Insufficient sympathetic regeneration leads to chronic functional deficiencies after traumatic peripheral nerve injury Restricted; Files Only
Tian, Tina (Spring 2025)
Abstract
Peripheral nerve injuries (PNIs) cause motor, sensory, and autonomic dysfunction that lead to lifelong functional deficits and disabilities. Electrical stimulation (ES) is a clinically-translatable intervention to enhance motor and sensory axon regeneration. However, its effects on sympathetic regeneration have not been previously characterized. The sympathetic nervous system is critical for maintaining bodily homeostasis, with functions that encompass every organ system.
In this dissertation, I describe the anatomy and the importance of the sympathetic nervous system, with special emphasis on its newfound roles in skeletal muscle function. I detail the localization of the mouse lumbar sympathetic ganglia and give an overview of peripheral nerve regeneration, reconstruction techniques, and neuronal activity-based interventions that enhance axon regeneration.
I evaluated the short-term and long-term effects of ES on sympathetic regeneration in a mouse sciatic nerve transection model. I also tested the effects of a conditioning lesion and sympathetic-specific targeting with bioluminescent optogenetics acutely after nerve injury. These regeneration-enhancing techniques do not enhance sympathetic regeneration, and the lag in sympathetic regeneration leads to persistent deficits in skeletal muscle energy charge.
I also investigated how conditioning ES (CES), a novel paradigm of ES affects sympathetic axon regeneration in acute and long-term settings. I find that CES has an inhibitory effect on sympathetic regeneration 1 week after nerve graft repair and no enhancement effect 2 weeks after direct nerve repair. At a longer time point after injury, there is no difference in the presence of sympathetic marker tyrosine hydroxylase at neuromuscular junctions.
Finally, I performed RNA sequencing on the lumbar sympathetic ganglia 3 days after sciatic nerve transection to uncover the molecular mechanisms governing sympathetic regeneration. I identified TrkA receptor activation via nerve growth factor (NGF) and pituitary adenylate cyclase-activating polypeptide (PACAP) as a possible pathway of interest. Although NGF increases sympathetic outgrowth and branching in vitro, PACAP-38 and its receptor inhibitor PACAP(6-38) do not significantly impact sympathetic growth. Increased branching, or sprouting, of sympathetic neurons in response to NGF is linked to pain, so an optimal window of NGF needs to be found to harness its potential to increase axonal growth.
In sum, postganglionic sympathetic axons do not respond to regeneration-enhancing interventions, and their regeneration lags behind their motor counterparts. This contributes to long-term functional deficits associated with PNIs. More studies are needed to better understand the molecular underpinnings of sympathetic regeneration.
Table of Contents
CHAPTER 1: Finding the balance – Introduction to the sympathetic nervous system.. 1
1.1. Sympathetic activity in skin. 1
1.2. Sympathetic signaling in skeletal muscle: more than just vessels 4
1.3. Zooming in: Anatomy of sympathetic nervous system.. 7
1.4. Clinical side effects of surgical sympathectomies. 10
1.5. Conclusions. 14
CHAPTER 2: Can I take that out? Surgical lumbar sympathectomy in mice. 15
2.1. Abstract 15
2.2. Introduction. 15
2.3. Protocol 18
2.4. Representative Results. 25
2.5. Discussion. 27
CHAPTER 3: Upping the ante – Enhancing peripheral nerve regeneration. 31
3.1. Regeneration after peripheral nerve injury. 31
3.2. Reconstructing peripheral nerves. 33
3.3. Conditioning lesion. 39
3.4. Perioperative electrical stimulation. 42
3.5. Conditioning electrical stimulation. 44
3.6. Bioluminescent optogenetics. 45
CHAPTER 4: A perspective on electrical stimulation and sympathetic regeneration in peripheral nerve injuries. 49
4.1. Abstract 49
4.2. Electrical stimulation in animal models. 49
4.3. Electrical stimulation in humans for peripheral nerve injury. 51
4.4. Anatomy of peripheral nerves. 54
4.5. Perspective on electrical stimulation for sympathetic axon regeneration. 55
4.6. Sympathetic sprouting and pain after peripheral nerve injury 57
4.7. Conclusions. 59
CHAPTER 5: Regenerative failure of sympathetic axons contributes to deficits in functional recovery after nerve injury. 61
5.1. Abstract 61
5.2. Introduction. 62
5.3. Materials and Methods. 63
5.3.1. Animals. 63
5.3.2. Surgical procedures. 66
5.3.3. Quantification of acute axon regeneration. 69
5.3.4. Bioluminescence quantification. 70
5.3.5. Local field potential recordings. 71
5.3.6. Lumbar sympathetic neuronal cell culture. 71
5.3.7. Fluorescent retrograde tracing. 73
5.3.8. Pilocarpine sweat assay. 74
5.3.9. Sweat gland reinnervation. 75
5.3.10. High performance liquid chromatography for purine quantification. 76
5.3.11. Electromyography and muscle collection. 77
5.3.12. Neuromuscular junction receptor quantification. 78
5.3.13. Bulk RNA sequencing of the lumbar sympathetic ganglia 79
5.3.14. Statistical analysis. 80
5.4. Results. 83
5.4.1. Neither conditioning lesion nor electrical stimulation enhances acute sympathetic axon regeneration. 83
5.4.2. Bioluminescent activation of sympathetic neurons inhibits their growth in vivo and in vitro. 86
5.4.3. ES fails to enhance recovery of sweating and anatomical sweat gland reinnervation. 90
5.4.4. ES enhances motor regeneration to the foot but not sympathetic regeneration. 93
5.4.5. Motor reinnervation of the tibialis anterior is complete by 12 weeks after injury, but sympathetic reinnervation remains incomplete. 96
5.4.6. ES does not remedy changes in skeletal muscle cellular energy charge after PNI and fails to reverse muscle atrophy. 96
5.4.7. ES rescues acetylcholine receptor content at the NMJ but not β2 adrenergic receptors. . 98
5.5. Discussion. 100
5.6. Conclusions. 106
CHAPTER 6: Conditioning electrical stimulation fails to enhance sympathetic axon regeneration. 107
6.1. Abstract 107
6.2. Introduction. 107
6.3. Materials and methods. 109
6.3.1. Animals. 109
6.3.2. Surgical procedures. 110
6.3.3. Axon profile quantification. 113
6.3.4. Tyrosine hydroxylase at the neuromuscular junction. 115
6.3.5. Retrograde labeling. 116
6.3.6. Statistical analysis. 117
6.4. Results. 117
6.4.1. Sympathetic axon regeneration into a nerve graft is acutely inhibited by CES. ...... 117
6.4.2. Sympathetic axon regeneration after direct nerve repair is not enhanced by CES. ...... 120
6.4.3. Long-term sympathetic reinnervation after injury is unaffected by CES. 120
6.5. Discussion. 123
6.6. Conclusions. 127
CHAPTER 7: Future explorations – Molecular mechanisms underlying sympathetic regeneration. 128
7.1. Introduction. 128
7.1.1. Nerve growth factor, TrkA, and regeneration. 129
7.1.2. Nerve growth factor, TrkA, and pain. 130
7.1.3. Pituitary adenylate cyclase-activating polypeptide - background. 133
7.2. Methods. 137
7.2.1. Animals. 137
7.2.2. Bulk RNA sequencing of the lumbar sympathetic ganglia after peripheral nerve
injury 137
7.2.3. Identifying gene targets in injured lumbar sympathetic neurons 139
7.2.4. Lumbar sympathetic neuron primary cell culture. 139
7.2.5. Immunohistochemistry. 142
7.2.6. Statistical analysis. 143
7.3. Results. 144
7.3.1. Peripheral nerve injury upregulates genes related to TrkA signaling in the lumbar sympathetic ganglia. 144
7.3.2. NGF increases sympathetic neurite elongation and branching 146
7.3.3. PACAP has no effect on sympathetic neurite growth. 146
7.3.4. Inhibition of PAC1R has no effect on the growth of sympathetic neurons. 148
7.3.5. The β subunit of the epithelial sodium channel is expressed in immune cells in the lumbar sympathetic ganglia. 148
7.4. Discussion. 151
7.4.1. Uncharted waters – new pathways to explore. 154
7.4.2. Immune cells and ENaC – an unlikely pair 157
7.5. Conclusions. 158
References. 160
Table of Figures
Figure 2.1........................................................................................... 25
Figure 2.2........................................................................................... 26
Figure 2.3........................................................................................... 28
Figure 4.1........................................................................................... 52
Figure 4.2........................................................................................... 54
Figure 4.3........................................................................................... 58
Figure 5.1........................................................................................... 66
Figure 5.2........................................................................................... 82
Figure 5.3........................................................................................... 84
Figure 5.4........................................................................................... 85
Figure 5.5........................................................................................... 87
Figure 5.6........................................................................................... 89
Figure 5.7........................................................................................... 91
Figure 5.8........................................................................................... 92
Figure 5.9........................................................................................... 94
Figure 5.10......................................................................................... 95
Figure 5.11......................................................................................... 97
Figure 5.12......................................................................................... 99
Figure 6.1......................................................................................... 112
Figure 6.2......................................................................................... 118
Figure 6.3......................................................................................... 119
Figure 6.4......................................................................................... 121
Figure 6.5......................................................................................... 122
Figure 7.1......................................................................................... 136
Figure 7.2......................................................................................... 144
Figure 7.3......................................................................................... 145
Figure 7.4......................................................................................... 147
Figure 7.5......................................................................................... 149
Figure 7.6......................................................................................... 150
Table of Tables
Table 5.1............................................................................................ 65
Table 5.2............................................................................................ 70
Table 6.1.......................................................................................... 110
Table 7.1.......................................................................................... 139
Table of Videos
Video 2.1............................................................................................ 30
Video 2.2............................................................................................ 30
Table of Supplemental Figures and Tables
Supplemental Figure 5.1.................................................................. 64
Supplemental Figure 5.2.................................................................. 68
Supplemental Figure 5.3................................................................ 103
Supplemental Figure 7.1................................................................ 155
Supplemental Table 7.1.................................................................. 156
Supplemental Table 7.2.................................................................. 157
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