Inhibition of Aquaporin-4 and its sub-cellular localization attenuates below-level central neuropathic pain via regulating astrocyte activation in a rat spinal cord injury model

doi:10.21203/rs.3.rs-3056661/v1

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Inhibition of Aquaporin-4 and its sub-cellular localization attenuates below-level central neuropathic pain via regulating astrocyte activation in a rat spinal cord injury model

https://doi.org/10.21203/rs.3.rs-3056661/v1

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The mechanisms of central neuropathic pain (CNP) caused by spinal cord injury have not been sufficiently studied. We have found that the up-regulation of astrocytic Aquaporin-4 (AQP4) aggravated peripheral neuropathic pain after spinal nerve ligation in rats. Using a T13 spinal cord hemisection model, we showed that spinal AQP4 was markedly up-regulated after SCI and mainly expressed in astrocytes in the spinal dorsal horn (SDH). Inhibition of AQP4 with TGN020 suppressed astrocytes activation, attenuated the development and maintenance of below-level CNP and promoted motor function recovery in vivo. In primary astrocyte cultures, TGN020 also changed cell morphology, diminished cell proliferation and suppressed astrocyte activation. Moreover, T13 spinal cord hemisection induced cell-surface abundance of AQP4 channel and the perivascular localization in the SDH. Targeted inhibition of AQP4 sub-cellular localization with trifluoperazine effectively diminished astrocytes activation in vitro and further ablated astrocytes activation, attenuated the development and maintenance of below-level CNP, and accelerated functional recovery in vivo. Together, these results provide mechanistic insights into the roles of AQP4 in the development and maintenance of below-level CNP. Intervening with AQP4, including targeting AQP4 subcellular localization, might emerges as a promising agent to prevent chronic CNP after SCI.

AQP4

Sub-cellular localization

Central neuropathic pain

Spinal cord injury

Astrocytes activation

Spinal cord injury (SCI) is a devastating neurological disease, with great physical, psychological and social burden [1]. Motor deficits usually acquired more attention, nevertheless, chronic pain cannot be neglected as well, in view of the fact that it has a significant impact on quality of life[2, 3]. This chronic pain after SCI belongs to central neuropathic pain(CNP), defined by the International Association for the Study of Pain[4]. SCI-induced CNP can be localized above-, at-, and below-levels, leading to spontaneous pain, hyperalgesia and allodynia[5]. However, conventional treatments or currently available pharmacotherapy has limited effificacy and serious adverse side effects on this debilitating CNP, due to the involvement of several pathophysiologic mechanisms [6].

Astrocytes play important roles in the maintenance of physiological homeostasis in the spinal cord[7]. A number of studies have reported the potential role of spinal astrocytes in both postoperative pain and neuropathic pain[8, 9]. Gwak and Hulsebosch found that activation of astrocytes modulates neuronal hyperexcitability and below-level CNP following SCI in rat[10]. Falnikar further demonstrated astrocyte expressed GLT1 associated with already-established CNP following cervical SCI[11]. Astrocytes could also contribute to hypofunction of GABA and persistent neuronal hyperexcitability, which plays a key role as a mechanism for neuropathic pain after SCI[12].

As the most abundantly subtype of Aquaporins, AQP4 plays crucial roles in CNS water homeostasis[13, 14]. Besides, AQP4 has been implicated in the processing of nociception through multiple mechanisms. AQP4 over-expression was found in the ipsilateral spinal dorsal horn upon peripheral nerve injury and SCI[15, 16]. AQP4 deficiency decreased the numbers of neurons responding to sensory stimulation and induced increase of pain thresholds in mice[17]. AQP4-mediated astrocyte reuptake of glutamate was also involved in the modulation of signaling pain in a mouse model of peripheral inflammation[18]Our recent studies have revealed that AQP4 aggravates peripheral neuropathic pain by alleviating astrocyte activation after spinal nerve ligation in rats[19]. All these findings suggest a potential role of AQP4 for the modulation of SCI induced CNP.

Since AQP4 is specifically localized to perivascular endfeet of astrocytes[20], the sub-cellular localization of AQP4 was found central to its physiological and pathological roles. In vitro, AQP4 underwent a rapid but reversible sub-cellular relocalization in primary cortical astrocytes in response to changes in the tonicity of the extracellular environment[21]. Douglas M .et al evaluated the perivascular AQP4 localization in the postmortem frontal cortex of cognitively healthy and histopathologically confirmed individuals with Alzheimer disease (AD) and found loss of perivascular AQP4 localization was significantly associated with AD status independent of age[22]. Focal brain ischemia triggers a redistribution of AQP4 away from the endfoot of astrocytes in grey matter[23]. Calcium signaling mechanisms was demonstrated required for AQP4 translocation[24]and the FDA-approved Trifluoperazine (TFP), a known calmodulin(CaM) inhibitor[25]could effectively reduce cerebral edema during the early acute phase in post-stroke mice using a photothrombotic stroke model[26]. Inhibiting SCI-mediated sub-cellular AQP4 localization by targeting CaM with TFP may therefore provide some therapeutic benefits for SCI- associated CNP.

In the current study, we investigated the expression of AQP4 protein, AQP4 immunoreactivity, and perivascular AQP4 localization after T13 spinal cord hemisection. We further examined the effects of AQP4 inhibition, especially blockade of AQP4 sub-cellular trafficking, on the development of SCI-induced below-level CNP and the recovery of motor function. We also investigated whether these effect was directly associated with astrocyte activation. Taken together, these observations suggest that interventions targeting astrocytic AQP4 and its sub-cellular localization potential are likely to be a potential therapeutic approaches for below-level central neuropathic pain after SCI.

Animals

All in vivo experiments were performed on Sprague-Dawley rats (male, 180-220g). They were maintained at 24°C ± 1°C and had free access to food and water under a natural light/dark cycle. All experimental procedures were reviewed and approved by the Animal Care and Use Committee of Jiangsu University. Care was taken to minimize animal discomfort and to limit the number of animals used.

Drugs and antibodies

AQP4 inhibitor TGN020 (T5102) was purchased from Targetmol (Boston, MA, USA). CaM inhibitor TFP(T8516) was purchased from Sigma (St. Louis, MO, USA). TNF-α Recombinant Protein ( #24095), IL-1α Recombinant Protein(#88205) were purchased from Cell Signaling Technology (CST) (Danvers, MA, USA). Component C1q Protein (MBS147399)was purchased from MedChemExpress (MCE) (Shanghai, China).

Antibodies against AQP4(16473-1-AP), C3(21337-1-AP), GFAP(60190-1) were purchased from Proteintech (Hubei, Wuhan, China). Antibodies against AQP4(ab259318), RECA-1 (ab264524), c-Fos (ab208942) and iba-1 (ab178847) were purchased from Abcam (Cambridge, MA, USA). Antibody against GFAP(#3670S), β-Actin (#4970), anti-mouse IgG (H + L) (#4408) and anti-Rabbit IgG (H + L) (#4413) were purchased from CST. Goat anti-rabbit IgG H&L (HRP) (ab6721), Goat anti-mouse IgG H&L (HRP)(ab205719) were purchased from Abcam.

Spinal cord injury

Spinal cord hemisection surgery was performed according to the method described by Christensen et al[27]. Briefly, rats were deeply anesthetized with 3% sodium pentobarbital (0.3 to 0.6mL each, I.P.). A laminectomy was performed between the T11 to T12 vertebral segments, and the lumbar enlargement region was identified. Then the spinal cord was unilaterally hemisected at thoracic T13 level without damaging the dorsal vessel[28, 29].In sham-operated animals, the spinal cord was exposed but not lesioned. Post-operative care included control of body temperature and housing individually with appropriate monitoring.

Primary astrocyte cultures

Astrocytes were obtained from the cerebral cortex of rat pups on postnatal day 3 as previously described[30]and grown to 70% confluence. Then the cells were passaged using 0.25% Trypsin and re-suspended with DMEM-F12 culture medium containing 10% serum.

Astrocytes was activated using TNF-a (30 ng/ml), IL-1α(3 ng/ml), c1q(400 ng/ml)[31]. For the effect of AQP4 and its sub-cellular localization on cell morphology and proliferation, astrocytes were also incubated with TGN020 (10 µmol/L) or TFP (12.7 µmol/L)[26]additionally for 24h or 48h. After that, the adherent cells were fixed with methanol for further immunocytochemical analysis. To explore the effect of AQP4 on cell proliferation and activation, the passaged astrocytes were activated until 70% fusion, and were pre-treated with TGN020 or TFP 30 minutes prior to activation. Astrocytes were collected or fixed for further immunocytochemistry (ICC) and western blot experiments after 24h.

Intrathecal injection

Intrathecal injection of TGN020, TFP or controlled DMSO and saline was performed based on a previously described protocol[32]. Briefly, using a microliter syringe (Hamilton) with a 30-gauge needle (BD), we delivered a total volume of 20 µL drug(s) to the subarachnoid space between the L4 and L5 levels. The success of the administration was verified by a tail‐ or paw‐flick response immediately after inserting the needle. Rats with signs of motor dysfunction were excluded from the experiments. Previously, we found that 15µg TGN020 was effective in producing anti-nociception when injected into subarachnoid space in a spinal nerve ligation model[19], and thus, we chose the same dose for evaluating the possible anti-nociceptive effects of intrathecal injection. The dose of TFP was chosen as 15µg and 45µg according to previous research[26]. Repetitive TGN020 or TFP and corresponding control injections during the induction phase were initiated on the third day post-SCI surgery and were then applied once a day for 7 consecutive days. During the maintenance phase, repetitive drugs was administered to SCI rats from Days 15 to 21 after surgery. For the effect of TGN020 on mRNA expression, single injection of drugs were applied right after SCI surgery.

Assessment of mechanical pain behaviors

All behavioral assessments were performed under the ethical guidelines set forth by the International Association for the Study of Pain (IASP). The temperature and humidity were kept stable for all experiments. Rats were allowed 30 minutes for acclimation before the examination. Pain behavior assessments were performed one day before hemisection surgery to obtain baseline values of withdrawal responses to mechanical stimulation. Then, rats were assigned randomly to each treatment group, and behavioral testing was subsequently performed blindly. Mechanical stimulations were done thrice for each hind paw at 5 min intervals. In all behavioral analysis experiments, six rats were used per group for each time point.

Mechanical hypersensitivity was evaluated using electronic Von Frey (e-VF, No.38450, Ugo Basile, Comerio, Italy). A continuously increased force was applied with a rigid metal tip until hind paw withdrawal was observed. A ratemeter was used to monitor the desired force and ensure us that the desired force was applied at a consistent rate as 10 g/s. The e-VF apparatus automatically recorded maximum force upon sudden hind paw retraction. These maximum values are referred to as paw withdrawal thresholds (PWT).

For the assessment of pain behaviors after SCI, tests were performed at the following time points after surgery: 5, 7, 10, 14, 21, 28, 35 and 42 days. For the effect of AQP4 on the induction of CNP (rats received TGN020 injections for 7 days since 3dpi), pain behavior tests were conducted at the following time points after SCI: 3, 7, 10, 14, 21, 35 and 42 days. For the effect of AQP4 on the maintenance of CNP (rats received TGN020 injections for 7 days since 14dpi), we assessed the pain behavior at the same time after SCI as mentioned above. Extra tests were conducted at the following time points after the first injection: 24, 48 and 72 hours.

Locomotor function

Recovery of hind limb motor function of was evaluated using the Basso, Beattie, and Bresnahan (BBB) score[33]. Briefly, the BBB scale ranges from 0 (no discernible hindlimb movement) to 21 (normal movement, including coordinated gait with parallel paw placement of the hindlimb and consistent trunk stability). The BBB scores were measured before and 3, 5, 7, 10, 14, 21, 28, 35 and 42 days after SCI. Only the scores of the ipsilateral hind limb on the hemisected side were recorded. Animals were excluded from the analysis when the score was lower than 10 was observed on the next fews days of SCI, because we are unable to conduct behavioral tests on rats with poor hind limb motor function. Finally, eight animals in each group were included.

Immunocytochemistry

At the appropriate time, the cells were rinsed thrice with PBS and fixed in methanol at -20°C for 15 min. Subsequently, they were blocked and permeabilized with 0.25% Triton X-100 for 20min. After further blocked in 3% BSA for 1 h, they were incubated overnight with primary antibodies rabbit anti-GFAP (1:1000, Abcam) or rabbit anti‐C3 (1:1000, Abcam) at 4°C. The cells were subsequently, washed thrice with PBST, and incubated with an appropriate fluorescence-conjugated second antibody (1:1000, CST) at room temperature. Finally,the cells were stained with DAPI to visualize the nucleus. The images were acquired using fluorescent microscopy and analyzed with ImageJ.

Immunohistochemistry

L4/5 spinal cord segments were prepared at appropriate survival times.

To explore the distribution and prove the cellular distribution of AQP4 in the SDH after SCI, spinal segments were collected at 7 days from after sham or SCI surgery. To explore the role of TGN020 or TFP on neuronal excitability and glia activation, inculding the effect of TFP on SCI driven perivascular AQP4 localization,segments were collected at 10 days after SCI since rats received consecutive drugs injections since 3dpi.

Briefly, rats were anesthetized and transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde. After perfusion, L4/5 spinal segment was removed immediately and post-fixed for overnight. After that, segments were dehydrated using graded sucrose. Transverse spinal sections (10 µm) were cut on a cryostat and prepared for immunofluorescence staining.The sections were incubated with: rabbit anti-AQP4 (1:500, Proteintech), Mouse anti-GFAP (astrocyte marker, 1:1000, Abcam), Mouse anti-c-Fos (A neuronal activation marker, 1:1500, Abcam), Mouse anti‐RECA-1 (a Endothelial Cell marker, 1:500, Abcam)and Mouse anti‐iba-1 (a microglial marker, 1:500, Abcam) at 4℃ overnight. The sections were then incubated with corresponding secondary antibodies conjugated with Alexa Fluor 488 or 555 (1:1000, CST).

A Nikon fluorescence microscope (DS-Qi2, Nikon Co., Tokyo, Japan) was used to analyze the stained spinal cord slices. Image J (NIH Image, Bethesda, MD, USA) was used to analyze the immunofluorescent images and Colocalization Finder plugins was used to calculate perivascular localization ratio of AQP4. Three representative images from 3 independent experiments, including the whole SDH in each group, were used for the semi-quantitative analysis.

Real-time PCR

Spinal cord samples were collected from rats with single I.T. of TGN020, TFP or controlled DMSO,saline at 1dpi. Animals were sacrificed by decapitation and the L4/5 spinal cord segments were harvested. To investigate the functional changes of the injuried spinal cord segments, the extracted spinal segments were divided into ipsilateral and contralateral halves. Subsequently, the ipsilateral spinal dorsal horns were used for real-time PCR analysis. Trizol reagent (Takara, Shiga, Japan) was used to extract total RNA. The RNA was then reverse transcribed into cDNA using PrimeScript RT reagent kit (Takara, Shiga, Japan). The cDNA was amplified after dilution using the specific primers shown in Table 1. The iTaqTM Universal SYBR Green Supermix (BIO-RAD, USA) was used for PCR amplification via the ABI Prism 7500 Fast sequence detection system (Applied Biosystems, Foster City, CA, USA). PCR conditions were: 95°C for 15 s, 35 cycles at 95°C for 10 s, 60°C for 30 s, and 95°C for 15 s. Three duplicate wells in each gene were used to compare the TGN020 or control groups, and the procedure was repeated three times. The relative mRNA expression was calculated using the Comparative CT Method.

Western Blot Assay

Animal protein samples were prepared in the same way as for PCR analysis. Briefly, the ipsilateral L4/5 segments of SDH were quickly isolated and temporarily stored at -80°C. To explore the temporal changes of AQP4/GFAP/iba-1 in the spinal cord after SCI, spinal segments were collected at 1, 5, 7, 14, 12, 28 and 35 days after SCI. To explore the role of TGN020 and TFP on astrocyte activation at the early phase of CNP, segments were collected at 7, 14, and 28 days after SCI since rats received consecutive drug injections since 3dpi. To explore the role of TGN020 and TFP at the later phase, segments were collected at 21 and 28 days after SCI since rats received consecutive injections since 14dpi. Then, the samples were homogenized in ice-cold RIPA lysis buffer. Cell protein samples were lysed by RIPA as well and collected by cytobrush. To explore the role of TGN020 and TFP on primary astrocyte activation, cell cultures that have grown to fusion were collected 24h after treatment.

After measurement of concentration, the tissue or cell protein samples were separated on 10% SDS-PAGE and electro‐transferred onto PVDF membranes. Protein samples were then incubated with antibodies for rabbit anti‐AQP4 (1:1000; Abcam), rabbit anti‐GFAP (1:1000, Abcam), rabbit anti‐C3 (1:1000, Abcam) and mouse anti‐Actin (1:2000; Santa). The membranes were then washed and incubated with specific HRP-conjugated secondary antibodies.The blots were visualized using the Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) and CLINX imaging system (CLINX Science Instrument, Shanghai, China) were used to visualize the blots. The intensity of protein bands was analyzed by Image J software (NIH Image, Bethesda, MD, USA).

Statistical analysis

Data were analyzed using GraphPad Prism, version 7.0 (GraphPad Software, Inc, San Diego, CA). Real-time PCR data and fluorescence intensity of GFAP between ipsilateral and contralateral sides were compared with Student’s t-test. IHC results of AQP4, GFAP, iba-1and c-Fos expression, including ICC results of GFAP and C3 were compared with one-way analysis of variance (ANOVA), followed by the Dunnett multiple comparison tests. IHC results of relative perivascular localization of AQP4 were compared with one-way ANOVA as well.The numerical data of western blotting were compared with Student’s t-test or one-way ANOVA. Data from the behavioral tests were analyzed by Two-way ANOVA with repeated measures followed by Bonferroni Multiple comparisons test. BBB score were analyzed by two-way ANOVA followed by Sidak's multiple comparisons test. All data were presented as means ± SEM. A p value of < 0.05 was considered to be statistically significant.

Mechanical hypersensitivity and locomotor dysfunction developed in the SCI model

First, we constructed a stable SCI model in rats. Consistent with previous studies, T13 spinal hemi-transection produced rapid and durable below-level mechanical pain hypersensitivity in both hind paws of rats. In addition, pain-related behavioral analysis revealed a clear reduction of PWT (Fig. 1) from 5 to 42 days in both ipsilateral paws (Fig. 1A) and contralateral paws (Fig. 1B) compared with sham groups, which reflected the development of below-level CNP.

To evaluate motor function after SCI, behavioral outcomes were assessed via the BBB scale, which has been widely used to evaluate the recovery of hindlimb motor function after SCI in rats. The sham group yielded a score of 21 at all time-points, reflecting normal motor function. Hemitransection of the left spinal cord in the T13 level caused serious motor dysfunction of ipsilateral hindlimb. Statistical analysis showed BBB scales of the hind paw decreased rapidly since day 5, compared with sham groups. Then the scales increased over time, with slow and continuous hindlimb functional recovery until the end of the evaluation period, achieving about 73.2% of the normal value (21) at the end of the 6th week (Fig. 1C).

SCI induces elevated astrocytic AQP4 expression in the L4/5 SDH

We detected the expression and distribution of AQP4 in the spinal cord after SCI. IHC analysis revealed a low AQP4 expression in the SDH of the sham group (Fig. 2C3). WB revealed that SCI induced a rapid-onset and long‐lasting upregulation of AQP4 protein versus sham surgery (Fig. 2A). Statistical analysis confirmed that this upregulation started at 3 d, peaked at 7 d, and remained at a high level until day 14 after SCI (Fig. 2B). The IHC results further showed that AQP4 expression was significantly increased in the SDH, and mainly distributed in the ipsilateral side at day 10 (Fig. 2C). Quantitative image analysis confirmed these changes, as shown in Fig. 2D. These results indicated that AQP4 was elevated in the spinal cord after SCI and predominately expressed in the ipsilateral SDH.

AQP4 was significantly expressed in astrocyte end-feet surrounding capillaries in the brain[13]. We have found AQP4 levels were also elevated mainly in astrocytes after SNL. Herein, we performed a double immunofluorescent labeling to confirm the astrocytic localization of AQP4 after SCI in the ipsilateral SDH. The IHC results showed a wide colocalization between AQP4 and GFAP (yellow) in the SDH (Fig. 2E3). The annular or irregular signals (AQP4, red) (Fig. 2E1)were mainly distributed around enlarged astrocytes terminal processes (GFAP, green) (Fig. 2E2), probably the end-feet of the astrocytes(Fig. 2F1-F3). These results showed that the AQP4 was expressed in activated astrocytes, and probably localized at the astrocytic endfeet in the SDH after SCI.

AQP4 blockade with TGN020 attenuates below-level CNP after SCI

We have previously shown that blocking AQP4 with TGN020 in the spinal cord level previously reduced post-SNL peripheral neuropathic pain[19]. To address whether changes in AQP4 regulation after SCI associated with CNP, rats were subjected to T13 hemitransection.

We first investigated the effect of SCI-induced upregulation of AQP4 in the established phase of CNP. The rats received intrathecal injection of 15µg AQP4 inhibitor TGN020 for 7 days since day 14 after SCI, at which time the central neuropathic pain had fully developed. The behavioral tests revealed that, compared with the control DMSO group, TGN020 partially reversed the reduction of PWT of both hind paws in SCI rats for up to 42 days after SCI (Fig. 3A and 3B). To evaluate the role of AQP4 in the development of CNP, 15 µg TGN020 was injected I.T. for 7 consecutive days at day 3 after SCI. Compared with the DMSO group, TGN020 partially reversed the SCI‐induced CNP and the effect was long-lasting. As shown in the Fig. 3C and 3D, mechanical hypersensitivity of hind paw was significantly relieved by the administration of TGN020. The difference of PWT in bilateral hind paws between TGN020 and control DMSO persisted until day 42, more than 1 month after the first administration.

Central sensitization of SDH, including neuron hypersensitivity, glia activation, and neuron-glial interactions constitute a fundamental regulatory mechanism of CNP after SCI[34, 35]. Therefore, we investigated whether intrathecal TGN020 could affect SCI-induced CNP by regulating the excitability of the spinal neuron and glia cells and the release of pain-related molecules in the spinal cord. In our IHC experiment, SCI induced a significantly increase of c-Fos, iba-1 fluorescence in the ipsilateral SDH 10 days after injury (Fig. 3E2 and G2), compared with the sham group (Fig. 3E1 and G1). However, 7 days successive intrathecal injections of TNG020, but not DMSO, since 3dpi significantly decreased enhanced fluorescence intensity of c-Fos and iba-1(Fig. 3E3,E4 and G3,G4). Semi-quantitative analysis furether confirmed this, as shown in Fig. 3F and H.

We found that the mRNA levels of c-Fos, CREB were significantly decreased in SCI‐injured rats treated with single I.T.of TGN020 compared to the DMSO group at one day after SCI (Fig. 3I). Besides, the same phenomenon was observed in the mRNA expression of GFAP, iba-1 and pro‐inflammatory cytokines (TNF‐α, IL‐1β and IL‐6) after TGN020 treatment (Fig. 3I). Collectively, these results suggest that AQP4 contributed to SCI‐induced CNP via suppressing central sensitization.

AQP4 blockade promoted functional locomotor recovery after SCI

To evaluate the effects of TGN020 on locomotor function, behavioral outcomes were assessed at days 5, 7, 10, 14, 21, 28 and 42 via the BBB scale. After received I.T. of 15µg TGN020 in the established phase of CNP, rats achieved better functional recovery, compare with the control group. Statistical analysis further revealed that BBB scores in the TGN020 group were significantly higher than they were in the DMSO group from day 21 to day 42 after SCI (p < 0.001 for day 21; p < 0.0001 for days 28 and 42) (Fig. 3K). Similarly, in the early stage of CNP, BBB scores in the TGN020 group were also significantly higher at all time-points from day 7 to day 42 after SCI (p < 0.001 for day 7, 10, 14 and 28; p < 0.0001 for days 21 and 42) (Fig. 3L). These results suggested that TGN020 administration could markedly promote the recovery of locomotor function in rats after SCI.

AQP4 blockade suppressed astrocytes activation after SCI

Astrocytes were involved in central sensitization, especially through glial-neuron interactions in neuropathic pain states[36]. We evaluated whether T13 level SCI affected astrocytes activation, which occur in rats after spinal nerve ligation injury[19].GFAP expressions at the ipsilateral L4/5 segments of SDH after SCI were evaluated by Western blotting. The results suggested that GFAP level was low in the sham group. However, there were significant increases in the expression of GFAP after SCI, especially in the maintenance period of CNP (Fig. 4A). Quantitative analysis revealed that the upregulation started on day 3, peaked on day 21, and remained significant until day 28 (Fig. 4B). Immunohistochemistry and subsequent quantification showed that, 10 days after injury, GFAP fluorescence intensity at the ipsilateral side was much higher than the contralateral sideat the L4/5 SDH (Fig. 4C and 4D). These results indicated that T13 SCI induced below-level astrocyte activation in the SDH, especially at the ipsilateral side, which could contribute to the development of below-level central neuropathic pain.

We further inhibited AQP4 activity with TGN020 to investigated the role of AQP4 in astrocyte activation in the late or early phase of CNP after SCI. L4/5 spinal cord segment was collected to investigated astrocyte activation. Rats received successive intrathecal injections of TNG020 (15 µg), (once daily since 14dpi ) or control DMSO for 7 days after SCI. WB analysis revealed a significantly lower level of AQP4 and GFAP expression upon TGN020 injection, but not DMSO, at 21 and 28 days (p < 0.01, p < 0.001) in SCI rats, respectively (Fig. 4G and 4H). Another groups of rats received successive intrathecal injections of TNG020 or DMSO for 7 days since 3dpi. WB analysis revealed a significantly lower level of GFAP expression upon TGN020 injection at 7(p < 0.05), 14(p < 0.01), and 28 days (p < 0.05) in SCI rats again, respectively(Fig. 4E and 4F). IHC evaluation showed a rarely expression of GFAP in sham rats (Fig. 4I1). SCI induced a significantly increase of GFAP fluorescence at 10 day after SCI (Fig. 4I2), which was markedly suppressed by TGN020 injection (Fig. 4I4-A). However, in the DMSO group, the treatment did not affected GFAP expression (Fig. 4I3-A). More details about the cellular morphology of astrocytes in the SDH after TGN020 or DMSO were shown with higher magnification in Fig. 4I3B and I4B. Semi-quantitative analysis confirmed this, as shown in Fig. 4J. Taken together, these results showed that upregulation of AQP4 promoted astrocyte activation in the SCI model, which might finally aggravate CNP in the early and late phase.

Astrocytes formed a highly interconnected network via gap junctions or glutamate transporter, which were highly was involved in nociceptive hyperalgesia[11, 37]. We examined if AQP4 could influence these astrocytic factors upon SCI. RT-qPCR analysis revealed that AQP4 blockade with TGN020 significantly suppressed mRNA expressions of the gap junction channels Cx30 but promoted the up-regulation of glutamate transporter GLT-1 and GLAST mRNA levels (Fig. 4K).

Aquaporin-4 and its sub-cellular localization have central roles in primary astrocyte proliferation and activation in vitro

We previously demonstrated by WB analysis that AQP4 played important role in TNF-α induced astrocyte activation[19]. Now we used a new model to investigate the potential effects of AQP4 expression and its sub-cellular translocation on astrocyte activation. Our fluorescent immunocytochemistry assay showed that, after passage and further 24h incubation, adherent astrocyte treated with TNF-α, IL-1α and c1q showed plumper cell soma and more cell processes (Fig. 5A2), compared with untreated astrocytes (Fig. 5A1). Moreover, 70% fusion astrocyte activated by TNF-α, IL-1α and c1q for 24h showed significantly elevated GFAP, C3 and AQP4 protein levels(Fig. 5B and 5C). Immunofluorescence assay also confirmed significantly stronger GFAP and C3 fluorescence intensity in astrocyte activated by TNF-α, IL-1α and c1q, as shown in Fig. 5D2 and 5F2. Quantitative analysis further confirmed these results as well(Fig. 5E and 5G). These results demonstrated that astrocyte activation can be recapitulatedin vitro by treating with TNF-α, IL-1α and c1q and activated astrocyte have a more mature cell morphology, stronger

Proliferation ability. Besides, the GFAP and AQP4 levels were significantly increased in activated astrocyte.

We next investigated the role of Aquaporin-4 and its sub-cellular localization in astrocyte activation. TGN-020, a well-known inhibitor of AQP4 and TFP, a FDA proven CaM antagonist, were chosen to inhibit AQP4 expression and sub-cellular translocalization from cytoplasm to cell membrane. Following TGN020 or TFP treatment, immunofluorescence assay proved that activated astrocyte showed a slimmer cell soma and less cell processes 24h after passage (Fig. 5A3 and 6A3). More details about the changes of astrocytes morphology after TGN020 or TFP pre-treatment were shown in higher magnification. Besides, when passaged primary cortical astrocytes were treated with TNF-α, IL-1α and c1q for 24h or 48h, GFAP fluorescence intensity significantly increased(Figure S1 A1,A2 and Figure S1 B1,B2) respectively, which can be partially reversed by pretreatment with TGN020 or TFP(Figure S1 A3,A4 and Figure S1 B3,B4). We also found that the GFAP intensity was even lower in TGN020 group than TFP group, especially at 24h. The same phenomenon occurs in C3 fluorescence intensity in passaged astrocytes after activation with/without pretreatment with TGN020 or TFP(Figure S2 A, B, C and D). Semi-quantitative analysis furether confirmed this, as shown in Figure S1 C,D and Figure S2 E, F.

When the passaged astrocytes grew into 70% fusion, WB result and quantitative analysis also confirmed TGN020 or TFP can reduce AQP4 protein level and significantly ablated the upregulation of GFAP and C3 in our activated astrocyte model, are shown in Fig. 5B, 5C and Fig. 6B, 6C. Immunofluorescence assay

further demonstrated that higher GFAP and C3 fluorescence intensity after 70% fusion were also ablated as well(Fig. 5D3, 5F3 and Fig. 6D3, 5F3). Quantitative analysis further confirmed these results, as shown in Fig. 5E, 5G and Fig. 6E, 6G, respectively.

These results suggested that AQP4 and its sub-cellular localization have central roles in primary astrocyte proliferation and activation in vitro.

SCI significantly increased the perivascular localization of AQP4 in the L4/5 SDH

We further evaluated perivascular AQP4 localization by double-labeling immunofluorescence, using AQP4 and endothelial cell marker RECA-1. In the sham group, there was a rare colocalization between AQP4 and RECA-1 (Fig. 7A3). On the other hand, SCI drove a wide co-localization of AQP4 and RECA-1(yellow) in the SDH at 10dpi (Fig. 7B3). In Fig. 7A4 and B4, amplified white rectangles further identify astrocyte processes physically associated with vascular endothelial cell, which were assigned as astrocyte endfeet, after SCI, compared with the sham group. Next, we investigate if TFP, a known CaM inhibitor[25], can reverse the perivascular AQP4 localization after T13 SCI. TFP was injected once a day for 7 days since 3dpi. Figure 7C3 and 7C4 showed that the colocalization of AQP4 and RECA-1 was obviously reduced at 10dpi, indicating CaM inhibition with TFP blocked T13 SCI- induced translocation of cytoplasm AQP4 to the cytomembrane at the L4/5 SDH.

Quantification of total AQP4-immunoreactivity(IR) showing increased AQP4 expression after SCI, compared with the sham group, while TFP injection did not affect the total expression level of AQP4 after SCI (Fig. 7D). On the other hand, relative perivascular localization ratio of AQP4, calculated by manders overlap coefficient (MOC) among different groups, showed that the relative MOC was elevated after SCI, and TFP injection could effectively reverse this phenomenon (Fig. 7E).

Taken together, these results indicated L4/5 AQP4 translocate to the plasma membrane of astrocytes in response to T13 spinal cord injury, which could blocked by CaM inhibitor TFP.

Targeted inhibition of AQP4 perivascular localization reduced astrocyte activation and attenuated of below-level CNP after SCI.

We next evaluated whether this sub-cellular translocation of AQP4 affect astrocyte activation and further facilitate the improvement of CNP after SCI in vivo.

Rats received successive intrathecal injections of TFP (45µg) or control saline once daily since 3dpi for 7 days. SCI induced increased GFAP immunofluorescence intensity and astrocyte hypertrophy at 10 days at L4/5 SDH(Fig. 8A2) as compared with sham-operated group(Fig. 8A1) and saline treatment had no effect on this change. Targeted inhibition of AQP4 perivascular localization by TFP treatment resulted in lower GFAP fluorescence strength and slenderer cell morphology of astrocyte, as shown in Fig. 8A3. Quantitative analysis further confirmed these results as well, as shown in Fig. 8B. Ipsilateral L4/5 spinal cord segment after TFP treatment were collected for WB analysis 7,14, and 28 days after SCI. Inhibition of AQP4 translocation resulted in reduced spinal cord GFAP expression at 7 dpi(p < 0.0001) and 14 dpi(p < 0.01) compared with injured animals (SCI + saline) and were still functionally impaired at 4 weeks(p < 0.05)after SCI (Fig. 8C ). Semi-quantitative analysis confirmed this, as shown in Fig. 8D. Another groups of rats received injections of TFP or saline for 7 days since 14 dpi. WB analysis revealed a significantly lower level of GFAP expression upon TFP injection at 21(p < 0.01),and 28 days (p < 0.0001) in SCI rats, respectively(Fig. 8E and 8F). Notably, the total AQP4 levels were also affected by TFP injections (Fig. 8D, 8G), and suggested that TFP may not only targeted the translocation of AQP4 from cytoplasm to cell membrane. All these results showed that perivascular localization of AQP4 promoted astrocyte activation in the SCI model both in the early (3 dpi) and late (14 dpi) phase of CNP after SCI.

We further determined whether inhibition of AQP4 perivascular localization can translate into improvements of hyperalgesia in TFP treated rats by behavioral tests. Rats were treated with TFP (15 µg or 45 µg) in the late-phase of CNP(14 dpi) and significant improvements in mechanical hypersensitivity were observed(Fig. 8H, 8I). The mean paw mechanical withdraw threshold in both ipsilateral and contralateral side were significantly decreased at 24, 48, and 72 h (p < 0.0001) after first injections. These hyperalgesia improvements were still distinguishable from control saline animals 4 weeks (15µgTFP )(p < 0.05) or 6 weeks (45 µg TFP)(p < 0.0001) post-injury. Additionally, this effect is dose-dependent and rats treated with 45 µg TFP showed a higher value of withdraw threshold until 42 dpi(p < 0.0001). In the early-phase of CNP (3dpi), TFP treatment (45µg ) attenuated mechanical hypersensitivity of both hind paws at 10dpi (p < 0.0001) but this phenomenon only last for 4 days(14dpi)(p < 0.0001) compared with the control group, as shown in Fig. 8J and 8K.

These results demonstrate that TFP suppresses astrocyte activation after SCI, which are likely to contribute to the improvements of hyperalgesia in the early and late phase of CNP.

Finally, we evaluated whether TFP treatment affected locomotor recovery after SCI via the BBB scale. In control animals, the mean BBB scores was 10 to15, with a slight improvement throughout the time period. After received I.T. of 45µg TFP in the established stage of CNP, significantly increased BBB scores were observed 1 week after treatment(Fig. 8L). Similarly, in the early stage of CNP, TFP treatment significantly increased BBB scores since 7 dpi as well. Besides, these improvements in rats treated with TFP were distinguishable from control animals (saline) throughout the 5-week assessment period(Fig. 8M).

Central neuropathic pain, which is pain caused by a lesion or disease of the central somatosensory nervous system, can result from spinal cord injury, stroke, multiple sclerosis and other diseases affecting the CNS[38]. Chronic CNP is a crucial complication in the course of SCI, entailing serious impacts on the individuals’ quality of life in later phases[39]. Effective treatment of SCI-related pain in general and of neuropathic pain in particular is challenging[40]. Central neuropathic pain can emerge above, at, or below the level of SCI. In the current study, we used a previously well-described T13 spinal hemitransection model to explore the pathological mechanisms of below-level CNP after spinal cord injury[27, 29], especially at the SDH level. As predicted, we found that SCI induced the development of mechanical allodynia in both the ipsilateral and contralateral hind paws. We chosed rats with similar BBB scores (> 10) 3–5 days after SCI to reduce the interference of motor dysfunction with behavioral assessments.

AQP4, the most abundant aquaporin in the CNS, plays an important role in maintaining the homeostasis of water and ions, and contribute to cytotoxic and vasogenic edema[41, 42]. Recent studies suggest that long-lasting AQP4 up-regulation was found in the lumbar region of the spinal cord after peripheral nerve injury, and might be associated with neuropathic pain[15]. we found that L4/5 AQP4 was significantly increased after T13 hemi-transection injury SCI, especially in the ipsilateral SDH. AQP4 absence resulted in sustained activation of spinal glutamatergic neurons in the superficial SDH[18]. Here, we demonstrated that AQP4 blockade suppressed neuronal excitability, microglia activation and reducing the release of pro-inflammatory cytokines, indicating that AQP4 up-regulation could affect central sensitization of the L4/5 SDH[43, 44, 45]. AQP4 has been shown recently to have anti-hyperalgesia effects in several pain models. We previously study also confirmed that inhibition of AQP4 attenuated peripheral neuropathic pain after spinal nerve ligation in rats[19]. In the present study, we observed that AQP4 blockade could partially reversed T13 spinal cord hemisection induced hind paw mechanical withdrawal threshold. Collectively, these results suggested that AQP4 was not only associate with CNS edema[46] but participated in SCI induced below-level CNP, which might provide a potential therapeutic approach of targeted AQP4 inhibition for this SCI driven chronic pain .

It is well known that astrocyte activation after injury was involved in chronic pain development and/or maintenance[36]. The role of spinal activated astrocytes in peripheral neuropathic pain states are widely explored and documented after diverse types of peripheral nerve injuries[47, 48, 49]. Recent studies began to focus on astrocyte contribution with respect to CNP after SCI. Below-level astrocyte activation was observed following rat cervical and thoracic spinal cord injury[10, 50]and accompanied by neuronal activation/hyperactivity and chronic at-level or below-level central neuropathic pain[51]. Matyas et al. Further found that trkB.T1-mediated astrocyte dysfunction contributes to CNP after a moderate spinal cord contusion injury[52]. Using a rat model of CNP, we showed that T13 spinal hemi-transection injury drive dramatically increased GFAP expression and trigger astrocyte hypertrophy in the lumbar spinal cord, when mechanical hypersensitivity was well established, implying that astrocyte activation contribute to central neuropathic pain after SCI.

AQP4 is predominantly present in the end-feet of perivascular astrocytes and in the plasma membrane of astrocytes in the CNS[46]. In the spinal cord, AQP4 was found exclusively localized at the astrocytes in both normal rats and rats with spinal cord injury [16, 53]. Several studies have reported that down-regulation of AQP4 was accompanied by a reduction in spinal GFAP expression after spinal cord injury[54, 55, 56]. our previous studies confirmed that blocking AQP4 suppressed spinal astrocyte activation both in rats after peripheral nerve ligation and in primary astrocyte cultures. AQP4 inhibition with TGN020 could also effectively inhibit astrocyte proliferation after T10 SCI in rats[57]. So we further explored whether AQP4 is relevant to astrocyte activation. Here, we showed AQP4 signals were mainly distributed around enlarged astrocytes terminal processes, probably the end-feet of astrocytes. AQP4 blockade with TGN020 could affected T13 SCI- induced activation of astrocytes in the lumbar SDH, both in the late phase or the early phase of CNP. Interestingly, AQP4 inhibition also influenced astrocytic gliotransmitters including Cx30,GLT1 and GLAST-1, which actively modulate neuronal excitability and synaptic efficacy after SCI[11, 51, 58]. These data constitute the first reported indication that up-regulation of Aquaporin4 could promote astrocyte activation and release of astrocytic gliotransmitters after SCI, and thereby contribute to the induction and maintenance of below-level CNP in rats.

Our present results and other studies suggest that AQP4 has central roles in development of CNS injury[59, 60]. Reversible inhibition of AQP4 function is a viable strategy to prevent CNS edema and CNP. However potential AQP4-blocking drugs has not been approved for the treatment of clinical application. Here, we focused on the subcellular localization of AQP4, not only AQP4 activity, and show a direct relationship between inhibition of AQP4 translocation to the cytomembrane or the endfeet of astrocyte and attenuation of CNP after SCI. Regulation by vesicular trafficking is a common biological mechanism controlling the function of many membrane protein families[61, 62, 63]. Here we present pathologically relevant AQP4 subcellular re-localization or perivascular localization as a functional regulatory mechanism. Using an activation model of astrocyte, we showed that total AQP4 expression and GFAP expression increased, and inhibition of cell-surface abundance of AQP4 by TFP blocked astrocyte proliferation and activation in vitro. Using a rat model of CNP, we show that T13 spinal cord injury induced the subcellular re-localization of AQP4 and the ratio of the perivascular AQP4 significantly elevated in the L4/5 SDH, which could blocked by CaM inhibitor TFP. We also demonstrate the central roles of TFP in astrocyte activation. Ablation of AQP4 subcellular relocalization in vivo are accompanied by attenuation of below-level CNP and functional recovery. All these results indicated a direct interaction between targeted inhibition of AQP4 perivascular localization and astrocyte activation that is necessary for sustained reversal of below-level CNP after SCI. Trifluoperazine (TFP), a CaM antagonist that is approved as an Antipsychotic drug by the US Food and Drug Administration(FDA) and the UK National Institute for Health and Care Excellence[25, 64], was used for treating human schizophrenia. This benefit of inhibiting AQP4 relocalization by TFP may therefore be a viable strategy for development of CNP therapies.

In conclusion, this study demonstrate for the first time that astrocytic AQP4 inhibition attenuated SCI-induced below-level central mechanical hypersensitivity, promoted functional locomotor recovery after SCI. Its potential mechanism is due to the reduction of compensatory astrocyte activation in the SDH. More importantly, we revealed that targeting AQP4 subcellular localization, a fundamental cellular process, by TFP following CNS injury has profound effects on the relief of subsequent CNP. To our knowledge, no effective intervention has been widely used for the treatment of SCI induced chronic CNP previously, has not been demonstrated. Overall, targeting spinal cord AQP4 activity, especially its subcellular localization, may serve as a potential strategy for the treatment of SCI-evoked central neuropathic pain.

Declaration of competing interests

All authors declare no conflicts of interest.

Acknowledgments

This study was supported by grants from the National Science Foundation of China (81901247), the Changzhou Sci&Tech Program (CJ20220010), the Medical Research Project of Jiangsu Health Commission (Z2022045), Open Project of Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University (No. XZSYSKF

2020044) and the Science and Technology Project of Changzhou Health Commission (WZ202227).

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figureS11.tif
Supplement Figure 1: AQP4 affected TNF-α, IL-1α and c1q -induced astrocyte proliferation in vitro. Fluorescent images showed that, after 24h, passaged primary astrocyte incubated with TNF-α, IL-1α and c1q showed a much higher GFAP fluorescence level than normal group(A1 and A2), which could be reduced by co-culture with TGN020 and TFP(A3, A4)(all images, scale bar, 100 μm). Quantitative analysis further confirmed these results as well, as shown in C. Similarly, blocking AQP4 expression and sub-cellular localization with TGN020 and TFP significantly reduced GFAP fluorescence intensity 48h after passage(B and D)(all images, scale bar, 100 μm). Data are presented as mean ± SEM. ****p < 0.0001, normal versus active. ##p < 0.01, ####p < 0.0001, active + TGN020 versus active, ѱѱѱp < 0.001, ѱѱѱѱp < 0.0001, active + TFP versus active.
figureS21.tif
Supplement Figure 2: AQP4 affected TNF-α, IL-1α and c1q -induced astrocytes activation in vitro. Fluorescent images showed that, after 24h, passaged primary astrocyte showed extremely low C3(red) fluorescence intensity (A1 and B1). Astrocytes incubated with TNF-α, IL-1α and c1q showed a much higher C3 fluorescence level (A2 and B2), which could be reduced by co-culture with TGN020 and TFP(A3, B3)(all images, scale bar, 100 μm). Quantitative analysis further confirmed these results as well, as shown in E. Similarly, blocking AQP4 expression and sub-cellular localization with TGN020 and TFP significantly reduced C3 fluorescence intensity 48h after passage(C, D and F))(all images, scale bar, 100 μm). Data are presented as mean ± SEM. **p < 0.01, ****p < 0.0001, normal versus active. ##p < 0.01, ###p < 0.001, active + TGN020 versus active, ѱp < 0.05, ѱѱp < 0.01, active + TFP versus active.

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Editorial decision: OA - Major Revision
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Inhibition of Aquaporin-4 and its sub-cellular localization attenuates below-level central neuropathic pain via regulating astrocyte activation in a rat spinal cord injury model

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Abstract

Figures

Introduction

Materials and methods

Animals

Drugs and antibodies

Spinal cord injury

Primary astrocyte cultures

Intrathecal injection

Assessment of mechanical pain behaviors

Locomotor function

Immunocytochemistry

Immunohistochemistry

Real-time PCR

Western Blot Assay

Statistical analysis

Results

Mechanical hypersensitivity and locomotor dysfunction developed in the SCI model

SCI induces elevated astrocytic AQP4 expression in the L4/5 SDH

AQP4 blockade with TGN020 attenuates below-level CNP after SCI

AQP4 blockade promoted functional locomotor recovery after SCI

AQP4 blockade suppressed astrocytes activation after SCI

SCI significantly increased the perivascular localization of AQP4 in the L4/5 SDH

Discussion

Declarations

References

Supplementary Files

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