Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector

Abstract

Background: Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD) after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn SOD or the control GFP transgenes 2 hours after intracortical N-methyl-D-aspartate (NMDA) administration, and daily functional evaluation was performed. Moreover, 3 days after, lesion volume, neuronal degeneration and nitrotyrosine immunoreactivity were evaluated.

Results: Overexpression of Cu/Zn SOD transgene after NMDA administration showed improved functional outcome and a reduced lesion volume at 3 days post lesion. In secondary degenerative areas, increased neuronal survival as well as decreased numbers of degenerating neurons and nitrotyrosine immunoreactivity was seen. Interestingly, injection of the NLSCt vector carrying the control GFP transgene also displayed a significant neuroprotective effect but less pronounced.

Conclusion: When the appropriate levels of Cu/Zn SOD are expressed transiently after injury using the non-viral modular protein vector NLSCt a neuroprotective effect is seen. Thus recombinant modular protein vectors may be suitable for in vivo gene therapy, and Cu/Zn SOD should be considered as an interesting therapeutic transgene.

Figure 1

Post-lesion over-expression of Cu/Zn SOD is neuroprotective. NMDA lesioned animals were not re-injected or re-injected 2 hours after with either saline solution, the NLSCt vector carrying the transgene for Cu/Zn SOD or the NLSCt vector carrying the transgene for EGFP (NMDA, NMDA+saline, NMDA+SOD and NMDA+GFP respectively). The percentage of total lesioned hemisphere observed in A show that animals injected with the transgene for Cu/Zn SOD displayed a significant reduction in lesion volume in comparison with NMDA+saline (*p < 0.05) or in comparison to NMDA alone (#p < 0.05). The lesion volume in NMDA+saline injected animals was 36.2 mm³. Noticeably, animals injected with the GFP transgene also displayed a significant reduction, though only when compared to NMDA+saline injected animals; a reduction that was less pronounced than those found in Cu/Zn SOD injected animals. The rostro-caudal percent of lesioned hemispheric area is shown in B. The overall lesion was reduced at all levels of the brain of Cu/Zn SOD and GFP injected animals. The injection site is highlighted in grey.

Figure 2

Post-lesion over-expression of Cu/Zn SOD increases neuronal survival. Nissl stained sections showing the lesion distribution in the animals post-injected with saline (NMDA+saline), NLSCt+Cu/Zn SOD (NMDA+SOD), or NLSCt+GFP (NMDA+GFP)(A). Quantification of Nissl stained neurons in the sensorimotor cortex and hippocampal CA1 penumbra showed increased cell survival in Cu/Zn SOD overexpressing animals (B). GFP overexpressing animals only showed an increase in neuronal number in hippocampal CA1 at a greater distance from the lesion point (B). Degenerating neurons were stained with Fluoro-Jade B staining (C), and quantitative analysis showed a significant reduction of degenerating neurons in Cu/Zn SOD overexpressing animals (D). (*p < 0.05 in relation to NMDA+saline injected animals and #p < 0.05 in relation to NMDA+GFP injected animals).

Figure 3

Post-lesion overexpression of Cu/Zn SOD decreases nitrotyrosine immunoreactivity. Sections from the caudal region of the CA1 hippocampal layer (bregma -2.43) of the contralateral side showed a basal nitrotyrosine immunoreactivity (A, Or: stratum oriens; Py; stratum pyramidalis; Ra: stratum radiatum). After NMDA injection, an increased immunorectivity was detected (B). However, animals overexpressing Cu/Zn SOD showed reduction in nitrotyrosine immunoreactivity (C), while animals overexpressing GFP showed a less pronounced reduction (D). Quantitative analysis confirmed these observations whereby, only animals overexpressing Cu/Zn SOD showed a significant (p < 0.05) decrease in nitrotyrosine immunoreactivity (E).

Figure 4

Functional evaluation of NLSCt-Cu/Zn SOD injected animals. At 24, 48 and 72 hours post-lesion, animals were subjected to several neurological tests including: the estimation of coordination skills by measuring the total climbing time until falling when placed on an inclinated grid (A); spontaneous turning behaviour in an open field (total turns recorded in 1.5 min.)(B); and spontaneous motor activity (C). Percentage of body weight increase was also followed (D). Lesioned animals injected with saline (NMDA+saline) showed a significant decrease in the time spent climbing on the inclinated grid in comparison to non-lesioned saline injected animals (Non lesioned)(A). Interestingly, only animals injected with the NLSCt vector carrying the Cu/Zn SOD transgene (NMDA+SOD) displayed a significant recovery in the time spent on the inclinated grid when compared to NMDA+saline animals (#p < 0.05). Animals injected with NMDA+saline or NMDA plus NLSCt vector carrying the EGFP transgene (NMDA+GFP) showed a significant increase in net turns compared to saline injected animals (*p < 0.05). In addition, only animals overexpressing Cu/Zn SOD showed a turning behaviour indistinguishable from non-lesioned control saline injected rats and significantly different from NMDA+saline (#p < 0.05) or NMDA+GFP injected animals (&p < 0,05)(B). There were no differences in the open field general motor activity of all experimental groups (C). Body weight of both NMDA+saline and NMDA+GFP injected animals showed a significant decrease in the developmentally physiological body weight increase in relation to saline injected animals (*p < 0.05). Overexpression of Cu/Zn SOD hindered this decrease and these animals showed an increase in body weight indistinguishable from control non-lesioned saline injected rats.