Sex-based differences of antioxidant enzyme nanoparticle effects following traumatic brain injury

Abstract

Following traumatic brain injury (TBI), reactive oxygen species (ROS) are released in excess, causing oxidative stress, carbonyl stress, and cell death, which induce the additional release of ROS. The limited accumulation and retention of small molecule antioxidants commonly used in clinical trials likely limit the target engagement and therapeutic effect in reducing secondary injury. Small molecule drugs also need to be administered every several hours to maintain bioavailability in the brain. Therefore, there is a need for a burst and sustained release system with high accumulation and retention in the injured brain. Here, we utilized Pro-NP™ with a size of 200 nm, which was designed to have a burst and sustained release of encapsulated antioxidants, Cu/Zn superoxide dismutase (SOD1) and catalase (CAT), to scavenge ROS for >24 h post-injection. Here, we utilized a controlled cortical impact (CCI) mouse model of TBI and found the accumulation of Pro-NP™ in the brain lesion was highest when injected immediately after injury, with a reduction in the accumulation with delayed administration of 1 h or more post-injury. Pro-NP™ treatment with 9000 U/kg SOD1 and 9800 U/kg CAT gave the highest reduction in ROS in both male and female mice. We found that Pro-NP™ treatment was effective in reducing carbonyl stress and necrosis at 1 d post-injury in the contralateral hemisphere in male mice, which showed a similar trend to untreated female mice. Although we found that male and female mice similarly benefit from Pro-NP™ treatment in reducing ROS levels 4 h post-injury, Pro-NP™ treatment did not significantly affect markers of post-traumatic oxidative stress in female CCI mice as compared to male CCI mice. These findings of protection by Pro-NP™ in male mice did not extend to 7 d post-injury, which suggests subsequent treatments with Pro-NP™ may be needed to afford protection into the chronic phase of injury. Overall, these different treatment effects of Pro-NP™ between male and female mice suggest important sex-based differences in response to antioxidant nanoparticle delivery and that there may exist a maximal benefit from local antioxidant activity in injured brain.

Keywords: Carbonyl stress; Lipid peroxidation products; PLGA nanoparticles; Reactive oxygen species; Traumatic brain injury; Treatment window.

Figure 1.

Identification of treatment window of Gd-Pro-NP^™. K^trans of Gd-Pro-NP^™ in male and female CCI mice in the lesion and contralateral hemisphere at various time points post-injury. (a) Representative K^trans mapping of Gd-Pro-NP^™. (b) Quantification of K^trans mapping in the lesion. Gd-Pro-NP^™ accumulated in the lesion the most when administered immediately following the injury (0 h post-injury). Data are shown as mean ± SD with n = 6 for male mice and n = 3 for female mice. * indicates a statistical difference compared to 0 h post-injury group, with two and three symbols indicating p < 0.01 and p < 0.001, respectively, as determined by one-way ANOVA and Dunnett’s post hoc test.

Figure 2.

Identifying the optimum SOD1:CAT activities of Pro-NP^™. In vivo DHE staining of the brains at 4 h post-injury was utilized to measure the ROS level in the acute phase of injury. Various concentrations and ratios of SOD1:CAT-Pro-NP^™ were administered to find the optimum concentration in reducing ROS. (a) Representative confocal microscopy images of control, MnTMPyP, and 4.5:4.9 SOD1:CAT-Pro-NP^™ following CCI in male mice. Scale bar is 1 mm. (b&c) Quantification of DHE fluorescence mean intensity at the perilesional normalized to the contralateral hemisphere with various SOD1:CAT-Pro-NP^™ ratios in male CCI mice (b), and various concentrations of 1:1 SOD1:CAT-Pro-NP^™ in male and female CCI mice (c). Data are shown as mean ± SD with n = 3 for female mice, and n = 3–9 for male mice. * indicates a statistical difference compared to the 0 mg/kg Pro-NP^™ group, with one, two, and three symbols indicating p < 0.05, p < 0.01, and p < 0.001, respectively, as determined by one-way ANOVA and Dunnett’s post hoc test.

Figure 3.

Analysis of carbonyl stress in the hippocampus. Western blot of 4-HNE and acrolein in the ipsi- and contralateral hippocampus for male and female mice at 1 and 3 d post-injury. The boxed area represents the ROI for quantification of 4-HNE and acrolein in the ipsilateral and contralateral hippocampus normalized to control mice. Data are shown as mean ± SD with n = 3 for each treatment group. ns indicates an insignificant different between treatment group. * indicates a significant different with p < 0.05, as determined by one-way ANOVA and Tukey’s post hoc test.

Figure 4.

Analysis of α-II-spectrin breakdown products (SBDPs) in the hippocampus. a) Representative Western blot of α-II-spectrin breakdown products (SBDPs) ipsi- (b,c) and contralateral (d,e) hippocampus for male and female mice at 1 and 3 d post-injury. Quantification of 145 kDa SBDP (proportional to necrosis) (b,d) and 120 kDa SBDP (proportional to apoptosis) (c,e) normalized to control mice, respectively. Data are shown as mean ± SD with n = 3 for each treatment group. ns indicates an insignificant different between treatment group. * and ** indicate a significant different with p < 0.05 and p < 0.01, respectively, as determined by one-way ANOVA and Tukey’s post hoc test.