Neuroprotective Potential of Tetrahydrobiopterin Using a Rat Model

Abstract

Introduction: Spinal cord ischemia (SCI) is one of the most devastating complications in thoracoabdominal aortic aneurysm surgery and the identification of neuroprotective substances to prevent this complication is of great interest. Tetrahydrobiopterin (BH4) is one of five essential cofactors of nitric oxide synthase (NOS) and is crucial for the conversion of L-arginine and oxygen to L-citrullin and NO. Ischemia reperfusion injury Is associated with intracellular BH4 depletion. This in turn, is known to be associated with the uncoupling of NOS leading to an increased production of hydrogen peroxide and superoxide anion resulting in endothelial dysfunction. Supplementation of BH4 has been shown to play a protective role in ischemia-reperfusion injury following solid organ transplantation. We therefore wanted to test its neuroprotective potential using a SCI model in rats. Methods: SCI has been induced in male Sprague Dawley rats (300 - 350g weight) by balloon occlusion of the descending thoracic aorta via insertion of a 2F Fogarty catheter. Blood pressure monitoring via the left carotid artery and the tail artery was performed to confirm sufficient balloon occlusion. Animals were subdivided into a sham group (n=12), a treatment group (50 mg/kg BH4 intramuscular 15 minutes before SCI; n=11) and a control group (intramuscular saline injection 15 minutes before SCI; n=12). Blinded neurofunctional testing was performed using the Bassio-Beattie-Breshnahan (BBB) locomotor rating scale ranging from 0 (no hind limb movement) to 21 (normal hind limb movement). After an observation period of 7 days, animals were sacrificed and tissue was harvested for further histopathological evaluation using H.E. staining and TUNEL assays. Results: The model was associated with unexpected high perioperative mortality due to hemodynamic and embolic complications. Within the control group, 3 animals (n=12; 25%) and within the treatment group 4 animals (n=11; 36%) survived until day 7. Pre-treatment with BH4 lead to significant better neurofunctional outcome 2 hours after awakening of animals (BBB Score treatment group 18.3 ± 3.4 vs. control group 15.3 ± 2.1; p=0.014). The difference of BBB Scores showed a trend towards a better outcome after pre-treatment with BH4 at day 7 (treatment group 21 ± 0 vs. 18.3 ± 3.8; p=0.205). Histopathological evaluation using H.E. staining revealed no difference between treatment and control group when counting the mean number of neurons in thoracic spinal cords. By using TUNEL assays for detection of vital neurons there was a clear trend towards a benefit of pre-treatment with BH4 (mean number of vital neurons treatment group 13.28 ± 4.77 vs control group 11.89 ± 4.04; p=0.117). Conclusion: We demonstrate a neuroprotective potential of BH4 as immediate postoperative neurofunctional outcome was significantly better in pre-treated animals. Due to the unexpected high postoperative mortality results of neurofunctional and histopathological evaluations at day 7 should be taken with caution. However, the observed trend towards a benefit of BH4 pre-treatment urges to repeat the study with a shorter postoperative observation period. Disclosure: Nothing to disclose