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To investigate whether caspase-1 and caspase-3 are activated in the mSOD1 mouse spinal cord, we performed immunohistochemical staining with an antibody specific for activated caspase-1 (provided by J. Yuan, Harvard Medical School, Boston, MA) and an antibody specific to activated caspase-3, which is also known as CM1 (provided by A. Srinivasan, Idun Pharmaceutical, La Jolla, CA). In addition, a neuron-specific marker, anti-NeuN antibody (Chemicon, Temecula, CA), was used in a double staining procedure to identify neuronal cells. At 70, 90, and 110 days of age, mice were killed and perfused with 4% paraformaldehyde. Spinal cords were removed under a dissecting microscope. Tissue was frozen in cold isopentane after cryoprotection in 30% sucrose. Frozen sections (10 μm thick) were washed with phosphate-buffered saline (PBS) containing 0.05% Tween-20 and were incubated with 5% normal goat serum. The sections were incubated overnight with rat polyclonal antibody to caspase-1 (1:5) or rabbit polyclonal antibody to caspase-3 (CM1) (1:2000), washed overnight, then washed three times with PBS, and finally probed with a biotinylated secondary antibody to rat immunogloblin G (IgG) or a biotinylated secondary antibody to rabbit IgG (1:300; Vector, Burlingame, CA) for 2 hours. Next, specimens were incubated in Fluorescein Avidin DCS (1:1000; Vector). Sections were also double stained with the NeuN antibody (1:100) followed by Texas Red antibody to mouse (1:200; Vector). Hoechst 33342 was used for counterstaining. The fluorescent stained sections were evaluated with epifluorescence microscopy.
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At 60 days of age, osmotic pumps (Alzet, Palo Alto, CA) were used for the intracerebroventricular delivery of zVAD-fmk (Enzyme Systems, Livermore, CA). Pumps were filled with vehicle (0.4% dimethyl sulfoxide, 0.1 M Pipes, pH 6.9), 100 μg of 2 VAG-fmk per 20g body weight, or 300 μg of zVAG-fmk per 20 g of body weight. Pumps continuously delivered the drug for 28 days and were then exchanged for new pumps filled with fresh drug or vehicle for an additional 28-day treatment. The investigator was blind to the identity of the drug or vehicle used in the pump until the death of all mice.
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A Rotarod (Columbus Instruments, Columbus, OH) was used to evaluate motor function. Mice were first evaluated the day before placement of the osmotic pumps and thereafter on a weekly basis. Mice were placed on the rotating rod at speeds of 5, 15, and 20 rpm. The time each mouse remained on the rod was registered automatically. If the mouse remained on the rod for 7 min, the test was completed and scored as 7 min.
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We thank A. Srinivasan for providing the CM1 antibody and J. Yuan for providing the caspase-1 antibody; M. Schoenebeck for expert preparation of the phrenic nerve samples; J. Yuan for insightful comments on the manuscript; and E. Friedlander for editorial assistance. Supported by the Muscular Dystrophy Association; the ALS Association; Project-ALS; the National Institute of Neurological Disorders and Stroke (grants R01 NS38586, R29 NS37345, and P50 NS38370); the U.S. Department of Defense (grant DAMD 17-99-1-9471); the Lowenstein Foundation; the Smart Foundation; and the Parkinson's Disease Foundation. C.G. is the recipient of a scholarship from the French ISERM. S.P. is a recipient of the Cotzias Award from the American Parkinson Disease Association.
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