The role of platelets in hepatic ischemia reperfusion injury in mice

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 The role of platelets in hepatic ischemia reperfusion injury in mice

Doctoral thesis for a doctoral degree
at the Graduate School of Life Sciences,
Julius-Maximilians-Universität Würzburg
Section Biomedicine
submitted by
Carina Gross
from Gronau/Westf.
Würzburg, 2020

Summary

Platelets are the second most abundant blood cells and their main function is maintenance of vascular integrity. In addition, platelets are increasingly recognized as cells with immune functions, as they participate in the recruitment of immune cells and modulate the progression and severity of an immune response. So-called lipid mediators, which are – besides other cells– released by activated platelets, influence the immune response. LTB4 is one of these potent lipid mediators and is able to activate neutrophils and induce their infiltration into injured tissue. In order to investigate the involvement of platelets in inflammatory processes, a murine model of hepatic ischemia reperfusion injury as well as confocal intravital microscopy of the liver were established. Both methods were used to analyze the influence of platelets on the inflammation that follows sterile liver inflammation. We found platelet function to be unaltered after three hours of reperfusion and platelet aggregation to be irrelevant for the outcome of hepatic ischemia reperfusion injury. However, a strong impact of the GPIb-vWF axis could be observed, as antibody mediated blockade of GPIb as well as vWF-deficiency significantly reduced liver damage markers and decreased neutrophil infiltration. GPIb-IL-4R mice were used to exclude the possibility that the protective effects of the anti-GPIbα antibody treatment (p0p/B) results from something else than blocking GPIb. Furthermore, the slope of neutrophil infiltration was decreased in p0p/B-treated mice, leading to overall decreased neutrophil numbers in the liver after three hours of reperfusion. Blockade of the integrin IIbβ3, however, showed no reduction in neutrophil infiltration into the post-ischemic liver, in line with unaltered liver damage. To study the role of leukotriene B4, conditional and constitutive knockout mice for the LTA4 hydrolase, which catalyzes the last step in LTB4 synthesis, were generated. Lta4h deficiency did not affect general platelet functionality in hemostasis and thrombosis. Interestingly, Lta4h mice were not protected from cellular damage following hepatic ischemia, despite lower neutrophil numbers in the post-ischemic liver. Intravital microscopy of the pancreas was established and revealed increased CD4+T cell numbers in GPVI-deficient animals compared to WT controls in line with the pre-diabetic phenotype of Gp6 mice that was revealed in Grzegorz Sumara’s group. Furthermore, platelet ‘behavior’ in pancreatic islets was observed following glucose injection. We found a high number platelets adherent islet sinusoids under basal conditions and rolling/decelerating of platelets following glucose injection. was accompanied by temporary sinusoidal constriction and stop of the blood flow. This phenomenon was not observed in control settings (injection of PBS, insulin or L-glucose). 

 

IV 

 

In a side project, which was carried out jointly with Tobias Heib, a side by side comparison of the classical syringe-based flushing and the centrifugation-based spinning method to isolate murine bone marrow was conducted. Flow cytometry revealed no differences in the distribution of hematopoietic stem cells and immune cells and functional analysis with primary and cultured megakaryocytes (MKs) showed comparable results in all conducted assays. Thus, our data demonstrated that the faster and more efficient spinning method can be used for the isolation of bone marrow cells.