Methods.

Peripheral nerves (phrenic and sciatic), muscle (diaphragm and gastrocnemius), and spinal cords from ALS (mSOD1 G93A) and wild-type (WT) mice on a B6/SJL background were evaluated by quantitative RT-PCR (qRT-PCR) at 10, 20, 55, and 77 days and end-stage disease (n= 3 for each time point) and fluorescent immunohistochemistry. Denervation was evaluated by qRT-PCR for the temporal changes in the mRNA levels of γ (fetal) and ε (adult) acetylcholine receptor (AChR) subunits. All experimental procedures involving animals were approved by The Methodist Research Institute's Institutional Animal Care and Use Committee in compliance with NIH guidelines. Data were analyzed using two-tailed Student t test and group means were plotted±SEM; p< 0.05 was considered statistically significant. Differences between groups were analyzed using a 2-way analysis of variance.

Results.

As evidence of denervation, the reappearance of the AChR fetal γ subunit was first noted in diaphragm and gastrocnemius muscles at 55 days (p= 0.033 for diaphragm and p= 0.041 for gastrocnemius, compared with their WT counterparts; figure, A and B) whereas the adult ε subunit remained unchanged in both muscles (not shown). The expression of CD68 mRNA, an inflammatory marker of phagocytic macrophages, was increased in mSOD1 mice muscle after 77 days compared with muscle from WT mice (p= 0.048 for diaphragm and p= 0.041 for gastrocnemius, figure, C and D); the mRNA levels for CCL2 mirrored the temporal expression levels of CD68 (not shown). Immunohistochemistry for CD68 confirmed that at 77 days inflammation was increased in phrenic nerves of mSOD1 mice compared with WT mice and was markedly increased in distal than proximal segments (figure, E) and although the sciatic nerves also showed increased CD68 signals, the proximal and distal segments were comparable; consistent with the qRT-PCR data, CD68 signal was not increased in mSOD1 mice at 55 days of age (not shown). The corresponding lumbar spinal cord level of the sciatic nerve showed increased CD68 signal in mSOD1 mice compared with WT mice at 77 days (p= 0.041, figure, F); CD68 mRNA was not increased in the corresponding cervical spinal cord level of the phrenic nerve in mSOD1 mice at this time point.