Body proportions as possible predictors for free gracilis one-stage facial reanimation.

BACKGROUND: Gracilis muscle and its motor nerve belongs to most commonly used flap for facial reanimation. However, it is performed in two steps, which is time consuming. One stage technique can be also performed, but the length of the motor nerve cannot be currently determined before surgery. AIM: The present study was conducted in order to evaluate the body composition on the length and suitability of the motor nerve of gracilis muscle for one stage facial reanimation. METHODS: The gracilis flaps along with the motoric nerve were dissected from 20 fresh cadavers (6 females, 14 males). The length of the lower extremity from superior iliac anterior spine to the bottom of the heel and BMI were measured. Regression analysis of lower extremity length and BMI to the actual length of the motor nerve of gracilis flap was performed. RESULTS: The linear regression analysis showed a positive correlation between the length of the lower limb and the size of the motor nerve length (r = 0.5060, p < 0.05), as well as between the BMI and the size of the motor nerve length (r = 0.5073, p < 0.05). Also, the males had longer motor nerve when compared to females by 13 % (p < 0.05). No difference between females and males in BMI was observed. CONCLUSION: The length from the superior iliac anterior spine, BMI and gender seemed to be potential factors that could help to predict the length of the gracilis flap motor nerve for the one stage facial reanimation. However, further studies evaluating other anatomical factors and validating the possible prediction rule for one stage reanimation success are needed (Fig. 3, Ref. 14).

Clinical outcome following transfer of the supinator motor branch to the posterior interosseous nerve in patients with C7-T1 brachial plexus palsy.

BACKGROUND: In cases of C7-T1 brachial plexus palsy, a reliable method for the reconstruction of the finger and thumb extension was not established until the transfer of the supinator motor branch to the posterior interosseous nerve was proposed. The long-term outcome of this new technique requires evaluation due to the limited number of cases and the shorter follow-up period of the previous study. OBJECTIVE: This study aims to evaluate the long-term effect of the transfer of the supinator motor branch to the posterior interosseous nerve and to determine the recovery time course for this new technique. METHODS: A retrospective review was conducted in 10 patients with lower brachial plexus injuries who underwent transfer of the supinator motor branch. Patients were followed up postoperatively for a minimum of 24 months, with all patients scheduled to receive a physical examination and electrophysiological testing every 3 months for the first 2 years. RESULTS: Nine patients (90%) recovered to the Medical Research Council (MRC) grade 3 or better for the extensor digitorum communis. The electrophysiologically documented recovery began at an average of 5.7 months after surgery, with the average time required for the first finger extension being 9.1 months (range 5-18 months), and the average time required for achieving MRC grade 3 being 14.3 months (range 9-24 months). Moreover, no complications or loss of supination was observed in any patient. CONCLUSION: The supinator motor branch transfer is a safe procedure that yields recovery of finger extension in C7-T1 brachial plexus palsies with encouraging long-term outcomes.

Electrical stimulation of transplanted motoneurons improves motor unit formation.

Motoneurons die following spinal cord trauma and with neurological disease. Intact axons reinnervate nearby muscle fibers to compensate for the death of motoneurons, but when an entire motoneuron pool dies, there is complete denervation. To reduce denervation atrophy, we have reinnervated muscles in Fisher rats from local transplants of embryonic motoneurons in peripheral nerve. Since growth of axons from embryonic neurons is activity dependent, our aim was to test whether brief electrical stimulation of the neurons immediately after transplantation altered motor unit numbers and muscle properties 10 wk later. All surgical procedures and recordings were done in anesthetized animals. The muscle consequences of motoneuron death were mimicked by unilateral sciatic nerve section. One week later, 200,000 embryonic day 14 and 15 ventral spinal cord cells, purified for motoneurons, were injected into the tibial nerve 10-15 mm from the gastrocnemii muscles as the only neuron source for muscle reinnervation. The cells were stimulated immediately after transplantation for up to 1 h using protocols designed to examine differential effects due to pulse number, stimulation frequency, pattern, and duration. Electrical stimulation that included short rests and lasted for 1 h resulted in higher motor unit counts. Muscles with higher motor unit counts had more reinnervated fibers and were stronger. Denervated muscles had to be stimulated directly to evoke contractions. These results show that brief electrical stimulation of embryonic neurons, in vivo, has long-term effects on motor unit formation and muscle force. This muscle reinnervation provides the opportunity to use patterned electrical stimulation to produce functional movements.

Inhibition of Sirt1 promotes neural progenitors toward motoneuron differentiation from human embryonic stem cells.

Several protocols direct human embryonic stem cells (hESCs) toward differentiation into functional motoneurons, but the efficiency of motoneuron generation varies based on the human ESC line used. We aimed to develop a novel protocol to increase the formation of motoneurons from human ESCs. In this study, we tested a nuclear histone deacetylase protein, Sirt1, to promote neural precursor cell (NPC) development during differentiation of human ESCs into motoneurons. A specific inhibitor of Sirt1, nicotinamide, dramatically increased motoneuron formation. We found that about 60% of the cells from the total NPCs expressed HB9 and ßIII-tubulin, commonly used motoneuronal markers found in neurons derived from ESCs following nicotinamide treatment. Motoneurons derived from ESC expressed choline acetyltransferase (ChAT), a positive marker of mature motoneuron. Moreover, we also examined the transcript levels of Mash1, Ngn2, and HB9 mRNA in the differentiated NPCs treated with the Sirt1 activator resveratrol (50 µM) or inhibitor nicotinamide (100 µM). The levels of Mash1, Ngn2, and HB9 mRNA were significantly increased after nicotinamide treatment compared with control groups, which used the traditional protocol. These results suggested that increasing Mash1 and Ngn2 levels by inhibiting Sirt1 could elevate HB9 expression, which promotes motoneuron differentiation. This study provides an alternative method for the production of transplantable motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease.

Transplanted oligodendrocytes and motoneuron progenitors generated from human embryonic stem cells promote locomotor recovery after spinal cord transection.

Human embryonic stem cells (hESC) hold great promise for the treatment of patients with many neurodegenerative diseases particularly those arising from cell loss or neural dysfunction including spinal cord injury. This study evaluates the therapeutic effects of transplanted hESC-derived oligodendrocyte progenitors (OPC) and/or motoneuron progenitors (MP) on axonal remyelination and functional recovery of adult rats after complete spinal cord transection. OPC and/or MP were grafted into the site of injury in the acute phase. Based on Basso-Beattie-Bresnahan scores recovery of locomotor function was significantly enhanced in rats treated with OPC and/or MP when compared with control animals. When transplanted into the spinal cord immediately after complete transection, OPC and MP survived, migrated, and differentiated into mature oligodendrocytes and neurons showing in vivo electrophysiological activity. Taken together, these results indicate that OPC and MP derived from hESC could be a useful therapeutic strategy to repair injured spinal cord.

Human motor neuron generation from embryonic stem cells and induced pluripotent stem cells.

Motor neuron diseases (MNDs) are a group of neurological disorders that selectively affect motor neurons. There are currently no cures or efficacious treatments for these diseases. In recent years, significant developments in stem cell research have been applied to MNDs, particularly regarding neuroprotection and cell replacement. However, a consistent source of motor neurons for cell replacement is required. Human embryonic stem cells (hESCs) could provide an inexhaustible supply of differentiated cell types, including motor neurons that could be used for MND therapies. Recently, it has been demonstrated that induced pluripotent stem (iPS) cells may serve as an alternative source of motor neurons, since they share ES characteristics, self-renewal, and the potential to differentiate into any somatic cell type. In this review, we discuss several reproducible methods by which hESCs or iPS cells are efficiently isolated and differentiated into functional motor neurons, and possible clinical applications.

BDNF-overexpressing human umbilical cord mesenchymal stem cell-derived motor neurons improve motor function and prolong survival in amyotrophic lateral sclerosis mice.

OBJECTIVE: To investigate the beneficial effect of brain-derived neurotrophic factor (BDNF) -overexpressing human umbilical cord mesenchymal stem cell (hUC-MSC)-derived motor neurons in the human Cu, Zn-superoxide dismutase1 (hSOD1)G93A amyotrophic lateral sclerosis (ALS) mice. METHODS: The BDNF gene was transfected into hUC-MSC-derived motor neurons by the lentivirus-mediated method. hSOD1G93A mice were assigned to the ALS, ALS/MN, and ALS/MN-BDNF groups, and intrathecally administrated phosphate-buffered saline (PBS), motor neurons, or motor neurons overexpressing BDNF, respectively. The control group included non-transgenic wild-type littermates administrated PBS. One month after transplantation, the motor function of the mice was assessed by the rotarod test, and the lumbar enlargements were then isolated to detect the expression of hSOD1 and BDNF by western blotting, and the expression of choline acetyltransferase (ChAT), homeobox protein 9 (HB9), major histocompatibility complex I (MHCI) and microtubule-associated protein-2 (MAP-2) by immunofluorescence assay. RESULTS: After transplantation, mice in the ALS/MN-BDNF and ALS/MN groups both exhibited longer latency to fall and longer survival than those in the ALS group (P < 0.01 vs. P < 0.05), and the improvement was more significant in the former than in the latter. However, cell transplantation did not delay disease onset. In the lumbar enlargements of the ALS/MN-BDNF and ALS/MN groups, the expression of hSOD1 was slightly reduced without statistical significance (P > 0.05), but the expression of BDNF, ChAT and HB9, and the co-expression of MHCI and MAP-2 were significantly greater than in the ALS group (P < 0.01), with the differences also being more prominent in the former group than in the latter. CONCLUSIONS: Transplantation of BDNF-overexpressing hUC-MSC-derived motor neurons can improve motor performance and prolong the survival of hSOD1G93A mice. Combining stem cell-derived motor neurons with BDNF might provide a new therapeutic strategy for ALS.

Optimal conditions for graft survival and reinnervation of denervated muscles after embryonic motoneuron transplantation into peripheral nerves undergoing Wallerian degeneration.

Motoneuron transplantation into peripheral nerves undergoing Wallerian degeneration may have applications in treating diseases causing muscle paralysis. We investigated whether functional reinnervation of denervated muscle could be achieved by early or delayed transplantation after denervation. Adult rats were assigned to six groups with increasing denervation periods (0, 1, 4, 8, 12, and 24 weeks) before inoculation with culture medium containing (transplantation group) or lacking (surgical control group) dissociated embryonic motoneurons into the peroneal nerve. Electrophysiological and tissue analyses were performed 3 months after transplantation. Reinnervation of denervated muscles significantly increased relative muscle weight in the transplantation group compared with the surgical control group for denervation periods of 1 week (0.042% ± 0.0031% vs. 0.032% ± 0.0020%, respectively; p = 0.009), 4 weeks (0.044% ± 0.0069% vs. 0.026% ± 0.0045%, respectively; p = 0.0023), and 8 weeks (0.044% ± 0.0029% vs. 0.026% ± 0.0008%, respectively; p = 0.0023). The ratios of reinnervated muscle contractile forces to naïve muscle in the 0, 1, 4, 8, and 12 weeks transplantation groups were 3.79%, 18.99%, 8.05%, 6.30%, and 5.80%, respectively, indicating that these forces were sufficient for walking. The optimal implantation time for transplantation of motoneurons into the peripheral nerve was 1 week after nerve transection. However, the neurons transplanted 24 weeks after denervation survived and regenerated axons. These results indicated that there is time for preparing cells for transplantation in regenerative medicine and suggested that our method may be useful for paralysed muscles that are not expected to recover with current treatment.

Motoneuron transplantation rescues the phenotype of SMARD1 (spinal muscular atrophy with respiratory distress type 1).

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a fatal form of infantile motoneuron disease. There is currently no effective treatment, although motor neuron replacement is a possible therapeutic strategy. We transplanted purified motor neurons into the spinal cord of nmd mice, an animal model of SMARD1. We also administered pharmacological treatment targeting the induction of axonal growth toward skeletal muscle target. At the end stage of the disease, donor-derived motor neurons were detected in the nmd anterior horns, extended axons into the ventral roots, and formed new neuromuscular junctions. These data correlated with improved neuromuscular function and increased life spans. The neuroprotective effect was associated with a reduction in proinflammatory molecules in treated spinal cords. This is the first report that functional restoration of motor units with transplanted motoneurons is feasible in an animal model of a human motoneuron disease, opening up new possibilities for therapeutic intervention.

Neurotrophic factors improve muscle reinnervation from embryonic neurons.

Motoneurons die in diseases like amyotrophic lateral sclerosis and after spinal cord trauma, inducing muscle denervation. We tested whether transplantation of embryonic cells with neurotrophic factors into peripheral nerve of adult rats improves muscle reinnervation and motor unit function more than cells alone. One week after sciatic nerve section, embryonic ventral spinal cord cells were transplanted into the tibial nerve with or without glial cell line-derived neurotrophic factor, hepatocyte growth factor, and insulin-like growth factor-1. These cells represented the only neuron source for muscle reinnervation. Ten weeks after transplantation, all medial gastrocnemius muscles contracted in response to electrical stimulation of cell transplants with factors. Only 80% of muscles responded with cells alone. Factors and cells resulted in survival of more motoneurons and reinnervation of more muscle fibers for a given axon (motor unit) number. Greater reinnervation from embryonic cells may enhance muscle excitation by patterned electrical stimulation.

Combined transplantation of neural stem cells and olfactory ensheathing cells improves the motor function of rats with intracerebral hemorrhage.

OBJECTIVE: To investigate the effects of combined transplantation of neural stem cells (NSC) and olfactory ensheathing cells (OEC) on the motor function of rats with intracerebral hemorrhage. METHODS: In three days after a rat model of caudate nucleus hemorrhage was established, NSCs and OEC, NSC, OEC (from embryos of Wistar rats) or normal saline were injected into hematomas of rats in combined transplantation group, NSC group, OEC group, and control group, respectively. Damage of neural function was scored before and in 3, 7, 14, 30 days after operation. Tissue after transplantation was observed by immunocytochemistry staining. RESULTS: The scores for the NSC, OEC and co-transplantation groups were significantly lower in 14 and 30 days after operation than in 3 days after operation (P < 0.05). The scores for the NSC and OEC groups were significantly lower than those for the control group only in 30 days after operation (P < 0.05), while the difference for the NSC-OEC group was significant in 14 days after operation (P < 0.05). Immunocytochemistry staining revealed that the transplanted OEC and NSC could survive, migrate and differentiate into neurons, astrocytes, and oligodendrocytes. The number of neural precursor cells was greater in the NSC and combined transplantation groups than in the control group. The number of neurons differentiated from NSC was significantly greater in the co-transplantation group than in the NSC group. CONCLUSION: Co-transplantation of NSC and OEC can promote the repair of injured tissue and improve the motor function of rats with intracerebral hemorrhage.

Transfer of supinator motor branches to the posterior interosseous nerve to reconstruct thumb and finger extension in tetraplegia: case report.

We treated a patient with tetraplegia who had paralysis of thumb and finger extension by transferring supinator motor branches to the posterior interosseous nerve. Surgery was performed bilaterally, 7 months after a spinal cord injury. Six months after surgery, with the wrist in neutral, extension of the thumb and finger were almost full, bilaterally. In tetraplegic patients with strong wrist extensors, supinator motor branch transfer is a promising new alternative for the reconstruction of thumb and finger extension.

Sequentially induced motor neurons from human fibroblasts facilitate locomotor recovery in a rodent spinal cord injury model.

Generation of autologous human motor neurons holds great promise for cell replacement therapy to treat spinal cord injury (SCI). Direct conversion allows generation of target cells from somatic cells, however, current protocols are not practicable for therapeutic purposes since converted cells are post-mitotic that are not scalable. Therefore, therapeutic effects of directly converted neurons have not been elucidated yet. Here, we show that human fibroblasts can be converted into induced motor neurons (iMNs) by sequentially inducing POU5F1(OCT4) and LHX3. Our strategy enables scalable production of pure iMNs because of the transient acquisition of proliferative iMN-intermediate cell stage which is distinct from neural progenitors. iMNs exhibited hallmarks of spinal motor neurons including transcriptional profiles, electrophysiological property, synaptic activity, and neuromuscular junction formation. Remarkably, transplantation of iMNs showed therapeutic effects, promoting locomotor functional recovery in rodent SCI model. Together, our advanced strategy will provide tools to acquire sufficient human iMNs that may represent a promising cell source for personalized cell therapy.

Hyperinnervation of skeletal muscle fibers: dependence on muscle activity.

After the motor nerve to the rat soleus muscle was blocked reversibly by local anesthesia, individual muscle fibers became innervated by a transplanted motor nerve without losing their original innervation. Such cross-innervation of the denervated soleus muscle by the same foreign nerve was largely reduced by direct electrical stimulation of the muscle. The results demonstrate the importance of muscle activity for synapse formation by a foreign motor nerve.

Determination of primary motoneuron identity in developing zebrafish embryos.

The developmental determination of primary motoneurons was investigated by transplanting identified motoneurons in embryonic zebrafish to new spinal cord positions. Some cells moved from the new positions in which they were placed back to their original positions, thus it was difficult to evaluate whether they were determined. Among cells that remained in their new positions, those transplanted about 1 hour before axogenesis developed axonal trajectories that were appropriate for their original soma positions, whereas those transplanted 2 to 3 hours before axogenesis developed morphologies appropriate for their new soma positions. These results suggest that motoneuronal identity is determined before axogenesis.

Xenotransplantation of embryonic stem cell-derived motor neurons into the developing chick spinal cord.

A growing number of specific cell types have been successfully derived from embryonic stem cells (ES cells), including a variety of neural cells. In vitro generated cells need to be extensively characterized to establish functional equivalency with their in vivo counterparts. The ultimate test for the ability of ES cell-derived neurons to functionally integrate into neural networks is transplantation into the developing central nervous system, a challenging technique limited by the poor accessibility of mammalian embryos. Here we describe xenotransplantation of mouse embryonic stem cell-derived motor neurons into the developing chick neural tube as an alternative for testing the ability of in vitro generated neurons to survive, integrate, extend axons, and form appropriate synaptic contacts with functionally relevant targets in vivo. Similar methods can be adapted to study functionality of other mammalian cells, including derivatives of human ES cells.

Repair of spinal cord injury by co-transplantation of embryonic stem cell-derived motor neuron and olfactory ensheathing cell.

BACKGROUND: The failure of regeneration after spinal cord injury (SCI) has been attributed to axonal demyelination and neuronal death. Cellular replacement and white matter regeneration are both necessary for SCI repair. In this study, we evaluated the co-transplantation of olfactory ensheathing cells (OEC) and embryonic stem (ES) cell-derived motor neurons (ESMN) on contused SCI. METHODS: OEC cultured from olfactory nerve rootlets and olfactory bulbs. ESMN was generated by exposing mouse ES cells to retinoic acid and sonic hedgehog. Thirty female rats were used to prepare SCI models in five groups. Control and medium-injected groups was subjected to induce lesion without cell transplantation. OEC or ESMN or both were transplanted into the site of the lesion in other groups. RESULTS: The purity of OEC culture was 95%. Motor neuron progenitor markers (Olig2, Nkx6.1 and Pax6) and motor neuron markers (Isl1, Isl2 and Hb9) were expressed. Histological analysis showed that significantly more (P<0.001) spinal tissue was spared in OEC, ESMN and OEC+ ESMN groups but the OEC+ ESMN group had a significantly greater percentage of spared tissue and myelination than other groups (P< 0.05). The numbers of ESMN in co-transplanted group were significantly higher than ESMN group (P<0.05). A significant (P<0.05) recovery of hindlimb function was observed in rats in the transplanted groups. CONCLUSION: We found that the co-transplantation of ESMN and OEC into an injured spinal cord has a synergistic effect, promoting neural regeneration, ESMN survival and partial functional recovery.

Upper Extremity Axon Counts and Clinical Implications for Motor Nerve Transfer.

BACKGROUND: Nerve transfers are planned based on the following parameters: location, number of branches, and axon count matching of the donor and recipient nerves. The authors have previously defined the former two in upper limb muscles. In the literature, axon counts are obtained from various sources, using different methods of histomorphometry. This study describes the axon counts of the same primary motor nerve branches from the authors' previous study using a uniform method of manual histomorphometry and completes the authors' blueprint of upper limb neuromuscular anatomy for reconstructive surgery. METHODS: The distal ends of the primary nerve branches of 23 upper limb muscles were harvested from 10 fresh frozen cadaveric upper limbs. Manual quantitative histomorphometry was performed by two independent investigators, and the average was reported. RESULTS: The primary nerve branches of the arm muscles had higher average axon counts (range, 882 to 1835) compared with those of the forearm muscles (range, 267 to 883). In the forearm, wrist flexor (range, 659 to 746) and extensor (range, 543 to 745) nerve branches had axons counts that were similar to those of potential donors (e.g., supinator, n = 602; pronator teres, n = 625; flexor digitorum superficialis, n = 883; and flexor digitorum profundus, n = 832). CONCLUSIONS: Apart from describing the axon counts of the upper limb, the authors have found that the forearm axon counts are very comparable. This insight, when combined with information on the location and number of primary nerve branches, will empower surgeons to tailor bespoke nerve transfers for every clinical situation.

Differentiation of human adipose-derived stem cells into neuron/motoneuron-like cells for cell replacement therapy of spinal cord injury.

Human adipose-derived stem cells (hADSCs) are increasingly presumed to be a prospective stem cell source for cell replacement therapy in various degenerative and/or traumatic diseases. The potential of trans-differentiating hADSCs into motor neuron cells indisputably provides an alternative way for spinal cord injury (SCI) treatment. In the present study, a stepwise and efficient hADSC trans-differentiation protocol with retinoic acid (RA), sonic hedgehog (SHH), and neurotrophic factors were developed. With this protocol hADSCs could be converted into electrophysiologically active motoneuron-like cells (hADSC-MNs), which expressed both a cohort of pan neuronal markers and motor neuron specific markers. Moreover, after being primed for neuronal differentiation with RA/SHH, hADSCs were transplanted into SCI mouse model and they survived, migrated, and integrated into injured site and led to partial functional recovery of SCI mice. When ablating the transplanted hADSC-MNs harboring HSV-TK-mCherry overexpression system with antivirial Ganciclovir (GCV), functional relapse was detected by motor-evoked potential (MEP) and BMS assays, implying that transplanted hADSC-MNs participated in rebuilding the neural circuits, which was further confirmed by retrograde neuronal tracing system (WGA). GFP-labeled hADSC-MNs were subjected to whole-cell patch-clamp recording in acute spinal cord slice preparation and both action potentials and synaptic activities were recorded, which further confirmed that those pre-conditioned hADSCs indeed became functionally active neurons in vivo. As well, transplanted hADSC-MNs largely prevented the formation of injury-induced cavities and exerted obvious immune-suppression effect as revealed by preventing astrocyte reactivation and favoring the secretion of a spectrum of anti-inflammatory cytokines and chemokines. Our work suggests that hADSCs can be readily transformed into MNs in vitro, and stay viable in spinal cord of the SCI mouse and exert multi-therapeutic effects by rebuilding the broken circuitry and optimizing the microenvironment through immunosuppression.