Potassium Chloride Cotransporter 2 Inhibits Neuropathic Pain and Future Development of Neurodegeneration

Abstract

Persistent neuropathic pain (NP) causes future development of neurodegenerative diseases, e.g., Alzheimer’ disease, and thus needs to be optimally treated. Surgically-induced neuropathic pain (SNPP) is a persistent pain that occurs in nearly half of the individuals after common operations. Here, we showed that specific activation of 5-hydroxytryptamine (5-HT) type 2A receptors by systemic administration of TCB-2 [(4-bromo-3,6-dimethoxybenzocyclobuten-1-yl) methylamine hydrobromide] improved the function of potassium chloride cotransporter 2 (KCC2), resulting in reduction in neuropathic pain after chronic constriction injury (CCI), a rat model that mimics SNPP. Moreover, TCB-2 administration attenuated both mechanical and thermal hyperalgesia, likely through augmentation of dorsal horn KCC2 levels, since this effect was abolished by intrathecal provision of dihydroindenyl oxy alkanoic acid (DIOA), which blocked the effects of KCC2. Furthermore, TCB-2-mediated re-activation of KCC2 likely reduces future development of neurodegeneration in rats. Together, our data support further studies on the possibility of using this strategy to reduce postoperative pain and future neurodegenerative disorders in clinic.

Keywords

Alzheimer’ disease neurodegeneration potassium chloride cotransporter 2 (KCC2)surgically-induced neuropathic pain

INTRODUCTION

Neurodegenerative diseases continue increasing in parallel to the elongation of the lifespan [1]. Motor and cognitive impairment in neurodegenerative diseases such as Alzheimer’s disease (AD) and other dementias is the major therapeutic target [2]. Interestingly, many AD patients appear to become more sensitive to surgically-induced neuropathic pain (SNPP), a persistent pain that occurs in up to half of the individuals who have received operations [3].

Postsurgical neuropathies can result from transection, stretching, contusion, or inflammation of the nerve [4]. Despite the majority of surgically-induced pain is believed to be neuropathic, the exact phenotype of the pain has not been specifically measured in the clinic with quantitative methods. Although the nature of the surgical procedure has a certain influence on the incidence of chronic neuropathic pain, a preexisting painful condition can typically alter the predisposition to SNPP [5, 6], e.g., in the case of AD patients [3].

An impaired GABAergic function hallmarks the occurrence of neuropathic pain [7], in which reduction of the potential of the inhibitory neurotransmitter that mediates a chloride-mediated hyperpolarizing current has been detected in dorsal horn neurons [8], concomitantly with expression of potassium chloride cotransporter 2 (KCC2) that keeps low concentration of intracellular chloride to promote influx of chloride at an activated GABA-A receptor [8]. KCC2 dysfunction induces a chloride gradient collapse, which attenuates the inhibitory action of GABA-A and glycine to induce neuropathic pain. Thus, re-establishment of KCC2 functionality can be a promising novel therapeutic strategy to SNPP [9]. A recent report showed that specific activation of 5-hydroxytryptamine (5-HT) type 2A (5-HT2A) receptors by a potent agonist, TCB-2 [(4-bromo-3,6-dimethoxybenzocyclobuten-1-yl) methylamine hydrobromide], improved the function of KCC2, resulting in reduction in neuropathic pain in a spinal cord injury (SCI) model [10], but not in a spared nerve injury (SNI) model. Here, we examined the effects of activation of this pathway on the pain after chronic constriction injury (CCI), a rat model that mimics SNPP.

We found that specific activation of 5-HT2A by systemic administration of TCB-2 improved the function of KCC2, resulting in reduction in neuropathic pain after CCl. Moreover, TCB-2 administration attenuated both mechanical and thermal hyperalgesia, likely through augmentation of dorsal horn KCC2 levels, since this effect was abolished by intrathecal provision of dihydroindenyl oxy alkanoic acid (DIOA), which blocked the effects of KCC2. Furthermore, TCB-2-mediated re-activation of KCC2 likely reduces future development of neurodegeneration in rats. Together, our data support further studies on the possibility of using this strategy to reduce postoperative pain and future neurodegenerative disorders in clinic.

MATERIALS AND METHODS

Animals

All experimental procedures were conducted according to the approved guideline from the local ethics committees at the First Affiliated Hospital of Zhengzhou University. The adult male Sprague Dawley (SD) rats (190–230 g) was provided by the Animal Experimental Science Department of the First Affiliated Hospital of Zhengzhou University, and the standard met the national standard for secondary laboratory animals. Animals were kept a week before surgery to adapt to the environment. The rats were housed in single cage under a 12-h light-dark cycle in a temperature animal care facility kept at 22±2 °C. The rats had free access to water and food from 7am through 7pm daily. Behavioral testing is conducted between 10am and 4pm daily.

Chronic constriction injury (CCI) model

The CCI model was induced in rats, according to the published method [11]. The surgical procedure was performed under anesthesia with ketamine (Imalgen, Merial, 50 mg/kg i.p.) and medetomidine (Domitor, Janssen, 0.25 mg/kg i.p.). The skin and muscle layer were incised on the left hind limb to expose the sciatic nerve of the rats, after which one loose ligature with a 4-0 braided silk suture was placed around the exposed nerve until exhibition of a brief twitch of the limb. The skin incision was then sutured to recover for 14 days. The sham-operated animals received same surgical procedure without ligature of the sciatic nerve.

Behavioral assessments

In the von Frey filament test, rats stayed in a test box fitted with a wire mesh backing. Von Frey microfilaments (from 8 g) were applied through the grid floor to the ventral surface of the hind paw of the injured hind limb of the rat. The filament is pressed until the filament is bent and then held for 3 s or until the animal withdraws the hind leg without moving. During each test, the filament series was presented after the incremental procedure and the 50% response threshold for each rat was calculated [12]. For the Hargreaves test, the thermal damage threshold for radiant heat was quantified using the paw withdrawal test [13]. Briefly, rats were placed without restriction in a plexiglass compartment. After adaptation, the infrared radiant heat source is focused on the midfoot surface of the hind paw. A 20-s cut-off stimulation time was used without response to avoid skin damage. The incubation period of paw withdrawal back to radiant heat is defined as the time from the onset of radiant heat to the removal of the hind paw of the rat. The average of three estimates was used to obtain the average latency of the paw withdrawal. The social recognition test (SRT) was used to assess the social recognition memory and novelty reaction in those rats, as described [14]. Briefly, an empty chamber was placed in the test cage with the rats allowed to spontaneously explore. The same inducer was placed inside a transparent acrylic chamber for 5 trials of 5 min each, separated by 10-min intervals. In the last trial (5th trial), a new inducer was placed in the same acrylic chamber and the time spent sniffing was quantified again. Plus-Maze discriminative avoidance task (PM-DAT) used a wood-made modified plus-maze, as described [14]. In the training session, rats were placed at the center of the apparatus, and received both the illumination of the 100 W light and blowing cold air when they entered the enclosed arm containing the lamp and the hair dryer. Twenty-four hours after the training, rats were placed in the same position in the same room for 3 min without these aversive stimuli when they entered the enclosed arm with presence of non-illuminated lamp and the hair dryer. The percentage of time spent in the aversive enclosed arm during training and testing was recorded respectively for assessment of learning and memory.

Western blotting

Tissue from the lumbar dorsal horn was obtained and homogenized in protein lysis buffer (Bio-rad, Beijing, China), followed by protein concentration assessment with a BCA protein assay kit (R&D systems, Beijing, China). Western blot was performed with the following primary antibodies: rabbit anti-KCC2 (Cell signaling, San Jose, CA, USA) and rabbit anti-GAPDH (Cell signaling). The secondary antibody was HRP-conjugated anti-rabbit (DAKO, Beijing, China). Image acquisition and densitometric analysis of the gels were performed with NIH ImageJ software (Bethesda, MA, USA).

Drug administration

TCB-2 and a selective KCC2 blocker, DIOA [15], were both purchased from Sigma Aldrich (St. Louis, MO, USA), and were diluted in distilled water or in 0.9% sodium chloride and sodic phosphate buffer of 300 mosm, pH 7.4 and DMSO (0.1% final concentration) mixed at a ratio of 3:1, respectively. Three main types of experimental treatments were performed. TCB-2 was i.p. injected at a dose of 0.3 mg/kg, and DIOA was intrathecally injected at a dose of 20μg in 15μl diluting solution. The intrathecal DIOA injection was performed 20 min before a systemic TCB-2 treatment. TCB-2 or distilled water was intraperitoneally given to rats. The injection was done daily for a continuous 7 days, starting at 2 h after injury. Mechanical thresholds were measured on day 3, day 7, and day 14 after surgery.

Statistics

Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Analysis was conducted by one-way or two-way ANOVA with a Bonferroni correction, followed by Fisher’s Exact Test upon necessity. All values are depicted as mean±standard deviation from 5 repeats and are considered significant if p < 0.05.

RESULTS

Establishment of a chronic constriction injury of the sciatic nerve (CCI) model in rats

First, we the CCI model was induced in rats. The rats were followed for recovery for 14 days. The sham-operated animals received the same surgical procedure without ligature of the sciatic nerve. The left loose ligature caused immediate paralysis of the rats, which was assessed by responsiveness to a clamp. The rats that had received CCI or sham-operation were further grouped. Beginning day 7, some rats received either vehicle or TCB-2 treatment till day 14. Group 1: sham-operated; Group 2: CCI; Group 3: CCI and vehicle; Group 4: CCI and TCB-2 treatment (Fig. 1).

Fig.1

Establishment of a chronic constriction injury of the sciatic nerve (CCI) model in rats. The CCI model was induced in rats, which recovered for 14 days. The sham-operated animals received the same surgical procedure without ligature of the sciatic nerve. The rats that had received CCI or sham-operation were further grouped. Beginning day 7, some rats received either vehicle or TCB-2 treatment till day 14. Group 1: sham-operated; Group 2: CCI; Group 3: CCI and vehicle; Group 4: CCI and TCB-2 treatment.

TCB-2 reduces neuropathic pain after CCI

The mechanical and thermal assessments were performed before surgery (day 0), and at day 1, 3, 7, and 14 after surgery. Only the ipsilesional side was examined. Vehicle-treated rats displayed mechanical allodynia, exhibiting a significant decrease in withdrawal threshold in the Von Frey filament test after CCI (Fig. 2A). After CCI, vehicle-treated rats also displayed thermal allodynia by a significant decrease in thermal-stimuli-responsive withdrawal latency of the paw (Fig. 2B). The mechanical and thermal allodynia were sustained till day 14 (Fig. 2A, B). Interestingly, both mechanical (Fig. 2A) and thermal (Fig. 2B) allodynia in CCI-rats were significantly attenuated by administration of TCB-2. Together, these data suggest that TCB-2 reduces neuropathic pain after CCI.

Fig.2

TCB-2 reduces neuropathic pain after CCI. The mechanical (A) and thermal assessments (B) were performed by Von Frey filament assay before surgery (day 0), and at day 1, 3, 7, and 14 after surgery. Only the ipsilesional side was examined. *p < 0.05. NS, non-significant. N = 5.

TCB-2 reactivates KCC2 through 5-HT2A receptor

The KCC2 levels in lumbar dorsal horn were assessed by western blotting. We found that compared to the vehicle-treated CCI-rats, the TCB-2-treated CCI-rats exhibited significantly higher levels of KCC2, suggesting that TCB-2 reactivates KCC2 through 5-HT2A receptor (Fig. 3).

Fig.3

TCB-2 reactivates KCC2 through 5-HT2A receptor. The KCC2 levels in lumbar dorsal horn were assessed by western blotting. *p < 0.05. NS, non-significant. N = 5.

KCC2 is required for the suppression of neuropathic pain by TCB-2

To figure out whether the reactivated KCC2 mediates the reduction of CCI-induced neuropathic pain, we included a new experimental group, in which CCI-rats received both TCB-2 and DIOA, while the latter was injected intrathecally 20 min prior the i.p. injection of TCB-2. We found that DIOA injection abolished the TCB-2-induced antinociception against mechanical (Fig. 4A) and thermal (Fig. 4B) stimuli, suggesting that TCB-2 may alleviate the pain through augmentation of KCC2. The KCC2 levels were determined at day 14 after CCI as a control, showing that the increases in TCB-2 levels by TCB-2 were abolished by DIOA (Fig. 4C).

Fig.4

KCC2 is required for the suppression of neuropathic pain by TCB-2. CCI-rats received both TCB-2 and DIOA, while the latter was injected intrathecally 20 min prior the i.p. injection of TCB-2, compared with CCI-rats that had received TCB-2 and vehicle. The mechanical (A) and thermal assessments (B) were performed by Von Frey filament assay before surgery (day 0), and at day 1, 3, 7, and 14 after surgery. Only the ipsilesional side was examined. C) The KCC2 levels in lumbar dorsal horn were assessed by western blotting. *p < 0.05. NS, non-significant. N = 5.

TCB-2-mediated re-activation of KCC2 reduces future development of neurodegeneration in rats

Four months after CCl/TCB-2 treatment, the SRT was performed on some rats that were kept long term. TCB-2 treatment significantly improved the behavior of the CCl-rats (Fig. 5A). Moreover, those rats were also assessed in with PM-DAT, showing significant improved behavior of the CCl-rats that had received TCB-2 (Fig. 5B, C). Thus, both recognition and memory of the CCl-rats were improved in the long run by TCB-2 treatment. Together, our data support further studies on the possibility of using this strategy to reduce postoperative pain and future neurodegenerative disorders in clinic (Fig. 6).

Fig.5

TCB-2-mediated re-activation of KCC2 reduces future development of neurodegeneration in rats. A–C) Four months after CCl/TCB-2, rats were analyzed for Social Recognition Test (A) and Plus-Maze Discriminative avoidance Task (B + C). *p < 0.05. N = 5.

Fig.6

Schematic of the model. CCI induces neuropathic pain through reduction of KCC2. TCB-2 administration increases dorsal horn KCC2 levels through 5-HT2A, which subsequently attenuates the neuropathic pain. DIOA contradicts the effects of TCB-2 by blocking the effects of KCC2. TCB-2 treatment reduces future chances for developing neurodegenerative diseases.

DISCUSSION

In this study, activation of 5-HT2A receptors by TCB-2 appeared to attenuate neuropathic pain through regaining of KCC2 expression on dorsal horn neurons after CCI. Since DIOA, a blocker of KCC2, abrogates the analgesic action of TCB-2, the major inhibitory effects of TCB-2 on CCI-induced neuropathic pain should be mediate through KCC2 signaling. Indeed, activation of GABA-A and glycine receptors can inhibit neurons through lowing intracellular chloride concentration, which is maintained almost exclusively by the potassium-chloride cotransporter KCC2. A reduction of KCC2 levels or functionality thus are associated with pathogenesis of neurological disorders, like chronic pain following SCI or operations. In line with these notions, a previous study has shown that re-activation of 5-HT2A receptors by TCB-2 hyperpolarizes the reversal potential of inhibitory postsynaptic potentials in spinal motoneurons, upregulates the cell membrane expression of KCC2, and reduces neuropathic pain following SCI [16]. In another report, KCC2 was found to decrease the intracellular chloride concentration in cultured neurons and attenuate calcium responses evoked by application of the GABA-A receptor agonist muscimol [17]. However, the effects of KCC2 re-activation on SNPP remains unknown and thus were addressed in the current study.

Here, we showed robust data that demonstrate anti-pain effects of re-activation of KCC2 by administration of TCB-2. Our results are consistent with reports on the function of this pathway in a rat SCI model, SNI model [10], and an incision pain model [18]. These reports and our findings could be resulting from the fact that KCC2 immunolabeling on membranes of motoneurons can be enhanced by TCB-2 administration [16]. Although here we did not assess the changes in chloride homeostasis, we assume that KCC2 upregulation by TCB-2 likely enhances the extrusion capacity of chloride in dorsal horn neurons and as a consequence strengthens the inhibitory synaptic transmission to alleviate hypersensitivity and reduce pain. Consistent with this hypothesis, DIOA-mediated inhibition of KCC2 dismissed the effects of TCB-2 on CCI-induced pain. These data are consistent with previous reports, suggesting an antinociceptive role of 5-HT2A receptor functioning at the spinal cord level [19].

Given the predominant postsynaptic localization of 5-HT2A receptors in the spinal cord [20], the pharmacological approach for using 5-HT2A receptor agonists to treat SNPP should be highly reasonable and promising, especially given that it reduces future chances for developing neurodegenerative diseases. However, a critical question remains, as it is unknown how the activation of 5-HT2A receptors promotes KCC2 function in the dorsal horn neurons. Efforts should be made to address this mechanistic question, since here we provide compelling evidence for application of 5-HT2A receptor agonists to the treatment of SNPP.

DISCLOSURE STATEMENT

Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/20-0027r1).

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