The role of neuroinflammation in motor circuit synaptic plasticity within the spinal cord following peripheral nerve injury Open Access
Rotterman, Travis (Spring 2018)
Abstract
Information regarding limb position is transmitted through Ia afferents and is integrated into spinal circuit networks to modulate motor output in response to environmental perturbations. However, following peripheral nerve injuries, a neuroinflammatory response occurs in the ventral horn that coincides with Ia afferent synapse degradation. The outcome after nerve regeneration depends on the type of injury; nerve transection causes permanent loss of Ia synapses and the reflex, while both recover after nerve crush. Our goal was to find causal relationships between spinal neuroinflamation and Ia synaptic losses. We further hypothesized that modulation of neuroinflammation properties could adjust synaptic plasticity to various levels according to injury severity. To test these hypotheses, we performed different types of nerve injuries in transgenic mouse models that allowed us to study resident microglia activation (CX3CR1-GFP mice) and infiltration of peripheral CCR2-expressing macrophages (CCR2-RFP mice), combined with various global and cell-specific knockout models to investigate signaling pathways that triggered the immune response and their relation to Ia synapse loss. Sciatic nerve transection results in long-lasting microglia activation and differentiation towards CCL2-relasing, pro-inflammatory phenotypes. This did not occur after sciatic nerve crush. Only after transection was there significant infiltration of CD45/CCR2 cells, including T-cells, monocyte/macrophages, and dendritic cells. CCL2-CCR2 signaling was especially critical for monocyte infiltration, some of which appear to transform into microglia-like cells by morphology and genetic lineage labeling. Lack of these cells in CCR2 global KOs correlated with better preservation of ventral horn Ia synapses after nerve transection. Moreover, CCR2 cell entry is graded to the location of injury with injuries to more distal nerves causing less CCR2 infiltration and less Ia synapse plasticity. In conclusion, promoting CCR2+ monocyte infiltration amplifies Ia synapse losses, and the properties of spinal neuroinflammation after different nerve injuries is under fine control by signaling between axotomized MNs, microglia and penetrating blood-derived CCR2+ immune cells.
Table of Contents
Table of Contents
Chapter 1: Introduction to spinal cord circuitry and plasticity following
peripheral nerve injury……………………………….………………......................1
1.1 Introduction…………………………………………………….……............2
1.2 Organization and function of spinal cord motor circuitry …………........3
1.2.1 Motoneurons……………………………………..…....................3
1.2.2 Ia afferent connections…….……………………………………..7
1.2.3 Stretch reflex and reciprocal inhibition……………..……….....11
1.3 Peripheral nerve injury……………………………..……………………...14
1.3.1 Type and prevalence of nerve injuries……...…......................16
1.3.2 Peripheral degeneration and reinnervation……………….......19
1.3.3 Clinical intervention and patient outcomes.…………..............24
1.3.4 Functional deficits in the Ia stretch circuit……………………..28
1.4 Synaptic stripping vs axon retraction and transganglionic
anterograde degeneration of sensory afferents injured in the periphery..35
1.4.1 History of synaptic stripping………..…………….…................37
1.4.2 Evidence of anterograde transganglionic degeneration of
sensory axons and synapses after PNI……………………………..39
1.4.3 Mechanisms involved…………………………………………...44
1.5 Neuroinflammation…………….…………….…………………………….51
1.5.1 Microglia………………………………………………………….53
1.5.2 Macrophage recruitment and infiltration into the CNS…...….63
1.5.3 Role of macrophages/microglia in synaptic stripping
and retraction……………………………………………………….…..68
1.5.4 Astrocytes…………………………………………………….......71
1.5.5 T-cells in PNI……………………………………………………..75
1.6 Dissertation overview………………………………………….................76
References…………………………………………………………………….. 83
Chapter 2: Normal distribution of VGLUT1 synapses on spinal
motoneuron dendrites and their reorganization after
nerve injury………………………………………..……………….………….....….104
2.1 Abstract…..…………………………………………………….…………105
2.2 Introduction……………….………………………………………………106
2.3 Methods……………………………………………….…………………..108
2.3.1 Peripheral nerve transection and repair…….……………….108
2.3.2 Electrophysiology………………………………………………110
2.3.3 Histological processing and immunohistochemistry………..111
2.3.4 Confocal imaging and neuron reconstruction……………….113
2.3.5 Neuron reconstructions………………………………………..114
2.3.6 Analyses………………………………………………………...114
2.3.7 Statistics………………………………………………………...117
2.4 Results…………………………………………………..………………...117
2.4.1 Basic physiological and morphological properties do not
differ between control and regenerated motoneurons…………....117
2.4.2 VGLUT1-IR synapses are lost in injured and regenerated
MG motoneurons…………………………………………………..... 121
2.4.3 VGLUT1 contacts accumulate on proximal dendrites……...123
2.4.4 Proximal VGLUT1 contacts are preferentially lost after
nerve injury……………………………………………………..….....125
2.4.5 VGLUT1 contact groupings are dispersed after peripheral
nerve injury……………………………………………………………128
2.4.6 VGLUT1-IR contacts show a preference for dendrite territories
located dorso-medially with respect to the cell body and this
preferred location becomes more apparent after injury…….........130
2.5 Discussion………………………………………………………………...134
2.5.1 VGLUT1 synapses concentrate in proximal dendrites…….134
2.5.2 Significance of VGLUT1 synapses remaining after
nerve injury…………………………………………………….....…..136
2.5.3 Relationship between synaptic reorganization and Ia afferent
input strength……………………………………………………….....138
References…………………………………………………………….………165
Chapter 3: Motor circuit synaptic plasticity after peripheral nerve injury
depends on a central neuroinflammatory response and a CCR2
mechanism. …………………………………………………….......................….. 169
3.1 Abstract……………………………………………………………………170
3.2 Introduction………………………………………………………….…… 171
3.3 Methods…………………………………………………………............. 173
3.3.1 Transgenic models……………………………………………..174
3.3.2 Motoneuron labeling and peripheral nerve injury
procedures ………………...………………………………………….176
3.3.3 Harvesting tissue for histological analysis…………………..178
3.3.4 Histological processing and immunohistochemistry………..178
3.3.5 Densitometric analysis of VGLUT1 and GFP
fluorescence…………………………………………………………..180
3.3.6 Analysis of VGLUT1 synaptic bouton densities on
motoneurons…………………………………………………………. 180
3.3.7 Quantification of microglia cells……………………………… 181
3.3.8 Microglia morphological analysis……………………………..182
3.3.9 Motoneuron cell body coverage by microglia cell processes
………………………………………………………………………….182
3.3.10 RFP cell quantification……………………………………….183
3.3.11 Quantification of Iba1 cells after injury that are not derived
from resident microglia…………………………………………...….183
3.3.12 Analysis of CCL2 expression inside the spinal cord after
nerve injuries………………………………..……………………...…184
3.3.13 Statistical analysis for histological analyses……………….185
3.3.14 Peripheral blood mononuclear cell (PBMC) isolation and
spinal cord tissue dissociation for myeloid cell analysis………….187
3.3.15 Flow cytometry………………………………………………..188
3.4 Results…………………………………………………………………….189
3.4.1 Animal model and experimental design……………………..189
3.4.2 VGLUT1 synapse depletions on motoneurons following tibial
or sciatic nerve injury in mice…………………………………..…..192
3.4.3 Microglia responses differ between tibial and sciatic
nerve injuries………………………………………………………..…197
3.4.4 Microglia undergo similar morphological changes after sciatic
or tibial nerve injuries but become more tightly associated to
motoneuron cell bodies after sciatic injuries……………………….199
3.4.5 The number of CCR2-RFP+ cells infiltrating the spinal cord
ventral horn depends on the type of injury and is tightly correlated to
overall VGLUT1 loss………………………………………………….201
3.4.6 Heterogeneity of CCR2+ cells infiltrating the spinal cord after
nerve injury…………………………………………………………… 203
3.4.7 Flow cytometry characterization of CCR2+ cells……………206
3.4.8 VGLUT1 synapses are preserved on motoneuron dendrites of
CCR2 knockout mice…………………………………………………209
3.4.9 CCL2 is upregulated inside the spinal cord by
axotomized motoneurons and activated microglia but with
different time courses……………………………………………….. 212
3.5 Discussion……………………………………………………….……......213
3.5.1 Different mechanisms control plasticity of VGLUT1
synapses on dendrites and cell bodies of motoneurons and
the role of microglia……………………………….………………….214
3.5.2 CCR2 monocyte entry is critical for VGLUT1
synapse plasticity…………………………...………………………. 217
References…………………………………………………………………… 248
Chapter 4: The effect of axon regeneration efficiency in spinal motor circuit plasticity following peripheral nerve injury and the role neuroinflammation plays…………………………………………………………………………………...256
4.1 Abstract…………………………………………………………………...257
4.2 Introduction………………………………………………………….…… 258
4.3 Methods…………………………………………………………..............261
4.3.1 Transgenic mouse models…………………………………… 261
4.3.2 Motoneuron labeling and peripheral nerve injury
procedures …………………...……………………………………….262
4.3.3 Harvesting tissue for histological analysis……………….…..263
4.3.4 Histological processing and immunohistochemistry………..264
4.3.5 Densitometric analysis of VGLUT1 and GFP immune-
fluorescence………………………………………………….…265
4.3.6 Neuromuscular junction analysis……………………………..266
4.3.7 Motoneuron reinnervation……………………………………..266
4.3.8 Edu injections and Ki-67 labeling……….…………………….267
4.3.9 Quantification of microglia cells……………………………….268
4.3.10 CCR2-RFP cell quantification……………………………….269
4.3.11 Experimental design and statistical analysis………………269
4.4 Results…………………………………………………………………….270
4.4.1 VGLUT1 synapse depletion on motoneurons following
sciatic nerve cut-repair or sciatic nerve crush……………….……..270
4.4.2 Rate and specificity of peripheral nerve regeneration
and reinnervation predict the permanency of VGLUT1 loss……..274
4.4.3 The activation and proliferation of microglia cells in the ventral
horn of the spinal cord after nerve injury…….…………..……….. 278
4.4.4 The release of CCL2 and the recruitment of peripheral
CCR2+ myeloid cells following sciatic injury……………..………..281
4.5 Discussion.…………………………………………………………..…...283
4.5.1 Success of peripheral regeneration determines
permanency of VGLUT1 loss……………………………………….283
4.5.2 CCL2 release from microglia results in a robust infiltration of
peripheral CD45+ cells……………………………………………....286
References…………………………………………………………………… 305
Chapter 5: General Discussion and Future Directions……………………….309
5.1 Discussion……………………………………………………………….. 310
5.1.1 Distribution of VGLUT1 synapses on motoneurons
and their permanent loss after nerve injur..……………………….. 312
5.1.2 The permanent removal of Ia afferents is dependent
on CCR2 mechanisms……………………………………………….316
5.1.3 The importance of peripheral nerve regeneration efficiency
and the permanency of Ia synaptic loss……………………………321
5.2 Future directions…………………………………………………….……324
5.2.1 CCL2 Removal from MNs and microglia…………………….324
5.2.2 Microglia activation by CSF1………………………………….325
5.2.3 CCR2 lineage tracing experiments…………………………..327
5.2.4 Live cell imaging………………………………………………..327
5.2.5 Physiology………………………………………………………328
References…………………………………………………………………… 331
Appendix I- VGLUT1 synapses and p-boutons on regenerating motoneurons
after nerve crush…………………………………………………………………... 335
6.1 Abstract……………………………………………………………………336
6.2 Introduction………………………………………………………….…… 337
6.3 Methods…………………………………………………………............. 342
6.3.1 Nerve injury and injections of retrograde tracers………….. 342
6.3.2 Histology and immunocytochemistry……………………….. 343
6.3.3 Antibody characterization……………………………………..344
6.3.4 Confocal imaging and neuron reconstruction……………….345
6.3.5 Surface-to-surface analysis…………………………………...346
6.3.6 Statistical analyses…………………………………………….347
6.3.7 Figure composition…………………………………………….347
6.4 Results…………………………………………………………………….348
6.4.1 Technical considerations and data interpretation…………..348
6.4.2 Nerve crush causes only a small loss of VGLUT1 contacts on
proximal dendrites and cell bodies of regenerating motoneuron
………………………………………………………………………….350
6.4.3 Reduction in P-bouton coverage of VGLUT1 boutons following
nerve crush……………………………………………………………354
6.5 Discussion………………………………………………………………...358
6.5.1 Differences in stretch reflex synaptic circuit plasticity after
different kinds of nerve injury………………………………………..359
6.5.2 Presynaptic inhibition of Ia afferents after nerve crush injuries
………………………………………………………………………….362
References…………………………………………………………………… 380
Appendix II- Two-photon live imaging of microglia dynamics after nerve
injury in an adult spinal cord slice ………………………………………...…...384
7.1 Abstract……………………………………………………………………385
7.2 Introduction………………………………………………………….…… 386
7.3 Methods…………………………………………………………............. 389
7.3.1 Animals………………………………………………………… 389
7.3.2 Injury model…………………………………………………… 390
7.3.3 aCSF preparation………………………………………………390
7.3.4 Spinal cord dissection………………………………………….391
7.3.5 Sectioning……………………………………………………… 391
7.3.6 Imaging parameters……………………………………………392
7.3.7 Analysis…………………………………………………………393
7.3.8 Statistics……………………………………………………….. 394
7.3.9 Tips and tricks………………………………………………….394
7.4 Results…………………………………………………………………….394
7.5 Discussion………………………………………………………………...398
References…………………………………………………………………… 408
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