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.

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. spindles. 3 Muscle spindles are involved in stretch reflexes and locomotor control. 4 Loss of sensory feedback from muscle spindles results in disruption of the proprioception of muscle movement.
Recent studies using cultured cells into nerve conduits have offered a novel approach for combining nerve repair and enhanced axonal regeneration. 5 Schwann cells are essential for peripheral nerve regeneration, and transplanted Schwann cells have been shown to enhance axonal regeneration. 6 Although there have been a few reports 2,7-9 investigating morphological changes and functional recovery of muscle spindles after preservation or repair of afferent innervation, to the best of our knowledge, there have been no reports that examined reinnervation of muscle spindles using cell transplantation procedures. Thus, we transplanted embryonic dorsal root ganglion (DRG) cells, including Schwann cells and sensory neurons, into a Walleriandegenerating nerve and evaluated muscle spindles, the proprioceptive receptors in the skeletal muscles. The purpose of this study was to investigate whether transplantation of DRG neurons into the peripheral nerve improves reinnervation of muscle spindles in the rat.

| Animal models
All animal maintenance procedures and experimental protocols used in this study were approved by the Animal Ethics Research Committee at Nagoya University. The recipients were 8-week-old male Fischer 344 rats (Japan SLC), which were assigned to three groups: naïve control (n = 12), surgical control (n = 12) and cell transplantation (n = 12). All surgical procedures were performed under a surgical microscope and 2% isoflurane anaesthesia. The naïve control group did not receive nerve transection or cell transplantation. In the surgical control and cell transplantation group, the sciatic nerve was transected at the mid-thigh. The proximal stump was ligated using 4-0 nylon and was sutured into the gluteus muscle to prevent natural regeneration. The distal stump of a sciatic nerve was divided into the tibial and peroneal nerves, which were ligated using 5-0 nylon.

| Cell preparation and transplantation
One week after nerve transection, embryonic DRG cells were prepared for injection into the distal stump of the tibial nerve. Three female Fischer 344 rats were used to obtain DRG cells from their 14-day-old embryos. The pregnant rats were anesthetized, and their embryos were removed from the uterus. DRGs were resected under a surgical microscope ( Figure 1A The harvested tibial nerve that was separated into proximal and distal halves at two and six months after cell transplantation anesthetized, and one million embryonic DRG cells in 10 μL culture medium were slowly injected into the distal stumps of the tibial nerve, using a Hamilton syringe with a 30-G needle; medium without any cells was injected into the surgical control side ( Figure 1B).
The injection point was about 20 mm proximal to the entry into the gastrocnemius and soleus muscles.

| Animal sacrifice
All rats were sacrificed under anaesthesia and perfused with 50 mL 0.9% saline, followed by 200 mL 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4). For chronological analysis, the entire tibial nerves and soleus muscles were frozen at 2 and 6 months after cell transplantation ( Figure 1C).

| Tibial nerve analysis
The harvested 20 mm of the tibial nerve was separated into proximal and distal halves ( Figure 1D). The tissues were cryoprotected in 30%

| Neuromuscular junctions and muscle spindles (intrafusal muscle fibres and capsules)
The triceps surae muscle was dissected from its origin and insertion. Then, it was divided into the soleus and gastrocnemius mus-

| Statistical analysis
Statistical analysis was performed using Ekuseru-Toukei 2010 software (Social Survey Research Information Co., Ltd). All data are reported as mean ± standard error of the mean. We performed statistical analysis with one-way ANOVA for multiple comparisons to determine the differences in the histological examination.
A value of P < 0.05 was considered statistically significant for all variables.

| Histological analysis in the transplantation site
In the cell transplantation group, nerve axons and cell bodies were identified by immunoreactivity to β3-tubulin. O4-positive tissue was also found within the transplantation site of the proximal tibial nerve   The average nerve fibre density was 125.1 ± 5.9 × 10 −4 µm 2 in the naïve control group. The density in the cell transplantation group (77.0 ± 9.4 × 10 −4 µm 2 ) was lower than that in the naïve control, but was improved by approximately 60% compared to the naïve control group.

| Neuromuscular junctions
Six months after cell transplantation, the average percentage of body wet weight of the soleus muscle was 0.092 ± 0.001, 0.021 ± 0.001 and 0.022 ± 0.001% in the naïve control, surgical control and in the cell transplantation groups, respectively.
There were no significant differences between surgical control and cell transplantation groups at 6 months after cell transplantation ( Figure 4A). The neuromuscular junctions were innervated with nerve fibres in the naive control group ( Figure 4B). In the cell transplantation group, nerve fibres reached into the muscle, but no neuromuscular junctions at the site of AChR clusterings were reinnervated ( Figure 4C).

| Muscle spindles
Two months after cell transplantation, all muscle spindles were innervated with nerve fibres in the naïve control group ( Figure 5A).
Almost half of muscle spindles were reinnervated with nerve fibres in the cell transplantation but not in the surgical control group ( Figure 5B,5C). The average number of muscle spindles was lower in the surgical control (10.2 ± 0.31) and cell transplantation groups (10.5 ± 0.22) than in the naïve control group (16.7 ± 0.96; Figure 5D).
Six months after cell transplantation, the average number of muscle spindles was 18.3 ± 1.52 in the naïve control group. There were no muscle spindles in the surgical control group. In contrast, in the cell transplantation group, some muscle spindles were reinnervated with nerve fibres, and their calbindin D-28k immunoreactivity in intrafusal muscle fibres was maintained for six months after denervation ( Figure 6A). The average number of muscle spindles was significantly higher in the cell transplantation (4.33 ± 0.62, P < 0.05) than in the surgical control group ( Figure 6C). Regenerated nerves showed reinnervation of muscle spindles in the skeletal muscle in the cell transplantation group. While most of the muscle spindles exhibited non-spiral and irregular morphology, some showed a spiral appearance ( Figure 6B).

| D ISCUSS I ON
The purpose of this study was to investigate whether transplanta- Muscle spindles are proprioceptive receptors in skeletal muscle that respond to the length and tension of the muscle. 10 It has been suggested that muscle spindles play an important role in the maintenance of postural stability and balance during locomotion 11,12 and that structural changes occur in them following peripheral nerve injury. 3 Almost all muscle spindles atrophy and finally disappear after denervation. 13 However, the number and morphologic deterioration of muscle spindles improve when the muscle is reinnervated. 7 Regeneration of muscle spindles is dependent upon survival of spindle capsules and intrafusal muscle fibres. 14 Muscle spindles are 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) innervated with γ-motorneurons and Ia and II sensory neurons. It is suggested that γ-motorneurons affect the maintenance of muscle spindle formation. However, motorneuron innervation is not essential for the formation and differentiation of muscle spindles in reinnervated muscles of neonatal rats. 15 In the present study, the mere transplantation of embryonic sensory neurons could maintain muscle spindle formation, as well as the preservation of spindle capsules and regeneration of intrafusal muscle fibres after denervation.
These results reveal that sensory nerve reinnervation is important to preserve the muscle spindles in regenerating mammalian skeletal muscle.
Regarding the morphological changes of muscle spindles, some authors have reported the influence of peripheral nerve injury, hindlimb unloading and age. 3,8,16,17 After nerve repair, sensory terminals of regenerated muscle spindles are tapered or have irregular forms, but the stretch reflex gradually recovers to normal levels. [18][19][20] Age causes remarkable changes in the structures, not only in the intrafusal muscle but also in sensory nerve endings in muscle spindles. 21 Sensory terminals of old rats appear somewhat irregular and less crowded. Primary endings of aged rat muscle spindles are less spiral or non-spiral in appearance. 22 Irregular innervation was also found in the muscle spindles of diabetic mice after Ia axonal degeneration and regeneration. 23 In the present study, we found that most muscle spindles in the rat are non-spiral and irregular in appearance after cell transplantation. This morphology may affect muscle spindle function, since sensory nerve terminals are important to mechanosensory function.
Calbindin D-28k is a vitamin D-dependent calcium-binding protein. 24 Calcium binding proteins buffer calcium, thus 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 preserving intracellular Ca 2+ homoeostasis. 25 Calbindin D-28k has been shown to have functions as Ca 2+ buffer, transporter and sensor. 26 (Schmidt H, 2012). Calbindin D-28k immunoreactivity has been demonstrated in central and peripheral neurons 25,27 and was also detected in the kidney and pancreas. 28 Furthermore, calbindin D-28k is found in sensory pathways such as cones and horizontal cells in the retina, cochlear and vestibular hair cells in the inner ear, intrafusal muscle fibres, the spindle capsule and the perineural sheath of nerves. [29][30][31] Calbindin D-28k is involved in adjustment of intracellular calcium levels and mediation of Ca 2+ dependent events, when intrafusal muscle fibres contract. In a rat denervation model, it was shown that calbindin D-28k immunoreactivity of intrafusal muscle fibres remains, although reduced, for 1 month. After two months of denervation, calbindin D-28k immunoreactivity is not seen in intrafusal muscle fibres, although it remains in the spindle capsule. 31 In the present study, we revealed that some muscle spindles were reinnervated with sensory nerve fibres and that intrafusal muscle fibres maintained calbindin D-28k immunoreactivity for six months in the sensory cell transplantation group. It is suggested that Ca 2+ homeostasis in the intrafusal muscle fibres is influenced by a trophic effect produced by the regenerated sensory nerves fibres. In Ia peripheral endings, synaptic vesicles contain a lot of glutamate and undergo recycling in a Ca 2+ -modulated manner to enhance terminal excitability during muscle stretch. 32 In our previous report, transplantation of neural stem cells into the peripheral nerve revealed a structure resembling spinal cord tissue, and all types of central nervous system cells, such as neurons, astrocytes and oligodendrocytes, were identified, by electron microscopy analysis. 33 In the present study, we could also find neurons surrounded by GFAP and O4 immunoreactive tissues in the transplantation site. While there is a possibility that support by cen-

CO N FLI C T O F I NTE R E S T
The authors have declared that there is no conflict of interest.

DATA AVA I L A B I L I T Y
The data that support the findings of this study are openly available in "figshare" at https ://doi.org/10.6084/m9.figsh are.78567 38.v1