A TRPV4-dependent neuroimmune axis in the spinal cord promotes neuropathic pain methods
Aim. Evidence-backed execution summary for A TRPV4-dependent neuroimmune axis in the spinal cord promotes neuropathic pain methods from A TRPV4-dependent neuroimmune axis in the spinal cord promotes neuropathic pain.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
SNI upregulated TRPV4 expression in the spinal cord resident microglia.
reagent used in the protocol.
- Use
- Next, we investigated how SNI affects TRPV4 expression in the spinal cord. Compared with naive or sham mice, Trpv4 mRNA transcripts were substantially upregulated in the ipsilateral side of the spinal dorsal horn starting within 1 day and lasting for at least 14 days after SNI ( ). RNAscope assay further revealed th...
Microglia-expressed TRPV4 mediated functional and structural plasticity of spinal excitatory neurons through LCN2.
reagent used in the protocol.
- Use
- Microglia communicate with other cell types in the CNS by releasing soluble factors (, ). We showed that many proinflammatory cytokines can be released from activated microglia after SNI (, A-E). Notably, published studies demonstrated that immune cell-derived proinflammatory cytokines generally either modul...
In vitro Ca 2+ imaging.
reagent used in the protocol.
- Use
- Primary cultured microglia were replated at 1 × 10 5 cells per well for at least 2 hours on multi-well glass-bottom dishes (Thermo Fisher Scientific) coated with 10 µg/mL poly l-lysine. Cells were loaded with Fura 2-AM (4 µM; Life Technologies) for 1 hour and subsequently washed and imaged in standard...
Ex vivo Ca 2+ imaging.
reagent used in the protocol.
- Use
- The Cx3cr1 CreER/+:tdTomato:GCaMP6s mice were used for Ca 2+ imaging of microglia in the spinal cord. The spinal slice preparation was performed identically to the electrophysiological recordings. The slices were placed in a recording chamber and microglial Ca 2+ signaling was measured using a Nikon 2-photon system...
Spinal slice preparation and patch-clamp electrophysiology.
reagent used in the protocol.
- Use
- For spinal cord slice preparations, 6- to 8-week-old mice were deeply anesthetized with 2% isoflurane and transcardially perfused with preoxygenated ice-cold sucrose-based artificial cerebrospinal fluid containing 80 mM NaCl, 2.5 mM KCl, 1.25 mM NaH 2 PO 4, 0.5 mM CaCl 2, 3.5 mM MgCl 2, 25 mM NaHCO 3, 75 mM D-su...
Spinal slice preparation and patch-clamp electrophysiology.
reagent used in the protocol.
- Use
- Whole-cell patch-clamp recording was used to record electrical and synaptic activities of the spinal lamina II neurons at 37°C. The resistance of the pipette electrode was 5-8 MΩ when filled with an internal solution containing 120 mM K-gluconate, 20 mM KCl, 2 mM MgCl 2, 2 mM Na 2 -ATP, 0.5 mM Na 2...
Spinal slice preparation and patch-clamp electrophysiology.
reagent used in the protocol.
- Use
- The spinal microglia labeled with GFP or tdTomato were recorded under whole-cell patch-clamp mode. The resistance of the pipette electrode was 8-10 MΩ when filled with an internal solution containing 140 mM CsCl, 1 mM MgCl 2, 0.5 mM EGTA, and 10 mM HEPES (pH 7.3; 310-320 mOsm). The membrane current...
TRPV4 was required for pain-related behaviors following spared nerve injury.
Besides reflexive pain-related behaviors, we also performed CatWalk gait analysis in mice subjected to SNI. This quantitative gait analysis represents an objective way to reflect changes in various walking parameters due to pain and motor impairment and has been successfully used in previous studies to assess nonref...
- Use
- Besides reflexive pain-related behaviors, we also performed CatWalk gait analysis in mice subjected to SNI. This quantitative gait analysis represents an objective way to reflect changes in various walking parameters due to pain and motor impairment and has been successfully used in previous studies to assess nonref...
SNI upregulated TRPV4 expression in the spinal cord resident microglia.
Next, we investigated how SNI affects TRPV4 expression in the spinal cord. Compared with naive or sham mice, Trpv4 mRNA transcripts were substantially upregulated in the ipsilateral side of the spinal dorsal horn starting within 1 day and lasting for at least 14 days after SNI ( ). RNAscope assay further revealed th...
- Use
- Next, we investigated how SNI affects TRPV4 expression in the spinal cord. Compared with naive or sham mice, Trpv4 mRNA transcripts were substantially upregulated in the ipsilateral side of the spinal dorsal horn starting within 1 day and lasting for at least 14 days after SNI ( ). RNAscope assay further revealed th...
SNI upregulated TRPV4 expression in the spinal cord resident microglia.
Interestingly, none of the TRPV4-eGFP + cells expressed CCR2 ( ), suggesting that TRPV4-eGFP + cells are a subpopulation of spinal resident microglia but not infiltrated blood-borne monocytes. Given that the contribution of infiltrating monocytes to nerve injury-induced microgliosis is still an area of controv...
- Use
- Interestingly, none of the TRPV4-eGFP + cells expressed CCR2 ( ), suggesting that TRPV4-eGFP + cells are a subpopulation of spinal resident microglia but not infiltrated blood-borne monocytes. Given that the contribution of infiltrating monocytes to nerve injury-induced microgliosis is still an area of controv...
In vitro Ca 2+ imaging.
Primary cultured microglia were replated at 1 × 10 5 cells per well for at least 2 hours on multi-well glass-bottom dishes (Thermo Fisher Scientific) coated with 10 µg/mL poly l-lysine. Cells were loaded with Fura 2-AM (4 µM; Life Technologies) for 1 hour and subsequently washed and imaged in standard...
- Use
- Primary cultured microglia were replated at 1 × 10 5 cells per well for at least 2 hours on multi-well glass-bottom dishes (Thermo Fisher Scientific) coated with 10 µg/mL poly l-lysine. Cells were loaded with Fura 2-AM (4 µM; Life Technologies) for 1 hour and subsequently washed and imaged in standard...
Ex vivo Ca 2+ imaging.
The Cx3cr1 CreER/+:tdTomato:GCaMP6s mice were used for Ca 2+ imaging of microglia in the spinal cord. The spinal slice preparation was performed identically to the electrophysiological recordings. The slices were placed in a recording chamber and microglial Ca 2+ signaling was measured using a Nikon 2-photon system...
- Use
- The Cx3cr1 CreER/+:tdTomato:GCaMP6s mice were used for Ca 2+ imaging of microglia in the spinal cord. The spinal slice preparation was performed identically to the electrophysiological recordings. The slices were placed in a recording chamber and microglial Ca 2+ signaling was measured using a Nikon 2-photon system...
In vivo 2-photon Ca 2+ imaging.
The GCaMP6s-expressing Vglut2 Cre/+ mice were anesthetized with 2% isoflurane. The skin was incised to expose the T12-L3 vertebrae and to remove the paravertebral muscle. A mouse spinal adaptor (World Precision Instruments) was attached to the vertebrae and a laminectomy was performed from T13-L1. A cust...
- Use
- The GCaMP6s-expressing Vglut2 Cre/+ mice were anesthetized with 2% isoflurane. The skin was incised to expose the T12-L3 vertebrae and to remove the paravertebral muscle. A mouse spinal adaptor (World Precision Instruments) was attached to the vertebrae and a laminectomy was performed from T13-L1. A cust...
Spinal slice preparation and patch-clamp electrophysiology.
For spinal cord slice preparations, 6- to 8-week-old mice were deeply anesthetized with 2% isoflurane and transcardially perfused with preoxygenated ice-cold sucrose-based artificial cerebrospinal fluid containing 80 mM NaCl, 2.5 mM KCl, 1.25 mM NaH 2 PO 4, 0.5 mM CaCl 2, 3.5 mM MgCl 2, 25 mM NaHCO 3, 75 mM D-su...
- Use
- For spinal cord slice preparations, 6- to 8-week-old mice were deeply anesthetized with 2% isoflurane and transcardially perfused with preoxygenated ice-cold sucrose-based artificial cerebrospinal fluid containing 80 mM NaCl, 2.5 mM KCl, 1.25 mM NaH 2 PO 4, 0.5 mM CaCl 2, 3.5 mM MgCl 2, 25 mM NaHCO 3, 75 mM D-su...
Spinal slice preparation and patch-clamp electrophysiology.
Whole-cell patch-clamp recording was used to record electrical and synaptic activities of the spinal lamina II neurons at 37°C. The resistance of the pipette electrode was 5-8 MΩ when filled with an internal solution containing 120 mM K-gluconate, 20 mM KCl, 2 mM MgCl 2, 2 mM Na 2 -ATP, 0.5 mM Na 2...
- Use
- Whole-cell patch-clamp recording was used to record electrical and synaptic activities of the spinal lamina II neurons at 37°C. The resistance of the pipette electrode was 5-8 MΩ when filled with an internal solution containing 120 mM K-gluconate, 20 mM KCl, 2 mM MgCl 2, 2 mM Na 2 -ATP, 0.5 mM Na 2...
Statistics.
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All values are reported as the mean ± SEM. Sample sizes were chosen based on the sample size determination ( ), analysis of relevant prior studies and our pilot studies, and considerations including technical restraints, resource availability, and ethics of animal use. Unless otherwise defined, n numbers in fig...
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TRPV4 was required for pain-related behaviors following spared nerve injury.
Although sensory TRP channels are generally considered critical molecular sensors for mechanical, thermal, and chemical cues and contribute to both pain and itch sensations (, - ), the role of centrally expressed TRPV4 in neuropathic pain remains poorly understood. To test this possibility, we generated a well-established mouse model of SNI ( ) in both Trpv4 -/- (global Trpv4 KO) and congenic WT C57BL/6J mice. Strikingly, compared with WT mice, Trpv4 -/- mice had a significantly improved paw withdrawal threshold, a hallmark of mechanical allodynia, in both male and female mice following SNI ( ). To complement genetic ablation studies, we administered TRPV4 antagonist GSK219 once daily through repeated i.p. injections for 7 consecutive days starting on day 1 after SNI in WT mice and observed a marked increase in paw withdrawal threshold in a dose- and tim...
SNI upregulated TRPV4 expression in the spinal cord resident microglia.
Next, we investigated how SNI affects TRPV4 expression in the spinal cord. Compared with naive or sham mice, Trpv4 mRNA transcripts were substantially upregulated in the ipsilateral side of the spinal dorsal horn starting within 1 day and lasting for at least 14 days after SNI ( ). RNAscope assay further revealed the presence of Trpv4 mRNA transcripts in CX3CR1 + microglia in the spinal cord of Cx3cr1 CreER/+:tdTomato mice on day 7 after SNI ( ). Moreover, flow cytometric analysis using Trpv4 eGFP mice with CD11b + CD45 + gating strategy revealed that,compared with the contralateral side, the number of TRPV4-eGFP + CX3CR1 + - but not TRPV4-eGFP + CCR2 + - cells was markedly increased in the ipsilateral side of the spinal cord on day 7 after SNI ( ). In line with the flow cytometry result, immunofluorescence revealed a marked increase in the number of TRPV4-eGFP + cells co...
SNI upregulated TRPV4 expression in the spinal cord resident microglia.
Interestingly, none of the TRPV4-eGFP + cells expressed CCR2 ( ), suggesting that TRPV4-eGFP + cells are a subpopulation of spinal resident microglia but not infiltrated blood-borne monocytes. Given that the contribution of infiltrating monocytes to nerve injury-induced microgliosis is still an area of controversy, we further verified whether there was an infiltration of TRPV4-expressing blood-borne monocytes in the spinal dorsal horn in response to SNI. We first crossed a double-transgenic Trpv4 eGFP: Ccr2 RFP/+ line and Trpv4 eGFP: Ccr2 CreER/+:tdTomato line bearing eGFP-labeled TRPV4 + cells and RFP/tdTomato-labeled CCR2 + monocytes and observed no infiltrated CCR2 + /IBA1 + monocytes or CCR2 + /TRPV4 + cells in the spinal dorsal horn on day 7 after SNI ( and ), which is consistent with our flow cytometry observation. Additionally, to exclude the possibility of infiltratin...
SNI upregulated TRPV4 expression in the spinal cord resident microglia.
Next, we performed i.p. injections of 5-ethynyl-20-deoxyuridine (EdU) to the Trpv4 eGFP mice following SNI surgery. As expected, most of the EdU + microglia in the ipsilateral side of the spinal dorsal horn on day 7 after SNI were eGFP + ( ). Collectively, these findings indicate that the increased number of the TRPV4-eGFP + cells in the ipsilateral side of the dorsal horn following SNI was due to the proliferation of resident microglia rather than the infiltration of blood-borne monocytes.
Spinal activation of TRPV4 was sufficient to promote excitatory neuronal hyperactivity and produce acute mechanical p...
Bidirectional microglia-neuron communications are critical to the generation and maintenance of neuropathic pain (, ). We reasoned that if TRPV4 was a critical mediator of SNI-induced mechanical allodynia and microglial activation, direct chemical activation of spinal TRPV4 alone should be sufficient to cause pain hypersensitivity. Indeed, a single i.t. injection of GSK101 markedly reduced the paw-withdrawal threshold, which was reversed by coapplied GSK219 ( ). Moreover, GSK101-induced mechanical allodynia was significantly abolished in global Trpv4 KO mice ( ) and microglia-specific Trpv4 cKO mice ( ), but not in endothelial cell-specific or neuron-specific Trpv4 cKO mice ( and ). Interestingly, the mechanical hypersensitivity induced by i.t. application of GSK101 was reversible between 24-48 hours after injection, suggesting an acute and reversible activation/sensitization o...
Methods
Adult (8-12 weeks) male and/or female C57BL/6J mice (The Jackson Laboratory), Trpv4 eGFP (The Mutant Mouse Resource and Research Center [MMRRC]), Trpv4 -/- (donated by Makoto Suzuki and Atsuko Mizuno [Jichi Medical School, Minamikawachi, Tochigi, Japan]), Trpv4 flox ( ), Cx3cr1 CreER (The Jackson Laboratory), Tmem119 CreER (The Jackson Laboratory), tdTomato (Ai9, The Jackson Laboratory), GCaMP6s (Ai96, The Jackson Laboratory), Cx3cr1 GFP (The Jackson Laboratory), Ccr2 RFP (The Jackson Laboratory), Ccr2 CreER (provided in-house), Trpv1 Cre (donated by Mark Hoon [National Institute of Dental and Craniofacial Research, Bethesda, Maryland, USA]), Cdh5 Cre (The Jackson Laboratory), Vglut2 Cre (The Jackson Laboratory), Vgat Cre (The Jackson Laboratory), Lcn2 -/- (The Jackson Laboratory), and Lcn2 flox (donated by Jack Cowland [Rigshospitalet, Copenhagen, Denmar...
Microglia-expressed TRPV4 was necessary for SNI-induced functional and structural plasticity of spinal excitatory neu...
Given that central sensitization also depends on the structural plasticity of spinal neurons in chronic pain states ( ), we sought to investigate whether peripheral nerve injury induced dendrite spine remodeling beyond functional plasticity in the spinal neurons and whether it relies on the function of microglia-expressed TRPV4. Indeed, neurons in spinal lamina IIo stained by microinjection of biocytin exhibited a significant increase in the density of dendrite spines on day 7 after SNI compared with neurons from the sham control mice (, A and B). This SNI-induced increase in dendrite spine density was reduced in both global Trpv4 KO (, C and D) and microglia-specific Trpv4 cKO (, E and F) mice, as well as mice treated with repeated i.p. injections of GSK219 for 7 days after SNI (, G and H) when compared with their respective controls. These findings demonstrate that the transitio...
Microglia-expressed TRPV4 mediated functional and structural plasticity of spinal excitatory neurons through LCN2.
Microglia communicate with other cell types in the CNS by releasing soluble factors (, ). We showed that many proinflammatory cytokines can be released from activated microglia after SNI (, A-E). Notably, published studies demonstrated that immune cell-derived proinflammatory cytokines generally either modulate or have no effect on dendritic spine density in the CNS ( - ). Interestingly, we found that TRPV4 activation in microglia also markedly upregulated the production of LCN2, a secreted protein that was shown to be an important regulator of neuronal excitability in the CNS (,, ). Further, LCN2 expression is significantly upregulated in a mouse model of chronic neuropathic pain caused by spinal nerve ligation ( ). Importantly, LCN2 expression and function are closely related to spine density and membrane excitability in the CNS in a region-specific manner (, ), pro...
Measurement outputs
What raw and processed outputs should exist?
Besides reflexive pain-related behaviors, we also performed CatWalk gait analysis in mice subjected to SNI. This quantitative gait analysis represents an objective way to reflec...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Although sensory TRP channels are generally considered critical molecular sensors for mechanical, thermal, and chemical cues and contribute to both pain and itch sensations (,...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
To confirm the absence of functional TRPV4 in mouse spinal neurons, we selectively knocked down TRPV4 function in the spinal neurons by intraspinal injection of AAV-hSyn-EGFP-Cr...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Next, we investigated how SNI affects TRPV4 expression in the spinal cord. Compared with naive or sham mice, Trpv4 mRNA transcripts were substantially upregulated in the ipsilat...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
Although sensory TRP channels are generally considered critical molecular sensors for mechanical, thermal, and chemical cues and contribute to both pain and itch sensations (, - ), the role of centrally expressed TRPV4 in neuropathic pain remains poorly understood.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Besides reflexive pain-related behaviors, we also performed CatWalk gait analysis in mice subjected to SNI. This quantitative gait analysis represents an objective way to reflec...; Although sensory TRP channels are generally considered critical molecular sensors for mechanical, thermal, and chemical cues and contribute to both pain and itch sensations (,...; To confirm the absence of functional TRPV4 in mouse spinal neurons, we selectively knocked down TRPV4 function in the spinal neurons by intraspinal injection of AAV-hSyn-EGFP-Cr...; Next, we investigated how SNI affects TRPV4 expression in the spinal cord. Compared with naive or sham mice, Trpv4 mRNA transcripts were substantially upregulated in the ipsilat....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
Although sensory TRP channels are generally considered critical molecular sensors for mechanical, thermal, and chemical cues and contribute to both pain and itch sensations (,...; Given that central sensitization also depends on the structural plasticity of spinal neurons in chronic pain states ( ), we sought to investigate whether peripheral nerve injury...; All values are reported as the mean ± SEM. Sample sizes were chosen based on the sample size determination ( ), analysis of relevant prior studies and our pilot studies, an...
from paperReporting output
Report representative outputs alongside summary comparisons for Besides reflexive pain-related behaviors, we also performed CatWalk gait analysis in mice subjected to SNI. This quantitative gait analysis represents an objective way to reflec..., Although sensory TRP channels are generally considered critical molecular sensors for mechanical, thermal, and chemical cues and contribute to both pain and itch sensations (,..., To confirm the absence of functional TRPV4 in mouse spinal neurons, we selectively knocked down TRPV4 function in the spinal neurons by intraspinal injection of AAV-hSyn-EGFP-Cr..., Next, we investigated how SNI affects TRPV4 expression in the spinal cord. Compared with naive or sham mice, Trpv4 mRNA transcripts were substantially upregulated in the ipsilat....
inferred from protocolStructured statistical methods
Although sensory TRP channels are generally considered critical molecular sensors for mechanical, thermal, and chemical cues and contribute to both pain and itch sensations (,...; Given that central sensitization also depends on the structural plasticity of spinal neurons in chronic pain states ( ), we sought to investigate whether peripheral nerve injury...; All values are reported as the mean ± SEM. Sample sizes were chosen based on the sample size determination ( ), analysis of relevant prior studies and our pilot studies, an...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Although sensory TRP channels are generally considered critical molecular sensors for mechanical, thermal, and chemical cues and contribute to both pain and itch sensations (, - ), the role of centrally expressed TRPV4 in neuropathic pain remains poorly understood. To test this possibility, we generated a well-established mouse model of SNI ( ) in both Trpv4 -/- (global Trpv4 KO) and congenic WT C57BL/6J mice. Strikingly, compared with WT mice, Trpv4 -/- mice had a significantly improved paw withdrawal threshold, a hallmark of mechanical allodynia, in both male and female mice following SNI ( ). To complement genetic ablation studies, we administered TRPV4 antagonist GSK219 once daily through repeated i.p. injections for 7 consecutive days starting on day 1 after SNI in WT mice and observed a marked increase in paw withdrawal threshold in a dose- and time-dependent manner ( ). Moreover, acute application of GSK219 through a single intrathecal (i.t.) injection on day 7 after SNI also produced a time- and dose-dependent attenuation of the decreased paw withdrawal threshold caused by SNI ( ), suggesting that TRPV4 in the spinal cord is involved in bot...
Next, we investigated how SNI affects TRPV4 expression in the spinal cord. Compared with naive or sham mice, Trpv4 mRNA transcripts were substantially upregulated in the ipsilateral side of the spinal dorsal horn starting within 1 day and lasting for at least 14 days after SNI ( ). RNAscope assay further revealed the presence of Trpv4 mRNA transcripts in CX3CR1 + microglia in the spinal cord of Cx3cr1 CreER/+:tdTomato mice on day 7 after SNI ( ). Moreover, flow cytometric analysis using Trpv4 eGFP mice with CD11b + CD45 + gating strategy revealed that,compared with the contralateral side, the number of TRPV4-eGFP + CX3CR1 + - but not TRPV4-eGFP + CCR2 + - cells was markedly increased in the ipsilateral side of the spinal cord on day 7 after SNI ( ). In line with the flow cytometry result, immunofluorescence revealed a marked increase in the number of TRPV4-eGFP + cells coexpressing IBA1 or CD31 in the ipsilateral side of the spinal cord on day 7 after SNI ( ). Of note, TRPV4-eGFP signals were only increased in IBA1 + microglia, but not in CD31 + endothelial cells ( ). We also crossed the Trpv4 eGFP mice with the Cx3cr1 CreER/+:tdTomato ( ) or Tmem119 CreER/+:tdTom...
Interestingly, none of the TRPV4-eGFP + cells expressed CCR2 ( ), suggesting that TRPV4-eGFP + cells are a subpopulation of spinal resident microglia but not infiltrated blood-borne monocytes. Given that the contribution of infiltrating monocytes to nerve injury-induced microgliosis is still an area of controversy, we further verified whether there was an infiltration of TRPV4-expressing blood-borne monocytes in the spinal dorsal horn in response to SNI. We first crossed a double-transgenic Trpv4 eGFP: Ccr2 RFP/+ line and Trpv4 eGFP: Ccr2 CreER/+:tdTomato line bearing eGFP-labeled TRPV4 + cells and RFP/tdTomato-labeled CCR2 + monocytes and observed no infiltrated CCR2 + /IBA1 + monocytes or CCR2 + /TRPV4 + cells in the spinal dorsal horn on day 7 after SNI ( and ), which is consistent with our flow cytometry observation. Additionally, to exclude the possibility of infiltrating CX3CR1 + monocytes in the spinal cord in response to peripheral nerve injury, we treated the Cx3cr1 CreER/+:tdTomato reporter mice with tamoxifen to label all CX3CR1 + myeloid cell populations, including spinal resident microglia and blood-borne monocytes. As the cell turnover rates differ betwee...
Next, we performed i.p. injections of 5-ethynyl-20-deoxyuridine (EdU) to the Trpv4 eGFP mice following SNI surgery. As expected, most of the EdU + microglia in the ipsilateral side of the spinal dorsal horn on day 7 after SNI were eGFP + ( ). Collectively, these findings indicate that the increased number of the TRPV4-eGFP + cells in the ipsilateral side of the dorsal horn following SNI was due to the proliferation of resident microglia rather than the infiltration of blood-borne monocytes.
Bidirectional microglia-neuron communications are critical to the generation and maintenance of neuropathic pain (, ). We reasoned that if TRPV4 was a critical mediator of SNI-induced mechanical allodynia and microglial activation, direct chemical activation of spinal TRPV4 alone should be sufficient to cause pain hypersensitivity. Indeed, a single i.t. injection of GSK101 markedly reduced the paw-withdrawal threshold, which was reversed by coapplied GSK219 ( ). Moreover, GSK101-induced mechanical allodynia was significantly abolished in global Trpv4 KO mice ( ) and microglia-specific Trpv4 cKO mice ( ), but not in endothelial cell-specific or neuron-specific Trpv4 cKO mice ( and ). Interestingly, the mechanical hypersensitivity induced by i.t. application of GSK101 was reversible between 24-48 hours after injection, suggesting an acute and reversible activation/sensitization of pain-transmitting neurons in the spinal cord.
Adult (8-12 weeks) male and/or female C57BL/6J mice (The Jackson Laboratory), Trpv4 eGFP (The Mutant Mouse Resource and Research Center [MMRRC]), Trpv4 -/- (donated by Makoto Suzuki and Atsuko Mizuno [Jichi Medical School, Minamikawachi, Tochigi, Japan]), Trpv4 flox ( ), Cx3cr1 CreER (The Jackson Laboratory), Tmem119 CreER (The Jackson Laboratory), tdTomato (Ai9, The Jackson Laboratory), GCaMP6s (Ai96, The Jackson Laboratory), Cx3cr1 GFP (The Jackson Laboratory), Ccr2 RFP (The Jackson Laboratory), Ccr2 CreER (provided in-house), Trpv1 Cre (donated by Mark Hoon [National Institute of Dental and Craniofacial Research, Bethesda, Maryland, USA]), Cdh5 Cre (The Jackson Laboratory), Vglut2 Cre (The Jackson Laboratory), Vgat Cre (The Jackson Laboratory), Lcn2 -/- (The Jackson Laboratory), and Lcn2 flox (donated by Jack Cowland [Rigshospitalet, Copenhagen, Denmark], Bin Gao [National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, USA], and Grace Guo [Rutgers University, Piscataway, New Jersey, USA]) mice were used for this study. Mice were bred and maintained (3-5 mice per cage) in individually ventilated cages on a 12 hour light/12 ho...
Given that central sensitization also depends on the structural plasticity of spinal neurons in chronic pain states ( ), we sought to investigate whether peripheral nerve injury induced dendrite spine remodeling beyond functional plasticity in the spinal neurons and whether it relies on the function of microglia-expressed TRPV4. Indeed, neurons in spinal lamina IIo stained by microinjection of biocytin exhibited a significant increase in the density of dendrite spines on day 7 after SNI compared with neurons from the sham control mice (, A and B). This SNI-induced increase in dendrite spine density was reduced in both global Trpv4 KO (, C and D) and microglia-specific Trpv4 cKO (, E and F) mice, as well as mice treated with repeated i.p. injections of GSK219 for 7 days after SNI (, G and H) when compared with their respective controls. These findings demonstrate that the transition of synaptic potentiation to structural alteration in the spinal cord pain circuit was dependent on the function of microglia-expressed TRPV4, which may underlie pain chronicity.
Microglia communicate with other cell types in the CNS by releasing soluble factors (, ). We showed that many proinflammatory cytokines can be released from activated microglia after SNI (, A-E). Notably, published studies demonstrated that immune cell-derived proinflammatory cytokines generally either modulate or have no effect on dendritic spine density in the CNS ( - ). Interestingly, we found that TRPV4 activation in microglia also markedly upregulated the production of LCN2, a secreted protein that was shown to be an important regulator of neuronal excitability in the CNS (,, ). Further, LCN2 expression is significantly upregulated in a mouse model of chronic neuropathic pain caused by spinal nerve ligation ( ). Importantly, LCN2 expression and function are closely related to spine density and membrane excitability in the CNS in a region-specific manner (, ), prompting us to hypothesize that LCN2 may be involved in TRPV4-mediated spinal synaptic plasticity and neuropathic pain. To test this hypothesis, we first checked LCN2 expression in the spinal cord and determined the role of TRPV4 in LCN2 production. Following SNI, LCN2 was specifically expressed in th...
Machine-readable layer
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"name": "TRPV4 was required for pain-related behaviors following spared nerve injury.",
"text": "Although sensory TRP channels are generally considered critical molecular sensors for mechanical, thermal, and chemical cues and contribute to both pain and itch sensations (, - ), the role of centrally expressed TRPV4 in neuropathic pain remains poorly understood. To test this possibility, we generated a well-established mouse model of SNI ( ) in both Trpv4 -/- (global Trpv4 KO) and congenic WT C57BL/6J mice. Strikingly, compared with WT mice, Trpv4 -/- mice had a significantly improved paw withdrawal threshold, a hallmark of mechanical allodynia, in both male and female mice following SNI ( ). To complement genetic ablation studies, we administered TRPV4 antagonist GSK219 once daily through repeated i.p. injections for 7 consecutive days starting on day 1 after SNI in WT mice and observed a marked increase in paw withdrawal threshold in a dose- and tim..."
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"name": "SNI upregulated TRPV4 expression in the spinal cord resident microglia.",
"text": "Next, we investigated how SNI affects TRPV4 expression in the spinal cord. Compared with naive or sham mice, Trpv4 mRNA transcripts were substantially upregulated in the ipsilateral side of the spinal dorsal horn starting within 1 day and lasting for at least 14 days after SNI ( ). RNAscope assay further revealed the presence of Trpv4 mRNA transcripts in CX3CR1 + microglia in the spinal cord of Cx3cr1 CreER/+:tdTomato mice on day 7 after SNI ( ). Moreover, flow cytometric analysis using Trpv4 eGFP mice with CD11b + CD45 + gating strategy revealed that,compared with the contralateral side, the number of TRPV4-eGFP + CX3CR1 + - but not TRPV4-eGFP + CCR2 + - cells was markedly increased in the ipsilateral side of the spinal cord on day 7 after SNI ( ). In line with the flow cytometry result, immunofluorescence revealed a marked increase in the number of TRPV4-eGFP + cells co..."
},
{
"@type": "HowToStep",
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"name": "SNI upregulated TRPV4 expression in the spinal cord resident microglia.",
"text": "Interestingly, none of the TRPV4-eGFP + cells expressed CCR2 ( ), suggesting that TRPV4-eGFP + cells are a subpopulation of spinal resident microglia but not infiltrated blood-borne monocytes. Given that the contribution of infiltrating monocytes to nerve injury-induced microgliosis is still an area of controversy, we further verified whether there was an infiltration of TRPV4-expressing blood-borne monocytes in the spinal dorsal horn in response to SNI. We first crossed a double-transgenic Trpv4 eGFP: Ccr2 RFP/+ line and Trpv4 eGFP: Ccr2 CreER/+:tdTomato line bearing eGFP-labeled TRPV4 + cells and RFP/tdTomato-labeled CCR2 + monocytes and observed no infiltrated CCR2 + /IBA1 + monocytes or CCR2 + /TRPV4 + cells in the spinal dorsal horn on day 7 after SNI ( and ), which is consistent with our flow cytometry observation. Additionally, to exclude the possibility of infiltratin..."
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"position": 4,
"name": "SNI upregulated TRPV4 expression in the spinal cord resident microglia.",
"text": "Next, we performed i.p. injections of 5-ethynyl-20-deoxyuridine (EdU) to the Trpv4 eGFP mice following SNI surgery. As expected, most of the EdU + microglia in the ipsilateral side of the spinal dorsal horn on day 7 after SNI were eGFP + ( ). Collectively, these findings indicate that the increased number of the TRPV4-eGFP + cells in the ipsilateral side of the dorsal horn following SNI was due to the proliferation of resident microglia rather than the infiltration of blood-borne monocytes."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Spinal activation of TRPV4 was sufficient to promote excitatory neuronal hyperactivity and produce acute mechanical p...",
"text": "Bidirectional microglia-neuron communications are critical to the generation and maintenance of neuropathic pain (, ). We reasoned that if TRPV4 was a critical mediator of SNI-induced mechanical allodynia and microglial activation, direct chemical activation of spinal TRPV4 alone should be sufficient to cause pain hypersensitivity. Indeed, a single i.t. injection of GSK101 markedly reduced the paw-withdrawal threshold, which was reversed by coapplied GSK219 ( ). Moreover, GSK101-induced mechanical allodynia was significantly abolished in global Trpv4 KO mice ( ) and microglia-specific Trpv4 cKO mice ( ), but not in endothelial cell-specific or neuron-specific Trpv4 cKO mice ( and ). Interestingly, the mechanical hypersensitivity induced by i.t. application of GSK101 was reversible between 24-48 hours after injection, suggesting an acute and reversible activation/sensitization o..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Methods",
"text": "Adult (8-12 weeks) male and/or female C57BL/6J mice (The Jackson Laboratory), Trpv4 eGFP (The Mutant Mouse Resource and Research Center [MMRRC]), Trpv4 -/- (donated by Makoto Suzuki and Atsuko Mizuno [Jichi Medical School, Minamikawachi, Tochigi, Japan]), Trpv4 flox ( ), Cx3cr1 CreER (The Jackson Laboratory), Tmem119 CreER (The Jackson Laboratory), tdTomato (Ai9, The Jackson Laboratory), GCaMP6s (Ai96, The Jackson Laboratory), Cx3cr1 GFP (The Jackson Laboratory), Ccr2 RFP (The Jackson Laboratory), Ccr2 CreER (provided in-house), Trpv1 Cre (donated by Mark Hoon [National Institute of Dental and Craniofacial Research, Bethesda, Maryland, USA]), Cdh5 Cre (The Jackson Laboratory), Vglut2 Cre (The Jackson Laboratory), Vgat Cre (The Jackson Laboratory), Lcn2 -/- (The Jackson Laboratory), and Lcn2 flox (donated by Jack Cowland [Rigshospitalet, Copenhagen, Denmar..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Microglia-expressed TRPV4 was necessary for SNI-induced functional and structural plasticity of spinal excitatory neu...",
"text": "Given that central sensitization also depends on the structural plasticity of spinal neurons in chronic pain states ( ), we sought to investigate whether peripheral nerve injury induced dendrite spine remodeling beyond functional plasticity in the spinal neurons and whether it relies on the function of microglia-expressed TRPV4. Indeed, neurons in spinal lamina IIo stained by microinjection of biocytin exhibited a significant increase in the density of dendrite spines on day 7 after SNI compared with neurons from the sham control mice (, A and B). This SNI-induced increase in dendrite spine density was reduced in both global Trpv4 KO (, C and D) and microglia-specific Trpv4 cKO (, E and F) mice, as well as mice treated with repeated i.p. injections of GSK219 for 7 days after SNI (, G and H) when compared with their respective controls. These findings demonstrate that the transitio..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Microglia-expressed TRPV4 mediated functional and structural plasticity of spinal excitatory neurons through LCN2.",
"text": "Microglia communicate with other cell types in the CNS by releasing soluble factors (, ). We showed that many proinflammatory cytokines can be released from activated microglia after SNI (, A-E). Notably, published studies demonstrated that immune cell-derived proinflammatory cytokines generally either modulate or have no effect on dendritic spine density in the CNS ( - ). Interestingly, we found that TRPV4 activation in microglia also markedly upregulated the production of LCN2, a secreted protein that was shown to be an important regulator of neuronal excitability in the CNS (,, ). Further, LCN2 expression is significantly upregulated in a mouse model of chronic neuropathic pain caused by spinal nerve ligation ( ). Importantly, LCN2 expression and function are closely related to spine density and membrane excitability in the CNS in a region-specific manner (, ), pro..."
}
],
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"@type": "HowToTool",
"name": "TRPV4 was required for pain-related behaviors following spared nerve injury."
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{
"@type": "HowToTool",
"name": "SNI upregulated TRPV4 expression in the spinal cord resident microglia."
},
{
"@type": "HowToTool",
"name": "SNI upregulated TRPV4 expression in the spinal cord resident microglia."
},
{
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"name": "In vitro Ca 2+ imaging."
},
{
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"name": "Ex vivo Ca 2+ imaging."
},
{
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"name": "In vivo 2-photon Ca 2+ imaging."
},
{
"@type": "HowToTool",
"name": "Spinal slice preparation and patch-clamp electrophysiology."
},
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"@type": "HowToTool",
"name": "Spinal slice preparation and patch-clamp electrophysiology."
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"@type": "HowToSupply",
"name": "SNI upregulated TRPV4 expression in the spinal cord resident microglia."
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"name": "Microglia-expressed TRPV4 mediated functional and structural plasticity of spinal excitatory neurons through LCN2."
},
{
"@type": "HowToSupply",
"name": "In vitro Ca 2+ imaging."
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{
"@type": "HowToSupply",
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"headline": "A TRPV4-dependent neuroimmune axis in the spinal cord promotes neuropathic pain",
"datePublished": "2023",
"author": [
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"@type": "Person",
"name": "Xueming Hu"
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"name": "Lixia Du"
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"name": "Shenbin Liu"
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"name": "Zhou Lan"
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"name": "Kaikai Zang"
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{
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"name": "Jing Feng"
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"name": "Xingliang Yang"
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