Intestinal insight into biochemical and cellular events during ischemia.

Intestinal ischemia reperfusion (IR) is a life-threateningcondition and is associated with a high morbidity and mortality rate. 1Ischemia, undersupply of blood to an organ, can occur as part of normalphysiology, for example during exercise, but the vast majority of ischemia is due to thromboembolic orartherothrombotic vaso-occlusive disease.2 IR injury represents a pathological condition in which restorationof blood supply (reperfusion) follows after deficient blood supply to tissues(ischemia). 3Discoveries over 30 years have brought more insight into biochemical andcellular events during ischemia. 4 However, theunderlying mechanisms of ischemia-reperfusion are still not completely known. The high mortality and morbidity rate of IR is aconsequence of not recognizing acute ischemia at an early stage and because oflimited effective therapies for IR.

Human intestinal IR models show damage of enterocytes in the villitips, but also apoptosis of Paneth cells in the crypts after IR. This damage ofthe epithelium results in an increased epithelial permeability andtranslocation of bacterial molecules or bacteria causing a dysfunctional innateimmune protection against pathogens, known to contribute to complications suchas inflammation and even sepsis. 56 In addition, Paneth cells secrete factors tosustain proper stem cell function, and thereby play indirectly a role inintestinal epithelial renewal. 7 To investigate how the intestine epithelium can beprotected during IR and how to recover the injured epithelium, we will validatea model for IR in human intestinal organoids.

Intestinal organoids are culturedfrom crypts which will generate villus-like epithelium with all differentiatedcell types. In addition, organoids have been shown to resemble in vivo self-renewal. 58 Developinga standardized experimental human IR model of the small intestinal epithelium ex vivo, is considered to be ofsignificant advantage to investigate the potential of therapeutics inprotecting the epithelium from damage and promote recovery after IR. The firststep is to characterise the organoids and validate the IR model in organoids. RNA sequencing data show that Notch signallingpathway was upregulated in human intestinal crypt cells afterischemia-reperfusion (unpublished data).

The Notch pathway isessential to maintain the intestinal stem cell self-renewal as well as thebalance between absorptive and secretory cell lineage differentiation. A study of Chen, G. et al showed thatthe Notch signalling pathway wasactivated and involved in the protection of intestinal epithelial cells fromintestinal IR injury in mice. 9 However, the role of Notchsignalling in limiting epithelial damage and stimulating regeneration afterintestinal IR in man needs further investigation.

 The main goal is therefore to study the role of Notch signalling inischemia-reperfusion crypt damage and regeneration. Toaccomplish the goal, the following research question will be answered. Namely,will stimulation or inhibition of the Notch signalling during IR recover orlimit the IR damage?  In the next section, background information on ischemia-reperfusioninjury, intestinal organoids and the Notch pathway will be discussed. Next the methodswill be discussed, and lastly a time schedule will be presented for theduration of this project.