Targeting Oxidative Stress with Antioxidant Duotherapy after Experimental Traumatic Brain Injury.

Jenni Kyyriäinen,Teresa Ravizza,Natallie Kajevu,Noora Puhakka,Ivette Bañuelos,Asla Pitkänen,Elina Hämäläinen,Anssi Lipponen,Leonardo Lara,Shalini Das Gupta,Mikko Hiltunen,Silvia Balosso,Teemu Natunen,Annamaria Vezzani

doi:10.3390/ijms221910555

Abstract

We assessed the effect of antioxidant therapy using the Food and Drug Administration-approved respiratory drug N-acetylcysteine (NAC) or sulforaphane (SFN) as monotherapies or duotherapy in vitro in neuron-BV2 microglial co-cultures and validated the results in a lateral fluid-percussion model of TBI in rats. As in vitro measures, we assessed neuronal viability by microtubule-associated-protein 2 immunostaining, neuroinflammation by monitoring tumor necrosis factor (TNF) levels, and neurotoxicity by measuring nitrite levels. In vitro, duotherapy with NAC and SFN reduced nitrite levels to 40% (p < 0.001) and neuroinflammation to –29% (p < 0.001) compared with untreated culture. The treatment also improved neuronal viability up to 72% of that in a positive control (p < 0.001). The effect of NAC was negligible, however, compared with SFN. In vivo, antioxidant duotherapy slightly improved performance in the beam walking test. Interestingly, duotherapy treatment decreased the plasma interleukin-6 and TNF levels in sham-operated controls (p < 0.05). After TBI, no treatment effect on HMGB1 or plasma cytokine levels was detected. Also, no treatment effects on the composite neuroscore or cortical lesion area were detected. The robust favorable effect of duotherapy on neuroprotection, neuroinflammation, and oxidative stress in neuron-BV2 microglial co-cultures translated to modest favorable in vivo effects in a severe TBI model.

Highlights

Traumatic brain injury (TBI) is defined as an alteration in brain function or other evidence of brain pathology caused by an external force [1]
We found that NAC+SFN duotherapy robustly reduced nitrite levels and neuroinflammation, and increased neuronal viability in vitro
Brains were dissected from JAXC57BL/6J mice on embryonic day 18 (E18) and the cerebral cortex was carefully stripped of the meninges

Summary

Introduction

Traumatic brain injury (TBI) is defined as an alteration in brain function or other evidence of brain pathology caused by an external force [1]. Approximately 2.5 million people in both Europe [2] and the United States [3] experience TBI. Type, and severity, TBI can result in different types of cognitive, emotional, and behavioral comorbidities [4,5]. Attempts to improve the TBI outcome are focused on mitigating the molecular cascades that lead to worsening of the secondary injury and/or on enhancing recovery [6,7]. Secondary injury comprises multifaceted temporally orchestrated pathologies, including apoptosis, neuroinflammation, and generation of reactive oxygen species (ROS) that lead to oxidative stress, and blood–brain barrier (BBB) dysfunction [8,9,10].

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