Complement activation after gut ischemia-reperfusion injury (GI I/R) in man

Gastro-intestinal ischemia-reperfusion (GI I/R) injury occurs when the gut is
temporarily deprived of blood supply and where restoration of the blood supply
triggers an intense inflammatory response. GI I/R remains a common major
clinical problem (partly as a result of the lack of diagnostic tools and poor
treatment options) with a reported mortality of 60-80% for patients with acute
mesenterial thrombosis. Furthermore, small and large intestinal ischemia is
considered a crucial factor associated with complications after abdominal and
(cardio-) vascular surgery, trauma and sepsis. It is seen as a major
contributor to the development of post-operative complications and multiple
organ failure.
Recent animal studies using complement-knockout mice showed an important role
of complement activation in the onset of inflammation and tissue injury after
GI I/R. To delineate the role of (three pathways of) complement activation in
human GI I/R injury, we will study complement activation peroperatively after
GI I/R.
Local tissue inflammation, as complement activation, is considered important to
resolve I/R induced damage, but derailment of the inflammatory response can
result in further complications.
Derailment of the inflammatory response can be triggered by the translocation
of highly pro-inflammatory intestinal intraluminal components, like endotoxin,
as a result of a compromised intestinal barrier function. 20 Therefore, the
next goal is to study the effect of intestinal ischemia and reperfusion on the
intestinal barrier function.

Apart from increasing knowledge on the molecular mechanisms involved in the
pathophysiology of intestinal IR, it is also of importance to get insight into
the macroscopic mucosal changes during intestinal IR. In this way, we can
improve early recognition of intestinal ischemic lesions using endoscopy.

To examine cellular damage, intestinal barrier function loss, activation of the
Complement system and inflammatory alterations induced by ischemia and
reperfusion of the human small and large intestine.
Second, we aim to study the consequences of human intestinal ischemia
reperfusion induced barrier function loss, i.e. the translocation of intestinal
intraluminal content into the circulation.
Third, we aim to study macroscopic mucosal changes during intestinal IR.
At last, we aim to study the consequences of human intestinal ischemia
reperfusion on gene expression patterns for identification of targets for
preventive and therapeutic strategies.

Small Intestinal I/R
In this study the effects of ischemia followed by reperfusion (I/R) of the
human small intestine will be investigated. To this end we will collect blood
and tissue samples during major upper abdominal surgery or major colorectal
surgery (model for large intestinal I/R). In some surgical procedures the
duodenum and duodenal-jejunal transition are resected and continuity of the
gastro-intestinal tract is restored by a gastro-jejunal or entero-enteric
anastomosis.
For research purposes a 3-6 centimeter small and isolated part of the jejunum
will be submitted to I/R during surgery. Because of the limited size of the
jejunal segment subjected to I/R, we suspect that the I/R induced changes have
no systemic implications, but will enable us to investigate the important
processes following GI I/R. The supplying small arterial branches (aa.
jejunales) and draining venous structures (vv. jejunales) of the specific
jejunal element are identified in the mesentery and subsequently clamped to
halt circulation. Following 15, 30, 45 or 60 minutes of ischemia the clamp,
occluding the vascular branches, is removed and the jejunum is inspected for
reperfusion. Reperfusion is continued for a subsequent two hours, after which
the tissue is removed along with the total resection specimen. The previously
identified venous mesenterial branches enable us to specifically draw blood
from the outflow of the isolated tissue segment. To analyze segment inflow
arterial samples can be taken from the arterial line, introduced for surgery.

To study the consequences on intestinal barrier function loss during ischemia
and reperfusion the differential sugar test is used. This test is in common use
in every day clinical practice and consists of the oral administration of two
sugars, lactulose and l-rhamnose, and their consequent measurement in the
plasma. This method is based on the fact that lactulose, a disaccharide, is
too large to cross a healthy intestinal barrier, but in case of barrier
function loss, lactulose is able to cross the barrier to the circulation. High
plasma levels of lactulose will therefore reflect increased intestinal
permeability. L-rhamnose is a monosaccharide which can cross the intestinal
barrier freely, and is added to the test to serve as an internal control for
confounders as gastric dilution and intestinal perfusion. Using high
performance liquid chromatography combined with mass spectrometry, both
saccharides can be detected in plasma.

To study macroscopic changes during small intestinal IR, a endo-videocapsule
will be inserted in the isolated gut-segment in a subgroup of 6 patients. This
small camera (size of a pill), will be inserted through an incision proximal of
the isolated segement. Directly after insertion, the part of intestine with the
incision-cut will be removed, to prevent leakage of intraluminal content
through the incision. To study IR-induced changes, the isolated gut-segment
will be exposed to 60 minutes of ischemia with 120 minutes of reperfusion,
which is in line with current IR-protocols.

The blood samples will be analyzed at the general surgery laboratories for
markers of cellular damage ILBP, I-FABP and L-FABP, inflammatory markers like
TNF-α, IL-6 and IL-10, neutrophil activation products (Calprotectin, MPO and
NGAL) and complement proteins like MBL and the terminal complement complex
(TCC), as well as for markers for intestinal permeability (lactulose and
L-rhamnose).
The tissue samples will be analyzed with various standard laboratory procedures
like western blot and immunohistochemistry, qPCR, electron microscopy, gene
expression analysis and other relevant techniques.

Another 3 cm of tissue is harvested from the already resected specimen and will
function as an internal healthy control. Essential in this procedure is the
fact that no extra tissue will be taken from the patient and all studies can be
performed on tissue already resected during the elective surgical procedure.

Large Intestinal I/R
Next to small intestinal ischemia reperfusion (IR), also large intestinal IR
will be studied. To this end we will collect blood and tissue samples during
major colorectal surgery, (i.e. Low Anterior resection or Hartmann*s
procedures). In this surgical procedure a part of the healthy colon is removed
for surgical/medical reasons. For research purposes a 3-6 centimeter isolated
colon segment will be submitted to IR during surgery in a similar manner as
already was performed in patients undergoing pancreaticoduodenectomies (for
studying small intestinal IR). This model is harmless for the patient, since we
take advantage of the fact that a part of healthy colon is resected for
surgical reasons. No extra tissue will be resected for research purposes.
Conform the study of the small intestine, the consequences of different
ischemic periods (15, 30, 45 and 60 minutes ischemia) and extent of the
ischemia (hypoperfusion) will be studied in the same manner as described for
small intestinal IR. Following ischemia, the clamp, occluding the vascular
branches, is removed and the colonic segment is inspected for reperfusion.
Reperfusion is continued for a subsequent two hours, after which the tissue is
removed along with the total resection specimen. Blood samples will be
collected at four different time points during surgery. The previously
identified venous mesenterial branches enable us to specifically draw blood
from the outflow of the isolated tissue segment. To analyze segment inflow
arterial samples can be taken from the arterial line, introduced for surgery.

The patients enrolled in this study will all undergo major abdominal surgery
with duration of at least 2 hours. Because I/R is applied on intestinal tissue
which will be resected anyway during the surgical procedure, this will not
interfere with standard surgical care. The only actions undertaken by the
surgeon solely related to this research proposal, is the isolation of 3-6 cm of
gut and apply 15, 30, 45 or 60 minutes ischemia to it prior to its removal
along with the rest of the gut which is to be resected for medical reasons.
During the operation, two sugars, lactulose and l-rhamnose, will be injected
into the intestine using a syringe with needle. The sugars will be administered
just after the part of intestine is isolated for the study protocol (see
above).Directly after injection, the part of isolated intestine with the
injection site will be removed using a linear cutting stapler to prevent
leakage of intraluminal content through the injection site (see also the
endo-videocapsule protocol). All materials are sterile. Lactulose and
L-rhamnose are both harmless saccharides which have been used since the 70s to
investigate intestinal permeability. Both saccharides have been extensively
used and can be administered without risk of adverse effects. These saccharides
are registered for oral use. In addition, it has to be taken into account that
these saccharides will only be introduced into the part of intestine, that will
be removed for surgical reasons anyway. Our research and clinical department
have experience with peri-operative administration of test substances into the
gut (see also MEC 03-032.5).
To study macroscopic changes during small intestinal IR, a endo-videocapsule
will be inserted in the isolated gut-segment in a subgroup of 6 patients. This
small camera (size of a pill), will be inserted through an incision proximal of
the isolated segement. Directly after insertion, the part of intestine with the
incision-cut will be removed, to prevent leakage of intraluminal content
through the incision. No extra part of intestine will be removed for this
procedure. The videocapsule is sterile.
During the operation the surgeon will sample blood from the vein of the
isolated gut segment. These actions will not add substantially to the total
duration of the operation. The additional risks for the patients are minimal
and will not increase the total risk of the operation. The arterial line used
for blood sampling is part of standard anesthetic care.

The technical protocol for large intestinal ischemia-reperfusion is the same as
the protocol for small intestinal ischemia reperfusion. There*s no interference
with the standard surgical procedure, and there are no additional risks. The
only actions undertaken by the surgeon solely related to this research
proposal, is the isolation of 3-6 cm of colon and apply ischemia to it prior to
its removal along with the rest of the gut which has to be resected for
surgical reasons.