At P30, mSOD1 containing cytoplasmic aggregates have been detected in MNs of G93A SOD1 mice (Johnston et al., 2000) and as mentioned previously, P90 is usually their approximate age of symptom onset (Gurney et al., 1996). also resulted in an increased quantity of innervated neuromuscular junctions compared with control tissue. Together these results suggest rhHsp70 may delay disease progression in the G93A SOD1 mouse via a yet to be identified peripheral mechanism. after trophic factor deprivation and oxidative stress (Robinson et al., 2005, 2007). Data from our lab also produced comparable results when mouse embryonic MNs were subjected to trophic factor withdrawal and supplemented with rhHsp70 (our unpublished observation). Systemic Lycorine chloride administration of rhHsp70 to the developing chick’s chorioallontoic membrane was capable of maintaining MN survival during the period of developmental programmed cell death (Robinson et al., 2005). These results Lycorine chloride provided evidence that rhHsp70 may be able to rescue MNs access to Riluzole in their water at a concentration of TNR 100 g/ml with the solution changed three times weekly (Gurney et al., 1996). rhHsp70-treated mice were injected three times weekly to recapitulate the access to new Riluzole treated water three times weekly. Nontransgenic littermates [wild-type (WT)] and G93A SOD1 mice were intraperitoneally injected with rhHsp70 (Assay Designs, Ann Arbor, MI; catalog #ESP-555; 20 g diluted in 100 l sterile saline). In our previous study we found that Lycorine chloride 10 g of rhHsp70 was effective for delaying naturally occurring MN death in chick embryos (Robinson et al., 2005). After accounting for excess weight and volume of the yolk and embryo, 20 g was estimated to be a comparable dose for P50 mice. Intraperitoneal injections of bovine serum albumin (BSA; 20 g in 100 l sterile saline), and saline alone were used as unfavorable controls. Because there were not any survival promoting effects of either BSA or saline alone, and there was no statistical difference between the two groups, both groups were combined into the single control group. Lycorine chloride A subset of animals was administered rhHsp70 beginning on day 30 or 90 to determine whether earlier or later initiation of treatment would influence lifespan. At P30, mSOD1 made up of cytoplasmic aggregates have been detected in MNs of G93A SOD1 mice (Johnston et al., 2000) and as mentioned previously, P90 is usually their approximate age of symptom onset (Gurney et al., 1996). Because the mean survival varies between male and female G93A SOD1 mice (Heiman-Patterson et al., 2005), we included comparable numbers of each gender in each treatment group. Behavioral assays. Disease progression was evaluated on each treatment day with weight determination, a leg extension test, and overall performance around the rotorod (San Diego Instruments, San Diego, CA) as explained previously (Gurney et al., 1996; Wong et al., 1998). As animals progress toward disease end stage, muscle mass atrophy, resulting in weight loss occurs (Kieran et al., 2004). End stage disease is considered when the mouse cannot right itself after 30 s when placed on its side (Gurney et al., 1996). When mice are suspended by the tail, the hind limbs are normally extended, whereas failure of extension and/or shaking of the hind limbs are considered signs of symptom onset (Gurney et al., 1996). The rotorod test examines motor coordination and may be sensitive to MN dysfunction and degeneration (Crawley, 1999); the rod was rotated at a gradually accelerating speed up to 11 rpm over a 2 min interval, and the animals’ latency to fall was recorded. Histology. Mice were killed at P90, P120, and at end stage, and the spinal cords were harvested and processed for MN counts. Briefly, mice were transcardially perfused with PBS (0.15 m NaCl, 2.98 mm Na2HPO4.7H2O, 1.03 Lycorine chloride mm KH2PO4) followed by Bouin’s fixative. The lumbar region of the spinal cord was removed and embedded in paraffin. 12 m sections were cut, and stained with a 5% thionin answer (Chu-Wang and Oppenheim, 1978). Only healthy MNs were counted in every tenth section of the lumbar spinal cord using a well established reliable method that has been validated against an optical.
