Reduction of lower motor neuron degeneration in wobbler mice by N-acetylcysteine

Jeffrey T. Henderson, Mohammed Javaheri, Susan Kopko and John C. Roder

Samuel Lunenfeld Research Institute, Mount Sinai Hospital,
Program in Molecular Biology and Cancer, 600 University Ave., Toronto, Ontario M5G-1X5

Materials and Methods

Animals:
Wobbler mice were obtained from the mating of confirmed heterozygous wr/+ mice. All animals received food and fluids ad libitum. Animals received drinking water supplemented with either 1% N-acetylcysteine, L-alanine or D-glucose, adjusted to the same pH (pH 3.5) and osmolarity. Fluids were changed every 48 hours. Wobbler mice were sacrificed for examination at postnatal day 63-66. All animals received food and water ad libitum, and were raised under identical conditions in the same room of our gnotobiotic animal facility. All experimental protocols conformed to Mount Sinai Hospital and University of Toronto animal colony care guidelines.

Sample preparation:
Animals were deeply anesthetized with sodium pentobarbital (Somnitol 80 mg/kg). Following the absence of twitch responses, animals were perfused transcardially with 15 mls of 100 mM phosphate buffered saline (PBS, pH 7.4), followed immediately by 50 mls of freshly prepared 4% paraformaldehyde in PBS at 4⁰C. At this point, a two millimeter segment representing the major and minor medial branches of the left facial nerve were dissected, along with the C2-C7 region of the spinal cord and several muscles of the left forelimb of each animal. Samples were then postfixed for a further 2 hours in 4% paraformaldehyde in PBS at 40C. At this point the spinal cords were further dissected to the C4-C7 region. Samples were subsequently processed for either cryostat, paraffin, or thin sections. At this point each sample was given a coded identification number so that data derived from them could be analyzed in a "blinded" manner.

Paraffin sections:
Samples were dehydrated by placing in an ascending series of ethanol/water and ethanol baths, and embedded into paraffin blocks according to standard procedures (Ausubel et al., 1994). Ten micron sections were then cut on a Reichert-Jung microtome, mounted on 2x gelatin coated slides and heated at 60⁰C for 1.5 hours. Slides were subsequently de-waxed and stained in thionin as described previously (Culling, 1974).

Thin sections:
Specimens were post-fixed in a solution of freshly prepared 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) for 4 hours at 4⁰C; then rinsed free of glutaraldehyde and fixed in 1% osmium tetroxide buffered in PBS for 1 hour. Samples were then dehydrated in a series of water/ethanol and ethanol/propylene oxide baths. Following removal of propylene oxide, samples were embedded in spurr resin and baked at 50⁰C for 36 hours. A series of one micron thick cross-sections were then cut through the entire nerve and stained with 1% toluidine blue according to standard procedures (Culling, 1974).

Cryostat sections / Immunohistochemistry:
Freshly post-fixed spinal cords were placed in 30% sucrose, 0.1 M PBS (pH 7.4) at 4'C until sunk (12-15 hours); then frozen in 2-methyl butane at -20⁰C. Ten micron serial cross-sections were subsequently obtained using a Reichardt-Jung Fridgocut cryostat. These were thaw-mounted onto 2x gelatin stubbed slides and either stained with thionin or processed for ChAT immunoreactivity using a Chemicon goat anti-ChAT antibody as previously described (Henderson et al., 1994).

Axon/nerve morphometry:
The morphometry of nerve and muscle cross-sections we