Blastema cell proliferation in vitro: Effects of limb amputation on the mitogenic activity of spinal cord extracts

Abstract

Primary cultures of mesenchymal cells of axolotl limb blastemas provide a very sensitive in vitro bioassay for studying nerve dependence of newt regeneration. These cells can be stimulated by crude spinal cord extracts of non-amputated animals in a dose-dependent manner up to 60 micrograms protein/ml of culture medium; at this concentration the mitotic index is increased 4-fold. Spinal cord extracts of axolotls 14 days after forelimb amputation (i.e., late bud stage) are more efficient in stimulating blastema cell proliferation (+50%) than extracts of axolotls 7 days after forelimb amputation (i.e., early bud stage) or of axolotls without amputation. In a similar manner, spinal cord extracts of young axolotls 14 days after forelimb amputation, are more stimulatory than older axolotls 14 d after forelimb amputation which regenerate only a very small blastema during the same time. It appears that spinal cord mitogenic activity is enhanced after limb amputation, probably in correlation with blastema cell requirements for limb regeneration.

Unilateral Forelimb Amputation Affects Protein Synthesis in Ipsilateral and Contralateral Spinal Cord and Spinal Ganglia Neurons of the Newt in vivo

“…Experimental work attempting to causally connect protein synthesis in spinal cord and spinal ganglia with the process of limb regeneration has been performed in the past, and has given interesting results [4,9,12,13], which differ from the present investigation in several critical points. One advantage of the study at hand is that it has been performed in vivo.…”

“…In this respect, it was early recognized that the presence of a regeneration blastema on a limb amputation stump prevents retro grade nerve degeneration and exerts other influences on the nerves as well [10,11]. The assumption that these effects might be due to several factors produced by the nervous tissue, the wounded organs and the blastema, has led to a search for such regulative molecules [4,5,9,12,13]. Thereby it was revealed that the central neural elements respond to limb amputation by augmentation in protein synthesis in vitro.…”

The influence of fundamental traits on mechanisms controlling appendage regeneration

“…In addition to body size, aging and metamorphosis may constrain regenerative capacity by inhibiting the ability of a nerve to produce the vital nerve-derived factor(s). Compared to old individuals, spinal cord extracts from young axolotls are more mitogenic than extracts from old axolotls when added to blastemal cells (in vitro) from a regenerating limb (Boilly & Albert, 1988). This suggests that there is some decrease in the neurotrophic influence of spinal cords across an age gradient in salamanders.…”

Advancements to the Axolotl Model for Regeneration and Aging

“…Interestingly, there is evidence that axolotl neurons are affected by age. It was found that spinal cord extracts from older axolotls were diminished in their capacity to stimulate cell proliferation compared to extracts from their younger counterparts, indicating that the nerves are producing less of these neurotrophic factors over time [34]. Moreover, axolotl nerve regeneration is vital for limb regeneration, and as with many vertebrates, the ability to regrow neurons decreases with time.…”