Hypoxia signaling in the equine small intestine: Expression and distribution of hypoxia inducible factors during experimental ischemia

Nicole Verhaar; Nicole de Buhr; Maren von Köckritz-Blickwede; Katrin Dümmer; Marion Hewicker-Trautwein; Christiane Pfarrer; Franziska Dengler; Sabine Kästner

doi:10.3389/fvets.2023.1110019

Hypoxia signaling in the equine small intestine: Expression and distribution of hypoxia inducible factors during experimental ischemia

Front Vet Sci. 2023 Feb 24:10:1110019. doi: 10.3389/fvets.2023.1110019. eCollection 2023.

Authors

Nicole Verhaar¹, Nicole de Buhr^{2

3}, Maren von Köckritz-Blickwede^{2

3}, Katrin Dümmer^{2

3}, Marion Hewicker-Trautwein⁴, Christiane Pfarrer⁵, Franziska Dengler⁶, Sabine Kästner^{1

7}

Affiliations

¹ Clinic for Horses, University of Veterinary Medicine Hannover, Hannover, Germany.
² Department of Biochemistry, University of Veterinary Medicine Hannover, Hannover, Germany.
³ Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Hannover, Germany.
⁴ Institute of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany.
⁵ Institute for Anatomy, University of Veterinary Medicine Hannover, Hannover, Germany.
⁶ Institute of Physiology, Pathophysiology and Biophysics, Department of Biomedical Sciences, University of Veterinary Medicine Vienna, Vienna, Austria.
⁷ Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, Germany.

Abstract

Introduction: Hypoxia inducible factors (HIF) are widely researched in human medicine for their role in different disease processes. The aim of this study was to investigate the expression and distribution of HIF in experimental small intestinal ischemia in the horse.

Methods: In 14 horses under general anesthesia, segmental jejunal ischemia with 90% reduction in blood flow was induced. The horses were randomly divided into two groups of seven horses, one subjected to ischemic postconditioning (IPoC) by delayed reperfusion, and a control group (group C) undergoing undelayed reperfusion. Intestinal samples were taken pre-ischemia, after ischemia and after reperfusion. Following immunohistochemical staining for HIF1α and -2α, the immunoreactivity pattern in the small intestine was evaluated by light microscopy, and the mucosal enterocyte and muscularis staining were semi-quantitatively scored. Additionally, mucosal HIF1α protein levels were determined by an Enzyme Linked Immunosorbent Assay (ELISA), and mRNA levels of HIF1α and its target genes by a two-step real-time Reverse Transcriptase Polymerase Chain Reaction. Statistical comparison was performed between the groups and time points using parametric and non-parametric tests (p < 0.05).

Results: All cell types exhibited cytoplasmic and nuclear immunoreactivity for HIF1α. After reperfusion, the cytoplasmic staining of the crypt and villus enterocytes as well as the villus nuclear staining significantly increased, whereas the perinuclear granules in the crypts decreased. The protein levels showed a significant decrease in group C at reperfusion, with lower HIF1α levels in group C compared to group IPoC during ischemia and reperfusion. No other group differences could be detected. In the HIF2α stained slides, mild to moderate cytoplasmic staining yet no nuclear immunoreactivity of the enterocytes was observed, and no significant changes over time were noted.

Discussion: the changes in HIF1α immunoreactivity pattern and expression over time suggest that this transcription factor plays a role in the intestinal response to ischemia in horses. However, the current study could not identify an effect of IPoC on HIF distribution or expression.

Keywords: HIF; horse; hypoxia; ischemia; jejunum; mucosa; postconditioning; strangulation.

Grants and funding

This study was funded by a research grant from the European College of Veterinary Surgeons (2019 LA, NV) and by a research grant from Stiftung ProPferd (2019/04, SK/NV). This Open Access publication was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)-−491094227 Open Access Publication Funding and the University of Veterinary Medicine Hannover, Foundation.