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 were analyzed using a Leitz wetzlar scope equipped with 25, 54 and 100 times objectives, a JVC model TK-1280U color video camera, and a 3600 rotating slide platform with X and Y controllers. Nerve areas were measured using a Leica Quantimet Q500MC system (Leica Canada, Willowdale, Ontario). The system was calibrated before and verified following each use, using a Leica 10 micron ruled calibration slide. Prior to analyzing each cross-section, a low resolution (25x) "map" was first generated and a hardcopy printed. This was then used as a reference to place each of the individual nerve (analyzed at 100x) or muscle (analyzed at 40x) sectors in a given morphometric analysis. In each case, data was gathered for the nerve cross section in its entirety; and data were analyzed in double-blinded manner.

Glutathione peroxidase assay:
Animals were sacrificed and flushed with 15 mls of 100 mM phosphate buffered saline (PBS, pH 7.4) at 4⁰C. Selected tissues were then dissected, washed in PBS at 4⁰C, their weights obtained, then frozen immediately in 2-methyl butane at -50⁰C, and stored at -70⁰C until used (72 hrs). Glutathione peroxidase (GPX) activity was determined by monitoring the rate of NADPH conversion to NADP+ in the presence of hydrogen peroxide as previously described (Doroshow et al., 1980). Assays were corrected for the non-enzymatic conversions of NADPH (consistently less than 11% of the enzymatic rates) and each sample was assayed in triplicate. Rates were expressed as nanomoles NADPH oxidized to NADP+per minute per milligram protein assuming an extinction coefficient for NADPH of 6.22 x 10³ mol-1 cm-1, and plotted as percent of NW control values.

Statistics:
Three analyses were performed on each group of data using one-way ANOVA, followed by Students t test, with Bonferroni adjustment for multiple comparisons (Howell, 1992). For each of the treatment groups (NW, NW(N), wr/wr, wr/wr(N)), three types of pre-planned pair-wise statistical comparisons were made: wr/wr versus NW; wr/wr versus wr/wr(N); and wr/wr(N) versus NW. The alpha level for all comparisons was set to 0.05 and all t values were compared with two-tailed critical t values. For each measure where significant pairwise comparisons are reported, ANOVA yielded significant results (alpha = 0.05).

Analysis of motor function:
Mice were scored at 7 weeks postnatal in a blinded manner for forelimb motor performance. Each limb was scored separately for each animal. Criteria were as follows: (1) no atrophy, limbs out-stretched when suspended, continuous grasping at proximal surface, normal inclination of the phalanges; (2) visible atrophy of limb, limb out-stretched when suspended, continuous grasping at proximal surface, normal inclination of phalanges; (3) significant atrophy of limb, limb not out-stretched when suspended, tentative grasping at proximal surface, abnormal (<60⁰) inclination of phalanges; (4) severe limb atrophy, limbs held closely to body when suspended, no grasping at proximal surface, abnormal (>90⁰) inclination of phalanges.

Abstract

Introduction

Materials and Methods

Results

Discussion

References