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

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Following peripheral nerve transection and microsurgical repair (PNIr) most patients retain significant sensorimotor impairments, a proportion of which also develop chronic neuropathic pain. Individual psychological factors may contribute to the development, intensity and duration of chronic pain. Furthermore, a large body of evidence has indentified beneficial and maladaptive cortical plasticity following disease or injury. The general aim of this thesis was to determine the extent of sensory and motor recovery, functional and structural brain changes, and the impact of chronic neuropathic pain on sensorimotor outcomes following upper limb PNIr. Towards this main aim a sensorimotor psychophysical assessment (that included psychological assessments), nerve conduction testing, and an MRI session that examined brain function and structure was performed in patients with peripipheral nerve injury induced neuropathic pain (PNI-P) and those with no neuropathic pain (PNI-NP). Nerve conduction testing demonstrated that all patients had incomplete peripheral nerve regeneration, and that PNI-P patients had worse sensory nerve regeneration. Psychophysical assessment confirmed that all PNIr patients had significant sensorimotor deficits. Additionally, deficits on tests of vibration detection, sensorimotor integration, and fine dexterity were significantly greater in PNI-P patients. Psychological measures clearly distinguished PNI-P from PNI-NP and healthy controls (HC). Vibrotactile stimulation of the deafferented territory in PNI-NP patients results in reduced BOLD activation within the primary and secondary somatosensory cortices. Interestingly, the regions of reduced BOLD corresponded with gray matter thinning which was negatively correlated with behavioural measures of sensory recovery. Structural abnormalities were also identified in the right insula. PNI-P patients had thinning within the right middle insula and a corresponding decrease in white matter pathways projecting into/out of that region. PNI-P patients also had white matter abnormalities in pathways feeding into/out of the contralesional primary somatosensory cortex and thalamus. In conclusion, PNIr is clearly associated with sensorimotor impairments and brain plasticity. Furthermore, neuropathic pain is associated with worse peripheral nerve regeneration, sensorimotor deficits, different psychological profiles, and structural alterations in brain regions involved in pain perception and somatosensation. These results provide insight into peripheral regeneration, the development of chronic pain, brain plasticity and structure-function-behavioural relationships following nerve injury and have important therapeutic implications.

Following peripheral nerve transection and microsurgical repair (PNIr) most patients retain significant sensorimotor impairments, a proportion of which also develop chronic neuropathic pain. Individual psychological factors may contribute to the development, intensity and duration of chronic pain. Furthermore, a large body of evidence has indentified beneficial and maladaptive cortical plasticity following disease or injury. The general aim of this thesis was to determine the extent of sensory and motor recovery, functional and structural brain changes, and the impact of chronic neuropathic pain on sensorimotor outcomes following upper limb PNIr. Towards this main aim a sensorimotor psychophysical assessment (that included psychological assessments), nerve conduction testing, and an MRI session that examined brain function and structure was performed in patients with peripipheral nerve injury induced neuropathic pain (PNI-P) and those with no neuropathic pain (PNI-NP). Nerve conduction testing demonstrated that all patients had incomplete peripheral nerve regeneration, and that PNI-P patients had worse sensory nerve regeneration. Psychophysical assessment confirmed that all PNIr patients had significant sensorimotor deficits. Additionally, deficits on tests of vibration detection, sensorimotor integration, and fine dexterity were significantly greater in PNI-P patients. Psychological measures clearly distinguished PNI-P from PNI-NP and healthy controls (HC). Vibrotactile stimulation of the deafferented territory in PNI-NP patients results in reduced BOLD activation within the primary and secondary somatosensory cortices. Interestingly, the regions of reduced BOLD corresponded with gray matter thinning which was negatively correlated with behavioural measures of sensory recovery. Structural abnormalities were also identified in the right insula. PNI-P patients had thinning within the right middle insula and a corresponding decrease in white matter pathways projecting into/out of that region. PNI-P patients also had white matter abnormalities in pathways feeding into/out of the contralesional primary somatosensory cortex and thalamus. In conclusion, PNIr is clearly associated with sensorimotor impairments and brain plasticity. Furthermore, neuropathic pain is associated with worse peripheral nerve regeneration, sensorimotor deficits, different psychological profiles, and structural alterations in brain regions involved in pain perception and somatosensation. These results provide insight into peripheral regeneration, the development of chronic pain, brain plasticity and structure-function-behavioural relationships following nerve injury and have important therapeutic implications.

<p>Schwann cells (SCs) are PNS glia with numerous neuron-supporting functions, including myelination of axons. Although lesser discussed, SCs also fulfill many important roles after peripheral nerve injury (PNI) contributing significantly to the PNS regeneration process. Clusters of congregated SCs (Bands of Bungner) precede axon regeneration and facilitate the growth of extending axons to their distal targets which is particularly important in the lesion area of severed nerves. While this phenomenon occurs naturally, recovery from PNI can still be inadequate, especially in nerve transection or large gap injuries. Current treatments for nerve transection injuries are limited to coaptation of the nerve via sutures or nerve grafts. However, poor functional outcomes or donor site morbidity remain unaddressed problems. At the cellular level, axon pathfinding and extension relies heavily on the interaction between SCs and axonal growth cones. Depletion or removal of SCs at the lesion has been implicated to poor functional outcomes. With their pivotal role throughout nerve regeneration, we theorize axon regeneration can be improved by augmenting the SC population at the site of injury by encouraging migration to the lesion and via expression of morphological phenotypes that imitate the Bands of Bungner. </p> <p>DC electric fields (EFs) have been well studied in the past as a method to modulate cell orientation and migration and within the context of the nervous system, have been used to promote regeneration in lesioned spinal cords. However, very little work has investigated the effects of electrical stimulation on glia, such as SCs. Existing literature is lacking with regards to various aspects of SC responses, including direction of alignment. We hypothesize electrical stimulation can modulate SC behavior to reinforce/replicate behaviors observed within Bands of Bungner, which may be developed into a treatment for victims suffering peripheral nerve injury. </p> <p>We begin the current study with a thorough investigation into electric field modulated SC behavior. Using conventional 2D cell culture we demonstrate SC sensitivity to EFs by analyzing alignment, morphology and migration data. We employed EFs within the physiologic range. Waveforms used were constant DC as well as a 50% duty cycle DC and an oscillating DC. The latter two may prove more appropriate <i>in vivo</i> due to reduced accumulation of cytotoxic byproducts generated at the electrode interfaces. </p> <p>Our results highlight the sensitivity of SCs to DC electric fields of varying waveforms. SCs showed a strong propensity to align perpendicular to the field and display some cathodal migration in 2D cultures. Additional studies with variable cell density revealed cell-cell interaction further enhanced the alignment response. To more closely replicate the nerve microenvironment, a 3D cell culture model of PNI was created. Embedded in matrices, we found SCs displayed weaker migratory and alignment responses compared to 2D results. The direction of galvanotaxis was reversed, with SCs migrating toward the anode. Both alignment and migratory responses have potential applications for PNI. The galvanotactic behavior of SCs could be used to boost the SC population, increasing the number of Bands of Bungner. Cell alignment would be particularly advantageous at the lesion where axon regeneration is most difficult without the physical guidance of endoneurial tubes.</p> <p>This study characterizes SC behavior in applied EFs using conventional 2D and 3D cell culture techniques. We found SCs are sensitive to electric stimulation, supporting the idea that applied EFs could be used to indirectly promote regeneration in damaged peripheral nerve by modulating SC response after injury. Potential applications include generating an EF across damaged nerves to align SCs, especially in the lesioned area, using EFs to induce SC migration to the lesion to increase the number of cells guiding severed axons, and pre-aligning SCs in synthetic nerve grafts.</p>

Following microsurgical reconstruction of injured peripheral nerves, severed axons are able to undergo spontaneous regeneration. However, the functional result is always unsatisfactory with poor sensory recovery and reduced motor function. One contributing factor is the retrograde neuronal death, which occurs in the dorsal root ganglia (DRG) and in the spinal cord. An additional clinical problem is the loss of nerve tissue that often occurs in the trauma zone and which requires “bridges” to reconnect separated nerve ends. The present thesis investigates the extent of retrograde degeneration in spinal motoneurons and cutaneous and muscular afferent DRG neurons after permanent axotomy and following treatment with N-acetyl-cysteine (NAC). In addition, it examines the survival and growth-promoting effects of nerve reconstructions performed by primary repair and peripheral nerve grafting in combination with NAC treatment. In adult rats, cutaneous sural and muscular medial gastrocnemius DRG neurons and spinal motoneurons were retrogradely labeled with fluorescent tracers from the homonymous transected nerves. Survival of labeled neurons was assessed at different time points after nerve transection, ventral root avulsion and ventral rhizotomy. Axonal regeneration was evaluated using fluorescent tracers after sciatic axotomy and immediate nerve repair. Intraperitoneal or intrathecal treatment with NAC was initiated immediately after nerve injury or was delayed for 1-2 weeks. Counts of labeled gastrocnemius DRG neurons did not reveal any significant retrograde cell death after nerve transection. Sural axotomy induced a delayed loss of DRG cells, which amounted to 43- 48% at 8-24 weeks postoperatively. Proximal transection of the sciatic nerve at 1 week after initial axonal injury did not further increase retrograde DRG degeneration, nor did it affect survival of corresponding motoneurons. In contrast, rhizotomy and ventral root avulsion induced marked 26- 53% cell loss among spinal motoneurons. Primary repair or peripheral nerve grafting supported regeneration of 53-60% of the motoneurons and 47-49% of the muscular gastrocnemius DRG neurons at 13 weeks postoperatively. For the cutaneous sural DRG neurons, primary repair or peripheral nerve grafting increased survival by 19-30% and promoted regeneration of 46-66% of the cells. Regenerating sural and medial gastrocnemius DRG neurons upregulate transcription of peripherin and activating transcription factor 3. The gene expression of the structural neurofilament proteins of high molecular weight was significantly downregulated following injury in both regenerating and non-regenerating sensory neurons. Treatment with NAC was neuroprotective for spinal motoneurons after ventral rhizotomy and avulsion, and sural DRG neurons after sciatic nerve injury. However, combined treatment with nerve graft and NAC had significant additive effect on neuronal survival and also increased the number of sensory neurons regenerating across the graft. In contrast, NAC treatment neither affected the number of regenerating motoneurons nor the number of myelinated axons in the nerve graft and in the distal nerve stump. In summary, the present results demonstrate that cutaneous sural sensory neurons are more sensitive to peripheral nerve injury than muscular gastrocnemius DRG cells. Moreover, the retrograde loss of cutaneous DRG cells taking place despite immediate nerve repair would still limit recovery of cutaneous sensory functions. Experimental data also show that NAC provides a highly significant degree of neuroprotection in animal models of adult nerve injury and could be combined with nerve grafting to further attenuate retrograde neuronal death and to promote functional regeneration.

Successful axon regeneration appears to depend on the development of an injury response. Dorsal root ganglion neurons exemplify the necessity of this injury response in a unique way. Peripheral nerve transection leads to development of an injury response and successful regeneration whereas central root transection does neither. The injury response may involve extracellular and intracellular pathways. To investigate the extraneuronal influences, we performed nerve transection of either the central or peripheral axon branches and studied the expression of GAP-43, a key growth associated protein, and the transcription factors ATF3, c-Jun, and STAT3. Our results show that the responses to peripheral versus central nerve transection are fundamentally different. Peripheral but not central nerve transection increases GAP-43, ATF3, and c-Jun expression. STAT3, however, is upregulated as a result of central but not peripheral nerve transection. To investigate potential intracellular signalling pathways, we applied FGF-2, an extracellular mitogen, or an analog of cAMP, an intracellular second messenger to the cut end of the peripheral axon. Our results indicate that FGF-2 and cAMP act as activators of GAP-43 expression. On the other hand, FGF-2 and cAMP act to downregulate the expression of ATF3. FGF-2 upregulates c-Jun and the activated form of STAT3. Paradoxically, the regulation of GAP-43 expression by cAMP or by FGF-2 in vivo shows opposing results from the previously reported in vitro studies. Our present results suggest that the peripheral nerve injury response may be governed by at least three different signalling pathways.

Successful axon regeneration appears to depend on the development of an injury response. Dorsal root ganglion neurons exemplify the necessity of this injury response in a unique way. Peripheral nerve transection leads to development of an injury response and successful regeneration whereas central root transection does neither. The injury response may involve extracellular and intracellular pathways. To investigate the extraneuronal influences, we performed nerve transection of either the central or peripheral axon branches and studied the expression of GAP-43, a key growth associated protein, and the transcription factors ATF3, c-Jun, and STAT3. Our results show that the responses to peripheral versus central nerve transection are fundamentally different. Peripheral but not central nerve transection increases GAP-43, ATF3, and c-Jun expression. STAT3, however, is upregulated as a result of central but not peripheral nerve transection. To investigate potential intracellular signalling pathways, we applied FGF-2, an extracellular mitogen, or an analog of cAMP, an intracellular second messenger to the cut end of the peripheral axon. Our results indicate that FGF-2 and cAMP act as activators of GAP-43 expression. On the other hand, FGF-2 and cAMP act to downregulate the expression of ATF3. FGF-2 upregulates c-Jun and the activated form of STAT3. Paradoxically, the regulation of GAP-43 expression by cAMP or by FGF-2 in vivo shows opposing results from the previously reported in vitro studies. Our present results suggest that the peripheral nerve injury response may be governed by at least three different signalling pathways.