Dorsal root ganglion macrophages contribute to both the initiation and persistence of neuropathic pain methods
Aim. Evidence-backed execution summary for Dorsal root ganglion macrophages contribute to both the initiation and persistence of neuropathic pain methods from Dorsal root ganglion macrophages contribute to both the initiation and persistence of neuropathic pain.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
DRG macrophages are depleted by systemic AP administration
reagent used in the protocol.
- Use
- To determine whether the increase in DRG macrophages contributes to neuropathic pain development, we chose the MAFIA transgenic line, which as noted above, expresses GFP and a drug-inducible Fas suicide gene under control of the CSF1R promoter (Supplementary Fig. ). The transgene is expressed in CNS microglia...
AP administration
reagent used in the protocol.
- Use
- The AP (Clontech, #635058) was diluted in a distilled water solution consisting of 4% ethanol, 10% PEG-400, and 1.7% Tween 20, and injected intraperitoneally.
In situ hybridization
reagent used in the protocol.
- Use
- We used the RNAscope Fluorescent Multiplex Reagent Kit (Advanced Cell Diagnostics) according to the manufacturer's instructions. Briefly, freshly dissected tissue was quickly frozen on dry ice, cryostat sectioned at 12 µm, and mounted on slides. The mounted sections were fixed in prechilled 10% neut...
Isolation of spinal microglia, DRG macrophages, and peripheral blood cells
reagent used in the protocol.
- Use
- Peripheral blood was drawn from a retro-orbital vein in isoflurane anesthetized mice using heparinized micropipettes (ThermoFisher Scientific #22362566). Red blood cells were lysed first before immunostaining. Fresh isolated spinal cord tissue was digested with collagenase, dissociated, and filtered through cell str...
Flow cytometric assays
reagent used in the protocol.
- Use
- Dissociated cells were briefly fixed in 4% paraformaldehyde for 15 min at room temperature and washed once with FACS staining buffer (5% fetal bovine serum (FBS) in PBS). Cells were then resuspended in 100 µl of saponin buffer (0.5% saponin and 2% FBS in PBS) and immunostained with anti-CD115-PE cy7...
Statistical analysis
Data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software). Student t -tests were used for single comparisons between two groups. Other data were analyzed using one-way or two-way analysis of variance. * P < 0.05, ** P ̴...
- Use
- Data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software). Student t -tests were used for single comparisons between two groups. Other data were analyzed using one-way or two-way analysis of variance. * P < 0.05, ** P ̴...
DRG macrophages are depleted by systemic AP administration
To determine whether the increase in DRG macrophages contributes to neuropathic pain development, we chose the MAFIA transgenic line, which as noted above, expresses GFP and a drug-inducible Fas suicide gene under control of the CSF1R promoter (Supplementary Fig. ). The transgene is expressed in CNS microglia...
- Use
- To determine whether the increase in DRG macrophages contributes to neuropathic pain development, we chose the MAFIA transgenic line, which as noted above, expresses GFP and a drug-inducible Fas suicide gene under control of the CSF1R promoter (Supplementary Fig. ). The transgene is expressed in CNS microglia...
DRG macrophages are depleted by systemic AP administration
Using the 3-day protocol, we next used different approaches to examine the fate of resident macrophages in the DRG and of blood monocytes. We monitored DRG macrophages by immunostaining for GFP and PU.1, a transcription factor expressed in myelomonocytic cells and in their immature precursors. By the third injectio...
- Use
- Using the 3-day protocol, we next used different approaches to examine the fate of resident macrophages in the DRG and of blood monocytes. We monitored DRG macrophages by immunostaining for GFP and PU.1, a transcription factor expressed in myelomonocytic cells and in their immature precursors. By the third injectio...
AP treatment does not alter spinal cord microglia number
Although previous studies concluded that AP does not cross the BBB, we considered it essential to rule out a direct CNS action of the AP treatment. Compared with uninjured, VEH-treated mice, we found no difference in the number of spinal cord microglia by FACS (Fig. ) in the lumbar cord of AP-treated mice. Im...
- Use
- Although previous studies concluded that AP does not cross the BBB, we considered it essential to rule out a direct CNS action of the AP treatment. Compared with uninjured, VEH-treated mice, we found no difference in the number of spinal cord microglia by FACS (Fig. ) in the lumbar cord of AP-treated mice. Im...
Nerve injury triggers a reciprocal interaction between DRG macrophages and sensory neurons
Recently, our laboratory demonstrated that axotomized DRG sensory neurons de novo express Csf1 after peripheral nerve injury. The CSF1, in turn, is transported to the spinal cord dorsal horn, where it activates microglia through the CSF1R, induces microglial proliferation, and promotes the neuropathic pain phenotyp...
- Use
- Recently, our laboratory demonstrated that axotomized DRG sensory neurons de novo express Csf1 after peripheral nerve injury. The CSF1, in turn, is transported to the spinal cord dorsal horn, where it activates microglia through the CSF1R, induces microglial proliferation, and promotes the neuropathic pain phenotyp...
The DRG macrophage expansion, but not the contribution to neuropathic pain initiation and maintenance, is sexually di...
In contrast to the evidence for a contribution of spinal microglia to neuropathic pain in male mice, Sorge et al. reported that ablating microglia in female mice does not prevent the development of mechanical hypersensitivity after peripheral nerve injury. Here we asked whether the contribution of DRG macrophages to...
- Use
- In contrast to the evidence for a contribution of spinal microglia to neuropathic pain in male mice, Sorge et al. reported that ablating microglia in female mice does not prevent the development of mechanical hypersensitivity after peripheral nerve injury. Here we asked whether the contribution of DRG macrophages to...
Bone marrow transplantation
Donor BM cells from CSF1R-GFP mice (CD45.2 + ) were collected at 4-5 weeks of age, enriched by immunomagnetic depletion of cells expressing mature hematopoietic lineage antigens defined by a cocktail of monoclonal antibodies: CD5 (Ly-1), CD11b (Mac-1), CD45R (B220), Gr-1, and TER119 (#19856 A, StemCell T...
- Use
- Donor BM cells from CSF1R-GFP mice (CD45.2 + ) were collected at 4-5 weeks of age, enriched by immunomagnetic depletion of cells expressing mature hematopoietic lineage antigens defined by a cocktail of monoclonal antibodies: CD5 (Ly-1), CD11b (Mac-1), CD45R (B220), Gr-1, and TER119 (#19856 A, StemCell T...
Immunohistochemistry
Mice were anesthetized with 2.5% Avertin and perfused transcardially with 1 × phosphate-buffered saline (PBS) followed by 4% formaldehyde. Dissected tissues were first post-fixed in the same fixative for 3 h, then preserved overnight in 30% sucrose in PBS before cryostat sectioning. The following antibodie...
- Use
- Mice were anesthetized with 2.5% Avertin and perfused transcardially with 1 × phosphate-buffered saline (PBS) followed by 4% formaldehyde. Dissected tissues were first post-fixed in the same fixative for 3 h, then preserved overnight in 30% sucrose in PBS before cryostat sectioning. The following antibodie...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software). Student t -tests were used for single comparisons between two groups. Other data were analyzed using one-way or two-way analysis of variance. * P < 0.05, ** P ̴...
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DRG macrophages are depleted by systemic AP administration
To determine whether the increase in DRG macrophages contributes to neuropathic pain development, we chose the MAFIA transgenic line, which as noted above, expresses GFP and a drug-inducible Fas suicide gene under control of the CSF1R promoter (Supplementary Fig. ). The transgene is expressed in CNS microglia (Supplementary Fig. ) and tissue macrophages, including DRG (Supplementary Fig. ). To deplete monocytic cells in the MAFIA mice, we began our studies with five daily intraperitoneal injections of AP (10 mg kg -1 ), a macrophage-depleting dose used in earlier studies. Supplementary Fig. shows that this dose was without effect on baseline mechanical threshold (S5A) and weight (S5B) in wild-type (WT) mice. By contrast, this regimen resulted in significant weight loss and also increased the baseline mechanical threshold 1 day after the last...
DRG macrophages are depleted by systemic AP administration
Using the 3-day protocol, we next used different approaches to examine the fate of resident macrophages in the DRG and of blood monocytes. We monitored DRG macrophages by immunostaining for GFP and PU.1, a transcription factor expressed in myelomonocytic cells and in their immature precursors. By the third injection, we recorded a significant loss of GFP + /PU.1 + cells (Fig. ) as well as GFP + /Iba1 + cells (Supplementary Fig. ). In a separate group of mice, 1 day after the 3-day AP regime (D3), FACS analysis demonstrated a significant, ~80% depletion of resident (GFP + ) macrophage in the L4 and L5 DRG, compared with VEH mice (Fig. ). At the same time point, FACS analysis of blood demonstrated an almost 90% depletion of mature (GFP hi or CSF1R hi ) circulating monocytes (GFP hi CD11b + or GFP + CSF1R hi ), with no change in the VEH-treated group (Supplementary Fig...
DRG macrophages are depleted by systemic AP administration
We next examined macrophages in the context of nerve injury. To monitor the early response of macrophages, we followed the expression of Cx3cr1 in the DRG by quantitative PCR (qPCR) 1 day after the nerve injury (POD1). We recorded a 2.1 ± 0.4-fold increase of Cx3cr1 expression in the L4/L5 DRG ipsilateral to the nerve injury in VEH-treated mice (Fig. ) compared with the uninjured contralateral DRG. On the other hand, AP treatment, prior to the nerve injury, not only reduced baseline Cx3cr1 expression in the uninjured, contralateral DRG but also prevented the injury-induced increase of Cx3cr1 expression in the ipsilateral DRG (Fig. ). By POD4 (5th day after the last AP treatment), macrophage number recovered to the level recorded in uninjured, VEH-treated mice (Fig. ). By POD9, the ipsilateral DRG expansion of macrophages reappeared and did not differ...
AP treatment does not alter spinal cord microglia number
Although previous studies concluded that AP does not cross the BBB, we considered it essential to rule out a direct CNS action of the AP treatment. Compared with uninjured, VEH-treated mice, we found no difference in the number of spinal cord microglia by FACS (Fig. ) in the lumbar cord of AP-treated mice. Immunostaining for microglia also did not differ (Fig. ). These results are consistent with the AP exerting a selective peripheral action. We also addressed the possibility that a compromised BBB following nerve injury could result in AP leakage into the cord,. Arguing against this possibility, both the nerve injury-induced upregulation of Cx3cr1 gene expression, evidence of microglia activation, 1 day after SNI (POD1; Fig. ) and microgliosis examined 4 days after SNI (POD4; Fig. ), did not differ between VEH- and AP-treated mice. Glial fibrillary acidic p...
DRG macrophages are required for initiation of nerve injury-induced mechanical allodynia
To investigate the contribution of DRG macrophages to nerve injury-induced mechanical hypersensitivity, we treated separate groups of mice with three daily injections of AP (1.0 mg kg -1 ) followed by SNI. In contrast to VEH-treated mice, which developed mechanical hypersensitivity within 24 h of the SNI, the 3-day AP treatment regimen significantly delayed development of the hypersensitivity, for at least 7 days after the nerve injury (Fig. ). Importantly, mice in which macrophage expansion was restored exhibited mechanical allodynia. Continuing the AP treatment into the day of nerve injury did not enhance the anti-allodynic effect (Supplementary Fig. ). These results indicate that the anti-allodynic effect of AP treatment correlates with the prevention of DRG macrophage expansion. Finally, as the AP-induced weight loss persisted in these mice (Sup...
DRG macrophages are required for initiation of nerve injury-induced mechanical allodynia
In a separate experiment we transplanted bone marrow (BM) progenitor cells (GFP + ) isolated from MAFIA mice into irradiated WT mice and confirmed the lack of effect of AP on spinal cord microglia. Specifically, 7 days after the SNI, in contrast to a complete replacement of peripheral host monocytic cells with GFP + donor cells (Supplementary Fig. ), we detected very few GFP + cells in the spinal cord (Supplementary Fig. ). We conclude that the majority of spinal cord microglia (PU1 + ) derived from the host (GFP - ), even after injury (Supplementary Fig. ). In a separate group of transplanted mice, we performed SNI after the 3-day AP or VEH treatment. Consistent with our findings in non-transplanted mice, we found that AP-mediated macrophage depletion in the DRG of the transplanted animals (Supplementary Fig. ) significantly delayed the development of ne...
Initiation of the neuropathic pain phenotype requires macrophage expansion in the DRG and is independent of infiltrat...
Next, we examined the relative contribution of macrophage expansion in the DRG and at the nerve injury site. As AP-mediated systemic macrophage depletion cannot distinguish their respective contribution, we designed the following experiment to deplete selectively macrophages at the injury site, leaving DRG macrophages intact. At the time of the nerve injury, we implanted a cannula and sutured it to muscles that overlay the injury site. Our intent was to deliver a low AP dose that only targeted the macrophages around the injury site, with limited to no systemic action. After serial dose titrations, we identified a systemic dose (0.8 µg in 20 µl) that, when administered daily for 3 days, did not influence nerve injury-induced mechanical allodynia (Supplementary Fig. ) or cause weight loss (Supplementary Fig. ). We administered the low-dose AP via the ca...
Macrophages in the DRG contribute to maintenance of nerve injury-induced mechanical allodynia
SNI-induced mechanical allodynia is typically long lasting and this behavioral phenotype is associated with prolonged microglial activation in the ipsilateral dorsal horn. Figure illustrates, e.g., that microglial activation persists for at least 4 weeks after peripheral nerve injury. At this time point, we also recorded a persistent increase in the number of macrophages in the ipsilateral DRG (Figs. a and ) and at the nerve injury site (Fig. ). Fig. 3 Microglia activation and peripheral macrophage infiltration persist at 28 days post SNI. a - c Immunostaining of Iba1 + microglia in the dorsal horn ( a ), CSF1R-GFP + macrophages (green) in the DRG ( b ), and at the nerve injury site ( c ), 28 days after SNI (POD28). NF200 (blue) labels neuronal cell bodies ( b ) and peripheral nerve myelinated axons ( c ). Dashed line in c denotes nerve ligature site. Scale bar...
Measurement outputs
What raw and processed outputs should exist?
Data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software). Student t -tests were used for single...
- 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 determine whether the increase in DRG macrophages contributes to neuropathic pain development, we chose the MAFIA transgenic line, which as noted above, expresses GFP and a...
- 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
Using the 3-day protocol, we next used different approaches to examine the fate of resident macrophages in the DRG and of blood monocytes. We monitored DRG macrophages by immuno...
- 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
We next examined macrophages in the context of nerve injury. To monitor the early response of macrophages, we followed the expression of Cx3cr1 in the DRG by quantitative PCR (q...
- 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
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inferred from protocolPreprocessing / cleaning
Data are expressed as mean ± SEM.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software). Student t -tests were used for single...; To determine whether the increase in DRG macrophages contributes to neuropathic pain development, we chose the MAFIA transgenic line, which as noted above, expresses GFP and a...; Using the 3-day protocol, we next used different approaches to examine the fate of resident macrophages in the DRG and of blood monocytes. We monitored DRG macrophages by immuno...; We next examined macrophages in the context of nerve injury. To monitor the early response of macrophages, we followed the expression of Cx3cr1 in the DRG by quantitative PCR (q....
from paperStatistical comparison
Data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software). Student t -tests were used for single...; To determine whether the increase in DRG macrophages contributes to neuropathic pain development, we chose the MAFIA transgenic line, which as noted above, expresses GFP and a...; Using the 3-day protocol, we next used different approaches to examine the fate of resident macrophages in the DRG and of blood monocytes. We monitored DRG macrophages by immuno...; Next, we examined the relative contribution of macrophage expansion in the DRG and at the nerve injury site. As AP-mediated systemic macrophage depletion cannot distinguish thei...
from paperReporting output
Report representative outputs alongside summary comparisons for Data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software). Student t -tests were used for single..., To determine whether the increase in DRG macrophages contributes to neuropathic pain development, we chose the MAFIA transgenic line, which as noted above, expresses GFP and a..., Using the 3-day protocol, we next used different approaches to examine the fate of resident macrophages in the DRG and of blood monocytes. We monitored DRG macrophages by immuno..., We next examined macrophages in the context of nerve injury. To monitor the early response of macrophages, we followed the expression of Cx3cr1 in the DRG by quantitative PCR (q....
inferred from protocolStructured statistical methods
Data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software). Student t -tests were used for single...; To determine whether the increase in DRG macrophages contributes to neuropathic pain development, we chose the MAFIA transgenic line, which as noted above, expresses GFP and a...; Using the 3-day protocol, we next used different approaches to examine the fate of resident macrophages in the DRG and of blood monocytes. We monitored DRG macrophages by immuno...; Next, we examined the relative contribution of macrophage expansion in the DRG and at the nerve injury site. As AP-mediated systemic macrophage depletion cannot distinguish thei...
source structuredSource and audit
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Evidence quotes (8)
To determine whether the increase in DRG macrophages contributes to neuropathic pain development, we chose the MAFIA transgenic line, which as noted above, expresses GFP and a drug-inducible Fas suicide gene under control of the CSF1R promoter (Supplementary Fig. ). The transgene is expressed in CNS microglia (Supplementary Fig. ) and tissue macrophages, including DRG (Supplementary Fig. ). To deplete monocytic cells in the MAFIA mice, we began our studies with five daily intraperitoneal injections of AP (10 mg kg -1 ), a macrophage-depleting dose used in earlier studies. Supplementary Fig. shows that this dose was without effect on baseline mechanical threshold (S5A) and weight (S5B) in wild-type (WT) mice. By contrast, this regimen resulted in significant weight loss and also increased the baseline mechanical threshold 1 day after the last injection in the MAFIA mice (Supplementary Fig. ). As these effects clearly complicated our proposed analysis, we considerably tapered the dose and determined that 3 daily 1.0 mg kg -1 injections did not affect baseline mechanical (Supplementary Fig. ) or thermal (heat...
Using the 3-day protocol, we next used different approaches to examine the fate of resident macrophages in the DRG and of blood monocytes. We monitored DRG macrophages by immunostaining for GFP and PU.1, a transcription factor expressed in myelomonocytic cells and in their immature precursors. By the third injection, we recorded a significant loss of GFP + /PU.1 + cells (Fig. ) as well as GFP + /Iba1 + cells (Supplementary Fig. ). In a separate group of mice, 1 day after the 3-day AP regime (D3), FACS analysis demonstrated a significant, ~80% depletion of resident (GFP + ) macrophage in the L4 and L5 DRG, compared with VEH mice (Fig. ). At the same time point, FACS analysis of blood demonstrated an almost 90% depletion of mature (GFP hi or CSF1R hi ) circulating monocytes (GFP hi CD11b + or GFP + CSF1R hi ), with no change in the VEH-treated group (Supplementary Fig. ). Importantly, at 4 days after the last AP injection (D6 in Supplementary Fig. ), the percentage of blood monocytes was largely restored, despite a persistent weight loss (Supplementary Fig. ). Taken together, these data demonstrate that resident macrophages in the DRG, as well...
We next examined macrophages in the context of nerve injury. To monitor the early response of macrophages, we followed the expression of Cx3cr1 in the DRG by quantitative PCR (qPCR) 1 day after the nerve injury (POD1). We recorded a 2.1 ± 0.4-fold increase of Cx3cr1 expression in the L4/L5 DRG ipsilateral to the nerve injury in VEH-treated mice (Fig. ) compared with the uninjured contralateral DRG. On the other hand, AP treatment, prior to the nerve injury, not only reduced baseline Cx3cr1 expression in the uninjured, contralateral DRG but also prevented the injury-induced increase of Cx3cr1 expression in the ipsilateral DRG (Fig. ). By POD4 (5th day after the last AP treatment), macrophage number recovered to the level recorded in uninjured, VEH-treated mice (Fig. ). By POD9, the ipsilateral DRG expansion of macrophages reappeared and did not differ from that recorded in VEH-treated SNI mice (Fig. ).
Although previous studies concluded that AP does not cross the BBB, we considered it essential to rule out a direct CNS action of the AP treatment. Compared with uninjured, VEH-treated mice, we found no difference in the number of spinal cord microglia by FACS (Fig. ) in the lumbar cord of AP-treated mice. Immunostaining for microglia also did not differ (Fig. ). These results are consistent with the AP exerting a selective peripheral action. We also addressed the possibility that a compromised BBB following nerve injury could result in AP leakage into the cord,. Arguing against this possibility, both the nerve injury-induced upregulation of Cx3cr1 gene expression, evidence of microglia activation, 1 day after SNI (POD1; Fig. ) and microgliosis examined 4 days after SNI (POD4; Fig. ), did not differ between VEH- and AP-treated mice. Glial fibrillary acidic protein (GFAP) immunostaining of astrocytes also did not differ (Supplementary Fig. ). Taken together, these data confirm that neither spinal microglia nor astrocytes are affected by the AP treatment, whether or not there was a nerve injury.
To investigate the contribution of DRG macrophages to nerve injury-induced mechanical hypersensitivity, we treated separate groups of mice with three daily injections of AP (1.0 mg kg -1 ) followed by SNI. In contrast to VEH-treated mice, which developed mechanical hypersensitivity within 24 h of the SNI, the 3-day AP treatment regimen significantly delayed development of the hypersensitivity, for at least 7 days after the nerve injury (Fig. ). Importantly, mice in which macrophage expansion was restored exhibited mechanical allodynia. Continuing the AP treatment into the day of nerve injury did not enhance the anti-allodynic effect (Supplementary Fig. ). These results indicate that the anti-allodynic effect of AP treatment correlates with the prevention of DRG macrophage expansion. Finally, as the AP-induced weight loss persisted in these mice (Supplementary Fig. ), we conclude that the AP effect on mechanical hypersensitivity was not secondary to the weight loss. Moreover, because thermal pain thresholds did not differ in the AP-treated mice (Supplementary Fig. ), we conclude that systemic side effects were not major contributors...
In a separate experiment we transplanted bone marrow (BM) progenitor cells (GFP + ) isolated from MAFIA mice into irradiated WT mice and confirmed the lack of effect of AP on spinal cord microglia. Specifically, 7 days after the SNI, in contrast to a complete replacement of peripheral host monocytic cells with GFP + donor cells (Supplementary Fig. ), we detected very few GFP + cells in the spinal cord (Supplementary Fig. ). We conclude that the majority of spinal cord microglia (PU1 + ) derived from the host (GFP - ), even after injury (Supplementary Fig. ). In a separate group of transplanted mice, we performed SNI after the 3-day AP or VEH treatment. Consistent with our findings in non-transplanted mice, we found that AP-mediated macrophage depletion in the DRG of the transplanted animals (Supplementary Fig. ) significantly delayed the development of nerve injury-induced mechanical hypersensitivity (Supplementary Fig. ).
Next, we examined the relative contribution of macrophage expansion in the DRG and at the nerve injury site. As AP-mediated systemic macrophage depletion cannot distinguish their respective contribution, we designed the following experiment to deplete selectively macrophages at the injury site, leaving DRG macrophages intact. At the time of the nerve injury, we implanted a cannula and sutured it to muscles that overlay the injury site. Our intent was to deliver a low AP dose that only targeted the macrophages around the injury site, with limited to no systemic action. After serial dose titrations, we identified a systemic dose (0.8 µg in 20 µl) that, when administered daily for 3 days, did not influence nerve injury-induced mechanical allodynia (Supplementary Fig. ) or cause weight loss (Supplementary Fig. ). We administered the low-dose AP via the cannula, prior to performing the nerve injury, and repeated the targeted injection for 2 more days. By quantifying GFP + cell density 1 day after the last injection, compared with VEH-treated mice (Fig. ), we found an almost 90% reduction of macrophages (GFP + ) at the nerve injury site in the A...
SNI-induced mechanical allodynia is typically long lasting and this behavioral phenotype is associated with prolonged microglial activation in the ipsilateral dorsal horn. Figure illustrates, e.g., that microglial activation persists for at least 4 weeks after peripheral nerve injury. At this time point, we also recorded a persistent increase in the number of macrophages in the ipsilateral DRG (Figs. a and ) and at the nerve injury site (Fig. ). Fig. 3 Microglia activation and peripheral macrophage infiltration persist at 28 days post SNI. a - c Immunostaining of Iba1 + microglia in the dorsal horn ( a ), CSF1R-GFP + macrophages (green) in the DRG ( b ), and at the nerve injury site ( c ), 28 days after SNI (POD28). NF200 (blue) labels neuronal cell bodies ( b ) and peripheral nerve myelinated axons ( c ). Dashed line in c denotes nerve ligature site. Scale bar: 50 µm in a; 15 µm in b, c.
Machine-readable layer
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"text": "Although previous studies concluded that AP does not cross the BBB, we considered it essential to rule out a direct CNS action of the AP treatment. Compared with uninjured, VEH-treated mice, we found no difference in the number of spinal cord microglia by FACS (Fig. ) in the lumbar cord of AP-treated mice. Immunostaining for microglia also did not differ (Fig. ). These results are consistent with the AP exerting a selective peripheral action. We also addressed the possibility that a compromised BBB following nerve injury could result in AP leakage into the cord,. Arguing against this possibility, both the nerve injury-induced upregulation of Cx3cr1 gene expression, evidence of microglia activation, 1 day after SNI (POD1; Fig. ) and microgliosis examined 4 days after SNI (POD4; Fig. ), did not differ between VEH- and AP-treated mice. Glial fibrillary acidic p..."
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"name": "DRG macrophages are required for initiation of nerve injury-induced mechanical allodynia",
"text": "To investigate the contribution of DRG macrophages to nerve injury-induced mechanical hypersensitivity, we treated separate groups of mice with three daily injections of AP (1.0 mg kg -1 ) followed by SNI. In contrast to VEH-treated mice, which developed mechanical hypersensitivity within 24 h of the SNI, the 3-day AP treatment regimen significantly delayed development of the hypersensitivity, for at least 7 days after the nerve injury (Fig. ). Importantly, mice in which macrophage expansion was restored exhibited mechanical allodynia. Continuing the AP treatment into the day of nerve injury did not enhance the anti-allodynic effect (Supplementary Fig. ). These results indicate that the anti-allodynic effect of AP treatment correlates with the prevention of DRG macrophage expansion. Finally, as the AP-induced weight loss persisted in these mice (Sup..."
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"name": "DRG macrophages are required for initiation of nerve injury-induced mechanical allodynia",
"text": "In a separate experiment we transplanted bone marrow (BM) progenitor cells (GFP + ) isolated from MAFIA mice into irradiated WT mice and confirmed the lack of effect of AP on spinal cord microglia. Specifically, 7 days after the SNI, in contrast to a complete replacement of peripheral host monocytic cells with GFP + donor cells (Supplementary Fig. ), we detected very few GFP + cells in the spinal cord (Supplementary Fig. ). We conclude that the majority of spinal cord microglia (PU1 + ) derived from the host (GFP - ), even after injury (Supplementary Fig. ). In a separate group of transplanted mice, we performed SNI after the 3-day AP or VEH treatment. Consistent with our findings in non-transplanted mice, we found that AP-mediated macrophage depletion in the DRG of the transplanted animals (Supplementary Fig. ) significantly delayed the development of ne..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Initiation of the neuropathic pain phenotype requires macrophage expansion in the DRG and is independent of infiltrat...",
"text": "Next, we examined the relative contribution of macrophage expansion in the DRG and at the nerve injury site. As AP-mediated systemic macrophage depletion cannot distinguish their respective contribution, we designed the following experiment to deplete selectively macrophages at the injury site, leaving DRG macrophages intact. At the time of the nerve injury, we implanted a cannula and sutured it to muscles that overlay the injury site. Our intent was to deliver a low AP dose that only targeted the macrophages around the injury site, with limited to no systemic action. After serial dose titrations, we identified a systemic dose (0.8 µg in 20 µl) that, when administered daily for 3 days, did not influence nerve injury-induced mechanical allodynia (Supplementary Fig. ) or cause weight loss (Supplementary Fig. ). We administered the low-dose AP via the ca..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Macrophages in the DRG contribute to maintenance of nerve injury-induced mechanical allodynia",
"text": "SNI-induced mechanical allodynia is typically long lasting and this behavioral phenotype is associated with prolonged microglial activation in the ipsilateral dorsal horn. Figure illustrates, e.g., that microglial activation persists for at least 4 weeks after peripheral nerve injury. At this time point, we also recorded a persistent increase in the number of macrophages in the ipsilateral DRG (Figs. a and ) and at the nerve injury site (Fig. ). Fig. 3 Microglia activation and peripheral macrophage infiltration persist at 28 days post SNI. a - c Immunostaining of Iba1 + microglia in the dorsal horn ( a ), CSF1R-GFP + macrophages (green) in the DRG ( b ), and at the nerve injury site ( c ), 28 days after SNI (POD28). NF200 (blue) labels neuronal cell bodies ( b ) and peripheral nerve myelinated axons ( c ). Dashed line in c denotes nerve ligature site. Scale bar..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Statistical analysis"
},
{
"@type": "HowToTool",
"name": "DRG macrophages are depleted by systemic AP administration"
},
{
"@type": "HowToTool",
"name": "DRG macrophages are depleted by systemic AP administration"
},
{
"@type": "HowToTool",
"name": "AP treatment does not alter spinal cord microglia number"
},
{
"@type": "HowToTool",
"name": "Nerve injury triggers a reciprocal interaction between DRG macrophages and sensory neurons"
},
{
"@type": "HowToTool",
"name": "The DRG macrophage expansion, but not the contribution to neuropathic pain initiation and maintenance, is sexually di..."
},
{
"@type": "HowToTool",
"name": "Bone marrow transplantation"
},
{
"@type": "HowToTool",
"name": "Immunohistochemistry"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "DRG macrophages are depleted by systemic AP administration"
},
{
"@type": "HowToSupply",
"name": "AP administration"
},
{
"@type": "HowToSupply",
"name": "In situ hybridization"
},
{
"@type": "HowToSupply",
"name": "Isolation of spinal microglia, DRG macrophages, and peripheral blood cells"
},
{
"@type": "HowToSupply",
"name": "Flow cytometric assays"
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],
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"headline": "Dorsal root ganglion macrophages contribute to both the initiation and persistence of neuropathic pain",
"datePublished": "2020",
"author": [
{
"@type": "Person",
"name": "Xiaobing Yu"
},
{
"@type": "Person",
"name": "Hongju Liu"
},
{
"@type": "Person",
"name": "Katherine A. Hamel"
},
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"@type": "Person",
"name": "Maelig G. Morvan"
},
{
"@type": "Person",
"name": "Stephen Yu"
},
{
"@type": "Person",
"name": "Jacqueline Leff"
},
{
"@type": "Person",
"name": "Zhonghui Guan"
},
{
"@type": "Person",
"name": "Joao M. Braz"
},
{
"@type": "Person",
"name": "Allan I. Basbaum"
}
],
"identifier": "10.1038/s41467-019-13839-2"
}
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