**2. Transplantation of embryonic neurons into peripheral nerve forms functional motor units**

### **2.1. Background and purpose**

There has been a rapid surge in clinical trials involving stem cell therapies over the last three to four years [5]. Those trials are establishing the clinical pathways for regenerative medicine, especially in nervous system. Since derivation of human embryonic stem cells (hESCs) in 1998 [6], hESCs have been thought a promising source of replacement cells for regenerative medicine. Although Geron Corporation has been no longer enrolling patients forthe trial, they conducted the first clinical trial in the United States to evaluate the safety of oligodendrocyte precursor cells derived from hESCs in patients with thoracic spinal cord injuries [7]. The company Advanced Cell Technology has reported promising results from the clinical trials on Stargardtʹs Macular Dystrophy with retinal pigment epithelium derived from hESCs [8]. However, neuronal replacement in central nervous system is still limited due to a number of cells required to reconstruct complex structures and narrow therapeutic time window [9]. There are still several hurdles to be overcome in order to establish clinical application of neuronal replacement therapies in central nervous systems.

One experimental approach to rescue denervated muscles from damage to lower motor neurons or axonal disconnection is transplantation of motoneurons into a peripheral nerve as a source of neurons for muscle reinnervation. Since Erb reported the reinnervation of dener‐ vated muscle by embryonic motoneurons transplanted into peripheral nerve in 1993 [10], several studies have investigated the factors that improve motoneuron survival in peripheral nerve [11‐13]. Considering the simplicity of neural network and wide windows of opportunity forthe treatment [14], peripheral nerve system can be an ideal target for neuronalreplacement therapy. This transplantation strategy may provide the potential to excite these muscles artificially with electrical stimulation. The aim of this study was to evaluate whether trans‐ planted rat embryonic motorneurons into adult rat peripheral nerve would survive, and whether transplanted motorneurons would form functional motor units.

### **2.2. Methods**

commands to the muscles. The other ends of the implanted neurons are connected by special types of electrodes to communicate with electric devices of motor controllers. The diameter of mammalian motor neurons is from 0.5 μm to 20 μm. There is a reason why our research requires bio‐compatible micro‐nano technologies for achieving such interfaces of new

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

Neuroprosthetic devices with interfaces detecting electromyogram (EMG) are in practical use [3]. Such EMG interfaces require to place the electrodes at the end plate of a motor neuron; thus, the paralyzed or amputated users can not control the paralyzed or amputated muscles with the same passes of motor neurons as before paralyzed or amputated. Moreover, EMG interfaces pickup the signals at end plate of a motor neuron; sensory signals from receptors are not obtainable via EMG. This means that two‐way communication by EMG interfaces is impossible. We can assemble sensors and stimulators into a neuroprosthetic device. If we have a way to send the signals from the assembled stimulators to sensory nervous system, ideal neuroprosthetic devices with two‐way communication will be achievable. The combination of EMG pickup and electrical stimulation with surface electrodes was proposed for two‐way communication [4]. However, there is no clear result on the amount of transmissive informa‐ tion through the afferent passes. In Section 3, we show a preliminary study on the possibility of sensory feedback via axial fibers of peripheral sensory neurons. In other words, we try to achieve an artificial afferent pass forfeeding signals back to the brain. A method forimproving on the amount of transmissive information has been proposed by using a configuration with

One application of PNIs is to achieve unconscious muscular movements such as walking, writing, dancing, playing musical instruments, and so on. For an example, in walking motions some neural networks generate the pattern of walking, and the steady behavioris closed within the peripheral nervous system and the pattern generators in spinal cord. The upper central nervous system provides the triggers of walking such as start/stop, speedup/slowdown, turn‐ right/turn‐left. The understanding on pattern generators for walking is now enough to simulate human walking or some animalsʹ walking. In Section 4, we give a walking simulation

**2. Transplantation of embryonic neurons into peripheral nerve forms**

There has been a rapid surge in clinical trials involving stem cell therapies over the last three to four years [5]. Those trials are establishing the clinical pathways for regenerative medicine, especially in nervous system. Since derivation of human embryonic stem cells (hESCs) in 1998 [6], hESCs have been thought a promising source of replacement cells for regenerative medicine. Although Geron Corporation has been no longer enrolling patients forthe trial, they conducted the first clinical trial in the United States to evaluate the safety of oligodendrocyte

reinnervation type electrodes.

Biomedical Engineering

14

multi‐channel electrodes.

**functional motor units**

**2.1. Background and purpose**

of ʺratʺ to show the possibility of practical usages of PNIs.

The sciatic nerves of adult Fischer 344 rats were transected; 1 week later, dissociated embryonic spinal neurons were transplanted into the distal stump of the tibial and peroneal nerves. Surgical controls underwent the same surgeries but had only medium injected into the peripheral nerves. Tissue analysis and measurement of ankle angles were performed twelve weeks after neural transplantation.

### *2.2.1. Cell preparation*

Ventral spinal cord cell were obtained from Fischer 344 rat embryos (Japan SLC,Inc., Shizuoka, Japan). After Fischer rats on day 14 of pregnancy were anesthetized with isoflurane, ventral spinal cords were resected from the fetuses using a surgical microscope and were cut into small pieces in ice‐cold Hanksʹ balanced salt solution. Ventral spinal neurons were dissociated using papain‐containing separation solution (MB‐X9901; Sumitomo Bakelite Co. Ltd, Tokyo, Japan). For implantation, dissociated neurons were suspended in Neurobasal medium (Gibco) containing B27 supplement (Gibco), Glutamax (Gibco), and N‐2 supplement (Gibco).

#### *2.2.2. Surgical procedures and transplantation*

All procedures were performed on 8‐week‐old male Fischer 344 rats (Japan SLC, Inc.) under isoflurane anesthesia.The sciatic nerves of Fischerrats were completely transectedat midthigh. The nerves were ligated on both ends and the proximal nerve stump was sutured into hip muscles to prevent reinnervation. Approximately 1 x 106 neurons contained within 10 μl of medium were injected into the distal stumps of the tibial and peroneal nerves after 1 week of

nerve transection using a Hamilton syringe. The injection site was 20 mm proximal to those entries into the lateral gastrocnemius and anterior tibialis muscles. Surgical controls under‐ went the same surgeries but had only medium injected into the peripheral nerves.

**2.3. Result**

*2.3.2. Axon regeneration*

*2.3.3. Analysis of muscle anatomy*

*2.3.4. Reproduction of ankle motions*

*2.3.1. Motoneuron survival and neuromuscular junction formations*

mius muscle of the rats that received cell transplants (Figure 1).

Transplanted motoneuron survival was observed up to 12 weeks in all of the cell‐transplanted tibial nerves. Neuromuscular junction formations were also present in the lateral gastrocne‐

Neural Interfaces: Bilateral Communication Between Peripheral Nerves and Electrical Control Devices 17

No myelinated axons were present in the surgical control group. The mean number of myelinated axons in the tibial nerves of animals that received cell transplantion was less than

No significant differences were found in muscle fiber cross‐sectional area between transplan‐ tation and surgical control groups (326μm2 ± 91 versus 176μm2 ± 105, p=0.28). The naive group

The reinnervatedmuscleoftransplantationgroupcouldreproduce ankledorsiflexionartificial‐ ly through electrical stimulation. Video‐based analysis revealed that the ankle angles in the neural transplantation group significantly decreased with electrical stimulation of the pero‐ neal nerve as compared to surgical control (30.4 ± 7.7 versus 106.7 ± 9.7, p < 0.001) (Figure 3).

**Figure 1.** Twelve weeks after transplantation, motoneurons survived in the tibial nerve. The ChAT (choline acetyltrans‐

ferase) positive axons and motoneurons (arrow) were observed in the tibial nerve.

, p<0.001)

half of uninjured animals (1198 ± 1218 versus 2510 ± 802, p=0.079) (Figure 2).

showed the largest gastrocnemius muscle fiber area (2873 ± 233μm2

#### *2.2.3. Reproduction of ankle motions*

The stainless steel wire electrodes were placed on the peroneal and tibial nerves of the neural transplantation group and those were covered with silicone gel for insulation and immobili‐ zation. The other ends of wires were passed through the dorsal neck skin and connected to the generator(Neuropack MEB‐5504; Nihon Kohden, Tokyo, Japan). Using video technique, ankle angles of the neural transplantation group were measured with electrical stimulation of the peroneal nerves at 60 Hz, 0.4 mA. The ankle angle is measured from the line connecting the knee and ankle joints, and the line connecting the ankle joint and the metatarsal head [15].

#### *2.2.4. Tissue analysis*

After the measurement of ankle angles, the rats were perfused through the left ventricle with 4% paraformaldehyde. The tibial nerves, gastrocnemius and anterior tibialis muscles were removed for immunohistochemical and histochemical analysis. The tibial nerve distal to the transplant was then fixed in 4% glutaraldehyde in phosphate buffer. The nerves were embed‐ ded in Epon and 1‐μm thick sections were stained with Toluidine blue (Sigma‐Aldrich, Germany) for light microscopic examination. The total number of myelinated axons was measured. The gastrocnemius muscles were dissected free from the origin and insertion and weighed immediately. Then the gastrocnemius muscles were embedded in paraffin, sectioned into 10‐μm cross sections and stained with hematoxylin and eosin. The mean muscle fiber area was measured at the middle of the lateral gastrocnemius muscle belly.

Neuron survival was examined in the cell transplantation group. The transplant sites of tibial nerves were cryoprotected in sucrose, and then frozen in dry‐ice‐cooled isopentane. The tibial nerve sections of 10‐μm thichness were stained with the antibodies against choline acetyl‐ transferase (ChAT, 1:50; Millipore, Billerica, MA, USA) in order to assess the survival of motoneurons and those axons in the tibial nerve. The anterior tibialis muscle were sectioned at 50μm to assess neuromuscular junctions using anti‐neurofilament H antibody (1:500; Millipore, Billerica, MA) and α‐bungarotoxin (1:200, Molecular Probes, Eugene, OR) which reveals terminal muscle innervation by staining motor end plates. Sections were stained using a full automatic Immunohistochemical staining system: Ventana HX system (Ventana, Yokohama, Japan) according to the manufacturerʹs instruction.

### *2.2.5. Statistics*

For statistical analysis, Student t test or ANOVA with Tukey post hoc comparisons was used, as appropriate. All statistical analyses were conducted using the Statistical Package for Social Science version 19.0 software (SPSS Inc, Chicago, IL, USA). The significance level was set at 0.05. All data are expressed as mean ± standard deviation.

### **2.3. Result**

nerve transection using a Hamilton syringe. The injection site was 20 mm proximal to those entries into the lateral gastrocnemius and anterior tibialis muscles. Surgical controls under‐

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

The stainless steel wire electrodes were placed on the peroneal and tibial nerves of the neural transplantation group and those were covered with silicone gel for insulation and immobili‐ zation. The other ends of wires were passed through the dorsal neck skin and connected to the generator(Neuropack MEB‐5504; Nihon Kohden, Tokyo, Japan). Using video technique, ankle angles of the neural transplantation group were measured with electrical stimulation of the peroneal nerves at 60 Hz, 0.4 mA. The ankle angle is measured from the line connecting the knee and ankle joints, and the line connecting the ankle joint and the metatarsal head [15].

After the measurement of ankle angles, the rats were perfused through the left ventricle with 4% paraformaldehyde. The tibial nerves, gastrocnemius and anterior tibialis muscles were removed for immunohistochemical and histochemical analysis. The tibial nerve distal to the transplant was then fixed in 4% glutaraldehyde in phosphate buffer. The nerves were embed‐ ded in Epon and 1‐μm thick sections were stained with Toluidine blue (Sigma‐Aldrich, Germany) for light microscopic examination. The total number of myelinated axons was measured. The gastrocnemius muscles were dissected free from the origin and insertion and weighed immediately. Then the gastrocnemius muscles were embedded in paraffin, sectioned into 10‐μm cross sections and stained with hematoxylin and eosin. The mean muscle fiber area

Neuron survival was examined in the cell transplantation group. The transplant sites of tibial nerves were cryoprotected in sucrose, and then frozen in dry‐ice‐cooled isopentane. The tibial nerve sections of 10‐μm thichness were stained with the antibodies against choline acetyl‐ transferase (ChAT, 1:50; Millipore, Billerica, MA, USA) in order to assess the survival of motoneurons and those axons in the tibial nerve. The anterior tibialis muscle were sectioned at 50μm to assess neuromuscular junctions using anti‐neurofilament H antibody (1:500; Millipore, Billerica, MA) and α‐bungarotoxin (1:200, Molecular Probes, Eugene, OR) which reveals terminal muscle innervation by staining motor end plates. Sections were stained using a full automatic Immunohistochemical staining system: Ventana HX system (Ventana,

For statistical analysis, Student t test or ANOVA with Tukey post hoc comparisons was used, as appropriate. All statistical analyses were conducted using the Statistical Package for Social Science version 19.0 software (SPSS Inc, Chicago, IL, USA). The significance level was set at

was measured at the middle of the lateral gastrocnemius muscle belly.

Yokohama, Japan) according to the manufacturerʹs instruction.

0.05. All data are expressed as mean ± standard deviation.

went the same surgeries but had only medium injected into the peripheral nerves.

*2.2.3. Reproduction of ankle motions*

*2.2.4. Tissue analysis*

Biomedical Engineering

16

*2.2.5. Statistics*

#### *2.3.1. Motoneuron survival and neuromuscular junction formations*

Transplanted motoneuron survival was observed up to 12 weeks in all of the cell‐transplanted tibial nerves. Neuromuscular junction formations were also present in the lateral gastrocne‐ mius muscle of the rats that received cell transplants (Figure 1).

#### *2.3.2. Axon regeneration*

No myelinated axons were present in the surgical control group. The mean number of myelinated axons in the tibial nerves of animals that received cell transplantion was less than half of uninjured animals (1198 ± 1218 versus 2510 ± 802, p=0.079) (Figure 2).

#### *2.3.3. Analysis of muscle anatomy*

No significant differences were found in muscle fiber cross‐sectional area between transplan‐ tation and surgical control groups (326μm2 ± 91 versus 176μm2 ± 105, p=0.28). The naive group showed the largest gastrocnemius muscle fiber area (2873 ± 233μm2 , p<0.001)

#### *2.3.4. Reproduction of ankle motions*

The reinnervatedmuscleoftransplantationgroupcouldreproduce ankledorsiflexionartificial‐ ly through electrical stimulation. Video‐based analysis revealed that the ankle angles in the neural transplantation group significantly decreased with electrical stimulation of the pero‐ neal nerve as compared to surgical control (30.4 ± 7.7 versus 106.7 ± 9.7, p < 0.001) (Figure 3).

**Figure 1.** Twelve weeks after transplantation, motoneurons survived in the tibial nerve. The ChAT (choline acetyltrans‐ ferase) positive axons and motoneurons (arrow) were observed in the tibial nerve.

> motoneuron with glial cells in the central nerve systems, such as astrocytes, may promote the survival of motoneurons. Further studies are needed to clarify the necessity of central nervous system derived cells to improve survivalrate of transplanted neurons in peripheral nerve.

Neural Interfaces: Bilateral Communication Between Peripheral Nerves and Electrical Control Devices 19

No significant differences were found in muscle fiber area between transplantation and surgical control groups. And the muscle fiber area was much smaller for the cell transplanta‐ tion group than for the naive group. Before neural connection between motoneurons and muscle was established, other treatment strategy had been needed to prevent muscle atrophy. After the neural connection, continuation of electrical stimulation and exercise beyond the

In clinical setting, several investigators have stated that performing reconstruction of dener‐ vated muscle with nerve transfer technique in peripheral nerve within 9 months notably improves functional outcome [16, 17]. Motoneurons can be placed very closely to neuromus‐ cular junction using the neural transplantation into peripheral nerve. Target muscles can be reinnervated in short‐term. Considering these facts, useful functional recovery of denervated muscle can be expected with this method even if the treatment was delayed until more than 6 months after Wallerian degeneration. Considering that generating neural cells from induced pluripotent stem cells (iPSCs) takes a few months, the wide windows of opportunity for the treatment can be advantage in propagating safe motoneurons derived from iPSCs made from

Another advantage of transplantation into peripheral nerve is that much less number of motoneurons will be required, compared with neural transplantation into the central nervous system. The iPSCs are likely to carry a higher risk of tumorigenicity than ES cells, due to the inappropriate reprogramming of these somatic cells, the activation of exogenous transcription factors, or other reasons [18, 19]. A larger number of transplanting cells are needed, the higher risk of tumorigenicity remains an obstacle for neural transplantation therapy. Presumably much less number of motoneurons are supposed to be required for neural transplantation into peripheral nerve, because the number of motor unit is usually about several hundred [20]. Considering these issues, transplantation of motoneurons into peripheral nerve can be an ideal

Transplantation of rat embryonic motoneurons into peripheral nerve provides reproduction of simple behaviors in conjunction with electrical stimulation. Recent breakthroughs in stem cell biology have raised possibilities of the clinical application of this treatment strategy. Our results lead us to believe that transplantation of iPS‐ or ES‐derived motoneurons into periph‐

The purpose of this section is to show the possibility of sensory feedback in nueroprosthetic devices. If we have a way to send the signals from the assembled sensors to sensory nervous

eral nerve changes treatment strategy for the diminished voluntary muscle function.

period of this study may improve muscle fiber area and the quality of gait.

the injured patientʹs own cells.

**3.1. Purpose**

target for regenerative therapy from a clinical viewpoint.

**3. Sensory feedback by electrical stimulation**

**Figure 2.** Myelinated axons in tibial nerve were assessed with toluidine blue stain.

**Figure 3.** Controlled reproduction of ankle motion and measurements of dorsiflexed angles. The ankle motions were restored with electrical stimulation of the peroneal nerve in neural transplantation group (A) as compared to surgical control group (B).

#### **2.4. Discussion**

The transplanted rat embryonic motoneurons could survive in the rat peripheral nerve, and transplanted motoneurons formed functional motor units. Transplantation of motoneurons into peripheral nerve provided the reproduction of ankle dorsiflexion. The transplantation of embryonic neurons into peripheral nerve could restore ankle motions in conjunction with electrical stimulation, even though no neural connection between central nervous system and muscle were present.

Althoughallratswere able toflex their ankle,the smallnumberof axonswaspresentinthe tibial nerves of cell transplantation group as compared with naive group. Endoneurial environment in peripheral nerve may not provide suitable condition for motoneurons. Transplanting motoneuron with glial cells in the central nerve systems, such as astrocytes, may promote the survival of motoneurons. Further studies are needed to clarify the necessity of central nervous system derived cells to improve survivalrate of transplanted neurons in peripheral nerve.

No significant differences were found in muscle fiber area between transplantation and surgical control groups. And the muscle fiber area was much smaller for the cell transplanta‐ tion group than for the naive group. Before neural connection between motoneurons and muscle was established, other treatment strategy had been needed to prevent muscle atrophy. After the neural connection, continuation of electrical stimulation and exercise beyond the period of this study may improve muscle fiber area and the quality of gait.

In clinical setting, several investigators have stated that performing reconstruction of dener‐ vated muscle with nerve transfer technique in peripheral nerve within 9 months notably improves functional outcome [16, 17]. Motoneurons can be placed very closely to neuromus‐ cular junction using the neural transplantation into peripheral nerve. Target muscles can be reinnervated in short‐term. Considering these facts, useful functional recovery of denervated muscle can be expected with this method even if the treatment was delayed until more than 6 months after Wallerian degeneration. Considering that generating neural cells from induced pluripotent stem cells (iPSCs) takes a few months, the wide windows of opportunity for the treatment can be advantage in propagating safe motoneurons derived from iPSCs made from the injured patientʹs own cells.

Another advantage of transplantation into peripheral nerve is that much less number of motoneurons will be required, compared with neural transplantation into the central nervous system. The iPSCs are likely to carry a higher risk of tumorigenicity than ES cells, due to the inappropriate reprogramming of these somatic cells, the activation of exogenous transcription factors, or other reasons [18, 19]. A larger number of transplanting cells are needed, the higher risk of tumorigenicity remains an obstacle for neural transplantation therapy. Presumably much less number of motoneurons are supposed to be required for neural transplantation into peripheral nerve, because the number of motor unit is usually about several hundred [20]. Considering these issues, transplantation of motoneurons into peripheral nerve can be an ideal target for regenerative therapy from a clinical viewpoint.

Transplantation of rat embryonic motoneurons into peripheral nerve provides reproduction of simple behaviors in conjunction with electrical stimulation. Recent breakthroughs in stem cell biology have raised possibilities of the clinical application of this treatment strategy. Our results lead us to believe that transplantation of iPS‐ or ES‐derived motoneurons into periph‐ eral nerve changes treatment strategy for the diminished voluntary muscle function.
