Muscle Reinnervation with Delayed or Immediate Transplant of Embryonic Ventral Spinal Cord Cells into Adult Rat Peripheral Nerve

Abstract

Muscle denervation is common in various neuromuscular diseases and after trauma. It induces skeletal muscle atrophy. Only muscle reinnervation leads to functional recovery. In previous studies, denervated adult rat muscles were rescued by transplantation of embryonic day 14–15 (E14–15) ventral spinal cord cells into a nearby peripheral nerve. In the present study, changes were made in the environment into which the cells were placed to test whether reinnervation was improved by: 1) prior nerve degeneration, induced by sciatic nerve transection 1 week before cell transplantation; 2) transplantation of 1 million versus 5 million cells; 3) addition of nerve growth factor (NGF) to the transplant. Ten weeks after cell transplantation, axons had grown from all of the transplants. The numbers of myelinated axons that regenerated into the tibial, medial (MG), and lateral gastrocnemius-soleus (LGS) nerves were similar across treatments. The mean diameters of large LGS axons (>6 μm) were significantly larger with nerve degeneration before transplantation. The mean diameters of MG and LGS axons were significantly larger with transplantation of 1 million versus 5 million cells. Silver-stained experimental and control lateral gastronemius (LG) muscles showed axons that terminated at motor end plates. Nodal and terminal sprouts were more common in reinnervated muscles (45–63% of all end plates) than in control muscles (10%). Electrical stimulation of the transplants induced weak contractions in 39 of 47 MG muscles (83%) and 33 of 46 LG muscles (72%) but at higher voltages than needed to excite control muscles. The threshold for MG contraction was lower with transplantation of 1 million cells, while LG thresholds were lower without NGF. The cross-sectional area of whole LG muscles was significantly larger with cell transplantation (immediate or delayed) than with media alone, but all of these muscle areas were reduced significantly compared with control muscle areas. These data suggest that delayed transplantation of fewer cells without NGF assists regeneration of larger diameter axons and prevents some muscle atrophy.

Acute Stimulation of Transplanted Neurons Improves Motoneuron Survival, Axon Growth, and Muscle Reinnervation

“…Acetylcholine receptors at motor end plates were detected with Alexa Fluor 568 conjugated a-Bungarotoxin (Invitrogen). 59 …”

“…Cell transplantation was delayed because this improved axon regeneration and a delay is a clinically feasible approach. 59 Physiological analysis of muscle function and tissue removal occurred 10 weeks after transplantation.…”

Transplantation of Embryonic Spinal Cord Derived Cells Helps to Prevent Muscle Atrophy after Peripheral Nerve Injury

“…After that, NPCs need to differentiate in the nerve, however the preferential differentiation is towards glial lineages. Few studies in the past that have used isolated fetal spinal cord cells have been shown to be efficient in assisting axonal regeneration and preventing muscle atrophy to a certain degree [46,50,51,52,53]. Taking this into consideration, we decided to use fetal P0 cells that did not need to be cultured and did not need to undergo further differentiation in the nerve.…”

“…So far, numerous studies have used various kinds of fetal or self-renewal stem/progenitor cells, including embryonic stem cells [21], Schwann cells [22,23], neural progenitor cells (NPCs) [24,25], induced pluripotent stem cells (iPSCs) [26], and stem cells from bone marrow [27,28,29,30], fat tissue [31,32,33], hair [34,35] and skin [36,37,38]. Many of these studies have shown some modest recovery after peripheral nerve injuries [21,30,35,36,38,39,40,41,42,43,44,45,46]. The underlying mechanisms are still unclear and many of the studies focus on speeding up the axonal regeneration rather than preventing the early muscle atrophy.…”

“…In order to avoid the differentiation concerns, we decided to also use fetal spinal cord cells directly as they have already differentiated into neurons at the isolation time. These few studies that have used fetal spinal cord cells have shown the cells to be able to both assist with the axonal regeneration and with preventing the muscle atrophy [46,50,51,52,53,54,55,56]. Our aim was to compare this kind of fetal cells with pure NPCs for their ability to survive in injured nerve and their ability to reinnervate the muscle and reduce the muscle atrophy.…”