveals a unique, endogenous clearing mechanism for injured enterocytes: rapid detachment of damaged apoptotic enterocytes into the lumen.This process is followed by repair of the epithelial continuity within an hour, resulting in a normal epithelial lining.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, School for Nutrition & Metabolism (NUTRIM), Maastricht University Medical Centre+, Maastricht, the Netherlands.

ABSTRACT

Background: Intestinal ischemia-reperfusion (IR) is a phenomenon related to physiological conditions (e.g. exercise, stress) and to pathophysiological events (e.g. acute mesenteric ischemia, aortic surgery). Although intestinal IR has been studied extensively in animals, results remain inconclusive and data on human intestinal IR are scarce. Therefore, an experimental harmless model for human intestinal IR was developed, enabling us to clarify the sequelae of human intestinal IR for the first time.

Methods and findings: In 30 patients undergoing pancreatico-duodenectomy we took advantage of the fact that in this procedure a variable length of jejunum is removed. Isolated jejunum (5 cm) was subjected to 30 minutes ischemia followed by reperfusion. Intestinal Fatty Acid Binding Protein (I-FABP) arteriovenous concentration differences across the bowel segment were measured before and after ischemia to assess epithelial cell damage. Tissue sections were collected after ischemia and at 25, 60 and 120 minutes reperfusion and stained with H&E, and for I-FABP and the apoptosis marker M30. Bonferroni's test was used to compare I-FABP differences. Mean (SEM) arteriovenous concentration gradients of I-FABP across the jejunum revealed rapidly developing epithelial cell damage. I-FABP release significantly increased from 290 (46) pg/ml before ischemia towards 3,997 (554) pg/ml immediately after ischemia (p<0.001) and declined gradually to 1,143 (237) pg/ml within 1 hour reperfusion (p<0.001). Directly after ischemia the intestinal epithelial lining was microscopically normal, while subepithelial spaces appeared at the villus tip. However, after 25 minutes reperfusion, enterocyte M30 immunostaining was observed at the villus tip accompanied by shedding of mature enterocytes into the lumen and loss of I-FABP staining. Interestingly, within 60 minutes reperfusion the epithelial barrier resealed, while debris of apoptotic, shedded epithelial cells was observed in the lumen. At the same time, M30 immunoreactivity was absent in intact epithelial lining.

Conclusions: This is the first human study to clarify intestinal IR induced cell damage and repair and its direct consequences. It reveals a unique, endogenous clearing mechanism for injured enterocytes: rapid detachment of damaged apoptotic enterocytes into the lumen. This process is followed by repair of the epithelial continuity within an hour, resulting in a normal epithelial lining.

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Immunolocalization of I-FABP in red (3-amino-9-ethylcarbazole, AEC) (100×) in the control jejunum not subjected to ischemia-reperfusion (A) shows an abundant cytosolic presence of I-FABP in the epithelial cells of the upper half of the villus.Upon 30 minutes ischemia (B), cytosolic I-FABP staining is decreased in mature enterocytes with abundant staining in the subepithelial spaces. A decreased cytosolic staining is still observed after 25 minutes reperfusion (C). Within 60 minutes reperfusion, I-FABP cytosolic positive cells are part of the resealed epithelial barrier (D), while shedded I-FABP containing enterocytes are found in the debris in the lumen.
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pone-0003428-g002: Immunolocalization of I-FABP in red (3-amino-9-ethylcarbazole, AEC) (100×) in the control jejunum not subjected to ischemia-reperfusion (A) shows an abundant cytosolic presence of I-FABP in the epithelial cells of the upper half of the villus.Upon 30 minutes ischemia (B), cytosolic I-FABP staining is decreased in mature enterocytes with abundant staining in the subepithelial spaces. A decreased cytosolic staining is still observed after 25 minutes reperfusion (C). Within 60 minutes reperfusion, I-FABP cytosolic positive cells are part of the resealed epithelial barrier (D), while shedded I-FABP containing enterocytes are found in the debris in the lumen.

Mentions: At the end of the ischemic period of 30 minutes, H&E sections of the jejunum showed microscopically normal epithelial lining (Figure 1A, B). However, subepithelial spaces appeared at the villus tip. To study the epithelial cells in more detail, immunohistochemical staining of I-FABP was performed, a small protein present in the cytoplasma of differentiated enterocytes [27]. Further, we stained ZO-1, a 225 kDa membrane bound protein, binding the transmembrane tight junction proteins occludin and claudins and linking them to cytoskeletal actin [28]. Intensive immunostaining for I-FABP was observed in the control jejunal epithelium, mainly in the cytoplasm of the mature enterocytes and goblet cells in the upper half of the villi, whereas cells in the crypts were not stained, as previously observed in experiments by our group (unpublished results) (Figure 2A). A decreased staining of I-FABP was observed in jejunal mature epithelial cells, after 30 minutes ischemia, while intense staining was found in the subepithelial spaces (Figure 2B), indicating early leakage of I-FABP from the cytoplasm of intestinal epithelial cells into the subepithelial space. ZO-1 was detected at the apical pole of the epithelial cells in both the control jejunum and the jejunum after an ischemic period of 30 minutes (data not shown).


Rapid reversal of human intestinal ischemia-reperfusion induced damage by shedding of injured enterocytes and reepithelialisation.

Derikx JP, Matthijsen RA, de Bruïne AP, van Bijnen AA, Heineman E, van Dam RM, Dejong CH, Buurman WA - PLoS ONE (2008)

Immunolocalization of I-FABP in red (3-amino-9-ethylcarbazole, AEC) (100×) in the control jejunum not subjected to ischemia-reperfusion (A) shows an abundant cytosolic presence of I-FABP in the epithelial cells of the upper half of the villus.Upon 30 minutes ischemia (B), cytosolic I-FABP staining is decreased in mature enterocytes with abundant staining in the subepithelial spaces. A decreased cytosolic staining is still observed after 25 minutes reperfusion (C). Within 60 minutes reperfusion, I-FABP cytosolic positive cells are part of the resealed epithelial barrier (D), while shedded I-FABP containing enterocytes are found in the debris in the lumen.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2561292&req=5

pone-0003428-g002: Immunolocalization of I-FABP in red (3-amino-9-ethylcarbazole, AEC) (100×) in the control jejunum not subjected to ischemia-reperfusion (A) shows an abundant cytosolic presence of I-FABP in the epithelial cells of the upper half of the villus.Upon 30 minutes ischemia (B), cytosolic I-FABP staining is decreased in mature enterocytes with abundant staining in the subepithelial spaces. A decreased cytosolic staining is still observed after 25 minutes reperfusion (C). Within 60 minutes reperfusion, I-FABP cytosolic positive cells are part of the resealed epithelial barrier (D), while shedded I-FABP containing enterocytes are found in the debris in the lumen.
Mentions: At the end of the ischemic period of 30 minutes, H&E sections of the jejunum showed microscopically normal epithelial lining (Figure 1A, B). However, subepithelial spaces appeared at the villus tip. To study the epithelial cells in more detail, immunohistochemical staining of I-FABP was performed, a small protein present in the cytoplasma of differentiated enterocytes [27]. Further, we stained ZO-1, a 225 kDa membrane bound protein, binding the transmembrane tight junction proteins occludin and claudins and linking them to cytoskeletal actin [28]. Intensive immunostaining for I-FABP was observed in the control jejunal epithelium, mainly in the cytoplasm of the mature enterocytes and goblet cells in the upper half of the villi, whereas cells in the crypts were not stained, as previously observed in experiments by our group (unpublished results) (Figure 2A). A decreased staining of I-FABP was observed in jejunal mature epithelial cells, after 30 minutes ischemia, while intense staining was found in the subepithelial spaces (Figure 2B), indicating early leakage of I-FABP from the cytoplasm of intestinal epithelial cells into the subepithelial space. ZO-1 was detected at the apical pole of the epithelial cells in both the control jejunum and the jejunum after an ischemic period of 30 minutes (data not shown).

Bottom Line: Although intestinal IR has been studied extensively in animals, results remain inconclusive and data on human intestinal IR are scarce.It reveals a unique, endogenous clearing mechanism for injured enterocytes: rapid detachment of damaged apoptotic enterocytes into the lumen.This process is followed by repair of the epithelial continuity within an hour, resulting in a normal epithelial lining.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, School for Nutrition & Metabolism (NUTRIM), Maastricht University Medical Centre+, Maastricht, the Netherlands.

ABSTRACT

Background: Intestinal ischemia-reperfusion (IR) is a phenomenon related to physiological conditions (e.g. exercise, stress) and to pathophysiological events (e.g. acute mesenteric ischemia, aortic surgery). Although intestinal IR has been studied extensively in animals, results remain inconclusive and data on human intestinal IR are scarce. Therefore, an experimental harmless model for human intestinal IR was developed, enabling us to clarify the sequelae of human intestinal IR for the first time.

Methods and findings: In 30 patients undergoing pancreatico-duodenectomy we took advantage of the fact that in this procedure a variable length of jejunum is removed. Isolated jejunum (5 cm) was subjected to 30 minutes ischemia followed by reperfusion. Intestinal Fatty Acid Binding Protein (I-FABP) arteriovenous concentration differences across the bowel segment were measured before and after ischemia to assess epithelial cell damage. Tissue sections were collected after ischemia and at 25, 60 and 120 minutes reperfusion and stained with H&E, and for I-FABP and the apoptosis marker M30. Bonferroni's test was used to compare I-FABP differences. Mean (SEM) arteriovenous concentration gradients of I-FABP across the jejunum revealed rapidly developing epithelial cell damage. I-FABP release significantly increased from 290 (46) pg/ml before ischemia towards 3,997 (554) pg/ml immediately after ischemia (p<0.001) and declined gradually to 1,143 (237) pg/ml within 1 hour reperfusion (p<0.001). Directly after ischemia the intestinal epithelial lining was microscopically normal, while subepithelial spaces appeared at the villus tip. However, after 25 minutes reperfusion, enterocyte M30 immunostaining was observed at the villus tip accompanied by shedding of mature enterocytes into the lumen and loss of I-FABP staining. Interestingly, within 60 minutes reperfusion the epithelial barrier resealed, while debris of apoptotic, shedded epithelial cells was observed in the lumen. At the same time, M30 immunoreactivity was absent in intact epithelial lining.

Conclusions: This is the first human study to clarify intestinal IR induced cell damage and repair and its direct consequences. It reveals a unique, endogenous clearing mechanism for injured enterocytes: rapid detachment of damaged apoptotic enterocytes into the lumen. This process is followed by repair of the epithelial continuity within an hour, resulting in a normal epithelial lining.

Show MeSH
Related in: MedlinePlus