Neuroprotective effect of Pycnogenol® following traumatic brain injury

Abstract

Traumatic brain injury (TBI) involves primary and secondary injury cascades that underlie delayed neuronal dysfunction and death. Oxidative stress is one of the most celebrated secondary injury mechanisms. A close relationship exists between levels of oxidative stress and the pathogenesis of TBI. However, other cascades, such as an increase in proinflammatory cytokines, also play important roles in the overall response to the trauma. Pharmacologic intervention, in order to be successful, requires a multifaceted approach. Naturally occurring flavonoids are unique in possessing not only tremendous free radical scavenging properties but also the ability to modulate cellular homeostasis leading to a reduction in inflammation and cell toxicity. This study evaluated the therapeutic role of Pycnogenol (PYC), a patented combinational bioflavonoid. Young adult Sprague-Dawley rats were subjected to a unilateral moderate cortical contusion and treated post injury with PYC or vehicle. At either 48 or 96 h post trauma, the animals were killed and the cortex and hippocampus analyzed for changes in enzymatic and non-enzymatic oxidative stress markers. In addition, possible changes in both pre- and post-synaptic proteins (synapsin-1, PSD-95, drebrin, synapse associated protein-97) were analyzed. Finally, a separate cohort of animals was used to evaluate two proinflammatory cytokines (IL-6, TNF-α). Following the trauma there was a significant increase in oxidative stress in both the injured cortex and the ipsilateral hippocampus. Animals treated with PYC significantly ameliorated levels of protein carbonyls, lipid peroxidation, and protein nitration. The PYC treatment also significantly reduced the loss of key pre- and post-synaptic proteins with some levels in the hippocampus of PYC treated animals not significantly different from sham operated controls. Although levels of the proinflammatory cytokines were significantly elevated in both injury groups, the cohort treated with PYC showed a significant reduction compared to vehicle treated controls. These results are the first to show a neuroprotective effect of PYC following TBI. They also suggest that the diverse effects of bioflavonoids may provide a unique avenue for possible therapeutic intervention following head trauma.

Fig. 1

Changes in levels of antioxidants and antioxidant enzymes in the injured cortex at 48h following a unilateral cortical contusion. In both the vehicle treated (Veh-48h) and pycnogonol treated (PYC-48h) there was a significant decline in levels of GSH compared to the sham operated (Sham) control group. Levels of several of the important antioxidant enzymes were also significantly decreased. PYC treatment significantly elevated levels of many antioxidants compared to the Veh-48h cohort. Each bar represents the group mean ± SD. * p < 0.05, ** p < 0.001 compared to Sham; # p < 0.05, ## p < 0.001 compared to Veh-48.

Fig. 2

Changes in the level of oxidative stress in both the cortex and hippocampus at 48h following a unilateral cortical contusion. There was a significant increase in oxidative stress in both brain regions following the injury. PYC treatment significantly ameliorated the oxidative stress compared to the vehicle treated cohort in both the cortex and hippocampus. Each bar represents the group mean ± SD. * p < 0.05, ** p < 0.001 compared to Sham; # p < 0.05, ## p < 0.001 compared to Veh-48.

Fig. 3

Changes in levels of antioxidants and antioxidant enzymes in the hippocampus ipsilateral to the cortical injury at 48h. In both the vehicle treated (Veh-48h) and pycnogonol treated (PYC-48h) there was a significant decline in levels of GSH compared to the sham operated (Sham) control group. Levels of several of the important antioxidant enzymes were also significantly decreased. PYC treatment significantly elevated levels of many antioxidants compared to the Veh-48h cohort. Each bar represents the group mean ± SD. * p < 0.05, ** p < 0.001 compared to Sham; # p < 0.05, ## p < 0.001 compared to Veh-48.

Fig. 4

Pre- and post-synaptic proteins in both the ipsilateral cortex (A) and hippocampus (B) were significantly affected at 96h following a unilateral cortical contusion. PYC treatment significantly increased levels of synaptic proteins in both the cortex and hippocampus. Ipsilateral and contralateral samples from eight animals/group were processed for immunobloting followed by Western-blot. Immunoblotes were developed with 5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium (alkaline phosphatase substrate; SIGMA FAST^™ BCIP/NBT tablets) and densities of bands were analyzed using Scion Image. Drebrin, synapse associated protein -97 (SAP-97), PSD-95, and synapsin-I, demonstrated a significant decrease in signal.

Fig. 5

Levels of both presynaptic (Synapsin-1) and post synaptic (SAP-97, PSD-95, Drebrin) protein markers were quantified in both the cortex (A) and hippocampus (B) following a unilateral cortical contusion. There was a significant injury related decline in cortex at 96h post trauma. Animals treated with PYC showed a significant elevation compared to the Veh-96h cohort. In the hippocampus, levels of synaptic proteins were significantly decreased in the vehicle treated animals. PYC treatment significantly spared the synaptic proteins. Each bar represents the group mean ± SD. * p < 0.05, ** p < 0.001 compared to Sham; # p < 0.05, ## p < 0.001 compared to Veh-96h.

Fig. 6

Pro-inflammatory cytokines are significantly attenuated with PYC treatment in both the cortex and hippocampus. There was a significant elevation in the cytokines (IL-6, TNF-α) following the unilateral cortical contusion. Levels were significantly lower in the PYC treatment group compared to the vehicle treated cohort. Each bar represents the group mean ± SD. * p < 0.05, ** p < 0.001 compared to Sham; # p < 0.05, ## p < 0.001 compared to Vehicle.