Peripheral nerve injuries are often associated with significant loss of nervous tissue leading to poor restoration of function following repair of injured nerves. Although the injury gap could be bridged by autologous nerve graft, the limited access to donor material and additional morbidity such as loss of sensation and scarring have prompted a search for biosynthetic nerve transplants. The present thesis investigates the effects of a synthetic matrix BD™ PuraMatrix™ peptide (BD)hydrogel, alginate/fibronectin gel and fibrin glue combined with cultured rat Schwann cells or human bone marrow derived mesenchymal stem cells (MSC) on neuronal regeneration and muscle recovery after peripheral nerve injury in adult rats. In a sciatic nerve injury model, after 3 weeks postoperatively, the regenerating axons grew significantly longer distances within the conduits filled with BD hydrogel if compared with the alginate/fibronectin gel. The addition of rat Schwann cells to the BD hydrogel drastically increased regeneration distance with axons crossing the injury gap and entering into the distal nerve stump. However, at 16 weeks the number of regenerating spinal motoneurons was decreased to 49% and 31% in the BD hydrogel and alginate/fibronectin groups respectively. The recovery of the gastrocnemius muscle was also inferior in both experimental groups if compared with the nerve graft. The addition of the cultured Schwann cells did not further improve the regeneration of motoneurons and muscle recovery. The growth-promoting effects of the tubular conduits prepared from fibrin glue were also studied following repair of short and long peripheral nerve gaps. Retrograde neuronal labeling demonstrated that fibrin glue conduit promoted regeneration of 60% of injured sensory neurons and 52% of motoneurons when compared with the autologous nerve graft. The total number of myelinated axons in the distal nerve stump in the fibrin conduit group reached 86% of the nerve graft control and the weight of gastrocnemius and soleus muscles recovered to 82% and 89%, respectively. When a fibrin conduit was used to bridge a 20 mm sciatic nerve gap, the weight of gastrocnemius muscle reached only 43% of the nerve graft control. The morphology of the muscle showed a more atrophic appearance and the mean area and diameter of fast type fibres were significantly worse than those of the corresponding 10 mm gap group. In contrast, both gap sizes treated with nerve graft showed similar fiber size. The combination of fibrin conduit with human MSC and daily injections of cyclosporine A enhanced the distance of regeneration by four fold and the area occupied by regenerating axons by three fold at 3 weeks after nerve injury and repair. In addition, the treatment also significantly reduced the ED1 macrophage reaction. At 12 weeks after nerve injury the treatment with cyclosporine A alone or cyclosporine A combined with hMSC induced recovery of the muscle weight and the size of fast type fibres to the control levels of the nerve graft group. In summary, these results show that a BD hydrogel supplemented with rat Schwann cells can support the initial phase of neuronal regeneration across the conduit. The data also demonstrate an advantage of tubular fibrin conduits combined with human MSC to promote axonal regeneration and muscle recovery after peripheral nerve injury.

The adaptation of inverse dynamics was performed to quantitatively examine the subtle locomotor changes, previously undetectable, in rodent locomotion following nervous system injury. The first experiment performed an injury with known effects, a unilateral lesion of the medial and lateral branches of the left tibial nerve of Long-Evans rats, and measured the resulting data via inverse dynamics. Special effort was made to account for skin movement artefacts using a global optimization method for marker digitization. The second experiment attempted to apply this technique to Long-Evans rats with spinal hemisections at spinal level T-10. After the peripheral nerve injury to the tibial nerve branches, the main findings were that ankle joint still produces an extensor moment and positive power without the active contraction of the gastrocnemius m. It is possible that this phenomenon is due to passive contractile elements of the muscle and tendon. In addition, the knee and hip of the lesion leg stiffen, resulting in substantial reductions in moment generation and nearly total losses of both negative and positive power production. There were also compensations made by the opposite hindlimb and contralateral forelimb. The spinal cord hemisection produced subtle, complicated intra and interlimb changes in both joint moment and joint power analysis that could not be seen by looking at joint angles alone.

Locomotion persists despite changes in external and internal circumstances. Motor responses to gait impairment exhibit commonalities across various taxa and types of injury, yet we lack a systematic understanding of compensation strategies. The objective of this dissertation is to uncover principles governing implicit goals within the control of locomotion. I propose that coordination of injured locomotion will demonstrate that these goals follow a hierarchical organization of the neuromuscular system. Accurate quantification of gait deficits in rodents demands sophisticated measurement techniques. I utilize X-ray technology to examine intralimb and interlimb coordination after unilateral injury in rats. My findings indicate that compensation to injury involves the coordination of lower-order motor elements to preserve the pre-injury behaviors of higher-order elements. Specifically I present evidence that preservation of limb angle and limb length are critical task goals that transcend injury states and afferent sensory feedback conditions. Broadening my investigation to include interlimb coordination revealed that task goals may change to satisfy the goals of a higher hierarchical level. This work is a necessary precursor to study locomotor coordination and injury compensation in more complex rodent injury models such as self-reinnervation, sciatic nerve, and spinal cord injury. These results could also translate to clinical gait rehabilitation through future protocols that address motor patterns of the entire limb over the behavior of individual joints.

Chronic denervation and chronic axotomy present independent barriers for axonal regeneration. Chronic denervation occurs when nerves are no longer connected to their neuronal cell bodies; chronic axotomy occurs when neurons are not connected to their targets for prolonged periods of time. The harmful effects of chronic denervation can be addressed by the side-to-side bridge surgical technique. Additionally, the negative effects of chronic axotomy can be reversed by GDNF delivery to the nerve. The experiments in this thesis were designed to evaluate nerve regeneration in a rat model of chronic injury after treatment with local GDNF delivery, side to-side bridge protection, or both. The GDNF delivery system consisted of poly(lactic-co-glycolic acid) microspheres embedded in fibrin for controlled delivery of GDNF. Overall, the side-to-side bridges technique was effective in protecting against the negative effects of chronic denervation regardless of treatment with or without GDNF. Local delivery of GDNF did not increase axonal regeneration or functional recovery.

Chronic denervation and chronic axotomy present independent barriers for axonal regeneration. Chronic denervation occurs when nerves are no longer connected to their neuronal cell bodies; chronic axotomy occurs when neurons are not connected to their targets for prolonged periods of time. The harmful effects of chronic denervation can be addressed by the side-to-side bridge surgical technique. Additionally, the negative effects of chronic axotomy can be reversed by GDNF delivery to the nerve. The experiments in this thesis were designed to evaluate nerve regeneration in a rat model of chronic injury after treatment with local GDNF delivery, side to-side bridge protection, or both. The GDNF delivery system consisted of poly(lactic-co-glycolic acid) microspheres embedded in fibrin for controlled delivery of GDNF. Overall, the side-to-side bridges technique was effective in protecting against the negative effects of chronic denervation regardless of treatment with or without GDNF. Local delivery of GDNF did not increase axonal regeneration or functional recovery.

Bone marrow-derived mesenchymal stem cells (MSC) have been shown to provide neuroprotection after transplantation into the injured nervous system. The present thesis investigates whether adult human and rat MSC differentiated along a Schwann cell lineage could increase their expression of neurotrophic factors and promote regeneration after transplantation into the injured peripheral nerve and spinal cord. Human and rat mesenchymal stem cells (hMSC and rMSC) expressed characteristic stem cell surface markers, mRNA transcripts for different neurotrophic factors and demonstrated multi-lineage differentiation potential. Following treatment with a cocktail of growth factors, the hMSC and rMSC expressed typical Schwann cells markers at both the transcriptional and translational level and significantly increased production of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Age and time in culture are of relevance for clinical settings and growth-promoting effects of hMSC from young donors (16-18 years) and old donors (67-75 years) were compared. Undifferentiated hMSC from both young and old donors increased total neurite length of cultured dorsal root ganglion (DRG) neurons. Differentiation of hMSC from the young donors, but not the eldery donors, further enhanced the neurite outgrowth. Undifferentiated hMSC were cultured for eleven weeks in order to examine the effect of in vitro expansion time on neurite outgrowth. hMSC from the young donors maintained their proliferation rate and their ability to enhance neurite outgrowth from DRG neurons. Using a sciatic nerve injury model, a 10mm gap was bridged with either an empty tubular fibrin glue conduit, or conduits containing hMSC, with and without cyclosporine treatment. Cells were labeled with PKH26 prior to transplantation. At 3 weeks after injury the conduits with cells and immunosuppression increased regeneration compared with an empty conduit. PKH26 labeled human cells survived in the rat model and the inflammatory reaction could be suppressed by cyclosporine. After cervical C4 hemisection, BrdU/GFP-labeled rMSC were injected into the lateral funiculus rostral and caudal to the spinal cord lesion site. Spinal cords were analyzed 2-8 weeks after transplantation. Transplanted MSC remained at the injection sites and in the trauma zone for several weeks and were often associated with numerous neurofilament-positive axons. Transplanted rMSC induced up-regulation of vascular endothelial growth factor in spinal cord tissue rostral to the injury site, but did not affect expression of brain-derived neurotrophic factor. Although rMSC provided neuroprotection for rubrospinal neurons and significantly attenuated astroglial and microglial reaction, cell transplantation caused aberrant sprouting of calcitonin gene-related peptide immunostained sensory axons in the dorsal horn. In summary these results demonstrate that both rat and human MSC can be differentiated towards the glial cell lineage, and show functional characteristics similar to Schwann cells. hMSC from the young donors represent a more favorable source for neurotransplantation since they maintain proliferation rate and preserve their growth-promoting effects in long-term cultures. The data also suggest that differentiated MSC increase expression of neurotrophic factors and support regeneration after peripheral nerve and spinal cord injury.

Schwann cells are the principal glial cell of the peripheral nervous system and are responsible for axon maintenance, regeneration and increasing saltatory conduction of neurons. Schwann cell differentiation and myelination is mediated by a core network of transcription factors and signalling pathways, which have been divided into two groups; positive and negative regulators. Sox10, NFATc4, Oct6, Krox20 and the ERK 1/2 signalling pathway have been characterised as positive regulators of Schwann cell differentiation and myelination; with Sox10 and Krox20 also playing critical roles in myelin maintenance. On the other hand, transcription factors cJun, Pax3, Id2 and signalling pathways Notch and p38 mitogen activated protein (MAP) kinases (MAPK) have been identified as negative regulators of Schwann cell differentiation and myelin formation. Recently, the HMG transcription factor Sox2 was identified as a negative regulator of Schwann cell myelination in vitro, however its role in Schwann cell myelination in vivo has not yet been studied. This study therefore aimed to examine the role of Sox2 overexpression in Schwann cells and how it effects Schwann cell differentiation and myelination during development and after injury. In addition, we aimed to investigate for the first time the specific role of p38α (the major isoform of p38 MAPK) in Schwann cell myelination in vivo, by generating Schwann cell specific p38α conditional knockout mice. Sox2 is highly expressed in immature Schwann cells, but is downregulated as Schwann cells being to mature and differentiate. This study shows that continued expression of Sox2 during development and after injury, impairs Schwann cell differentiation and myelination by directly downregulating the expression of two core transcription factors; Sox10 and Krox20, as well as myelin proteins, P0 and MBP. In addition, we observe that continued Sox2 expression significantly increases Schwann cell proliferation and maintains Schwann cells in an immature state. Unexpectedly, we also observed that continued Sox2 expression significantly increases the number of macrophages present in the nerves of Sox2 overexpressing mice at both P60 and 21 days post injury. Phenotypically, Sox2 overexpressing mice 6 show signs of a peripheral neuropathy and animals have impaired motor and sensory function. These findings confirm that Sox2 is a negative regulator of Schwann cell myelination and suggests that continued Sox2 expression is sufficient to drive the progressive development of a peripheral nerve disorder which may resemble Charcot-Marie-Tooth type 1 demyelinating neuropathy and congenital hypomyelinating neuropathy. As a negative regulator of Schwann cell myelination, activity of the p38 MAPK pathway has been shown to inhibit myelin formation in vitro and to also induce the Schwann cell injury response; by driving Schwann cell dedifferentiation and demyelination following injury. Here we show that specific removal of the p38α isoform in Schwann cells leads to an increase in myelin thickness at early developmental time-points, along with an elevated expression of myelin proteins, P0 and MBP. Further analysis following nerve injury revealed that removal of p38α results in an initial decrease in Schwann cell demyelination, yet improves axon remyelination at 21 days post injury. These results demonstrate the specific role of p38α in regulating Schwann cell myelination, and how it could be a direct therapeutic target for improving nerve repair after injury.

Repair of peripheral nerve injury often results in poor functional motor recovery. This deficit has previously been attributed to the failure of axons to regenerate into the muscle. However, we have recently reported that following nerve injury in mice, axons have regenerated to the motor end plate in animals with poor recovery. We proposed that following axonal injury, there is a critical period during which the axon must reach the muscle in order to form a functional neuromuscular junction. We have developed a mouse model of prolonged denervation, in which the sciatic nerve is crushed repeatedly every few days, preventing regenerating axons from reaching the muscle. This multiple crush model allows us to vary the period of denervation by modifying the number of crushes. Motor recovery as assessed using the toe-spreading score occurs after 3 or 4 multiple crushes every 7 days (24 or 31 days of denervation) but not after 5 crushes (38 days). Immunostaining for alpha-bungarotoxin and neurofilament confirmed end plate reinnervation. Thus following denervation > 38 days, a motor deficit persists despite end plate reinnervation. Although the mechanism for the deficit requires investigation, these results suggest that functional neuromuscular junction reestablishment more than end plate reinnervation and that there is a time limit for functional synapse reformation.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23056号 / 医博第4683号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸口田 淳也, 教授 森本 尚樹, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

INTRODUCTION: Injury to the facial nerve can lead to functional and aesthetic sequelae in patients. Though surgical interventions are available to restore lost motor and sensory function, outcomes are often suboptimal due to inadequate or disorganized axonal regeneration. While engineering improvements to the standard of care are underway, gaps remain in our molecular understanding of peripheral nerve injury to translate these efforts clinically. Over the last few decades, advancements in intravital imaging such as the development of fluorescent reporter mice and use of multiphoton excitation techniques have allowed for markedly enhanced characterization of biological phenomena at higher resolutions, at greater depths, and for longer timescales. Challenges in reliably and serially imaging in vivo within murine models have been overcome through the development of chronic imaging windows in various settings of the body. However, there are very few techniques available presently for imaging the peripheral nerve microenvironment and no prior work detailing use in the facial nerve setting. OBJECTIVE: Longitudinal studies employing intravital imaging techniques carry potential to improve understanding of peripheral nerve regeneration and function. Using multiphoton microscopy and fluorescent reporter mice, we propose a prototype, surgical protocol of implantation, and initial safety and efficacy testing of a facial nerve window to enable chronic imaging for enhanced characterization of the peripheral nerve microenvironment. METHODS: A stainless-steel implant with an affixed glass coverslip and aluminum external fixation component was developed for implantation in a transgenic reporter mouse model to enable chronic intravital imaging of the facial nerve buccal and marginal mandibular branches. A qualitative observational study and clinical assessment scoring study was performed post-surgical implantation to monitor behavior, physical appearance, weight loss, and reactivity to animal handling over the typical time-course of nerve regeneration. Segments of facial nerve branches were harvested from control and window-implanted mice and imaged using widefield epifluorescence microscopy for axon quantification to determine any adverse effects from window compression onto axonal fibers. Two-photon microscopy (2PM) and Simulated Raman Scattering (SRS) were also performed through the window to visualize axon tracts, myelin sheaths, and surrounding collagen matrix in wild-type and transgenic mice models. RESULTS: Qualitative serial observational studies and assessment scoring indicated no obvious functional deficits over the time-course of typical nerve regeneration and normal scores for weight, behavior, physical appearance, and reactivity. Neural histomorphometric analysis indicated no significant difference in mean myelinated axon count of buccal (mean ± SD; control buccal, 947.6 ± 129.9; window-implanted buccal, 799.3 ± 128.6; p = .136) and marginal mandibular branches (control marginal mandibular, 801.3 ± 145.1; window-implanted marginal mandibular, 738.0 ± 197.2; p = .599) between control and window-implanted mice, suggesting that neuropathy was not induced from the window itself. High-resolution images of nerve morphology in healthy and injured transgenic and wild-type mice were obtained using 2PM and SRS. CONCLUSION: Herein, we describe a novel and replicable platform for longitudinal intravital imaging of murine facial nerve. Future studies will evaluate viability of this model for imaging the facial nerve microenvironment, particularly Schwann cell-axon interactions, in the setting of severe nerve injury over a period of several weeks to months. Improved understanding gained through such studies of the structural peripheral nerve microenvironment may allow for advancements in viral vector therapeutics, nerve graft scaffold design, as well as advanced injury diagnostics and tracking. / 2022-06-02T00:00:00Z