Central nervous system responses to pressure and pollutants in the hyperbaric work environment

Underwater workers involved in welding in closed hyperbaric chambers, are exposed to potentially toxic levels of oxygen and to welding dust containing particles and metals that can be toxic to the central nervous system (CNS). In previous analyses on dived rats, we have observed altered brain perfusion and temporary permeability of the blood-brain barrier (BBB) after decompression, but histology revealed no structural changes. Therefore, we hypothesise that hyperbaric exposure causes altered brain perfusion and BBB physiology that may increase the passage of pollutants in the work environment into the brain, subsequently causing injury. To address this, we need to establish the CNS response to hyperbaric exposure per se, before exposing rats to hyperbaric dust. The main methods will be MRI and histology. MRI allows for in vivo evaluation of injury on a structural, molecular and functional level, and will be repeated to give insight in the dynamics of injury development. BBB permeability will be evaluated by determining extravasation of Evans blue and horseradish peroxidase tracers administered at different times to determine for how long BBB integrity is disturbed. Brain perfusion and blood-brain barrier integrity will be studied using dynamic contrast enhanced MRI. T1- and T2-weighted imaging together with diffusion tensor imaging (figure 2) will be used to assess tissue injury, edema and inflammatory response as well as white matter function at several time points after exposure to hyperbaric welding dust. Brain tissue will be sampled at several time points after the exposure and examined by immunohistochemistry for reactive astrocytosis (GFAP and S100), inflammatory response and neuronal injury and death. We will use immunohistochemistry to study neuronal integrity (Anti-MAP-2), reactive astrocytes (anti-GFAP), myelin (anti-MBP), myelin integrity (Luxol fast blue) and activated microglia/macrophages (anti-CD68). Primary areas for investigation are cortex, hippocampus, thalamus, basal ganglia and brain stem. Atomic absorption spectroscopy will be used to determine brain tissue concentrations of potential toxic metals (thorium, zirconium, manganese and iron) found in welding dust.