N4638

MNs loss was determined by neutral red staining as previously reported [17 (link)]. 1% neutral red (N4638, Sigma–Aldrich, USA) in 0.1 M acetic acid (pH 4.8) was used to stain sections for 2 h and then followed by a series of dehydration in graded concentrations of ethanol. The images were captured Nikon light microscope using the bright field feature. The counting of motor neurons was conducted by 2 laboratory personnel blinded to the injury or treatment conditions of the mice. The MNs loss was determined by its survival rate (ratio of ipsilateral/contralateral MNs number). Only large soma neurons bearing a visible nucleolus and located in lamina IX of Rexed were enumerated.

Apoptotic cells were revealed in 2-days-old live embryos by adding the acridine orange vital dye (N-4638; Sigma) to the embryo water at a final concentration of 5 mg/mL. Embryos were then incubated in the dark at 28 °C for 1.5–2 h, rinsed, anesthetized and observed under a fluorescent stereomicroscope Olympus (Shinjuku, Tokyo 163-0914, Japan).

Lysosomal membrane stability was measured on freshly cut sponge tissue following standard methods^{52 , 53} modified for sponges^{54 (link)}. Approximately 1 cm³ of choanosome was excised from each explant, rinsed and stored in seawater for a maximum of 1 h. Tissue was further minced into 1 mm³ pieces which were placed into a single well of a 24-well culture plate with 1 ml of Ca²⁺/Mg²⁺ free saline (CMFS) and agitated in a shaker at 120 rpm for 30 min. The tissue homogenate was filtered through a 40 µm nylon mesh into a 2 ml microcentrifuge tube. Samples were centrifuged at 300 g, 10 °C for 5 min, before the supernatant was discarded and the pellets resuspended in 1 ml CMFS and centrifuged again at 300 g for 5 min. The supernatant was again discarded and the pellet resuspended in 50 µl CMFS. Fifty µl of neutral red solution (N4638, Sigma-Aldrich) was added and mixed in the sample, following storage for 1 h in a light protected humidified chamber at room temperature. A small aliquot of the cell suspension was placed onto a microscope slide and viewed under a light microscope (400x magnification). A total of 50 cells were assessed for lysosomal integrity. Cells with neutral red contained discretely within the lysosomes were scored as stable, while cells with neutral red leaking into the cytoplasm were scored as destabilised.