Original Research Papers
ASIC3 in the cerebrospinal fluid-contacting nucleus of brain parenchyma contributes to inflammatory pain in rats X. Y. Wang*, W. W. Yan*, X. L. Zhang*, H. Liu, L. C. Zhang Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, China Objective: The study explores the role of acid-sensitive ion channel 3 (ASIC3) in the cerebrospinal fluid (CSF)-contacting nucleus of the parenchyma of rat brain, in inflammatory pain. Methods: We injected rats subcutaneously with complete Freund’s adjuvant (CFA) into the plantar surface of the left hind paw. We then measured the thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT). After horseradish peroxidase-conjugated toxin subunit B (CB-HRP) retrograde tracing of one of the rats’ lateral ventricles, we used immunohistochemistry, to observe the distribution and expression of ASIC3 in CSF-contacting nucleus with double labeling of CB-HRP and ASIC3. We injected a selective ASIC3 antagonist, APETx2, into one of the rat’s lateral ventricles, to test for inhibition of ASIC3 activation and the effects on heat and mechanical hyperalgesia, in response to inflammation. Results: The CSF-contacting nucleus is always located in a specific region of parenchyma in rat brain, consistent with previous findings. The CSF-contacting nucleus has ASIC3, which increased significantly in persistent inflammation following injection of CFA. The intracerebroventricular injection (ICV) administration of specific ASIC3 antagonist APETx2 suppressed the heat and mechanical hyperalgesia following CFA injection. Conclusion: The CSF-contacting nucleus regulates inflammatory pain in the central nervous system (CNS) via increased expression of ASIC3, providing a new molecular basis for pain transmission and regulation. Keywords: CSF-contacting nucleus, ASIC3, Inflammatory pain
Introduction Cerebrospinal fluid-contacting neurons (CSF-CNs) are divided into three types: (1) an intraependymal neuron, which projects into the ventricle lumen and the central canal of the spinal cord, (2) a supraependymal cell, which is subjacent to the ependyma, and (3) a distal CSF-contacting neuron, with a body in the parenchyma of the brain and processes extending into the CSF in the cavity of the ventricle in the central nervous system (CNS).1 Zhang et al. successfully used horseradish peroxidase-conjugated toxin subunit B (CBHRP) retrograde tracing to show that CB-HRP does not pass through the spaces of the ependyma and diffuse into parenchyma. Thus, any labeled structures found in the ventricle or the parenchyma of the brain are the CSF-contacting structures. The distal CSFCNs have functions distinct from the subependymal and ependymal CSF-CNs and are found in different parts of the parenchyma, mainly in the ventral periaqueductal central gray (PAG) of the brainstem.2,3 *These authors contributed equally to this work. Correspondence to: L. C. Zhang, Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, 209 Tongshan Road, Xuzhou City 221002, Jiangsu Province, China. Email: [email protected]
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Based on this anatomical feature, we named this group of neurons as CSF-contacting nucleus.4 It plays an important role in signal modulation and substance transport between the brain and CSF.3 Our previous studies offered significant evidence indicating that the CSF-contacting nucleus mediated by the cold sensation receptor channel, TRPM8 as the receptor, was closely linked to the transmission of pain sensation and pain regulation.5 The TRPV1 expression in the CSF-contacting nucleus increased in neuropathic pain, which suggests that the CSF-contacting nucleus in rat brain parenchyma partly contributed to pain behaviors.6 In addition, substance P is expressed in the CSF-contacting nucleus in the formalin-induced orofacial inflammatory pain.7 Until now, the function of this special nucleus has not been fully deciphered. Acid-sensing ion channels (ASICs) that belong to the degenerin/epithelial sodium channel (DEG/ ENaC) superfamily are cationic channels activated by extracellular protons.8–10 Seven subunits encoded by four genes (ASIC1a, ASIC1b, ASIC1b2, ASIC2a, ASIC2b, ASIC3, and ASIC4) have been identified so far in mammals.11,12 Among the ASIC subunits, ASIC3 is the most sensitive to changes in pH, even as
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small as 0.2 units.10,13,14 Multiple studies have demonstrated that peripheral ASIC3 channels are particularly important in inflammatory pain. Chronic inflammatory pain elevates the expression of ASIC3 in dorsal root ganglia (DRG), an analgesic effect that is blocked by concurrent treatment with anti-inflammatory drugs.15,16 Subcutaneous injection of the ASIC3-selective blocker APETx2 also alleviates the inflammation-induced thermal and mechanical hyperalgesia.17,18 Until now, the expression of ASIC3 has been confirmed in hypothalamus, suprachiasmatic nucleus, and brainstem,19–21 playing a role in modulating brain systems involved in emotion.22,23 Unlike the well-established distribution and function of ASIC3 in peripheral nervous system (PNS), the role of ASIC3 in the CNS is still unclear. The present study was therefore designed to determine the expression of ASIC3 in CSF-contacting nucleus and explore the role of ASIC3 in inflammatory pain.
Materials and Methods Animal care and treatment The experimental protocols were approved by the Committee for the Ethical Use of Laboratory Animals, Xuzhou Medical College. Rats were used only once. Male Sprague-Dawley rats (250¡50 g) (n 5 6, each) were maintained in climate- and lightcontrolled conditions (23¡1uC, 12/12 hours dark/ light cycle with light on at 08:00 hour) for at least 1 week prior to the experiments. After determining the baseline threshold, the rats in complete Freund’s adjuvant (CFA) group received subcutaneous injection of CFA (1 mg ml21, 100 ml, Sigma) into the plantar surface of the left hind paw, and were subsequently returned to their cages. Following CFA injection, the rats were tested for thermal and mechanical hypersensitivity. The rats in control group were injected with saline, 100 ml. All experiments were conducted in accordance with the guidelines of the International Association for the Study of Pain (IASP).
Pain behavioral assessment Pain response was measured in a blinded fashion over a specific period of time, indoor temperature, and humidity. Rats were tested for basic thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) before injecting the hind paw with CFA, and the same procedure was repeated on days 1, 3, and 7 after injection. Rats were placed in a 3-mmthick glass plate and allowed to acclimatize for 30 minutes before testing. Thermal withdrawal latency was measured using the Plantar Test apparatus (Hargreaves method) (IITC series 8-390, Institute of Biological Medicine, Academy of Medical Science Beijing, China). Latency of the reflex was measured from the onset of radiant heat until hind-paw withdrawal. The cut-off time was set at 25 seconds in order to avoid tissue damage.
Role of ASIC3 in rat brain
Stimulation intensity was kept constant during the whole experiment. Each animal was tested five times at intervals of 5 minutes, and the mean TWL value of the last three measurements was used for analysis. The MWT on the plantar surface of the hind paw was measured with a set of Von Frey hairs (VFHs; Semmes-Weinstein Monofilaments, North Coast Medical, Morgan Hill, CA, USA) in ascending order of force (0.16, 0.4, 0.6, 1, 1.4, 2, 4, 6, 8, 10, 15, 26g). The VFH was applied to the paw for 6 seconds, or until a withdrawal response, when the paw was retested, starting with the next descending VFH until no response was noted. The lowest amount of force required to elicit a response was recorded as MWT in g. A significant reduction in TWL and MWT compared with normal baseline was interpreted as thermal and mechanical hyperalgesia. Rats that did not demonstrate hyperalgesia were excluded from the study. After recording the values of MWT and TWL over 3 days after injection of CFA, APETx2 was administered as an ICV injection of saline solution vehicle.
Intracerebroventricular injection Rats were anesthetized with 10% chloral hydrate (300 mg/kg, i.p.) and the head fixed in a stereotaxic instrument (Narishige Scientific Instruments, Tokyo, Japan). A 3 ml volume of 30% CB-HRP (Sigma) was injected into one of the rats’ lateral ventricles, according to stereotaxic coordinates (bregma: 21.2¡0.4 mm, depth: 3.2¡0.4 mm, right of median sagittal plane: 1.4¡0.2 mm). The ASIC3 specific inhibitor APETx2 (Alomone labs, Jerusalem, Israel) was diluted in physiological saline and administrated at 3 mM (3 ml) 3 days after injection of CFA as a unilateral ICV infusion, with a saline (3 ml) solution vehicle.
Tissue processing After 48 hours of tracer injection, under anesthesia with 10% chloral hydrate (300 mg/kg, i.p.), rats were perfused with 150 ml of phosphate buffered saline (PBS) (0.01 M PBS, pH 7.4), followed by 4% paraformaldehyde in 0.2 M phosphate buffer (300 ml, pH 7.4) without interruption. The brainstem was removed immediately and post-fixed for 4–6 hours at 4uC, and then immersed overnight in a 30% sucrose solution at 4uC. The brain tissue was embedded with OCT tissue freezing medium, at 220uC and sectioned in a cryostat (Leica CM1900, Germany) at 40 mm in the transverse plane. The frozen sections were collected in PBS. Following three washes in PBS, sections were incubated in PBS with 0.3% Triton X-100 (TBS) for 48–72 hours at 4uC with a goat anti-cholera toxin B-subunit (1 : 200, Sigma, St. Louis, MO, USA) and rabbit anti-ASIC3 (1 : 200, Alpha Diagnostic International, Inc., San Antonio, USA). After rinsing in PBS, sections were
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considering the intensity of the staining. Phosphate buffered saline was added to sections instead of primary antibody as negative control, and no ASIC3-labeled neurons were found (Fig. 1).
Statistical analysis Software Image-Pro Plus Version 6.0 (Media Cybernetics, Bethesda, MD, USA) was used to count the number of neurons. Four sections with centralized CB-HRP positive neurons were chosen from the same aspect of brain parenchyma in each rat. Data were expressed as mean¡SD. The data were subjected to a one-way analysis of variance (ANOVA) followed by Tukey’s t-test (or just a t-test when there were only two groups). P , 0.05 was considered to indicate statistical significance.
Results Behavioral assessment of CFA injection Figure 1 No immunosignal was detected when phosphate buffered saline (PBS) was added to section instead of primary antibody. (Scale bar: 100 mm.)
incubated in donkey anti-goat IgG conjugated to Cy3 (1 : 200, Millipore, Temecula, CA, USA) and in donkey anti-rabbit IgG conjugated to Fluorescein isothiocyanate (FITC) (1 : 100, Millipore) in the dark for 2 hours at room temperature. Finally, sections were rinsed, mounted, and coverslipped with glycerol and stored at 220uC in the dark. Tissue sections were examined using laser scanning confocal microscopy (TCS SP2, Leica, Wetzlar, Germany) to identify CSFcontacting nucleus neurons labeled with Cy3 and ASIC3 labeled with FITC. We selected four brain sections per animal, and each section had the greatest number of positive neurons. We recorded the total number of positive neurons in the brain of each animal. All positive neurons were counted without
In our experiments, the TWL and MWT in response to heat or mechanical stimulation were significantly decreased 1 day after CFA injection and the effect persisted for at least 3 days in CFA-treated rats. After gradual increase, complete recovery from foot swelling, walking difficulty, and protected behaviors such as lifting-affected foot took 2 weeks. However, no significant changes in TWL and MWT of rats in salinetreated control group were seen (Fig. 2A and B).
Expression of ASIC3 in CSF-contacting nucleus Positive labeling of CB-HRP-traced neurons in the ventral aqueduct region was mainly seen in cytoplasm, with most of the neurons multipolar and round or oval in shape. The cell bodies of CSF-contacting nucleus were in brain parenchyma, and the processes extended into CSF. A tissue section with CB-HRP immunoreactivity is illustrated in Fig. 3A and ASIC3-positive neurons (FITC, green) in Fig. 3B. The CB-HRP/ ASIC3 double-labeled neurons in yellow are shown in
Figure 2 Time course of development of thermal and mechanical hypersensitivity in rats after intraplantar injection with complete Freund’s adjuvant (CFA) or saline. Thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) are shown as mean¡SD. A significant reduction in TWL and MWT in CFA group compared with saline group: *P , 0.05, **P , 0.01 compared with saline group; #P , 0.05, ##P , 0.01 compared with 0 day (n 5 6).
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Figure 3 Acid-sensitive ion channel 3 (ASIC3) expression in cerebrospinal fluid (CSF)-contacting nucleus of normal rats. (A) Horseradish peroxidase-conjugated toxin subunit B (CBHRP) positive neurons (red), (B) ASIC3-positive neurons (green), and (C) CB-HRP/ASIC3 double-labeled neurons (yellow) (scale bar: 100 mm).
Fig. 3C. Immunofluorescence analysis indicated the localization of ASIC3 in the CSF-contacting nucleus.
Changes in ASIC3 expression in CSF-contacting nucleus Neurons positive for CB-HRP, ASIC3, and CB-HRP/ ASIC3 existed in CFA group. The ASIC3-immunoreactive levels were higher in CFA group (Fig. 4C–F) compared with normal group (Fig. 4A and B) following 3 and 7 days after injection of CFA. The number of CB-HRP/ASIC3 double-labeled neurons in CFA treatment group significantly increased, compared with that of normal group, which is positively relevant to signs of pain behavior (Fig. 4G).
Behavioral assessment of APETx2 injection The ICV administration of APETx2 (3 mM, 3 ml) 3 days after CFA injection significantly attenuated both thermal and mechanical hypersensitivities compared with the vehicle group. Time to reach the maximal anti-hyperalgesia was 15 minutes after administration of APETx2, and the effects decayed gradually over the subsequent 60 minutes (Fig. 5A and B). In contrast to what we observed in inflamed rats, injection of APETx2 did not affect either TWL or MWT in normal rats (Fig. 5C and D).
Discussion The present study has shown that the CSF-contacting nucleus is always located in a specific region of the rat brain parenchyma, which is consistent with previous investigations,3–7 and ASIC3 existed in CSF-contacting nucleus. The expression of ASIC3 in CSF-contacting nucleus increased following inflammatory pain. Furthermore, ICV administration of the specific ASIC3 antagonist, APETx2, exhibited analgesic effect. The result was consistent with the hypothesis that ASIC3 in CSF-contacting nucleus participates in central regulation of pain.
Figure 4 Acid-sensitive ion channel 3 (ASIC3) expression in rats with inflammatory pain. Photomicrograph (A) depicts ASIC3 expression in normal group; Photomicrographs (C, E) show ASIC3 expression 3 and 7 days following injection with complete Freund’s adjuvant (CFA); photomicrographs (B, D, F) show the enlargement of the rectangle in (A), (C), and (E); graph (G) illustrates the number of neurons labeled with ASIC3 or horseradish peroxidase-conjugated toxin subunit B (CB-HRP) in every six sections of three groups. Data are presented as mean¡SD. The numbers of ASIC3/CB-HRP neurons at 3 and 7 days are significantly higher than the normal group, #P , 0.05, **P , 0.01 compared with normal group (n 5 6). (Scale bars represent 100 mm.)
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Figure 5 Time course of the effect of ICV injection of APETx2 on thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT). Photomicrographs (A and B) depict time course of the effect of ICV injection of APETx2 on the complete Freund’s adjuvant (CFA)-induced reduction of TWL and MWT, 3 days after CFA injection. Injection of APETx2 elevated TWL and MWT to a level close to the normal level. A significant difference was observed between vehicle and APETx2 groups. Data are presented as mean¡SD, *P , 0.05, **P , 0.01 compared with vehicle group (n 5 6). Photomicrographs (C and D) depict the effect of injection of APETx2 in normal rats. Blockade of ASIC3 did not affect normal thermal and mechanical nociception. Data are presented as mean¡SD, P . 0.05 compared with vehicle group (n 5 6).
The CSF-contacting nucleus is a particular cell type in the CNS, whose bodies are in the brain parenchyma and processes extend into CSF. In view of their anatomical characteristics, they transmit signals between brain parenchyma and CSF,3 but the exact biological function was not clear. Acid-sensitive ion channel 3 has been reported to be predominantly distributed in the PNS.10,14,23 However, the expression of ASIC3 in the CNS was still unclear. Neurons in the hypothalamus, suprachiasmatic nucleus, and brainstem contain ASIC3 transcripts, and were immunoreactive for ASIC3.19–21 The characterization of the ASIC3 in brain was designed to explore its functional role in physiological and pathological processes of the CNS. We first confirmed that only a small part of CSF-contacting nucleus was immunoreactive for ASIC3. It suggested that in normal physiological conditions, ASIC3 may mediate the information transfer in CSF-contacting nucleus, which was similar to the DRG neurons serving multiple sensory modalities. Rats that were not injected CFA showed no differences in behavioral response before and after ICV injection of APETx2. Voilley
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et al. similarly showed that non-steroid anti-inflammatory drugs (NSAIDs) did not alter ASIC expression in normal sensory neurons.16 Therefore, ASIC3 channels in CSF-contacting nucleus specifically contribute to pathological but not physiological pain. The management of inflammatory pain is a clinical challenge. Efforts are mainly focused on the relationship between neuronal pathway and pain instead of brain-CSF neurohumoral circuit, which renders it difficult to fully clarify the pathogenesis of pain. Recent studies have documented that inflammatory pain elicits an inflammatory response in CSF, at least partly attributed to chemical changes in CSF.24,25 Complete Freund’s adjuvant-induced inflammation is a commonly used experimental animal model of persistent inflammatory pain. Animals showed thermal and mechanical hyperalgesia, and the expression level of ASIC3 significantly increased in CSFcontacting nucleus, 3 days after injection. The data are similar to a previous study that showed increased ASIC3 expression in L4 and L5 DRG neurons following CFA injection into hind paw.26
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We have also reported that ICV administration of APETx2 produced antihyperalgesia in rats with inflammatory pain; in that it reduced previously established thermal and mechanical hyperalgesia 3 days after CFA injection. In the PNS, the ASIC3 antagonist has a proven role in alleviating inflammatory pain.17,18 Therefore, our observations suggest that ASIC3 in CSF-contacting nucleus participates in the central regulation of pain. Indeed, for the first time, our findings confirm that AISC3 is involved in pain transmission at the superspinal level. In summary, we found that only a proportion of neurons in CSF-contacting nucleus were labeled for ASIC3 in normal rats. Inflammatory pain upregulated ASIC3 expression in CSF-contacting nucleus. Furthermore, ICV administration of APETx2 exhibited an antihyperalgesic effect. Our findings are consistent with the hypothesis that CSF-contacting nucleus neurons regulate inflammatory pain in the CNS via increased expression of ASIC3, thereby providing a new molecular basis for pain transmission and regulation.
Acknowledgements This article is supported by National Natural Science Foundation of China, numbers 81371243 and 81300957, and Natural Science Foundation of Jiangsu Province, number BK2012580.
References 1 Vı´gh B, Manzanoe, Silva MJ, Frank CL. The system of cerebrospinal fluid-contacting neurons. It supposed role in the non-synaptic signal transmission of the brain. Histol Histopathol. 2004;19:607–28. 2 Wang MS, Zhang LC. Methodological comparison on the tracing CSF-CNs with HRP and CB-HRP. Acad Med. 1992;12:286–87. 3 Zhang LC, Zeng YM, Ting J. The distributions and signaling directions of the cerebrospinal fluid contacting neurons in the parenchyma of a rat brain. Brain Res. 2003;989:1–8. 4 Lu XF, Li YY, Zhang LC. Substance P in the cerebrospinal fluid-contacting nucleus contributes to morphine physical dependence in rats. Neurosci. Let. 2011;488:188–92. 5 Du J, Yang XW, Zhang LC, Zeng YM. Expression of TRPM8 in the distal cerebrospinal fluid-contacting neurons in the brain mesencephalon of rats. Cerebrospinal Fluid Res. 2009;3:1–7. 6 Xu C, Zhao ZJ, Wu TT. Distribution of TRPV1 in CSF contacting nucleus of rat brain parenchyma and its expression in neuropathic pain. J Neurol Neurophysiol. 2011;2:3. 7 Lu XF, Geng XJ, Zhang LC. Substance P expression in the distal cerebrospinal fluid-contacting neurons and spinal trigeminal
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nucleus in formalin-induced the orofacial inflammatory pain in rats. Brain Res Bull. 2008;78:139–45. Waldmann R, Champigny G, Bassilana F. A proton-gated cation channel involved in acid-sensing. Nature. 1997;386:173– 77. Krishtal O. The ASICs: signaling molecules? Modulators? Trends Neurosci. 2003;269:477–83. Deval E, Gasull X, Noel J, Salinas M, Baron A, Diochot S, et al. Acid sensing ion channels (ASICs): Pharmacology and implication in pain. Pharmacol Ther. 2010;128:549–58. Waldmann R, Lazdunski M. Hz-gated cation channels: neuronal acid sensors in the NaC/DEG family of ion channels. Curr Opin Neurobiol. 1998;8:418–24. Sakai H, Lingueglia E, Champigny G. Cloning and functional expression of a novel degenerin-like Naz channel gene in mammals. J Physiol. 1999;519:323–33. Yagi J, Wenk HN, Naves LA. Sustained currents through ASIC3 ion channels at the modest pH changes that occur during myocardial ischemia. Circ Res. 2006;99:501–9. Li WG, Xu TL. ASIC3 channels in multimodal sensory perception. ACS Chem Neurosci. 2011;2:26–37. Mamet J, Baron A, Lazdunski M. Proinflammatory mediators, stimulators of sensory neuron excitability via the expression of acid-sensing ion channels. J Neurosci. 2002;22:10662–70. Voilley N, Weille J, Mamet J. Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors. J Neurosci. 2001;21:8026–33. Diochot S, Baron A, Rash LD, Deval E, Escoubas P, Scarzello SA. New sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons. EMBO J. 2004;23:1516–25. Karczewski J, Spencer RH, Garsky VM. Reversal of acidinduced and inflammatory pain by the selective ASIC3 inhibitor, APETx2. Br J Pharmacol. 2010;161:950–60. Meng QY, Wang W, Chen XN. Distribution of acid-sensing ion channel 3 in the rat hypothalamus. Neuroscience. 2009;159: 1126–34. Chen CH, Hsu YT, Chen CC. Acid-sensing ion channels in neurons of the rat suprachiasmatic nucleus. J Physiol. 2009;587:1727–37. Cao XL, Chen Q, Zhou H, Tang YH, Xu JG, Zheng Y. Expression of acid-sensing ion channels in neurons of trapezoid body and lateral paragigantocellular nuclei in rat brain, and effects of intermittent hypoxia on their expression. Sichuan Da Xue Xue Bao Yi Xue Ban. 2009;40:662–66. Wu WL, Lin YW, Min MY. Mice lacking Asic3 show reduced anxiety-like behavior on the elevated plus maze and reduced aggression. Genes Brain Behav. 2010;9:603–14. Wu WL, Cheng CF, Sun WH. Targeting ASIC3 for pain, anxiety, and insulin resistance. Pharmacol Ther. 2012;134:127– 38. Lingueglia E. Acid-sensing ion channels in sensory perception. J Biol Chem. 2007;282:17325–29. Strittmatter M, Grauer M, Isenberg E. Cerebrospinal fluid neuropeptides and monoaminergic transmitters in patients with trigeminal neuralgia. Headache. 1997;37:211–16. Bianchi M, Martucci C, Ferrario P. Increased tumor necrosis factor-alpha and prostaglandin E2 concentrations in the cerebrospinal fluid of rats with inflammatory hyperalgesia: the effects of analgesic drugs. Anesth Analg. 2007;104:949–54.
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ASIC3 in the cerebrospinal fluid-contacting nucleus of brain parenchyma contributes to inflammatory pain in rats X. Y. Wang*, W. W. Yan*, X. L. Zhang*, H. Liu, L. C. Zhang Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, China Objective: The study explores the role of acid-sensitive ion channel 3 (ASIC3) in the cerebrospinal fluid (CSF)-contacting nucleus of the parenchyma of rat brain, in inflammatory pain. Methods: We injected rats subcutaneously with complete Freund’s adjuvant (CFA) into the plantar surface of the left hind paw. We then measured the thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT). After horseradish peroxidase-conjugated toxin subunit B (CB-HRP) retrograde tracing of one of the rats’ lateral ventricles, we used immunohistochemistry, to observe the distribution and expression of ASIC3 in CSF-contacting nucleus with double labeling of CB-HRP and ASIC3. We injected a selective ASIC3 antagonist, APETx2, into one of the rat’s lateral ventricles, to test for inhibition of ASIC3 activation and the effects on heat and mechanical hyperalgesia, in response to inflammation. Results: The CSF-contacting nucleus is always located in a specific region of parenchyma in rat brain, consistent with previous findings. The CSF-contacting nucleus has ASIC3, which increased significantly in persistent inflammation following injection of CFA. The intracerebroventricular injection (ICV) administration of specific ASIC3 antagonist APETx2 suppressed the heat and mechanical hyperalgesia following CFA injection. Conclusion: The CSF-contacting nucleus regulates inflammatory pain in the central nervous system (CNS) via increased expression of ASIC3, providing a new molecular basis for pain transmission and regulation. Keywords: CSF-contacting nucleus, ASIC3, Inflammatory pain
Introduction Cerebrospinal fluid-contacting neurons (CSF-CNs) are divided into three types: (1) an intraependymal neuron, which projects into the ventricle lumen and the central canal of the spinal cord, (2) a supraependymal cell, which is subjacent to the ependyma, and (3) a distal CSF-contacting neuron, with a body in the parenchyma of the brain and processes extending into the CSF in the cavity of the ventricle in the central nervous system (CNS).1 Zhang et al. successfully used horseradish peroxidase-conjugated toxin subunit B (CBHRP) retrograde tracing to show that CB-HRP does not pass through the spaces of the ependyma and diffuse into parenchyma. Thus, any labeled structures found in the ventricle or the parenchyma of the brain are the CSF-contacting structures. The distal CSFCNs have functions distinct from the subependymal and ependymal CSF-CNs and are found in different parts of the parenchyma, mainly in the ventral periaqueductal central gray (PAG) of the brainstem.2,3 *These authors contributed equally to this work. Correspondence to: L. C. Zhang, Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, 209 Tongshan Road, Xuzhou City 221002, Jiangsu Province, China. Email: [email protected]
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Based on this anatomical feature, we named this group of neurons as CSF-contacting nucleus.4 It plays an important role in signal modulation and substance transport between the brain and CSF.3 Our previous studies offered significant evidence indicating that the CSF-contacting nucleus mediated by the cold sensation receptor channel, TRPM8 as the receptor, was closely linked to the transmission of pain sensation and pain regulation.5 The TRPV1 expression in the CSF-contacting nucleus increased in neuropathic pain, which suggests that the CSF-contacting nucleus in rat brain parenchyma partly contributed to pain behaviors.6 In addition, substance P is expressed in the CSF-contacting nucleus in the formalin-induced orofacial inflammatory pain.7 Until now, the function of this special nucleus has not been fully deciphered. Acid-sensing ion channels (ASICs) that belong to the degenerin/epithelial sodium channel (DEG/ ENaC) superfamily are cationic channels activated by extracellular protons.8–10 Seven subunits encoded by four genes (ASIC1a, ASIC1b, ASIC1b2, ASIC2a, ASIC2b, ASIC3, and ASIC4) have been identified so far in mammals.11,12 Among the ASIC subunits, ASIC3 is the most sensitive to changes in pH, even as
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small as 0.2 units.10,13,14 Multiple studies have demonstrated that peripheral ASIC3 channels are particularly important in inflammatory pain. Chronic inflammatory pain elevates the expression of ASIC3 in dorsal root ganglia (DRG), an analgesic effect that is blocked by concurrent treatment with anti-inflammatory drugs.15,16 Subcutaneous injection of the ASIC3-selective blocker APETx2 also alleviates the inflammation-induced thermal and mechanical hyperalgesia.17,18 Until now, the expression of ASIC3 has been confirmed in hypothalamus, suprachiasmatic nucleus, and brainstem,19–21 playing a role in modulating brain systems involved in emotion.22,23 Unlike the well-established distribution and function of ASIC3 in peripheral nervous system (PNS), the role of ASIC3 in the CNS is still unclear. The present study was therefore designed to determine the expression of ASIC3 in CSF-contacting nucleus and explore the role of ASIC3 in inflammatory pain.
Materials and Methods Animal care and treatment The experimental protocols were approved by the Committee for the Ethical Use of Laboratory Animals, Xuzhou Medical College. Rats were used only once. Male Sprague-Dawley rats (250¡50 g) (n 5 6, each) were maintained in climate- and lightcontrolled conditions (23¡1uC, 12/12 hours dark/ light cycle with light on at 08:00 hour) for at least 1 week prior to the experiments. After determining the baseline threshold, the rats in complete Freund’s adjuvant (CFA) group received subcutaneous injection of CFA (1 mg ml21, 100 ml, Sigma) into the plantar surface of the left hind paw, and were subsequently returned to their cages. Following CFA injection, the rats were tested for thermal and mechanical hypersensitivity. The rats in control group were injected with saline, 100 ml. All experiments were conducted in accordance with the guidelines of the International Association for the Study of Pain (IASP).
Pain behavioral assessment Pain response was measured in a blinded fashion over a specific period of time, indoor temperature, and humidity. Rats were tested for basic thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) before injecting the hind paw with CFA, and the same procedure was repeated on days 1, 3, and 7 after injection. Rats were placed in a 3-mmthick glass plate and allowed to acclimatize for 30 minutes before testing. Thermal withdrawal latency was measured using the Plantar Test apparatus (Hargreaves method) (IITC series 8-390, Institute of Biological Medicine, Academy of Medical Science Beijing, China). Latency of the reflex was measured from the onset of radiant heat until hind-paw withdrawal. The cut-off time was set at 25 seconds in order to avoid tissue damage.
Role of ASIC3 in rat brain
Stimulation intensity was kept constant during the whole experiment. Each animal was tested five times at intervals of 5 minutes, and the mean TWL value of the last three measurements was used for analysis. The MWT on the plantar surface of the hind paw was measured with a set of Von Frey hairs (VFHs; Semmes-Weinstein Monofilaments, North Coast Medical, Morgan Hill, CA, USA) in ascending order of force (0.16, 0.4, 0.6, 1, 1.4, 2, 4, 6, 8, 10, 15, 26g). The VFH was applied to the paw for 6 seconds, or until a withdrawal response, when the paw was retested, starting with the next descending VFH until no response was noted. The lowest amount of force required to elicit a response was recorded as MWT in g. A significant reduction in TWL and MWT compared with normal baseline was interpreted as thermal and mechanical hyperalgesia. Rats that did not demonstrate hyperalgesia were excluded from the study. After recording the values of MWT and TWL over 3 days after injection of CFA, APETx2 was administered as an ICV injection of saline solution vehicle.
Intracerebroventricular injection Rats were anesthetized with 10% chloral hydrate (300 mg/kg, i.p.) and the head fixed in a stereotaxic instrument (Narishige Scientific Instruments, Tokyo, Japan). A 3 ml volume of 30% CB-HRP (Sigma) was injected into one of the rats’ lateral ventricles, according to stereotaxic coordinates (bregma: 21.2¡0.4 mm, depth: 3.2¡0.4 mm, right of median sagittal plane: 1.4¡0.2 mm). The ASIC3 specific inhibitor APETx2 (Alomone labs, Jerusalem, Israel) was diluted in physiological saline and administrated at 3 mM (3 ml) 3 days after injection of CFA as a unilateral ICV infusion, with a saline (3 ml) solution vehicle.
Tissue processing After 48 hours of tracer injection, under anesthesia with 10% chloral hydrate (300 mg/kg, i.p.), rats were perfused with 150 ml of phosphate buffered saline (PBS) (0.01 M PBS, pH 7.4), followed by 4% paraformaldehyde in 0.2 M phosphate buffer (300 ml, pH 7.4) without interruption. The brainstem was removed immediately and post-fixed for 4–6 hours at 4uC, and then immersed overnight in a 30% sucrose solution at 4uC. The brain tissue was embedded with OCT tissue freezing medium, at 220uC and sectioned in a cryostat (Leica CM1900, Germany) at 40 mm in the transverse plane. The frozen sections were collected in PBS. Following three washes in PBS, sections were incubated in PBS with 0.3% Triton X-100 (TBS) for 48–72 hours at 4uC with a goat anti-cholera toxin B-subunit (1 : 200, Sigma, St. Louis, MO, USA) and rabbit anti-ASIC3 (1 : 200, Alpha Diagnostic International, Inc., San Antonio, USA). After rinsing in PBS, sections were
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considering the intensity of the staining. Phosphate buffered saline was added to sections instead of primary antibody as negative control, and no ASIC3-labeled neurons were found (Fig. 1).
Statistical analysis Software Image-Pro Plus Version 6.0 (Media Cybernetics, Bethesda, MD, USA) was used to count the number of neurons. Four sections with centralized CB-HRP positive neurons were chosen from the same aspect of brain parenchyma in each rat. Data were expressed as mean¡SD. The data were subjected to a one-way analysis of variance (ANOVA) followed by Tukey’s t-test (or just a t-test when there were only two groups). P , 0.05 was considered to indicate statistical significance.
Results Behavioral assessment of CFA injection Figure 1 No immunosignal was detected when phosphate buffered saline (PBS) was added to section instead of primary antibody. (Scale bar: 100 mm.)
incubated in donkey anti-goat IgG conjugated to Cy3 (1 : 200, Millipore, Temecula, CA, USA) and in donkey anti-rabbit IgG conjugated to Fluorescein isothiocyanate (FITC) (1 : 100, Millipore) in the dark for 2 hours at room temperature. Finally, sections were rinsed, mounted, and coverslipped with glycerol and stored at 220uC in the dark. Tissue sections were examined using laser scanning confocal microscopy (TCS SP2, Leica, Wetzlar, Germany) to identify CSFcontacting nucleus neurons labeled with Cy3 and ASIC3 labeled with FITC. We selected four brain sections per animal, and each section had the greatest number of positive neurons. We recorded the total number of positive neurons in the brain of each animal. All positive neurons were counted without
In our experiments, the TWL and MWT in response to heat or mechanical stimulation were significantly decreased 1 day after CFA injection and the effect persisted for at least 3 days in CFA-treated rats. After gradual increase, complete recovery from foot swelling, walking difficulty, and protected behaviors such as lifting-affected foot took 2 weeks. However, no significant changes in TWL and MWT of rats in salinetreated control group were seen (Fig. 2A and B).
Expression of ASIC3 in CSF-contacting nucleus Positive labeling of CB-HRP-traced neurons in the ventral aqueduct region was mainly seen in cytoplasm, with most of the neurons multipolar and round or oval in shape. The cell bodies of CSF-contacting nucleus were in brain parenchyma, and the processes extended into CSF. A tissue section with CB-HRP immunoreactivity is illustrated in Fig. 3A and ASIC3-positive neurons (FITC, green) in Fig. 3B. The CB-HRP/ ASIC3 double-labeled neurons in yellow are shown in
Figure 2 Time course of development of thermal and mechanical hypersensitivity in rats after intraplantar injection with complete Freund’s adjuvant (CFA) or saline. Thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) are shown as mean¡SD. A significant reduction in TWL and MWT in CFA group compared with saline group: *P , 0.05, **P , 0.01 compared with saline group; #P , 0.05, ##P , 0.01 compared with 0 day (n 5 6).
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Figure 3 Acid-sensitive ion channel 3 (ASIC3) expression in cerebrospinal fluid (CSF)-contacting nucleus of normal rats. (A) Horseradish peroxidase-conjugated toxin subunit B (CBHRP) positive neurons (red), (B) ASIC3-positive neurons (green), and (C) CB-HRP/ASIC3 double-labeled neurons (yellow) (scale bar: 100 mm).
Fig. 3C. Immunofluorescence analysis indicated the localization of ASIC3 in the CSF-contacting nucleus.
Changes in ASIC3 expression in CSF-contacting nucleus Neurons positive for CB-HRP, ASIC3, and CB-HRP/ ASIC3 existed in CFA group. The ASIC3-immunoreactive levels were higher in CFA group (Fig. 4C–F) compared with normal group (Fig. 4A and B) following 3 and 7 days after injection of CFA. The number of CB-HRP/ASIC3 double-labeled neurons in CFA treatment group significantly increased, compared with that of normal group, which is positively relevant to signs of pain behavior (Fig. 4G).
Behavioral assessment of APETx2 injection The ICV administration of APETx2 (3 mM, 3 ml) 3 days after CFA injection significantly attenuated both thermal and mechanical hypersensitivities compared with the vehicle group. Time to reach the maximal anti-hyperalgesia was 15 minutes after administration of APETx2, and the effects decayed gradually over the subsequent 60 minutes (Fig. 5A and B). In contrast to what we observed in inflamed rats, injection of APETx2 did not affect either TWL or MWT in normal rats (Fig. 5C and D).
Discussion The present study has shown that the CSF-contacting nucleus is always located in a specific region of the rat brain parenchyma, which is consistent with previous investigations,3–7 and ASIC3 existed in CSF-contacting nucleus. The expression of ASIC3 in CSF-contacting nucleus increased following inflammatory pain. Furthermore, ICV administration of the specific ASIC3 antagonist, APETx2, exhibited analgesic effect. The result was consistent with the hypothesis that ASIC3 in CSF-contacting nucleus participates in central regulation of pain.
Figure 4 Acid-sensitive ion channel 3 (ASIC3) expression in rats with inflammatory pain. Photomicrograph (A) depicts ASIC3 expression in normal group; Photomicrographs (C, E) show ASIC3 expression 3 and 7 days following injection with complete Freund’s adjuvant (CFA); photomicrographs (B, D, F) show the enlargement of the rectangle in (A), (C), and (E); graph (G) illustrates the number of neurons labeled with ASIC3 or horseradish peroxidase-conjugated toxin subunit B (CB-HRP) in every six sections of three groups. Data are presented as mean¡SD. The numbers of ASIC3/CB-HRP neurons at 3 and 7 days are significantly higher than the normal group, #P , 0.05, **P , 0.01 compared with normal group (n 5 6). (Scale bars represent 100 mm.)
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Figure 5 Time course of the effect of ICV injection of APETx2 on thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT). Photomicrographs (A and B) depict time course of the effect of ICV injection of APETx2 on the complete Freund’s adjuvant (CFA)-induced reduction of TWL and MWT, 3 days after CFA injection. Injection of APETx2 elevated TWL and MWT to a level close to the normal level. A significant difference was observed between vehicle and APETx2 groups. Data are presented as mean¡SD, *P , 0.05, **P , 0.01 compared with vehicle group (n 5 6). Photomicrographs (C and D) depict the effect of injection of APETx2 in normal rats. Blockade of ASIC3 did not affect normal thermal and mechanical nociception. Data are presented as mean¡SD, P . 0.05 compared with vehicle group (n 5 6).
The CSF-contacting nucleus is a particular cell type in the CNS, whose bodies are in the brain parenchyma and processes extend into CSF. In view of their anatomical characteristics, they transmit signals between brain parenchyma and CSF,3 but the exact biological function was not clear. Acid-sensitive ion channel 3 has been reported to be predominantly distributed in the PNS.10,14,23 However, the expression of ASIC3 in the CNS was still unclear. Neurons in the hypothalamus, suprachiasmatic nucleus, and brainstem contain ASIC3 transcripts, and were immunoreactive for ASIC3.19–21 The characterization of the ASIC3 in brain was designed to explore its functional role in physiological and pathological processes of the CNS. We first confirmed that only a small part of CSF-contacting nucleus was immunoreactive for ASIC3. It suggested that in normal physiological conditions, ASIC3 may mediate the information transfer in CSF-contacting nucleus, which was similar to the DRG neurons serving multiple sensory modalities. Rats that were not injected CFA showed no differences in behavioral response before and after ICV injection of APETx2. Voilley
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et al. similarly showed that non-steroid anti-inflammatory drugs (NSAIDs) did not alter ASIC expression in normal sensory neurons.16 Therefore, ASIC3 channels in CSF-contacting nucleus specifically contribute to pathological but not physiological pain. The management of inflammatory pain is a clinical challenge. Efforts are mainly focused on the relationship between neuronal pathway and pain instead of brain-CSF neurohumoral circuit, which renders it difficult to fully clarify the pathogenesis of pain. Recent studies have documented that inflammatory pain elicits an inflammatory response in CSF, at least partly attributed to chemical changes in CSF.24,25 Complete Freund’s adjuvant-induced inflammation is a commonly used experimental animal model of persistent inflammatory pain. Animals showed thermal and mechanical hyperalgesia, and the expression level of ASIC3 significantly increased in CSFcontacting nucleus, 3 days after injection. The data are similar to a previous study that showed increased ASIC3 expression in L4 and L5 DRG neurons following CFA injection into hind paw.26
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We have also reported that ICV administration of APETx2 produced antihyperalgesia in rats with inflammatory pain; in that it reduced previously established thermal and mechanical hyperalgesia 3 days after CFA injection. In the PNS, the ASIC3 antagonist has a proven role in alleviating inflammatory pain.17,18 Therefore, our observations suggest that ASIC3 in CSF-contacting nucleus participates in the central regulation of pain. Indeed, for the first time, our findings confirm that AISC3 is involved in pain transmission at the superspinal level. In summary, we found that only a proportion of neurons in CSF-contacting nucleus were labeled for ASIC3 in normal rats. Inflammatory pain upregulated ASIC3 expression in CSF-contacting nucleus. Furthermore, ICV administration of APETx2 exhibited an antihyperalgesic effect. Our findings are consistent with the hypothesis that CSF-contacting nucleus neurons regulate inflammatory pain in the CNS via increased expression of ASIC3, thereby providing a new molecular basis for pain transmission and regulation.
Acknowledgements This article is supported by National Natural Science Foundation of China, numbers 81371243 and 81300957, and Natural Science Foundation of Jiangsu Province, number BK2012580.
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