and the ratio of cleaved capase 9/caspase 9, signifying heightened apoptosis in this animal model and diminished protection from cellular stresses, such as inflammation, as a result of the elevated oxidative damage within the G93A mouse model. Materials and Methods Animals Male B6SJL-TgN(SOD1-G93A)1Gur hemizygous mice (No. 002726) were harem-bred with female B6SJL nonaffected control mice (Jackson Laboratory, Bar Harbor, ME). The presence of the human G93A transgene was confirmed using PCR amplification of DNA extracted from tail samples as outlined by Jackson Laboratories. All animals were housed three to five per cage in a 12-h light/dark cycle. The experimental protocol strictly followed guidelines put forth by Canadian Council of Animal Care and McMaster University Animal Research Ethics Board. All necessary steps were taken to ameliorate suffering to animals involved in the study. Study Design Eighty G93A mice (49 females, 31 males) were fed ad libitum (AL) after weaning (21 d) until the study commenced at age 40 d. At age 35 d, the mice were housed in individual cages. At age 40 d, 27 G93A mice (13 females, 14 males) were allocated to a surgery group and subsequently divided into two groups: the first group (AL; 7 females, 7 males) was provided food AL with a standard rodent diet (Harlan Teklad 22/5 Rodent Diet (W), product #8640); the second group (CR; 6 females, 7 males) was provided with 60% of the average intake of the AL group. The rest of the mice were also divided into AL (15 females, 7 males) and CR (21 females, 10 males) groups and carried to endpoint. The CR group diet (NIH-31/NIA Fortified Diet) was fortified with vitamins to ensure intakes similar to those of the AL group. The CR mice were provided with food equivalent to 60% of the average intake of the AL group on a daily basis. When mice attained a clinical score of 2.5 or started losing weight, food and calorie-free gel (Harlan-Gel, Harlan Teklad, Madison, WI) were placed on the floor of the cage to fulfill the requirements of the ethics committee. The calorie-free gel contained synthetic polymers (WATER LOCKH superabsorbent polymer G-400, G- 430, G-500, G-530; ,95% by weight) and methanol (,4.5% by weight). All measurements were conducted by two researchers. The researchers were not blinded to the treatment protocol. The inter-researcher coefficients of variation are 3.2% for body condition, 2.2% for ability to move and 0.24% for clinical score. Food Intake Food intake measurements were initiated at age 43 d for all mice and recorded until age 95 d. The CR group was provided 60% of the average intake of the AL group, sex specific, following all measurements and testing. Body Weight and Body Condition Body weights of mice were logged starting at age 43 d twice per week until age 99 d. Body weight and body condition for mice in the CR group were recorded before daily food portions were provided. Starting at age 43 d, body condition was assessed following a 5-point scale: 5 = obese mice, 4 = overconditioned mice (spine is a continuous column and the vertebrae are palpable only with firm pressure), 3 = well-conditioned mice (the vertebrae and dorsal pelvis are not prominent and are palpable with slight pressure), 2 = underconditioned mice (the segmentation of the vertebral column is evident and the dorsal pelvic bones are easily Caloric Restriction and ALS palpable), and 1 = emaciated mice (the skeletal structure is extremely prominent and the vertebrae are distinctly segmented). Ability to Move Starting at age 43 d, ability to move was assessed following a 5- point scale: 4 = normal mobility, 3 = move with limited use of the hindlegs, 2 = move with the use of the forelegs, 1 = move only for a short period with the use of the forelegs, and 0 = unable to move. For the CR group, ability to move was recorded before mice were provided with their daily food portions. Paw Grip Endurance Paw grip endurance (PaGE) was measured twice per week starting at age 70 d using the modified hanging wire test [12,13]. Each mouse was placed on the wire-lid of a housing cage, placed at a height of 40 cm, and allowed to tightly hold on as induced by a gentle shake of the lid. Next, the lid was inverted and the time was recorded until the mouse fell off the lid, for a maximum of 90 seconds. This test was completed in triplicate, with the best result recorded. For the CR group, PaGE was recorded before mice were provided with food. Voluntary Activity This study was part of a larger study investigating the effect of CR on oxidative stress. At age 100 d, muscle and tissue samples were harvested and stored at 280uC. This allowed us to measure voluntary activity in a representative subset of mice. Hence, in a subset of 64 mice (16 each for AL females, AL males, CR females and CR males), voluntary activity was recorded at age 72–89 d using mice activity wheels with living chambers (Single Activity Wheel Chamber Systerm Model 80820, Lafayette Instrument, Lafayette, IN). The mice were individually placed in the chambers and provided with food and water until they were returned to their cages. The activity wheel counters were interfaced with a computer which recorded cumulative activity in total revolutions over a 24-h period. Each 2.5 revolutions are equivalent to 1 m. Clinical Score Using an 8-point scale, the clinical score for each mouse was assessed starting at age 81 d until age 95 d. The clinical score was dependent on signs exhibited to identify the severity of the disease: 0 = no evidence of disease, 1 = shaking of the hind legs or splaying of the hind legs when suspended by the tail (an indication of weakness in the hind legs), 1.5 = weakness in one hindlimb (compensation for footdrop), 2 = change in gait (used as clinical onset when attained on two consecutive days), 2.5 = extreme weakness in one hindlimb (inability to dorsiflex), 3 = extreme weakness in both hindlimbs, 3.5 = functional paralysis in one hindlimb, 4 = functional paralysis in both hind legs but can right themselves in less than 20 seconds after being placed on their side, and 5 = cannot right themselves within 20 seconds after being placed on their sides (clinical endpoint). For the CR group, clinical score was recorded before the mice were provided with their daily food portions. Animal Sacrifice and Tissue Collection Twenty seven G93A mice were sacrificed at ,99 days of age, while the rest of the mice (n = 53) were led to endpoint. Mice were anesthetized with gaseous isoflurane and maintained under general anesthesia as the tissue was collected. Skeletal muscle (red and white gastrocnemius, and quadriceps) was removed and placed into individual sterile polyethylene tubes for immediate freezing in liquid nitrogen. All samples were stored at 286uC until analysis. PLoS ONE | www.plosone.org 2 February 2010 | Volume 5 | Issue 2 | e9386