Death, Destruction, and Destiny: Microglia-Motoneuron Interactions in the Spinal Cord After Peripheral Nerve Injury Open Access
Pottorf, Tana (Fall 2025)
Abstract
Motoneuron (MN) degeneration and loss of the stretch reflex are common hallmarks of Peripheral Nerve Injuries (PNI). PNI can induce MN death with varying severity. Additionally, in severe cases of PNI (e.g., a nerve transection), proprioceptive circuits, which are necessary for reflexive movements and therefore basic human functions, are permanently lost (demonstrated by retraction of primary afferents from the ventral horn, and loss of Vesicular Glutamate Transporter 1 (VGluT1) synapses). The permanent loss of MNs and altered stretch reflex circuitry both limit a patient's ability for a full functional recovery. Therefore, the identification of mechanisms that govern selective MN loss and altered circuitry following PNI is crucial for therapeutic advancement and may have implications for other MN pathologies. Herein, I utilize a PNI mouse model to investigate diverse microglia interactions with MNs of varying health states. Within the first few days after PNI, injured MNs release Colony Stimulating Factor 1 (CSF1), activating local microglia. Microglia proliferate, migrate, and extend processes towards MNs, and thereafter, adhere to and scan the MN surface with dynamic filopodia. I have identified different microglia morphologies as they interact with MNs of differing health states (e.g., regenerating or degenerating), and identified differential expression of the microglia receptor, Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), whereby TREM2 is upregulated in microglia that associate with regenerating MNs compared to microglia around dying MNs. Further, TREM2 levels correlate with increases in the phagocytic marker, CD68, and therefore may be associated with synapse removal and/or degenerating MN removal. By using both global and microglia-specific conditional TREM2 knockout mouse models, I investigate TREM2’s role in microglia activation and response to PNI-induced MN cell death, muscle reinnervation, and functional recovery. My results provide novel insights about the regulation of microglia activation and function around injured MNs and suggest possible therapeutics to increase MN survival and circuitry preservation in pathological conditions.
Table of Contents
Table of Contents
CHAPTER 1: INTRODUCTION TO MOTONEURONS AND THE STRETCH REFLEX ............................. 1
1.1 INTRODUCTION .............................................................................................................................. 2
1.2 MOTONEURONS AND SPINAL MOTOR CIRCUITRY....................................................................................... 3
1.2.1 Motoneurons ...................................................................................................................... 3
1.2.2 The Stretch Reflex Pre- and Post-Injury .............................................................................. 7
1.2.3 Motoneuron Response to Axotomy and Cell Death ........................................................... 12
1.3 ABBREVIATIONS ........................................................................................................................... 17
CHAPTER 2: THE ROLE OF MICROGLIA IN NEUROINFLAMMATION OF THE SPINAL CORD AFTER PERIPHERAL NERVE INJURY ................................................................................................... 19
2.1 ABSTRACT ......................................................................................................................................... 20
2.2 KEYWORDS ........................................................................................................................................ 20
2.3 INTRODUCTION .................................................................................................................................. 20
2.3.1 Peripheral Nerve Injuries .................................................................................................. 22
2.3.2 Microgliosis around axotomized motoneurons................................................................... 24
2.3.3 Microgliosis in the dorsal horn around the central projections of sensory afferent axotomized in the periphery ......................................................................................................................... 25
2.3.4 Scope of the present review ............................................................................................. 27
2.4 MECHANISMS OF SPINAL MICROGLIAL ACTIVATION, MOTILITY, AND RECRUITMENT......................................... 28
2.4.1 Microglia activation dynamics in the spinal cord after peripheral nerve injury ..................... 28
2.4.2 Colony Stimulating Factor 1 (CSF1) and other DNAX Activating Protein of 12kDa (DAP12) activating pathways ................................................................................................................... 32
2.4.3 C-C chemokine receptor type 2 (CCR2) and Fractalkine receptor (CX3CR1) .................... 35
2.4.4 Toll-Like Receptors (TLRs) ............................................................................................... 42
2.4.5 Activation of microglia by purinergic receptors .................................................................. 45
2.5 THEORIES ABOUT THE FUNCTION OF MICROGLIA AROUND AXOTOMIZED MOTONEURONS ................................ 50
2.5.1 Neuroprotection or Neurodegeneration ............................................................................. 50
2.5.2 Synaptic plasticity around axotomized motoneurons. ........................................................ 55
2.5.3 Microglia and immune system responses around axotomized motoneurons ...................... 57
2.5.4 Motor Axon Regeneration ................................................................................................. 61
2.6 THEORIES BEHIND MICROGLIOSIS AROUND THE CENTRAL BRANCHES OF SENSORY AFFERENTS INJURED IN THE PERIPHERAL NERVE .................................................................................................................................... 64
2.6.1 Complement as a targeting mechanism for synaptic pruning ............................................. 68
2.7 CONCLUSION ..................................................................................................................................... 69
2.8 AUTHOR CONTRIBUTIONS .................................................................................................................... 70
2.9 FUNDING .................................................................................................................................... 70
2.10 ACKNOWLEDGEMENTS ...................................................................................................................... 70
2.12 ABBREVIATION: ................................................................................................................................ 70
CHAPTER 3: IS TREM2 A STRETCH? IMPLICATIONS OF TREM2 ALONG THE SPINAL REFLEX CIRCUITS IN HEALTH, AGING, INJURY, AND DISEASE. ............................................................................. 74
3.1 ABSTRACT: ........................................................................................................................................ 75
3.2 KEYWORDS: ....................................................................................................................................... 75
3.3 INTRODUCTION .................................................................................................................................. 76
3.4 TRIGGERING RECEPTOR EXPRESSED ON MYELOID CELLS 2 (TREM2) .......................................................... 79
3.4.1 What and Where is TREM2? ............................................................................................ 79
3.4.2 TREM2 Ligands and Signaling Cascade ........................................................................... 80
3.4.3 sTREM2 ........................................................................................................................... 83
3.4.4 TREM2/DAP12 Mutations in Disease ................................................................................. 84
3.4.5 Different Mouse Models May Contribute to Conflicting TREM2 Results ............................. 86
3.5 THE DORSAL ROOT GANGLION ............................................................................................................. 87
3.5.1 Anatomical Overview of the Dorsal Root Ganglion ............................................................ 87
3.5.2 TREM2 in the Dorsal Root Ganglion ................................................................................. 88
3.6 TREM2 IN THE SPINAL CORD ............................................................................................................... 88
3.6.1 Anatomical Overview of the Spinal Cord ........................................................................... 88
3.6.2 TREM2 in Microglia .......................................................................................................... 91
3.6.3 TREM2 In the Dorsal Horn ............................................................................................... 95
3.6.4 TREM2 in the Ventral Horn ............................................................................................. 102
3.6.5 Spinal Cord Injury ........................................................................................................... 104
3.7 TREM2 ALONG PERIPHERAL AXONS ...................................................................................................108
3.7.1 Anatomical Overview of Peripheral Nerves ..................................................................... 108
3.7.2 TREM2 in Schwann Cells ............................................................................................... 109
3.7.3 TREM2 in Peripheral Macrophages ................................................................................ 110
3.8 TREM2 IN MUSCLE .........................................................................................................................113
3.8.1 Anatomical Overview of Skeletal Muscle ......................................................................... 113
3.8.2 Overview of Neuromuscular Junction Denervation/Reinnervation.................................... 114
3.8.3 TREM2 and the Neuromuscular Junction ........................................................................ 115
3.9 TREM2 AS A THERAPEUTIC TARGET ....................................................................................................116
3.10 CONCLUDING REMARKS...................................................................................................................118
3.11 AUTHOR CONTRIBUTIONS: ...............................................................................................................118
3.12 FUNDING: .....................................................................................................................................118
3.13 ACKNOWLEDGEMENTS ....................................................................................................................119
3.14 ABBREVIATIONS..............................................................................................................................119
CHAPTER 4: THE DUAL ROLE OF MICROGLIAL TREM2 IN NEURONAL DEGENERATION AND REGENERATION AFTER AXOTOMY ....................................................................................... 122
4.1 ABSTRACT .......................................................................................................................................123
4.2 KEYWORDS ......................................................................................................................................124
4.3 MAIN POINTS ..................................................................................................................................124
4.4 ABBREVIATIONS ................................................................................................................................124
4.5 INTRODUCTION ................................................................................................................................125
4.6 MATERIALS AND METHODS ................................................................................................................128
4.6.1 Ethical Statement ........................................................................................................... 128
4.6.2 Experimental Animals ..................................................................................................... 129
4.6.3 Tamoxifen Injections....................................................................................................... 131
4.6.4 Intramuscular Injections for Retrograde Motoneuron Labeling ......................................... 131
4.6.5 Sciatic Cut Ligation and Sham Surgeries ........................................................................ 132
4.6.6 Sciatic Cut Repair With or Without Fast Blue Nerve Soak ............................................... 132
4.6.7 Transcardial Perfusion and Tissue Collection ................................................................. 133
4.6.8 Immunohistochemistry and Confocal Microscopy ............................................................ 133
4.6.9 Microglia Sholl Analysis .................................................................................................. 135
4.6.10 Ex Vivo Spinal Cord Slice Preparation and Two-Photon Time Lapse Imaging ............... 135
4.6.11 Analysis of TREM2 Immunofluorescence ...................................................................... 137
4.6.12 CX3CR1 trem2 RNAscope Co-detection and Analysis .................................................. 138
4.6.13 P-Syk Immunofluorescence Analysis ............................................................................ 140
4.6.14 Ventral Horn Microglia Number and Territories in Sham Animals .................................. 141
4.6.15 Time course of microglia proliferation and TREM2 expression in WT, GKO, and CKO .. 141
4.6.16 Number of Death Clusters comparison in WT, GKO, and CKO...................................... 142
4.6.17 Microglia morphologies in GKO mice ............................................................................ 143
4.6.18 Microglia-Motoneuron Interactions ................................................................................ 143
4.6.19 CD68 Quantification ..................................................................................................... 144
4.6.20 Analyses of motoneuron cross-sectional areas ............................................................. 145
4.6.21 Statistics....................................................................................................................... 146
4.7 RESULTS .....................................................................................................................................149
4.7.1 Activated microglia display two morphologically distinct phenotypes 14 days post sciatic nerve transection. ............................................................................................................................. 149
4.7.2 Morphologically distinct phenotypes show different interaction dynamics with motoneurons. ............................................................................................................................................... 153
4.7.3 TREM2 expression increases in activated microglia after PNI, with Death Cluster microglia containing the highest levels. .................................................................................................. 156
4.7.4 TREM2 knock-out validation and suppression of downstream p-SYK .............................. 165
4.7.5 TREM2 knockout does not affect the number, territories, or process lengths and branching of non-activated surveying microglia in sham animals.................................................................. 170
4.7.6 TREM2 Knockout does not affect microglia proliferation or the time course of microglia activation following PNI. .......................................................................................................... 173
4.7.7 TREM2 KO alters microglia morphology mainly in Death Cluster microglia with little effect on the number of microglia-MN interactions.................................................................................. 179
4.7.8 Death Clusters display the greatest phagocytic capacity assessed by CD68. TREM2 KO reduces CD68 expression, but this is selective to females. ...................................................... 184
4.7.9 Microglia TREM2 is necessary for normal motoneuron cell body swelling during the chromatolytic reaction following axotomy. ................................................................................ 193
4.8 DISCUSSION .....................................................................................................................................199
4.8.1 TREM2 deficiency preferentially decreased CD68 in microglia from females and in death clusters. .................................................................................................................................. 200
4.8.2 TREM2 deficiency prevents motoneuron cell body swelling, which correlates with reduced muscle reinnervation ............................................................................................................... 201
4.8.3 Limitations of the Study .................................................................................................. 203
4.9 CONCLUSIONS ..................................................................................................................................204
4.10 AUTHOR CONTRIBUTIONS ................................................................................................................205
4.11 ACKNOWLEDGMENTS...............................................................................................................205
CHAPTER 5: FUTURE DIRECTIONS - UNCOUPLING THE EFFECTS OF CENTRAL VERSUS PERIPHERAL TREM2 DURING PERIPHERAL NERVE REGENERATION ........................................................... 251
5.1 INTRODUCTION ................................................................................................................................252
5.2 METHODS .......................................................................................................................................254
5.2.1 Ethical Statement ........................................................................................................... 254
5.2.2 Experimental Animals ..................................................................................................... 254
5.2.3 Intramuscular Injection Motoneuron Labeling .................................................................. 255
5.2.4 Naïve, Sham, and Sciatic Cut-Repairs ............................................................................ 255
5.2.5 Beam Walks ................................................................................................................... 255
5.2.6 Lateral Gastrocnemius (LG) Muscle Electromyography (EMG) ....................................... 256
5.2.7 Transcardial Perfusion and Tissue Collection ................................................................. 257
5.2.8 Immunohistochemistry and Confocal Microscopy ............................................................ 258
5.2.9 Extrafusal Muscle Fiber Innervation ................................................................................ 261
5.2.10 Statistics....................................................................................................................... 261
5.3 RESULTS ..........................................................................................................................................262
5.3.1 Characterization of TREM2 Conditional Knock Out (CKO) Mouse Peripheral Labeling .... 262
5.3.2 TREM2 Knockout (KO) reduces muscle reinnervation eight weeks post injury (wpi) ........ 264
5.3.3 TREM2 Global Knockout (GKO) significantly reduces muscle function. ........................... 267
5.3.4 TREM2 knockout (KO) does not significantly alter beam walk traverse performance ....... 270
5.4 DISCUSSION .....................................................................................................................................274
5.4.1 Muscle Histology Limitations and Technical Difficulties ................................................... 275
5.4.2 Electromyography (EMG) Limitations and Technical Difficulties ...................................... 276
5.4.3 Beam Walk Limitations and Technical Difficulties ............................................................ 277
5.4.4 Future Directions ............................................................................................................ 278
5.5 CONCLUSIONS ..................................................................................................................................281
5.6 ABBREVIATIONS ................................................................................................................................281
CHAPTER 6: INTRAMUSCULAR INJECTION OF TRADITIONAL RETROGRADE TRACERS IN ADULT MICE PREFERENTIALLY LABELS ALPHA MOTONEURONS................................................................. 284
6.1 ABSTRACT .......................................................................................................................................285
6.2 INTRODUCTION ................................................................................................................................285
6.3 MATERIALS AND METHODS: ...............................................................................................................287
6.3.1 Experimental animals: .................................................................................................... 287
6.3.2 Retrograde tracing procedures ....................................................................................... 288
6.3.3 Tissue collection and Immunohistochemistry (IHC) ......................................................... 289
6.3.4 Motoneuron cell body size quantification......................................................................... 290
6.3.5 Statistics ........................................................................................................................ 291
6.4 RESULTS ..........................................................................................................................................291
6.4.1 Choline Acetyltransferase (ChAT) immunohistochemistry labels both - and -MNs with expected ratios........................................................................................................................ 291
6.4.2 Fast Blue (FB) preferentially labels a-MNs in adult mice ................................................. 293
6.4.3 Cholera Toxin subunit b conjugated to Alexa Fluor 555TM (CTb-555) minimally labels y-MNs in adult mice............................................................................................................................ 295
6.4.4 Adeno-Associated Virus serotype 1 (AAV1)-tdTomato labels both a- and y-MNs, but with inadequate representation of expected MN ratios .................................................................... 297
6.5 CONCLUSIONS ..................................................................................................................................299
REFERENCES ...................................................................................................................... 300
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