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. 2019 Mar 30;16(1):67.
doi: 10.1186/s12974-019-1456-x.

Hyperactivation of Proprioceptors Induces Microglia-Mediated Long-Lasting Pain in a Rat Model of Chronic Fatigue Syndrome

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Free PMC article

Hyperactivation of Proprioceptors Induces Microglia-Mediated Long-Lasting Pain in a Rat Model of Chronic Fatigue Syndrome

Masaya Yasui et al. J Neuroinflammation. .
Free PMC article

Abstract

Background: Patients diagnosed with chronic fatigue syndrome (CFS) or fibromyalgia experience chronic pain. Concomitantly, the rat model of CFS exhibits microglial activation in the lumbar spinal cord and pain behavior without peripheral tissue damage and/or inflammation. The present study addressed the mechanism underlying the association between pain and chronic stress using this rat model.

Methods: Chronic or continuous stress-loading (CS) model rats, housed in a cage with a thin level of water (1.5 cm in depth), were used. The von Frey test and pressure pain test were employed to measure pain behavior. The neuronal and microglial activations were immunohistochemically demonstrated with antibodies against ATF3 and Iba1. Electromyography was used to evaluate muscle activity.

Results: The expression of ATF3, a marker of neuronal hyperactivity or injury, was first observed in the lumbar dorsal root ganglion (DRG) neurons 2 days after CS initiation. More than 50% of ATF3-positive neurons simultaneously expressed the proprioceptor markers TrkC or VGluT1, whereas the co-expression rates for TrkA, TrkB, IB4, and CGRP were lower than 20%. Retrograde labeling using fluorogold showed that ATF3-positive proprioceptive DRG neurons mainly projected to the soleus. Substantial microglial accumulation was observed in the medial part of the dorsal horn on the fifth CS day. Microglial accumulation was observed around a subset of motor neurons in the dorsal part of the ventral horn on the sixth CS day. The motor neurons surrounded by microglia were ATF3-positive and mainly projected to the soleus. Electromyographic activity in the soleus was two to three times higher in the CS group than in the control group. These results suggest that chronic proprioceptor activation induces the sequential activation of neurons along the spinal reflex arc, and the neuronal activation further activates microglia along the arc. Proprioceptor suppression by ankle joint immobilization significantly suppressed the accumulation of microglia in the spinal cord, as well as the pain behavior.

Conclusion: Our results indicate that proprioceptor-induced microglial activation may be a key player in the initiation and maintenance of abnormal pain in patients with CFS.

Keywords: Chronic fatigue syndrome; Chronic stress; Fibromyalgia; Microglia; Pain; Proprioceptor.

Conflict of interest statement

Ethics approval and consent to participate

This study was approved by the Institutional Animal Care and Use Committees of Nagoya University (Approval No. 24294) and Aichi Medical University (Approval No. 2016–27). The animals were handled in accordance with the guidelines established by the Institutional Animal Care and Use Committees of Nagoya University and Aichi Medical University.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Six days of stress loading significantly prolonged pain behaviors. a Pressure pain threshold of the muscle. b Mechanical withdrawal threshold of the skin (von Frey test (VFT)). Note the significant decreases in pain threshold for 10 days (pressure pain threshold test) and skin pain for 8 days (VFT). NCS, no continuous stress; right, right leg or paw; left, left leg or paw. n = 5 (each group); **p < 0.01, ***p < 0.001 vs. NCS left; ††p < 0.01, †††p < 0.001 vs. NCS right. Two-way repeated measures ANOVA followed by the Holm-Sidak multiple comparisons test
Fig. 2
Fig. 2
Appearance of ATF3-positive signal in L5 dorsal root ganglion (DRG) neurons during stress loading. a–f ATF3 immunoreactivity was first observed in the L5 DRG at day 2 (b). The number of the ATF3-positive cells increased as CS progressed (c–f). Arrows indicate ATF3-positive nuclei. Scale bar 200 μm
Fig. 3
Fig. 3
Identification of cell types among ATF3-positive neurons. Double immunofluorescence staining was performed using L5 dorsal root ganglion (DRG) tissues from rats subjected to continuous stress-loading (CS). a–r Representative double immunofluorescence labeling for ATF3 (b, e, h, k, n, q) with the markers TrkA (a), TrkB (d), TrkC (g), IB4 (j), CGRP (m), and VGluT1 (p). c, f, i, l, o, r Merged images of the respective two photos on the left. Arrowheads indicate co-expressing cells. Scale bar 50 μm. s The co-expression rate of each marker in ATF3-positive cells. n = 4. ***p < 0.001 vs. TrkC. ****p < 0.0001 vs. TrkC. †p < 0.05 vs. VGluT1. ††p < 0.01 vs. VGluT1. One-way ANOVA with Tukey’s test
Fig. 4
Fig. 4
A significant number of ATF3-positive dorsal root ganglion (DRG) neurons projected to the soleus. a–c Double staining for ATF3 (green) and fluorogold (FG)-labeled (blue) neurons in the L5 DRG of rats subjected to continuous stress-loading (CS). FG was injected into the soleus (a), tibialis anterior (b), or gastrocnemius (c). d The proportion of FG-labeled neurons among ATF3-positive cells after FG injection: Soleus, 57.7 ± 7.3%; tibialis anterior, 42.7 ± 14.7%; and gastrocnemius, 14.0 ± 4.1%. Soleus (n = 8), tibialis anterior (n = 4), and gastrocnemius (n = 6). *p < 0.05, **p < 0.01. One-way ANOVA with Tukey’s test. e A representative photo of the triple labeling for VGluT1 (green), ATF3 (red), and FG (blue) in the L5 DRG of CS rats. Arrowheads indicate co-expression
Fig. 5
Fig. 5
Profile of microglial activation and accumulation during continuous stress-loading (CS) in the L5 spinal cord. a–e Iba-1 positive cells (microglia) in the L5 spinal cord in the no-CS (NCS) (a) group and at 3 days (b), 4 days (c), 5 days (d), and 6 days (e) after the initiation of CS. f A representative dot plot indicating microglial localization in the L5 spinal cord during 6 days of CS. Areas in which microglial activation and accumulation were observed are surrounded by black dotted lines. Scale bar 200 μm
Fig. 6
Fig. 6
Neurons surrounded by activated microglia after 6 days of CS were α-motor neurons. a–f Double labeling images of the dorsal region of the ventral horn of the L5 spinal cord on day 6 of CS. a OX42 (activated microglia, green)/ATF3 (red). b Iba1 (microglia, green)/ChAT (motor neurons, red). c Iba1 (microglia, green)/DINE (motor neurons, red). d ATF3 (green)/ChAT (motor neurons, red). e Iba1 (microglia, green)/NeuN (α-motor neurons, red). f Iba1 (microglia, green)/Err3 (nuclear staining of γ-motor neurons, red). Arrowheads indicate α-motor neurons, while arrows indicate γ-motor neurons. All scale bars 100 μm
Fig. 7
Fig. 7
Motor neurons surrounded by microglia projected to the soleus. a A map indicating the localization of retrogradely labeled FG-positive cells after FG injection into the soleus. The FG-positive motor neurons are plotted on a standardized chart. b FG (blue)-positive motor neurons were attracted to Iba1-positive microglia (green) at L5. The white dotted line in b indicates the border between white matter and gray matter
Fig. 8
Fig. 8
Electromyogram of the soleus muscle during stress loading. a A representative chart of electromyographic activity for the no-CS (NCS/Pre) and CS groups on day 6 of CS. b Profile of muscle activity prior to and during stress loading. Muscle activity was obtained via the integration of spike power for 6 diurnal hours (12:00–18:00) and 6 nocturnal hours (0:00–6:00) for each CS day. The activity observed during the 6 diurnal hours (12:00–18:00) of the pre-stress-loading period (Pre-d) was defined as 100%. Pre-n, the 6 nocturnal hours (0:00–6:00) of activity prior to stress loading. All points represent the average +/− standard error of the mean (SEM) from four animals. White circle, % index of diurnal muscle activity; black circle, % index for nocturnal muscle activity. n = 4. *p < 0.05, **p < 0.01 vs. Pre-d. One-way ANOVA
Fig. 9
Fig. 9
Ankle joint immobilization suppressed microglial activation. a Unilateral arthrodesis of the right side was performed to immobilize the ankle joint (i.e., left = non-arthrodesis side). Clear microglial accumulation was observed in the L5 spinal cord in the medial region of the dorsal horn and the dorsal region of the ventral horn (non-arthrodesis side), whereas no such accumulation was observed on the arthrodesis side (arthrodesis side). b Quantification of microglial cell number/unit area (10,000 μm2) in the medial region of the L5 dorsal horn. c Quantification of microglial cell number/unit area (10,000 μm2) in the dorsal region of the L5 ventral horn. d Quantification of microglial occupied area/unit area in the medial region of the L5 dorsal horn. e Quantification of microglial occupied area/unit area in the dorsal region of the L5 ventral horn. f ATF3 immunoreactivity (green) was observed in ChAT-positive cells (motor neurons, red) in the rectangular inset region of the non-arthrodesis side in a. g ATF3 immunoreactivity was not observed in ChAT-positive cells (motor neurons, red) in the inset region of the arthrodesis side in a. h Expression of ATF3 (green) was observed in the L5 dorsal root ganglion (DRG) of the non-arthrodesis side. i Expression of ATF3 was suppressed in the L5 DRG of the arthrodesis side. Scale bar 200 μm. j ATF3 expression was significantly suppressed in the L5 DRG of the arthrodesis side. k Changes in the pressure pain threshold of the muscle after CS on the non-arthrodesis and arthrodesis sides. The pain threshold increased on the arthrodesis side. n = 4. *p < 0.05. **p < 0.01. ***p < 0.001. Paired t test

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