Hyperbaric oxygen therapy improves early posttransplant islet function

Abstract

Objective: This study investigates the therapeutic potential of hyperbaric oxygen therapy (HBO) in reducing hypoxia and improving engraftment of intraportal islet transplants by promoting angiogenesis.

Methods: Diabetic BALB/c mice were transplanted with 500 syngeneic islets intraportally and received six consecutive twice-daily HBO treatments (n = 9; 100% oxygen for 1 h at 2.5 atmospheres absolute) after transplantation. Dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) was used to assess new vessel formation at postoperative days (POD) 3, 7, and 14. Liver tissue was recovered at the same time points for correlative histology, including: hematoxylin and eosin, hypoxia-inducible factor (HIF1α), Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL), vascular endothelial growth factor (VEGF), and von Willebrand Factor immunohistochemistry.

Results: HBO therapy significantly reduced HIF-1α, TUNEL and VEGF expression in islets at POD 7. In the non-HBO transplants, liver enhancement on DCE MRI peaked at POD 7 consistent with less mature vasculature but this enhancement was suppressed at POD 7 in the HBO-treated group. The number of new peri-islet vessels at POD 7 was not significantly different between HBO and control groups.

Conclusion: These results are consistent with a hyperbaric oxygen-mediated decrease in hypoxia that appeared to enhance vessel maturation in the critical days following intraportal islet transplantation.

Fig. 1

Dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) enhancement after intraportal liver islet transplantation. Liver DCE MRI in an islet transplanted mouse at POD 7 after HBO therapy demonstrates enhancement after gadolinium (Gd) injection. Liver images prior (0 min) and after (1, 2, 4, 20, and 30 min) Gd injection demonstrated peak enhancement at 4 min. The increased concentration of contrast agent in the intravascular space can be visualized by enhancement of the portal vein (arrows).

Fig. 2

Quantification of dynamic contrast-enhanced magnetic resonance imaging. (A–C) Normalized DCE enhancement curves at POD 3 (A), 7 (B), and 14 (C). HBO inhibited the increase in contrast enhancement found in controls (no HBO) at POD 7. (D) The DCE ratio at POD 7 or 14 compared to POD 3 revealed a significant decrease in enhancement at POD 7 after HBO therapy. *p < 0.05.

Fig. 3

Islets stained with von Willebrand Factor (vWF) for new vessel formation. At POD 3 islets (dotted line) were stained with vWF (indicated as arrow) but there were few newly formed vessels (A). At POD 3 vWF stained areas were considered as coagula. Vessel formation around islets (arrows) were more prominent at POD 7 (B) and 14 (C) (see expanded insets). Figures are from control mice. (D) Quantified vWF staining was significantly increased at POD 7 and 14 compared to POD 3 in both groups (*p < 0.0001 in HBO group and **p < 0.0001 in control group, Dunnet test). Calibration bars = 100 μm.

Fig. 4

Histological findings at POD 3, 7, and 14. Insulin staining revealed the location of the islets in liver tissues. Necrosis (H & E), hypoxia (HIF-1α) and apoptosis (TUNEL) of the islets and surrounding liver tissues were prominent at POD 3 but decreased after POD 7. Vascular endothelial growth factor (VEGF) was also prominent at POD 3 and decrement at POD7 and 14. Figures are from control mice. Arrows indicate apoptotic cells. Calibration bar = 100 μm.

Fig. 5

Quantification of histological assessment at POD 7. (A) Hypoxic islet cells (HIF-1α positive staining) were significantly decreased in animals undergoing HBO therapy. (*p = 0.02). (B) Apoptotic islet cells (TUNEL positive) were also significantly decreased after transplantation of islets in mice that underwent HBO therapy. (*p = 0.01). (C) Vascular endothelial growth factor (VEGF) positive islet cells were prominently decreased in HBO group at POD 7 (*p = 0.01). Decreased HIF-1α may contribute to a decrease in VEGF expression.

Fig. 6

HBO improves functional outcomes after intraportal islet transplant. (A) A significant increase in serum insulin levels was observed in HBO-treated animals at POD 7. (*p = 0.04). (B) Glucose tolerance test (GTT) was also significantly improved after HBO therapy at POD 7. (*p = 0.04). The GTT insert illustrates a typical time course response.