Long-term expression of IGF-1 and GFP in lumbar spinal cord of SOD1G93A mice following AAV2 delivery
GFP () and IGF-1 (, C) were expressed in SOD1G93A lumbar cord at high levels even at disease end-stage, up to 90 days post-injection. Similar to GFP, IGF-1 expression was observed in continuous distinct regions in gray matter, likely related to each individual injection. GFP expression was observed in distinct individual cells, the neuropil, in neuronal cell bodies of gray matter at the level of injection (including motor neurons: -inset), and in neuritic processes in white matter both at the level of injection and in regions rostral and caudal to injection sites. The rostrocaudal extent of GFP expression for each injection was 0.6–1.2 mm, and the estimated volume of distribution of GFP product in gray matter was 0.04–0.21 mm3. Expression of GFP was not seen in IGF-1 injected cords, and similar long-term GFP expression was found following AAV2 GFP injections into lumbar spinal cord of wild-type mice (not shown). IGF-1 expression was found in neuronal cell bodies and neuritic processes of gray matter at injection sites, was specifically noted in ventral horn motor neurons ( - arrows), but was seen to a lesser extent in surrounding white matter. The rostrocaudal extent for each IGF-1 injection site was 2.4–2.6 mm, and the estimated volume of distribution of IGF-1 product in gray matter was 0.13–0.21 mm3 for each site. Expression of human IGF-1 was not seen in formulation buffer-injected cords (). Unfortunately, tissue was unavailable to measure total IGF-1 levels in lumbar spinal cord.
AAV2 IGF-1 delivery results in long-term IGF-1 expression and rescues motor neurons in the lumbar spinal cord of SOD1G93A mice
To determine the identity of AAV2-infected spinal cord cells, double-immuno labeling of GFP with specific phenotypic markers was employed. GFP co-localized with NeuN+ neurons (not shown); however, no co-localization of GFP with GFAP+ astrocytes was observed ().
AAV2 IGF-1 delivery partially rescued motor neuron loss in SOD1G93A mice
AAV2 GFP (14.7 ± 0.7 motor neurons/section; n = 4) and AAV2 IGF-1 (18.7 ± 1.6/section; n = 5) SOD1G93A mice had greatly reduced numbers of motor neurons compared to wild-type age-matched mice (30.9 ± 1.3/section; n = 4) at 110 days of age (). Compared to AAV2 GFP injected controls, AAV2 IGF-1 animals had a significantly greater number of motor neurons at 110 days of age (p < 0.01; ), demonstrating that IGF-1 delivery partially rescued motor neuron loss in SOD1G93A mice. While the number of mice for sex-specific analysis of motor neuron survival was small, no gender differences were noted within the AAV GFP (males: 14.8 ± 0.8 motor neurons/section; n = 2; females: 14.4 ± 0.5 motor neurons/section; n = 2) or AAV IGF-1 (males: 18.7 ± 2.2 motor neurons/section; n = 2; females: 18.8 ± 0.4 motor neurons/section; n = 3) treatment groups.
To access a potential mechanism of neuroprotection, the host microglial response was examined. No differences in the response of Iba1+ microglia was noted between lumbar spinal cord of AAV2 GFP and AAV2 IGF-1 mice (), suggesting that therapeutic effects of IGF-1 were not mediated by reduction in the host immune response.
AAV2 IGF-1 and AAV2 GFP delivery had no adverse behavioral effects
Injections of either AAV2 GFP or AAV2 IGF-1 did not result in weight loss or decline in hindlimb grip strength in wild-type mice (; p > 0.05 for all comparisons). Considering that multiple intraparenchymal spinal cord injections is a relatively invasive technique, these results are important in demonstrating that the injection procedure itself had no apparent adverse behavioral effects. Furthermore, there were no differences in weight or hindlimb grip strength values between AAV2 GFP and AAV2 IGF-1 mice (; p > 0.05 for all comparisons), suggesting that IGF-1 exerted no adverse behavioral effects on normal animals.
AAV2 IGF-1 delivery delayed hindlimb grip strength decline and weight loss selectively in male SOD1G93A mice
AAV2 IGF-1 delivery partially slowed hindlimb grip strength decline selectively in male SOD1G93A mice
Compared to AAV2 GFP controls, AAV2 IGF-1 SOD1G93A mice injected with AAV2 IGF-1 showed a significant delay in the decline in hindlimb grip strength when all animals from both sexes were analyzed together (p < 0.05 at 3, 4, 5 and 7 weeks post-injection; data not shown). No change in hindlimb grip strength decline was noted for female mice (p > 0.05 for all comparisons; ), while a significant delay in grip strength decline was seen in male mice (p < 0.05 at 4, 5 and 7 weeks post-injection; ).
AAV2 IGF-1 delivery partially slowed weight decline selectively in male SOD1G93A mice
AAV2 IGF-1 significantly delayed weight decline compared to AAV2 GFP controls at 8, 9 and 10 weeks post-injection in male mice (; p < 0.05), but no effect was found in female mice (; p > 0.05 for all comparisons). Motor neuron rescue by AAV2 IGF-1 likely slowed muscle atrophy and consequent weight loss. In addition, because AAV2 IGF-1 mice maintained greater hindlimb motor function than AAV2 GFP controls, weight was likely maintained in AAV-2 IGF-1 mice because they were better able to feed.
AAV2 IGF-1 delivery delayed disease onset selectively in male SOD1G93A mice
Compared to AAV2 GFP controls, AAV2 IGF-1 mice showed a significant delay in disease onset of 11.9 days (102.9 ± 10.9 days vs 114.7 ± 8.2 days; p < 0.05; data not shown) when all animals from both sexes were analyzed together. No change in onset was noted for female mice (111.3 days ± 14.6 vs 115.0 ± 9.4 days; p > 0.05; ), while a significant increase in onset of 19.0 days was seen in male mice (97.8 days ± 3.8 vs 116.8 ± 8.1 days; p < 0.05; ).
AAV2 IGF-1 delivery delayed disease onset and extended survival selectively in male SOD1G93A mice
AAV2 IGF-1 delivery did not extend disease duration in male and female SOD1G93A mice
No change was observed in disease duration, the time between disease onset and end-stage, in male mice (28.6 days ± 5.5 vs 26.3 ± 9.7 days; p > 0.05; ), female mice (21.7 days ± 4.6 vs 22.2 ± 8.4 days; p > 0.05; ) or when all animals from both sexes were analyzed together (26.0 ± 6.0 days vs 22.2 ± 9.8 days; p > 0.05; data not shown).
AAV2 IGF-1 delivery extended survival selectively in male SOD1G93A mice
When male and female SOD1G93A mice were analyzed together, AAV2 IGF-1 increased survival by 6.6 days (127.8 ± 11.7 days vs 134.4 ± 10.8 days; p > 0.05; data not shown) compared to AAV2 GFP; however, this increase was not significant. AAV2 IGF-1 significantly increased survival in male mice by 12.3 days (121.7 ± 11.4 days vs 134.0 ± 7.7 days; p < 0.05) compared to the AAV2 GFP group (), while no effect was found in females (134.8 ± 7.6 days vs 134.8 ± 13.4 days; p > 0.05; ). Interestingly, AAV2 IGF-1 delivery was only able to extend male survival to the level of AAV2 GFP female controls.