Intestinal apoptosis of Paneth cells in the

Intestinal ischemia reperfusion (IR) is associated
with a high mortality rate. 1 Ischemia,
undersupply of blood to an organ, can occur as part of normal physiology, for
example during exercise, but the
vast majority of ischemia is due to thromboembolic or artherothrombotic
vaso-occlusive disease. 2 IR
injury represents a pathological condition in which restoration of blood supply
(reperfusion) follows after deficient blood supply to tissues (ischemia). 3

Discoveries over 30 years have brought more insight into biochemical and
cellular events during ischemia. 4 However, the
underlying mechanisms of ischemia-reperfusion are still not completely known.

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The high mortality and morbidity rate of IR is a
consequence of not recognizing acute ischemia at an early stage and because of
limited effective therapies for IR. Human intestinal IR models show damage of enterocytes in the villi
tips, but also apoptosis of Paneth cells in the crypts after IR. This damage of
the epithelium results in an increased epithelial permeability and
translocation of bacterial molecules or bacteria causing a dysfunctional innate
immune protection against pathogens, known to contribute to complications such
as inflammation and even sepsis. 56 In addition, Paneth cells secrete factors to
sustain proper stem cell function, and thereby play indirectly a role in
intestinal epithelial renewal. 7


To investigate how the intestine epithelium can be
protected during IR and how to recover the injured epithelium, we will validate
a model for IR in human intestinal organoids. Intestinal organoids are cultured
from crypts which will generate villus-like epithelium with all differentiated
cell types. In addition, organoids have been shown to resemble in vivo self-renewal. 58 Developing
a standardized experimental human IR model of the small intestinal epithelium ex vivo, is considered to be of
significant advantage to investigate the potential of therapeutics in
protecting the epithelium from damage and promote recovery after IR. The first
step is to characterise the organoids and validate the IR model in organoids.


RNA sequencing data show that Notch signalling
pathway was upregulated in human intestinal crypt cells after
ischemia-reperfusion (unpublished data). The Notch pathway is
essential to maintain the intestinal stem cell self-renewal as well as the
balance between absorptive and secretory cell lineage differentiation. A study of Chen, G. et al showed that
the Notch signalling pathway was
activated and involved in the protection of intestinal epithelial cells from
intestinal IR injury in mice. 9 However, the role of Notch
signalling in limiting epithelial damage and stimulating regeneration after
intestinal IR in man needs further investigation.


The main goal is therefore to study the role of Notch signalling in
ischemia-reperfusion crypt damage and regeneration. To
accomplish the goal, the following research question will be answered. Namely,
will stimulation or inhibition of the Notch signalling during IR recover or
limit the IR damage?


In the next section, background information on ischemia-reperfusion
injury, intestinal organoids and the Notch pathway will be discussed. Next the methods
will be discussed, and lastly a time schedule will be presented for the
duration of this project. 


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