Muscle spindle reinnervation using transplanted embryonic dorsal root ganglion cells after peripheral nerve transection in rats

Abstract

Objectives: Muscle spindles are proprioceptive receptors in the skeletal muscle. Peripheral nerve injury results in a decreased number of muscle spindles and their morphologic deterioration. However, the muscle spindles recover when skeletal muscles are reinnervated with surgical procedures, such as nerve suture or nerve transfer. Morphological changes in muscle spindles by cell transplantation procedure have not been reported so far. Therefore, we hypothesized that transplantation of embryonic sensory neurons may improve sensory neurons in the skeletal muscle and reinnervate the muscle spindles.

Materials and methods: We collected sensory neurons from dorsal root ganglions of 14-day-old rat embryos and prepared a rat model of peripheral nerve injury by performing sciatic nerve transection and allowing for a period of one week before which we performed the cell transplantations. Six months later, the morphological changes of muscle spindles in the cell transplantation group were compared with the naïve control and surgical control groups.

Results: Our results demonstrated that transplantation of embryonic dorsal root ganglion cells induced regeneration of sensory nerve fibre and reinnervation of muscle spindles in the skeletal muscle. Moreover, calbindin D-28k immunoreactivity in intrafusal muscle fibres was maintained for six months after denervation in the cell transplantation group, whereas it disappeared in the surgical control group.

Conclusions: Cell transplantation therapies could serve as selective targets to modulate mechanosensory function in the skeletal muscle.

Figure 1

Cell preparation and transplantation. (A) Image showing the dissected DRGs obtained from a 14‐day rat embryo. (B) Image showing DRG cells injection into the distal stumps of the tibial nerve. (C) Flow chart depicting experimental steps. (D) The harvested tibial nerve that was separated into proximal and distal halves at two and six months after cell transplantation

Figure 2

Properties of cells in the transplantation site. (A) Longitudinal sections of the proximal tibial nerve isolated from rats in the surgical control and cell transplantation groups, stained with anti‐β3‐tubulin to mark neurons, anti‐oligodendrocyte marker O4 and Hoechst. White = β3‐tubulin; red = oligodendrocyte marker; blue = Hoechst. Scale bars are 100 μm. (B) Confocal image showing neurons surrounded by GFAP or O4 immunoreactive tissues in the transplantation site. White = β3‐tubulin; red = oligodendrocyte marker; blue = Hoechst; green = glial fibrillary acidic protein. Scale bars are 100 μm

Figure 3

Tibial nerve innervation improves after sensory cell transplantation. (A‐F) Representative axial (A,C,E) and longitudinal sections (B,D,F) of the tibial nerve from naïve control (A,B), surgical control (C,D) and cell transplantation (E,F) rats stained with the FluoroPan neuronal marker. Green = FluoroPan. Scale bars are 100 μm. (G) Quantification of fibre density in the tibial nerve six months after cell transplantation ((n = 6 per group) *P < 0.001). The average nerve fibre density in the cell transplantation group was lower than that in the naïve control (P = 0.0003), but was higher than that in the surgical control group (P = 0.000006)

Figure 4

Neuromuscular junctions and the weight of soleus muscle. (A) Quantification of the weight of soleus muscle in the three groups at six months after cell transplantation. (n = 6 per group). There were no significant differences between the weight of soleus muscle in the surgical control and that in the cell transplantation groups (P = 0.85). (B,C) Longitudinal sections of the soleus muscle stained with α‐bungarotoxin, calbindin D‐28k and FluoroPan neuronal marker in naïve control (B) and cell transplantation (C) groups. Red = α‐bungarotoxin; green = FluoroPan; purple = calbindin D‐28k. Scale bars are 100 μm

Figure 5

Number of muscle spindles at two months after cell transplantation. (A‐C) Longitudinal sections of the soleus muscle stained with α‐bungarotoxin, calbindin D‐28k, and FluoroPan neuronal marker in naïve control (A), surgical control (B) and cell transplantation (C) groups. Red = α‐bungarotoxin; green = FluoroPan; purple = calbindin D‐28k. Scale bars are 100 μm. (D) Quantification of the number of muscle spindles in the three groups at two months after cell transplantation ((n = 6 per group) *P < 0.001). The average number of muscle spindles was lower in the surgical control (P = 0.000005) and cell transplantation groups (P = 0.000008) than in the naïve control group

Figure 6

Number of muscle spindles at six months after cell transplantation. (A) Longitudinal sections of the soleus muscle stained with α‐bungarotoxin, calbindin D‐28k and FluoroPan neuronal marker in the cell transplantation group. Red = α‐bungarotoxin; green = FluoroPan; purple = calbindin D‐28k. Scale bar is 100 μm. (B) Confocal image showing muscle spindles reinnervated with nerve terminals. Red = α‐bungarotoxin; green = FluoroPan; purple = calbindin D‐28k. Scale bar is 100 μm. (C) Quantification of the number of muscle spindles in the three groups at six months after cell transplantation ((n = 6 per group) *P < 0.05). The average number of muscle spindles was significantly higher in the cell transplantation (P = 0.014) than in the surgical control group