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Hypoalgesia in mice lacking aquaporin-4 water channels
Brain Research Bulletin 83 (2010) 298–303
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Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Hypoalgesia in mice lacking aquaporin-4 water channels Feng Bao, Mengling Chen, Yuqiu Zhang, Zhiqi Zhao ∗ Institute of Neurobiology, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yi-Xue-Yuan Road, Shanghai 200032, PR China
a r t i c l e
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Article history: Received 13 January 2010 Received in revised form 14 August 2010 Accepted 27 August 2010 Available online 22 September 2010 Keywords: Aquaporin-4 Astrocyte Knockout mice Nociception Spinal cord
a b s t r a c t Previous studies have demonstrated the involvement of astrocytes in the modulation of pain. The water channel aquaporin-4, which is expressed in astrocytes but not neurons, has also been demonstrated to function in sensory processing, including hearing, vision, and olfaction. In the present study, we investigated a possible role of aquaporin-4 in the processing of nociception by measuring behavioral responses to noxious stimulation in aquaporin-4 knockout mice. Pain thresholds were increased in knockout mice, when compared to wild-type mice, with thermal and chemical stimulation but not mechanical stimulation. Aquaporin-4 knockout mice presented normal locomotor activity and basal skin temperature. Likewise, the electrophysiological recordings showed a significant decrease in the number of dorsal horn neurons sensitive to noxious thermal stimuli in aquaporin-4 knockout mice. Moreover, latencies to thermal stimuli were significantly prolonged in a subset of dorsal horn wide-dynamic-range neurons. Taken together, these results suggest that aquaporin-4 plays a role in the processing of nociception. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Our previous study demonstrated that intrathecal administration of fluorocitrate (FC), an astrocyte metabolic inhibitor, blocked both the long-term potentiation (LTP) of C-fiber-evoked field potentials and persistent pain induced by the tetanic stimulation of the sciatic nerve [19]. Likewise, the spinal LTP was blocked by the astrocyte glutamate transporter-1 (GLT-1) selective inhibitor, dihydrokainate (DHK) [31]. These results and other studies suggest an involvement of spinal astrocytes in the modulation of pain [5,19,29,31,34]. Aquaporin-4 (AQP4) is a water-selective channel specifically expressed in astrocytes and ependymal cells in the central nervous system. Aquaporin-4 is co-expressed with the astrocytic marker, glial fibrillary acidic protein (GFAP), in the spinal cord [14,24]. A line of evidence shows that AQP4 takes part in many physiological and pathological functions in the central nervous system. Studies in AQP4 knockout (KO) mice suggest the involvement of AQP4 in the movement of water into and out of the brain [1,20] and in the migration and proliferation of astrocytes [11,28]. Moreover, mice lacking AQP4 produce decreased electroretinogram potentials [15],
Abbreviations: AQP4, aquaporin-4; AQP4 KO, aquaporin-4 knockout; AQP4 WT, wild-type; Cx43, connexin43; DHK, dihydrokainate; FC, fluorocitrate; GFAP, glial fibrillary acidic protein; GLT-1, glutamate transporter-1; KO, knockout; LT, low threshold; LTP, long-term potentiation; NS, nociceptive specific; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; TMS, thermal-mechano-sensitive; WDR neuron, wide dynamic range neuron; WT, wild-type. ∗ Corresponding author. Tel.: +86 21 54237634; fax: +86 21 54237647. E-mail address: [email protected] (Z.Q. Zhao). 0361-9230/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2010.08.015
impaired hearing [16,21], reduced seizure susceptibility [4], slowed dynamics of cortical spreading depression [25], and impaired olfaction [17]. However, whether AQP4 contributes to the processing of nociception has not been explored. The present study was to investigate the possible roles of AQP4 in nociceptive processing by means of behavioral pain tests and extracellular recording in the spinal dorsal horn of AQP4 KO mice. 2. Materials and methods 2.1. Animals Both AQP4 KO mice and AQP4 wild type (WT) mice in a CD1 (out-bred) genetic background were provided by Dr. Gang Hu, Nanjing Medical University. AQP4 KO mice were generated by targeted gene disruption as described [7]. Six-to eight-month-old weight-matched WT and AQP4 KO mice were used. Mice were maintained in air-filtered cages and fed normal mouse chow. This study was conducted in concordance with the guidelines of the Ethical Standards of the International Association for the Study of Pain (IASP) [35]. 2.2. Western blot analysis Mice were sacrificed and the lumbar spinal cords (L4–6) were removed quickly. Collected tissues were homogenized in a lysis buffer (12.5 l/mg tissue) containing a mixture of protease inhibitors (Roche) and PMSF (Sigma). Protein samples were separated on SDS-PAGE (5–12% gels, Bio-Rad) and transferred onto PVDF membranes (Millipore). The membranes were blocked at room temperature for 2 h with 5% non-fat milk dissolved in Tris-buffered saline containing 0.05% Tween-20 (TBST) and incubated at 4 ◦ C overnight with a primary antibody (rabbit anti-AQP4, 1:2000, Chemicon). After being washed three times with TBST, the membranes were incubated at room temperature for 2 h with the corresponding HRP-conjugated secondary antibody (1:1000, Santa Cruz). The blots were then visualized in ECL solution (Pierce) and exposed onto X-films. The developed X-films were scanned for data analysis. Each sample was repeated three times under the same conditions. Nega-
F. Bao et al. / Brain Research Bulletin 83 (2010) 298–303 tive control lanes lacking primary antibody were performed in parallel. GAPDH was used as a loading control. 2.3. Behavioral tests 2.3.1. Rota-Rod test The Rota-Rod test is used to determine deficits in motor function. This test was performed as described [12] with several modifications. The mouse was required to walk on a rotating rod of 3.5 cm in diameter that gradually increased in speed up to 25 rpm until the animal can no longer maintain its position. The test was performed in the following sequence: a mouse was placed on the Rota-Rod; the stepper motor and timer was initiated. In order to remain in a stationary position, the mouse must walk or run on the Rota-Rod. The time required for the mouse to fall from the rotating rod was determined and taken as a measure of motor function. The mean of three trials was determined for each mouse. 2.3.2. Beam balance/walking test Deficits in fine motor coordination can be assessed by using a beam balance or walking task. This test was done as described [13] with several modifications. A beam approximately 8 mm wide and 200 mm in length was suspended about 250 mm above a foam pad. The mouse was placed on one end of the beam and encouraged to walk along the beam to reach the opposite end. Once it reached the end, the mouse turned 180◦ and continued to walk back towards the opposite end. The number of foot-faults were counted as the mouse walked the beam. The number of foot-faults are defined as the number of times the forepaws and/or hindpaws slip from the horizontal surface of the beam over 50 steps. Each mouse was allowed 5 min to complete the task. 2.4. Hindpaw radiant heat test (Hargreaves test) Mice were placed in dark plastic chambers (7 cm × 7 cm) on a glass surface maintained at 22–23 ◦ C by air condition system. A radiant heat source (BME-410C, Int. of Biomedical Engineering, CAMS, China) was focused on the hindpaw. The latency of hindpaw withdrawal was recorded and the average of three trials was calculated. The cut-off time was set at 15 s to avoid tissue damage. 2.5. Tail flick test Tail radiant heat test was performed on both genotypes of mice under identical condition. The mice were acclimated to the testing conditions for 3 days. The radiant heat source (BME-410C, Int. of Biomedical Engineering, CAMS, China) was placed just beneath the tail about 2.5 cm away from the tip. An intensive flick or obvious upward bending of the tail was considered a nocifensive response. The latency of tail withdrawal per animal was measured a minimum of 5 min apart. Cut-off time was 7 s to avoid tissue damage. In order to minimize the stress caused by restricting the movement of the mouse, the tail immersion test was done under light gas anesthesia by diethyl ether. The mice were anaesthetized by inhaling diethyl ether for less than 5 s. Corneal reflex was measured to ensure the mice were not deeply anaesthetized. A time window of 20-s is available to apply hot water on the pendulous tail before autonomic functions recovered. About 1/3 of the tail was immersed, and the latency until an obvious tail flick was recorded. 2.6. Hot plate test Mice were placed on a hot plate and the time latencies to lick/flip the forepaw/hindpaw or to jump were recorded. Each mouse was measured once a day at an interval of at least 1 day. The cut-off time was set at 30 s to avoid tissue damage.
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2.8. Formalin test Mice were tested individually in plastic chambers on a room-temperature glass surface. After intraplantar (i.pl.) formalin injection (2.5%, 5 l), the time of withdrawal and licking was recorded every 5 min. Each mouse was observed for a total time of 1 h. The formalin score was calculated based on the following formula: Formalin score =
Timewithdrawal + (Timelicking × 2) 300
2.9. Electrophysiological recording in the spinal cord Experiments were performed in adult AQP4 WT and KO mice (20–30 g). Animals were anesthetized with urethane (25%, 0.6 ml/100 g) intraperitoneally (i.p.). Endotracheal intubation was done before laminectomy to expose the L4 and L5 segments of the spinal cord. The animals were suspended by clamping the rostral and caudal vertebral segments (STS – 7, NARISHIGE, Japan) to minimize movement of the spinal cord. A well surrounding the spinal cord was filled with mineral oil. The animal was covered with a matched cloth to maintain permanent core temperature. Respiratory function was not altered during the experiment. Extracellular discharges of wide-dynamic-range (WDR) neurons were recorded 200–500 m from the surface of the spinal cord using a glass microelectrode with an impedance of ∼8 M at 100 Hz. Data were acquired and processed using Micro 1401 and Spike 2 software. Search stimuli for neurons consisted of applying soft pressure or brushing to the plantar surface of the hindpaw. Once a neuron was isolated, responses to brush, pinch, or noxious heat (43, 47, 50, or 52 ◦ C) were recorded. A lighting stimulator delivered thermal stimuli with instant heating to the preset temperature. The heat stimuli were applied for 10 s and separated by at least 3 min. The number of WT and KO mice was 14 and 16, respectively (oscilloscope: SSI – 2220, SAMPO, Taiwan; amplifier: AC/DC Differential Amplifier Model 3000, A-M Systems, Inc., USA; stimulator: Isolated Pulse Stimulator Model 2100, A-M Systems, Inc., USA). 2.10. Statistical analysis All values were expressed as the means ± S.E.M. Differences among means were analyzed using one- or two-way ANOVA with time, treatment or genotype as the independent factors. When ANOVA showed significant differences, pair-wise comparisons between means were performed with two-tailed indirect Student’s t-test. In all analyses, the level of statistical significance was defined as P < 0.05.
3. Results 3.1. General appearance of AQP4 knockout mice The experiments were performed on adult AQP4 WT and KO mice (22–28 g). Western blot analysis showed complete deletion of AQP4 (Fig. 1A). Both the hindpaw skin and tail temperature displayed no differences between the two genotypes (22.1 ± 0.9 ◦ C, n = 8, WT mice vs. 22.3 ± 1.1 ◦ C, n = 8, KO mice). Spontaneous locomotor activities were examined in both genotypes of mice with two methods. In the Rota-Rod test, AQP4 KO mice exhibited better locomotor activities than paired WT mice (Fig. 1C). Also, AQP4 KO mice showed the same performance as the WT mice did in beam balance/walking test (Fig. 1B). Foot-faults were counted over 5 min periods for each mouse.
2.7. von Frey test for mechanical threshold The mechanical threshold was measured by probing von Frey filaments (von Frey hairs, Stoelting, USA) on the hindpaws with bending forces ranging from 1 to 4 g. Each mouse was placed in a chamber (10 cm × 10 cm × 15 cm) on a metal-wire platform with 1 mm diameter holes. Each mouse was allowed to acclimate to the testing conditions for approximately 30 min. After acclimation, a series of von Frey filament stimuli were delivered with increasing bending force to the central region of the plantar surface of the hindpaw. Each filament was applied until buckling of the filament occurred and was maintained for approximately 2 s. A withdrawal response was considered valid only if the hindpaw was completely removed from the platform. A trial consisted of a von Frey filament application to the hindpaw five times at 15 s intervals. When the hind paw withdrew from a particular filament four or five times out of the five applications, the value of the filament in grams was considered to be the “paw withdrawal threshold” (PWT) to mechanical stimulation. After a 5 min rest period, the mechanical threshold was confirmed with the previously effective von Frey filament and was reconfirmed after another 5 min. If the withdrawal threshold in the second or third session did not match the previous one, this procedure was repeated with progressively larger von Frey filament until the withdrawal threshold in three successive trials were consistent.
3.2. AQP4 deficiency-induced increase of pain thresholds in different behavioral tests 3.2.1. Tail flick to radiant heat The tail radiant heat test was performed on both genotypes of mice under identical conditions. As shown in Fig. 2, AQP4 KO mice displayed a significant increase in tail flick latencies (3.3 ± 0.05 s, n = 55) compared to WT mice (2.7 ± 0.05 s, n = 54), indicating an increase in pain threshold. 3.2.2. Tail water-immersion test The tail flick latencies were significantly increased in AQP4 KO mice compared to WT mice in both 47 ◦ C (11.82 ± 0.83 s, n = 34, vs. 1.93 ± 0.24 s, n = 41) and 53 ◦ C (1.19 ± 0.22 s, n = 16, vs. 0.59 ± 0.05 s, n = 10) in the water-immersion test (Fig. 3A and B).
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Fig. 1. No impairment of locomotor activities in aquaporin-4 (AQP4) null mice. (A) Western blot analysis showed complete knockout of AQP4. (B) Locomotor activities of wild-type (WT) (n = 8) and knockout (KO) mice (n = 7) in the beam walking test showed no impairment of fine motor coordination of mutant mice. The number of foot-faults were counted over 5 min (P > 0.05, Student’s t-test). (C) Locomotor activities of WT and KO mice in the Rota-Rod test showed no impairment of gross motor functions of knockout mice. Rolling speed was fixed on 25 rpm (n = 7, *P < 0.05, Student’s t-test).
3.2.3. Paw withdrawal test Radiant heat (50 ◦ C) was applied unilaterally to the hindpaw. The thermal stimuli-induced paw withdrawal latency represents the pain threshold. In AQP4 KO mice, the latencies were significantly prolonged (11.01 ± 0.78 s, n = 10) compared to WT mice (5.44 ± 0.28 s, n = 13) (Fig. 3C). 3.2.4. Hot plate test The temperature of the hot plate was set at 47, 52 or 54 ◦ C. The latencies of the escaping behavior were measured after placing the mice onto the hot plate. As shown in Fig. 3D, AQP4 KO mice displayed significantly prolonged latencies when compared to the WT mice at all three temperatures. As the temperature increased, the latencies were shortened in both AQP4 KO and WT mice.
3.2.5. Chemical pain: formalin test The formalin-induced licking behaviors were used as an indication of spontaneous pain. After intraplantar injection of formalin (2.5%, 5 l), two phases of spontaneous pain occurred at 5 min and 15–45 min. As shown in Fig. 4, pain behaviors in both genotypes displayed no significant difference at the first phase. But the curve of second phase had an obvious rightward shift and a lower peak in AQP4 KO mice. The peak of the curve in AQP4 WT mice was 85% higher than that in KO mice. Meanwhile, the WT mice displayed spontaneous pain for longer time at the second phase (n = 8, WT mice, n = 9, KO mice). 3.2.6. Mechanical pain test The paw withdrawal threshold induced by von Frey filaments represents the mechanical pain threshold. There was no difference in mechanical pain thresholds between WT mice (2.34 ± 0.21 g, n = 14) and KO mice (2.07 ± 0.25 g, n = 11) (P = 0.41, Student’s t-test). 3.3. Electrophysiological recording: decrease in the numbers of neurons responding to sensory stimulation in AQP4 KO mice
Fig. 2. Prolongation of tail flick latencies of AQP4 KO mice in radiant heat test. Reponses to noxious thermal stimuli were measured by radiant heat-induced tail flick latencies. KO mice (n = 55) displayed increase in tail flick latencies compared to WT mice (n = 54) (***P < 0.001, Student’s t-test).
A total of 58 neurons (wild-type) and 55 neurons (knockout) were extracellularly recorded in the superficial dorsal horn of the spinal cord in vivo, including: nociceptive specific (NS) neurons, low threshold (LT) neurons, and wide-dynamic-range (WDR) neurons. WDR neurons included thermal-mechano-sensitive (TMS) neurons, sensitive to both thermal and mechanical stimuli, and mechano-sensitive (MS) neurons, sensitive to mechanical stimuli. The proportion of TMS-WDR neurons and LT neurons made up 24% (n = 58, WT) vs. 11% (n = 55, KO) and 38% (n = 58, WT) vs. 25% (n = 55, KO), respectively. Likewise, several NS neurons that selectively responded to noxious pinch and/or heat stimulation were found in wild-type mice, whereas none were recorded in AQP4 KO mice. However, firing frequencies of TMS-WDR neurons to thermal stimulation at 50 ◦ C were not changed in both WT mice (26 ± 1.5 Hz, n = 6) and KO mice (25 ± 2.9 Hz, n = 6). Noticeably, a few TMS-WDR neurons exhibited significantly longer latencies to noxious thermal stimulation in four AQP4 KO mice. The latencies were 7.35 ± 1.73 s (n = 5), compared to WT mice (0.74 ± 0.03 s, n = 6). The latencies were shortened with an increase in stimuli temperature from 43 to 50 ◦ C (Fig. 5).
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Fig. 3. Increase of thermal pain thresholds of AQP4 KO mice in tail immersion test, paw withdrawal test, and hot plate test. Responses to noxious thermal stimuli were measured by hot-water-immersion-induced tail flick latencies. KO mice (n = 34) showed a longer latency than WT mice (n = 41) in 47 ◦ C hot water (A). Both the WT mice (n = 10) and mutant mice (n = 16) showed very short latencies when 53 ◦ C water was applied to the tails (B) (*P < 0.05, ***P < 0.001, Student’s t-test). (C) Reponses to noxious thermal stimuli were measured by radiant heat-induced hindpaw withdrawal latencies. KO mice (n = 10) displayed significantly increased pain thresholds compared to WT mice (n = 13) (***P < 0.001, Student’s t-test). (D) Hot plate tests were applied to WT and KO mice with varied temperatures: 47 ◦ C, 52 ◦ C and 54 ◦ C. All the asterisks showed a significant difference between wild-type and mutant mice at each temperature (**P < 0.01, ***P < 0.001, Student’s t-test).
4. Discussion Increasing evidence has revealed that astrocytes play a crucial role in the processing of pain in the spinal cord [5,32]. Many signaling molecules related to synaptic transmission and modulation are expressed in astrocytes [2,26]. The signaling molecules in astrocytes that are involved in nociceptive processing are closely followed with interest. Out of them, AQP4 is expressed in astrocytes but not in neurons in the central nervous system. Therefore, AQP4 knockout mice are an ideal model to examine possible roles of AQP4 in the processing of nociception. This is the first study to
Fig. 4. AQP4 KO mice displayed delayed and impaired responses in the second phase of the formalin test. 2.5% formalin-induced spontaneous pain was measured by the formalin score in WT mice (n = 8) and KO mice (n = 9). AQP4 KO mice showed significant differences at the second phase. Arrow: formalin injection. Asterisk showed comparison between wild-type mice and mutant ones 20/25/45 min after formalin injection (*P < 0.05, ***P < 0.001, Student’s t-test).
show that an AQP4 deficiency produces an increase in behavioral pain thresholds. Except for mechanical stimulation, pain responses to thermal and chemical stimulation were attenuated. Consistent with the increase in pain thresholds, the number of neurons responsive to noxious thermal stimulation was reduced and latencies to thermal stimulation were significantly prolonged in a subset of dorsal horn WDR neurons. Taken together, the results imply that AQP4 deficiency induce hypoalgesia. To support a role of AQP4 in nociceptive processing, a study showed that AQP4 expression was highly increased in astrocytes following rat spinal cord injury with central neuropathic pain [22]. Given that there were no impairments of locomotor activities or change in skin temperature, it is conceivable that pain behavioral changes were attributable to the functional alteration due to AQP4 gene deletion. It is very interesting that the KO mice displayed better motor function in the Rota-Rod test compared to WT mice. It is well-known that dopamine is a very important neurotransmitter in motor function. An increase in dopamine is closely related to enhanced locomotor activity [3] and the speed of running [9]. A previous report demonstrated that the basal extracellular levels of dopamine and its metabolites (dihydroxyphenylacetic acid, DO-PAC, and homovanillic acid, HVA) were significantly increased in mice after AQP4 gene deletion [6]. This result might provide one explanation for the improved motor performance of the aquaporin-4 knockout mice. The mechanisms underlying AQP4 deficiency-induced changes in nociceptive processing are poorly understood. However, two possibilities can be considered: (1) in the central nervous system, AQP4 in astrocytes is commonly thought to facilitate K+ fluxes associated with adjacent neurons [30] in spite of having a contradictory report [27]. In AQP4 KO mice, K+ channels could be responsible for the delayed reuptake of excess extracellular K+ after a depolarizing stimulus [15,16]. It has been demonstrated that AQP4 in astrocytes contribute to hearing, vision and olfaction via modulation of
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Fig. 5. A subset of TMS-WDR neurons in KO mice had prolonged latencies. Firing samples of TMS-WDR neurons had prolonged latencies in KO mice compared to WT mice. A and B represented firing samples in WT mice after noxious heat application. C and D represented firing samples in KO mice. Five neurons responding to all stimuli from four KO mice responded normally to touch and noxious mechanical stimulation but they displayed prolonged latencies to noxious heat stimulation applied for 10 s when compared to WT mice.
extracellular space K+ buffering [15–17,21]. Therefore, it is possible that the increase in the pain threshold could also result from impaired extracellular K+ buffering in AQP4 KO mice. Meanwhile, AQP4 KO mice display an expansion in the extracellular space of the brain that is associated with enhanced solute diffusion. This suggests that a volume dilution mechanism contributes to the delayed solute and transmitter reuptake [33]. (2) Connexin 43 (Cx43) is a modulator of extracellular neurotransmitter reuptake and astrocytic networks [10]. AQP4 siRNA produced a down-regulation of AQP4 and Cx43 [23]. Cx43’s dysfunction decreased the glutamate release from astrocytes [8]. Accordingly, Cx43 might be involved in AQP4 deficiency-induced changes in nociception. Unexpectedly, the mechanical pain threshold was not changed after AQP4 deletion. Given that the non-neuronal cells and corpuscles in the skin are considered the primary mechano-transducers in mechanical nociceptive processing [18], it might be that mechanotransducers are insensitive to changes in the ambient buffering. In conclusion, AQP4 deficiency altered the modulatory function of astrocytes, resulting in the increase of pain thresholds via the glia-neuron communication. It is suggested that AQP4 is involved in the processing of nociception. Acknowledgements Special thanks to Dr. Gang Hu from Nanjing Medical University and Dr. Tonghui Ma from Northeast Normal University for mice provide. We thank Dr. Bryan Chai from Department of Neural and Pain Sciences, University of Maryland Dental School for manuscript editing. This work was supported by grant from the National Basic Research Program of China Grant 2006CB500807 and 2007CB512502 and the National Natural Science Fund of China 30830044. References [1] P. Agre, L.S. King, M. Yasui, W.B. Guggino, O.P. Ottersen, Y. Fujiyoshi, A. Engel, S. Nielsen, Aquaporin water channels—from atomic structure to clinical medicine, J. Physiol. 542 (2002) 3–16. [2] A. Araque, Astrocytes process synaptic information, Neuron Glia Biol. 4 (2008) 3–10. [3] R.J. Beninger, The role of dopamine in locomotor activity and learning, Brain Res. 287 (1983) 173–196. [4] A.D. Bragg, M. Amiry-Moghaddam, O.P. Ottersen, M.E. Adams, S.C. Froehner, Assembly of a perivascular astrocyte protein scaffold at the mammalian blood–brain barrier is dependent on alpha-syntrophin, Glia 53 (2006) 879–890. [5] H. Cao, Y.Q. Zhang, Spinal glial activation contributes to pathological pain states, Neurosci. Biobehav. Rev. 32 (2008) 972–983. [6] J.H. Ding, L.L. Sha, J. Chang, X.Q. Zhou, Y. Fan, G. Hu, Alterations of striatal neurotransmitter release in aquaporin-4 deficient mice: an in vivo microdialysis study, Neurosci. Lett. 422 (2007) 175–180.
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Hypoalgesia in mice lacking aquaporin-4 water channels Feng Bao, Mengling Chen, Yuqiu Zhang, Zhiqi Zhao ∗ Institute of Neurobiology, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yi-Xue-Yuan Road, Shanghai 200032, PR China
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Article history: Received 13 January 2010 Received in revised form 14 August 2010 Accepted 27 August 2010 Available online 22 September 2010 Keywords: Aquaporin-4 Astrocyte Knockout mice Nociception Spinal cord
a b s t r a c t Previous studies have demonstrated the involvement of astrocytes in the modulation of pain. The water channel aquaporin-4, which is expressed in astrocytes but not neurons, has also been demonstrated to function in sensory processing, including hearing, vision, and olfaction. In the present study, we investigated a possible role of aquaporin-4 in the processing of nociception by measuring behavioral responses to noxious stimulation in aquaporin-4 knockout mice. Pain thresholds were increased in knockout mice, when compared to wild-type mice, with thermal and chemical stimulation but not mechanical stimulation. Aquaporin-4 knockout mice presented normal locomotor activity and basal skin temperature. Likewise, the electrophysiological recordings showed a significant decrease in the number of dorsal horn neurons sensitive to noxious thermal stimuli in aquaporin-4 knockout mice. Moreover, latencies to thermal stimuli were significantly prolonged in a subset of dorsal horn wide-dynamic-range neurons. Taken together, these results suggest that aquaporin-4 plays a role in the processing of nociception. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Our previous study demonstrated that intrathecal administration of fluorocitrate (FC), an astrocyte metabolic inhibitor, blocked both the long-term potentiation (LTP) of C-fiber-evoked field potentials and persistent pain induced by the tetanic stimulation of the sciatic nerve [19]. Likewise, the spinal LTP was blocked by the astrocyte glutamate transporter-1 (GLT-1) selective inhibitor, dihydrokainate (DHK) [31]. These results and other studies suggest an involvement of spinal astrocytes in the modulation of pain [5,19,29,31,34]. Aquaporin-4 (AQP4) is a water-selective channel specifically expressed in astrocytes and ependymal cells in the central nervous system. Aquaporin-4 is co-expressed with the astrocytic marker, glial fibrillary acidic protein (GFAP), in the spinal cord [14,24]. A line of evidence shows that AQP4 takes part in many physiological and pathological functions in the central nervous system. Studies in AQP4 knockout (KO) mice suggest the involvement of AQP4 in the movement of water into and out of the brain [1,20] and in the migration and proliferation of astrocytes [11,28]. Moreover, mice lacking AQP4 produce decreased electroretinogram potentials [15],
Abbreviations: AQP4, aquaporin-4; AQP4 KO, aquaporin-4 knockout; AQP4 WT, wild-type; Cx43, connexin43; DHK, dihydrokainate; FC, fluorocitrate; GFAP, glial fibrillary acidic protein; GLT-1, glutamate transporter-1; KO, knockout; LT, low threshold; LTP, long-term potentiation; NS, nociceptive specific; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; TMS, thermal-mechano-sensitive; WDR neuron, wide dynamic range neuron; WT, wild-type. ∗ Corresponding author. Tel.: +86 21 54237634; fax: +86 21 54237647. E-mail address: [email protected] (Z.Q. Zhao). 0361-9230/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2010.08.015
impaired hearing [16,21], reduced seizure susceptibility [4], slowed dynamics of cortical spreading depression [25], and impaired olfaction [17]. However, whether AQP4 contributes to the processing of nociception has not been explored. The present study was to investigate the possible roles of AQP4 in nociceptive processing by means of behavioral pain tests and extracellular recording in the spinal dorsal horn of AQP4 KO mice. 2. Materials and methods 2.1. Animals Both AQP4 KO mice and AQP4 wild type (WT) mice in a CD1 (out-bred) genetic background were provided by Dr. Gang Hu, Nanjing Medical University. AQP4 KO mice were generated by targeted gene disruption as described [7]. Six-to eight-month-old weight-matched WT and AQP4 KO mice were used. Mice were maintained in air-filtered cages and fed normal mouse chow. This study was conducted in concordance with the guidelines of the Ethical Standards of the International Association for the Study of Pain (IASP) [35]. 2.2. Western blot analysis Mice were sacrificed and the lumbar spinal cords (L4–6) were removed quickly. Collected tissues were homogenized in a lysis buffer (12.5 l/mg tissue) containing a mixture of protease inhibitors (Roche) and PMSF (Sigma). Protein samples were separated on SDS-PAGE (5–12% gels, Bio-Rad) and transferred onto PVDF membranes (Millipore). The membranes were blocked at room temperature for 2 h with 5% non-fat milk dissolved in Tris-buffered saline containing 0.05% Tween-20 (TBST) and incubated at 4 ◦ C overnight with a primary antibody (rabbit anti-AQP4, 1:2000, Chemicon). After being washed three times with TBST, the membranes were incubated at room temperature for 2 h with the corresponding HRP-conjugated secondary antibody (1:1000, Santa Cruz). The blots were then visualized in ECL solution (Pierce) and exposed onto X-films. The developed X-films were scanned for data analysis. Each sample was repeated three times under the same conditions. Nega-
F. Bao et al. / Brain Research Bulletin 83 (2010) 298–303 tive control lanes lacking primary antibody were performed in parallel. GAPDH was used as a loading control. 2.3. Behavioral tests 2.3.1. Rota-Rod test The Rota-Rod test is used to determine deficits in motor function. This test was performed as described [12] with several modifications. The mouse was required to walk on a rotating rod of 3.5 cm in diameter that gradually increased in speed up to 25 rpm until the animal can no longer maintain its position. The test was performed in the following sequence: a mouse was placed on the Rota-Rod; the stepper motor and timer was initiated. In order to remain in a stationary position, the mouse must walk or run on the Rota-Rod. The time required for the mouse to fall from the rotating rod was determined and taken as a measure of motor function. The mean of three trials was determined for each mouse. 2.3.2. Beam balance/walking test Deficits in fine motor coordination can be assessed by using a beam balance or walking task. This test was done as described [13] with several modifications. A beam approximately 8 mm wide and 200 mm in length was suspended about 250 mm above a foam pad. The mouse was placed on one end of the beam and encouraged to walk along the beam to reach the opposite end. Once it reached the end, the mouse turned 180◦ and continued to walk back towards the opposite end. The number of foot-faults were counted as the mouse walked the beam. The number of foot-faults are defined as the number of times the forepaws and/or hindpaws slip from the horizontal surface of the beam over 50 steps. Each mouse was allowed 5 min to complete the task. 2.4. Hindpaw radiant heat test (Hargreaves test) Mice were placed in dark plastic chambers (7 cm × 7 cm) on a glass surface maintained at 22–23 ◦ C by air condition system. A radiant heat source (BME-410C, Int. of Biomedical Engineering, CAMS, China) was focused on the hindpaw. The latency of hindpaw withdrawal was recorded and the average of three trials was calculated. The cut-off time was set at 15 s to avoid tissue damage. 2.5. Tail flick test Tail radiant heat test was performed on both genotypes of mice under identical condition. The mice were acclimated to the testing conditions for 3 days. The radiant heat source (BME-410C, Int. of Biomedical Engineering, CAMS, China) was placed just beneath the tail about 2.5 cm away from the tip. An intensive flick or obvious upward bending of the tail was considered a nocifensive response. The latency of tail withdrawal per animal was measured a minimum of 5 min apart. Cut-off time was 7 s to avoid tissue damage. In order to minimize the stress caused by restricting the movement of the mouse, the tail immersion test was done under light gas anesthesia by diethyl ether. The mice were anaesthetized by inhaling diethyl ether for less than 5 s. Corneal reflex was measured to ensure the mice were not deeply anaesthetized. A time window of 20-s is available to apply hot water on the pendulous tail before autonomic functions recovered. About 1/3 of the tail was immersed, and the latency until an obvious tail flick was recorded. 2.6. Hot plate test Mice were placed on a hot plate and the time latencies to lick/flip the forepaw/hindpaw or to jump were recorded. Each mouse was measured once a day at an interval of at least 1 day. The cut-off time was set at 30 s to avoid tissue damage.
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2.8. Formalin test Mice were tested individually in plastic chambers on a room-temperature glass surface. After intraplantar (i.pl.) formalin injection (2.5%, 5 l), the time of withdrawal and licking was recorded every 5 min. Each mouse was observed for a total time of 1 h. The formalin score was calculated based on the following formula: Formalin score =
Timewithdrawal + (Timelicking × 2) 300
2.9. Electrophysiological recording in the spinal cord Experiments were performed in adult AQP4 WT and KO mice (20–30 g). Animals were anesthetized with urethane (25%, 0.6 ml/100 g) intraperitoneally (i.p.). Endotracheal intubation was done before laminectomy to expose the L4 and L5 segments of the spinal cord. The animals were suspended by clamping the rostral and caudal vertebral segments (STS – 7, NARISHIGE, Japan) to minimize movement of the spinal cord. A well surrounding the spinal cord was filled with mineral oil. The animal was covered with a matched cloth to maintain permanent core temperature. Respiratory function was not altered during the experiment. Extracellular discharges of wide-dynamic-range (WDR) neurons were recorded 200–500 m from the surface of the spinal cord using a glass microelectrode with an impedance of ∼8 M at 100 Hz. Data were acquired and processed using Micro 1401 and Spike 2 software. Search stimuli for neurons consisted of applying soft pressure or brushing to the plantar surface of the hindpaw. Once a neuron was isolated, responses to brush, pinch, or noxious heat (43, 47, 50, or 52 ◦ C) were recorded. A lighting stimulator delivered thermal stimuli with instant heating to the preset temperature. The heat stimuli were applied for 10 s and separated by at least 3 min. The number of WT and KO mice was 14 and 16, respectively (oscilloscope: SSI – 2220, SAMPO, Taiwan; amplifier: AC/DC Differential Amplifier Model 3000, A-M Systems, Inc., USA; stimulator: Isolated Pulse Stimulator Model 2100, A-M Systems, Inc., USA). 2.10. Statistical analysis All values were expressed as the means ± S.E.M. Differences among means were analyzed using one- or two-way ANOVA with time, treatment or genotype as the independent factors. When ANOVA showed significant differences, pair-wise comparisons between means were performed with two-tailed indirect Student’s t-test. In all analyses, the level of statistical significance was defined as P < 0.05.
3. Results 3.1. General appearance of AQP4 knockout mice The experiments were performed on adult AQP4 WT and KO mice (22–28 g). Western blot analysis showed complete deletion of AQP4 (Fig. 1A). Both the hindpaw skin and tail temperature displayed no differences between the two genotypes (22.1 ± 0.9 ◦ C, n = 8, WT mice vs. 22.3 ± 1.1 ◦ C, n = 8, KO mice). Spontaneous locomotor activities were examined in both genotypes of mice with two methods. In the Rota-Rod test, AQP4 KO mice exhibited better locomotor activities than paired WT mice (Fig. 1C). Also, AQP4 KO mice showed the same performance as the WT mice did in beam balance/walking test (Fig. 1B). Foot-faults were counted over 5 min periods for each mouse.
2.7. von Frey test for mechanical threshold The mechanical threshold was measured by probing von Frey filaments (von Frey hairs, Stoelting, USA) on the hindpaws with bending forces ranging from 1 to 4 g. Each mouse was placed in a chamber (10 cm × 10 cm × 15 cm) on a metal-wire platform with 1 mm diameter holes. Each mouse was allowed to acclimate to the testing conditions for approximately 30 min. After acclimation, a series of von Frey filament stimuli were delivered with increasing bending force to the central region of the plantar surface of the hindpaw. Each filament was applied until buckling of the filament occurred and was maintained for approximately 2 s. A withdrawal response was considered valid only if the hindpaw was completely removed from the platform. A trial consisted of a von Frey filament application to the hindpaw five times at 15 s intervals. When the hind paw withdrew from a particular filament four or five times out of the five applications, the value of the filament in grams was considered to be the “paw withdrawal threshold” (PWT) to mechanical stimulation. After a 5 min rest period, the mechanical threshold was confirmed with the previously effective von Frey filament and was reconfirmed after another 5 min. If the withdrawal threshold in the second or third session did not match the previous one, this procedure was repeated with progressively larger von Frey filament until the withdrawal threshold in three successive trials were consistent.
3.2. AQP4 deficiency-induced increase of pain thresholds in different behavioral tests 3.2.1. Tail flick to radiant heat The tail radiant heat test was performed on both genotypes of mice under identical conditions. As shown in Fig. 2, AQP4 KO mice displayed a significant increase in tail flick latencies (3.3 ± 0.05 s, n = 55) compared to WT mice (2.7 ± 0.05 s, n = 54), indicating an increase in pain threshold. 3.2.2. Tail water-immersion test The tail flick latencies were significantly increased in AQP4 KO mice compared to WT mice in both 47 ◦ C (11.82 ± 0.83 s, n = 34, vs. 1.93 ± 0.24 s, n = 41) and 53 ◦ C (1.19 ± 0.22 s, n = 16, vs. 0.59 ± 0.05 s, n = 10) in the water-immersion test (Fig. 3A and B).
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Fig. 1. No impairment of locomotor activities in aquaporin-4 (AQP4) null mice. (A) Western blot analysis showed complete knockout of AQP4. (B) Locomotor activities of wild-type (WT) (n = 8) and knockout (KO) mice (n = 7) in the beam walking test showed no impairment of fine motor coordination of mutant mice. The number of foot-faults were counted over 5 min (P > 0.05, Student’s t-test). (C) Locomotor activities of WT and KO mice in the Rota-Rod test showed no impairment of gross motor functions of knockout mice. Rolling speed was fixed on 25 rpm (n = 7, *P < 0.05, Student’s t-test).
3.2.3. Paw withdrawal test Radiant heat (50 ◦ C) was applied unilaterally to the hindpaw. The thermal stimuli-induced paw withdrawal latency represents the pain threshold. In AQP4 KO mice, the latencies were significantly prolonged (11.01 ± 0.78 s, n = 10) compared to WT mice (5.44 ± 0.28 s, n = 13) (Fig. 3C). 3.2.4. Hot plate test The temperature of the hot plate was set at 47, 52 or 54 ◦ C. The latencies of the escaping behavior were measured after placing the mice onto the hot plate. As shown in Fig. 3D, AQP4 KO mice displayed significantly prolonged latencies when compared to the WT mice at all three temperatures. As the temperature increased, the latencies were shortened in both AQP4 KO and WT mice.
3.2.5. Chemical pain: formalin test The formalin-induced licking behaviors were used as an indication of spontaneous pain. After intraplantar injection of formalin (2.5%, 5 l), two phases of spontaneous pain occurred at 5 min and 15–45 min. As shown in Fig. 4, pain behaviors in both genotypes displayed no significant difference at the first phase. But the curve of second phase had an obvious rightward shift and a lower peak in AQP4 KO mice. The peak of the curve in AQP4 WT mice was 85% higher than that in KO mice. Meanwhile, the WT mice displayed spontaneous pain for longer time at the second phase (n = 8, WT mice, n = 9, KO mice). 3.2.6. Mechanical pain test The paw withdrawal threshold induced by von Frey filaments represents the mechanical pain threshold. There was no difference in mechanical pain thresholds between WT mice (2.34 ± 0.21 g, n = 14) and KO mice (2.07 ± 0.25 g, n = 11) (P = 0.41, Student’s t-test). 3.3. Electrophysiological recording: decrease in the numbers of neurons responding to sensory stimulation in AQP4 KO mice
Fig. 2. Prolongation of tail flick latencies of AQP4 KO mice in radiant heat test. Reponses to noxious thermal stimuli were measured by radiant heat-induced tail flick latencies. KO mice (n = 55) displayed increase in tail flick latencies compared to WT mice (n = 54) (***P < 0.001, Student’s t-test).
A total of 58 neurons (wild-type) and 55 neurons (knockout) were extracellularly recorded in the superficial dorsal horn of the spinal cord in vivo, including: nociceptive specific (NS) neurons, low threshold (LT) neurons, and wide-dynamic-range (WDR) neurons. WDR neurons included thermal-mechano-sensitive (TMS) neurons, sensitive to both thermal and mechanical stimuli, and mechano-sensitive (MS) neurons, sensitive to mechanical stimuli. The proportion of TMS-WDR neurons and LT neurons made up 24% (n = 58, WT) vs. 11% (n = 55, KO) and 38% (n = 58, WT) vs. 25% (n = 55, KO), respectively. Likewise, several NS neurons that selectively responded to noxious pinch and/or heat stimulation were found in wild-type mice, whereas none were recorded in AQP4 KO mice. However, firing frequencies of TMS-WDR neurons to thermal stimulation at 50 ◦ C were not changed in both WT mice (26 ± 1.5 Hz, n = 6) and KO mice (25 ± 2.9 Hz, n = 6). Noticeably, a few TMS-WDR neurons exhibited significantly longer latencies to noxious thermal stimulation in four AQP4 KO mice. The latencies were 7.35 ± 1.73 s (n = 5), compared to WT mice (0.74 ± 0.03 s, n = 6). The latencies were shortened with an increase in stimuli temperature from 43 to 50 ◦ C (Fig. 5).
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Fig. 3. Increase of thermal pain thresholds of AQP4 KO mice in tail immersion test, paw withdrawal test, and hot plate test. Responses to noxious thermal stimuli were measured by hot-water-immersion-induced tail flick latencies. KO mice (n = 34) showed a longer latency than WT mice (n = 41) in 47 ◦ C hot water (A). Both the WT mice (n = 10) and mutant mice (n = 16) showed very short latencies when 53 ◦ C water was applied to the tails (B) (*P < 0.05, ***P < 0.001, Student’s t-test). (C) Reponses to noxious thermal stimuli were measured by radiant heat-induced hindpaw withdrawal latencies. KO mice (n = 10) displayed significantly increased pain thresholds compared to WT mice (n = 13) (***P < 0.001, Student’s t-test). (D) Hot plate tests were applied to WT and KO mice with varied temperatures: 47 ◦ C, 52 ◦ C and 54 ◦ C. All the asterisks showed a significant difference between wild-type and mutant mice at each temperature (**P < 0.01, ***P < 0.001, Student’s t-test).
4. Discussion Increasing evidence has revealed that astrocytes play a crucial role in the processing of pain in the spinal cord [5,32]. Many signaling molecules related to synaptic transmission and modulation are expressed in astrocytes [2,26]. The signaling molecules in astrocytes that are involved in nociceptive processing are closely followed with interest. Out of them, AQP4 is expressed in astrocytes but not in neurons in the central nervous system. Therefore, AQP4 knockout mice are an ideal model to examine possible roles of AQP4 in the processing of nociception. This is the first study to
Fig. 4. AQP4 KO mice displayed delayed and impaired responses in the second phase of the formalin test. 2.5% formalin-induced spontaneous pain was measured by the formalin score in WT mice (n = 8) and KO mice (n = 9). AQP4 KO mice showed significant differences at the second phase. Arrow: formalin injection. Asterisk showed comparison between wild-type mice and mutant ones 20/25/45 min after formalin injection (*P < 0.05, ***P < 0.001, Student’s t-test).
show that an AQP4 deficiency produces an increase in behavioral pain thresholds. Except for mechanical stimulation, pain responses to thermal and chemical stimulation were attenuated. Consistent with the increase in pain thresholds, the number of neurons responsive to noxious thermal stimulation was reduced and latencies to thermal stimulation were significantly prolonged in a subset of dorsal horn WDR neurons. Taken together, the results imply that AQP4 deficiency induce hypoalgesia. To support a role of AQP4 in nociceptive processing, a study showed that AQP4 expression was highly increased in astrocytes following rat spinal cord injury with central neuropathic pain [22]. Given that there were no impairments of locomotor activities or change in skin temperature, it is conceivable that pain behavioral changes were attributable to the functional alteration due to AQP4 gene deletion. It is very interesting that the KO mice displayed better motor function in the Rota-Rod test compared to WT mice. It is well-known that dopamine is a very important neurotransmitter in motor function. An increase in dopamine is closely related to enhanced locomotor activity [3] and the speed of running [9]. A previous report demonstrated that the basal extracellular levels of dopamine and its metabolites (dihydroxyphenylacetic acid, DO-PAC, and homovanillic acid, HVA) were significantly increased in mice after AQP4 gene deletion [6]. This result might provide one explanation for the improved motor performance of the aquaporin-4 knockout mice. The mechanisms underlying AQP4 deficiency-induced changes in nociceptive processing are poorly understood. However, two possibilities can be considered: (1) in the central nervous system, AQP4 in astrocytes is commonly thought to facilitate K+ fluxes associated with adjacent neurons [30] in spite of having a contradictory report [27]. In AQP4 KO mice, K+ channels could be responsible for the delayed reuptake of excess extracellular K+ after a depolarizing stimulus [15,16]. It has been demonstrated that AQP4 in astrocytes contribute to hearing, vision and olfaction via modulation of
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Fig. 5. A subset of TMS-WDR neurons in KO mice had prolonged latencies. Firing samples of TMS-WDR neurons had prolonged latencies in KO mice compared to WT mice. A and B represented firing samples in WT mice after noxious heat application. C and D represented firing samples in KO mice. Five neurons responding to all stimuli from four KO mice responded normally to touch and noxious mechanical stimulation but they displayed prolonged latencies to noxious heat stimulation applied for 10 s when compared to WT mice.
extracellular space K+ buffering [15–17,21]. Therefore, it is possible that the increase in the pain threshold could also result from impaired extracellular K+ buffering in AQP4 KO mice. Meanwhile, AQP4 KO mice display an expansion in the extracellular space of the brain that is associated with enhanced solute diffusion. This suggests that a volume dilution mechanism contributes to the delayed solute and transmitter reuptake [33]. (2) Connexin 43 (Cx43) is a modulator of extracellular neurotransmitter reuptake and astrocytic networks [10]. AQP4 siRNA produced a down-regulation of AQP4 and Cx43 [23]. Cx43’s dysfunction decreased the glutamate release from astrocytes [8]. Accordingly, Cx43 might be involved in AQP4 deficiency-induced changes in nociception. Unexpectedly, the mechanical pain threshold was not changed after AQP4 deletion. Given that the non-neuronal cells and corpuscles in the skin are considered the primary mechano-transducers in mechanical nociceptive processing [18], it might be that mechanotransducers are insensitive to changes in the ambient buffering. In conclusion, AQP4 deficiency altered the modulatory function of astrocytes, resulting in the increase of pain thresholds via the glia-neuron communication. It is suggested that AQP4 is involved in the processing of nociception. Acknowledgements Special thanks to Dr. Gang Hu from Nanjing Medical University and Dr. Tonghui Ma from Northeast Normal University for mice provide. We thank Dr. Bryan Chai from Department of Neural and Pain Sciences, University of Maryland Dental School for manuscript editing. This work was supported by grant from the National Basic Research Program of China Grant 2006CB500807 and 2007CB512502 and the National Natural Science Fund of China 30830044. References [1] P. Agre, L.S. King, M. Yasui, W.B. Guggino, O.P. Ottersen, Y. Fujiyoshi, A. Engel, S. Nielsen, Aquaporin water channels—from atomic structure to clinical medicine, J. Physiol. 542 (2002) 3–16. [2] A. Araque, Astrocytes process synaptic information, Neuron Glia Biol. 4 (2008) 3–10. [3] R.J. Beninger, The role of dopamine in locomotor activity and learning, Brain Res. 287 (1983) 173–196. [4] A.D. Bragg, M. Amiry-Moghaddam, O.P. Ottersen, M.E. Adams, S.C. Froehner, Assembly of a perivascular astrocyte protein scaffold at the mammalian blood–brain barrier is dependent on alpha-syntrophin, Glia 53 (2006) 879–890. [5] H. Cao, Y.Q. Zhang, Spinal glial activation contributes to pathological pain states, Neurosci. Biobehav. Rev. 32 (2008) 972–983. [6] J.H. Ding, L.L. Sha, J. Chang, X.Q. Zhou, Y. Fan, G. Hu, Alterations of striatal neurotransmitter release in aquaporin-4 deficient mice: an in vivo microdialysis study, Neurosci. Lett. 422 (2007) 175–180.
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