Journal: CNS Neuroscience & Therapeutics

Article Title: Combined use of CLP290 and bumetanide alleviates neuropathic pain and its mechanism after spinal cord injury in rats

doi: 10.1111/cns.70045

Figure Lengend Snippet: Experimental design. Rats were randomly divided into five groups: (1) Sham: Removal of the lamina of the T10 vertebrae without any other intervention; (2) SCI + vehicle: Moderate spinal cord contusion was performed at the T10 vertebral level and rats were treated with control vehicle; (3) SCI + CLP290: Administration of CLP290 at 50 mg/kg/day, intragastrically (i.g.) after SCI; (4) SCI + bumetanide: Administration of bumetanide at 30 mg/kg/day, intraperitoneally (i.p.) after SCI; and (5) SCI + combination: Combined administration of CLP290 at 50 mg/kg/day i.g. and bumetanide at 30 mg/kg/day i.p. after SCI. CLP290 was administered i.g. and bumetanide was administered i.p. once daily for 14 consecutive days from 7 dpi. A total of 102 adult female Sprague–Dawley rats (8‐week‐olds weighing 220 ± 30 g) were used in the study. Among them, 56 rats (Sham, n = 8; SCI + different intervention groups, n = 12) underwent behavioral assessments multiple times after surgery (0, 7, 21, 35, 56 dpi), and electrophysiological assessments were performed at 21 and 56 dpi ( n = 8/group). Additionally, 20 rats were euthanized at 21 dpi for immunoblotting tests ( n = 4/group), and another 20 were euthanized at 21 dpi for immunofluorescence assay ( n = 4/group). At the end of the experiment, 20 rats used for behavioral measurements were euthanized at 56 dpi for immunoblotting ( n = 4/group). A total of six rats were excluded: three rats died due to excessively low body weight and infection during the experiment, and three rats were excluded due to excessive asymmetry in the degree of paralysis in both lower extremities after the impact.

Article Snippet: The KCC2 agonist CLP290 (HY‐103023, MCE) was dissolved in a solution containing 50% PEG300 + 50% saline at a concentration of 50 mg/kg and administered intragastrically.

Techniques: Control, Western Blot, Immunofluorescence, Infection

Journal: CNS Neuroscience & Therapeutics

Article Title: Combined use of CLP290 and bumetanide alleviates neuropathic pain and its mechanism after spinal cord injury in rats

doi: 10.1111/cns.70045

Figure Lengend Snippet: Effects of CLP290 and bumetanide, alone or in combination, on SCI‐associated hypersensitivity and locomotor function. (A) Von Frey evaluation model diagram. Rats were placed in transparent plastic cages (18 × 8 × 8 cm) on an elevated wire mesh surface and allowed to acclimate for approximately 30 min. To assess mechanical allodynia, threshold levels to harmless mechanical stimulation were measured using 0.4–15 g of calibrated von Frey hairs. (B) Von‐Frey fiber test area. The central plantar surface area of rat hind paw. (C) Effects of CLP290 and bumetanide, alone or combination, on SCI‐associated mechanical allodynia. * p < 0.05, ** p < 0.01, *** p < 0.001 for SCI + combination versus SCI + vehicle; # p < 0.05, ## p < 0.01 for SCI + bumetanide versus SCI + vehicle; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 for SCI + CLP290 versus SCI + vehicle; ^ p < 0.05 for SCI + combination versus Sham; & p < 0.05 for SCI + combination versus SCI + CLP290 or bumetanide. (D) Effects of CLP290 and bumetanide, alone or combination, on SCI‐associated heat hyperalgesia. ** p < 0.01, *** p < 0.001 for SCI + combination versus SCI + vehicle; ### p < 0.001 for SCI + bumetanide versus SCI + vehicle; $$ p < 0.01 for SCI + CLP290 versus SCI + vehicle; ^ p < 0.001 for SCI + combination versus SCI + CLP290; ns, p > 0.05. (E) Effects of CLP290 and bumetanide, alone or combination, on SCI‐associated cold allodynia. * p < 0.05, ** p < 0.01 for SCI+ combination versus SCI + vehicle; # p < 0.05 for SCI+ bumetanide versus SCI+ vehicle; $ p < 0.05 for SCI + CLP290 versus SCI+ vehicle; ^ p < 0.05 for SCI+ bumetanide versus sham; && p < 0.01 for SCI + CLP290 versus sham; ns, p > 0.05. (F) Effects of CLP290 and bumetanide, alone or in combination, on SCI‐associated locomotor function. * p < 0.05, ** p < 0.01, *** p < 0.001 for SCI + combination vs SCI + vehicle; ### p < 0.001 for SCI+ CLP290, SCI+ bumetanide, SCI+ combination versus sham. Repeated measures two‐way ANOVA with Student Neuman–Keuls post‐hoc test. Values are mean ± SEM, Sham n = 8, SCI + different intervention groups n = 12.

Article Snippet: The KCC2 agonist CLP290 (HY‐103023, MCE) was dissolved in a solution containing 50% PEG300 + 50% saline at a concentration of 50 mg/kg and administered intragastrically.

Techniques:

Journal: CNS Neuroscience & Therapeutics

Article Title: Combined use of CLP290 and bumetanide alleviates neuropathic pain and its mechanism after spinal cord injury in rats

doi: 10.1111/cns.70045

Figure Lengend Snippet: Effects of CLP290 and bumetanide, alone or in combination, on improving anxiety, depression, and activity levels following SCI. (A, B) Comparison of the total distance and average speed of horizontal movement at 56 dpi in different experimental groups. (C) Comparison of the central area entries (Bouts) at 56 dpi in different experimental groups. (D) Representative movement trajectories of different experimental groups of rats in the open field test. ** p < 0.01, *** p < 0.001 (four intervention groups vs. sham); # p < 0.05, ## p < 0.01 (three drug treatment groups vs. SCI + vehicle); one‐way ANOVA, Tukey post‐hoc test. Values are mean ± SEM, Sham, n = 8, SCI + different intervention groups, n = 12.

Article Snippet: The KCC2 agonist CLP290 (HY‐103023, MCE) was dissolved in a solution containing 50% PEG300 + 50% saline at a concentration of 50 mg/kg and administered intragastrically.

Techniques: Activity Assay, Comparison

Journal: CNS Neuroscience & Therapeutics

Article Title: Combined use of CLP290 and bumetanide alleviates neuropathic pain and its mechanism after spinal cord injury in rats

doi: 10.1111/cns.70045

Figure Lengend Snippet: Effects of CLP290 and bumetanide, alone or in combination, on H‐reflex RDD levels. (A) Schematic diagram of the rate‐dependent depression (RDD) of the H‐reflex in the spinal cord. Under continuous electrical stimulation, the amplitude of the spinal cord Hoffman reflex gradually decreases with an increase in stimulation frequency. The % depression is calculated as the change in amplitude of the second recorded H‐reflex (H2) compared to the first in the train (H1). The M wave is generated by the orthodromic propagation of the stimulus along motor fibers. In contrast, the H‐reflex is a monosynaptic reflex transmitted by Ia afferent fibers and modulated by inhibitory interneurons (GABAergic neurons) in the spinal cord. (B, E) The RDD of the M wave in the experimental groups is shown with different stimulation rates at 21 and 56 dpi. (C) The RDD of the H wave in the experimental groups at 21 dpi. * p < 0.05, ** p < 0.01 for SCI + combination versus SCI + vehicle; # p < 0.05, ## p < 0.01 for SCI + combination versus SCI + bumetanide. (F) The RDD of the H wave in the experimental groups at 56 dpi. * p < 0.05, ** p < 0.01 for SCI + combination versus SCI + vehicle; # p < 0.05 for SCI + bumetanide versus SCI + vehicle; ^ p < 0.05 for SCI + CLP290 versus SCI + vehicle. (D, G) Trajectories of H‐reflexes evoked by stimulation of the tibial nerve at 0.1 Hz (black), 0.5 Hz (green), 1 Hz (blue), 2 Hz (orange), and 5 Hz (red). Repeated measures two‐way ANOVA with Student Neuman‐Keuls post‐hoc test. Values are mean ± SEM, n = 8/group.

Article Snippet: The KCC2 agonist CLP290 (HY‐103023, MCE) was dissolved in a solution containing 50% PEG300 + 50% saline at a concentration of 50 mg/kg and administered intragastrically.

Techniques: Generated

Journal: CNS Neuroscience & Therapeutics

Article Title: Combined use of CLP290 and bumetanide alleviates neuropathic pain and its mechanism after spinal cord injury in rats

doi: 10.1111/cns.70045

Figure Lengend Snippet: Effects of CLP290 and bumetanide, alone or in combination, on KCC2 and NKCC1 protein expression following SCI at 21 and 56 dpi. (A) Expression of KCC2 in the lumbar enlargement of the spinal cord in different experimental groups at 21 dpi. (B) The expression of NKCC1 in the lumbar enlargement of the spinal cord in different experimental groups at 21 dpi. (C) The KCC2/NKCC1 ratio in different experimental groups at 21 dpi. (D) Expression of KCC2 in the lumbar enlargement of the spinal cord in different experimental groups at 56 dpi. (E) The expression of NKCC1 in the lumbar enlargement of the spinal cord in different experimental groups at 56 dpi. (F) The KCC2/NKCC1 ratio in different experimental groups at 56 dpi. * p < 0.05 (different treatment groups or sham vs SCI + vehicle). One‐way ANOVA, Tukey post‐hoc test. Values are mean ± SEM, n = 4/group.

Article Snippet: The KCC2 agonist CLP290 (HY‐103023, MCE) was dissolved in a solution containing 50% PEG300 + 50% saline at a concentration of 50 mg/kg and administered intragastrically.

Techniques: Expressing

Journal: CNS Neuroscience & Therapeutics

Article Title: Combined use of CLP290 and bumetanide alleviates neuropathic pain and its mechanism after spinal cord injury in rats

doi: 10.1111/cns.70045

Figure Lengend Snippet: Effects of CLP290 and bumetanide, alone or in combination, on KCC2 and NKCC1 immunofluorescence intensity following SCI at 21 dpi. (A)The expression and distribution of KCC2 and NKCC1 in the dorsal horn of the lumbar enlargement after SCI was visualized by immunofluorescence staining. KCC2 protein expression was found to be uniform and intact on neuronal membranes in the sham group and in the three different drug treatment groups but not in the SCI + vehicle. In the latter group, KCC2 immunofluorescence distribution on neuronal membranes was discontinuous, and dot‐like internalization of KCC2 was observed in the cytoplasm. Conversely, NKCC1 protein expression was expressed evenly and completely on the neuronal cell membrane in the SCI + vehicle but less intensively and discontinuously in the sham and other three treatment groups. (B) Mean value of KCC2 fluorescence intensity in the dorsal horn of the lumbar enlargement in different experimental groups at 21 dpi. (C) Mean value of NKCC1 fluorescence intensity in the dorsal horn of the lumbar enlargement in different experimental groups at 21 dpi. (D) The ratio of KCC2/NKCC1 fluorescence intensity in different experimental groups at 21 dpi: * p < 0.05, ** p < 0.01 (different treatment groups or sham vs SCI + vehicle). One‐way ANOVA, Tukey post‐hoc test. Values are mean ± SEM, n = 4/group.

Article Snippet: The KCC2 agonist CLP290 (HY‐103023, MCE) was dissolved in a solution containing 50% PEG300 + 50% saline at a concentration of 50 mg/kg and administered intragastrically.

Techniques: Immunofluorescence, Expressing, Staining, Membrane, Fluorescence

Journal: Cell

Article Title: Reactivation of Dormant Relay Pathways in Injured Spinal Cord by KCC2 Manipulations

doi: 10.1016/j.cell.2018.06.005

Figure Lengend Snippet: (A) Schematics of transverse spinal cord sections showing c-Fos expression patterns in T8/9 segments after 1 hour of continuous locomotion in intact mice and injured mice with treatment of vehicle, CLP290, AAV-PHP.B-syn-HA-KCC2 or L838,417. Each spot represents a cell positively stained with both c-Fos and NeuN. Representative raw images are shown in Figure S5A.

Article Snippet: METHOD DETAILS Chemicals and Antibodies For systemic administration (i.p.): Quipazine [Sigma (Q1004), 0.2 mg/kg)] and 8-OH-DPAT [Tocris (0529), 0.1 mg/kg)] were suspended in 0.9% NaCl; Baclofen [Tocris (0417), 1 mg/kg)] was suspended in 100mM NaOH and then 0.9% NaCl; CP101606 [Sigma (SML0053), 10 mg/kg)] was suspended in DMSO and then 0.9% NaCl; CLP290 [synthesized by PharmaBlock, 35mg/kg] was suspended in DMSO and then 20% 2-hydroxypropyl-β-cyclodextrin; L838,417 [synthesized by PharmaBlock, 1mg/kg] was suspended in 0.5% methylcellulose and 0.9% NaCl; and Bumetanide [Tocris, (3108), 0.3 mg/kg)] was suspended in 15% DMSO.

Techniques: Expressing, Staining

Journal: ASN NEURO

Article Title: Restoration of KCC2 Membrane Localization in Striatal Dopamine D2 Receptor-Expressing Medium Spiny Neurons Rescues Locomotor Deficits in HIV Tat-Transgenic Mice

doi: 10.1177/17590914211022089

Figure Lengend Snippet: CLP290 Administration Restores Phosphorylation of S940 and Membrane Localization of KCC2 in the Striatum. Two-way analysis revealed a main effect of Tat expression for 2 weeks to reduce total KCC2 (**, p < .01, n = 12–18) suggesting that CLP290 failed to rescue total KCC2 levels (a). Examination of phosphorylation of S940 revealed a significant decrease in the Tat+/Vehicle group compared to control (** p < .01, n = 8–12) and rescue with CLP290 (* p < .05, n = 8–12) (b) suggesting that membrane-bound KCC2 may be reduced, which was confirmed by western blots on membrane fractions (** p < .01; * p < .05, n = 8–9) (c). Blots for total and pS940-KCC2 are represented as intensity relative to GAPDH normalized to Tat–/Vehicle controls (a and b). Blots for membrane bound KCC2 are relative to total lane protein normalized to controls (c). Data are presented as mean ± SEM.

Article Snippet: Tat+ and Tat– mice were treated with either 50 mg/kg of the KCC2 enhancer, CLP290 (Aobious, Gloucester, MA), freshly suspended in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO) and 20% 2-hydroxypropyl-β-cyclodextrin (HPCD; Tocris, Bristol, UK), or vehicle (20% HPCD in DMSO), once per day (morning, p.o., 200 μL total volume per mouse) for seven days during the second week of DOX administration.

Techniques: Phospho-proteomics, Membrane, Expressing, Control, Western Blot

Journal: ASN NEURO

Article Title: Restoration of KCC2 Membrane Localization in Striatal Dopamine D2 Receptor-Expressing Medium Spiny Neurons Rescues Locomotor Deficits in HIV Tat-Transgenic Mice

doi: 10.1177/17590914211022089

Figure Lengend Snippet: CLP290 Reverses Hyperactive Locomotion of Tat+ Mice in the Open Field Assay. Tat+ mice given vehicle showed increased motor activity compared to controls in the open field assay, measured by both total distance travelled (a) and rearing number (b) (**, p < .01, n = 8–9) during the 20 min open field testing period. 50 mg/kg CLP290 administration significantly decreased total distance travelled and rearing number in Tat+ mice (*, p < .05, n = 8–9), suggesting that restoration of KCC2 function was sufficient to rescue abnormal locomotor activity in these mice. We also examined time spent in the center zone during the open field assay as a measure of anxiety-like behavior and found no significant differences ( n = 8–9) (c). Data from rotarod testing showed no significant differences in latency to fall between any group ( n = 8–9), suggesting that Tat induction or CLP290 administration did not affect motor coordination or sedation (d). Data are presented as mean ± SEM.

Article Snippet: Tat+ and Tat– mice were treated with either 50 mg/kg of the KCC2 enhancer, CLP290 (Aobious, Gloucester, MA), freshly suspended in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO) and 20% 2-hydroxypropyl-β-cyclodextrin (HPCD; Tocris, Bristol, UK), or vehicle (20% HPCD in DMSO), once per day (morning, p.o., 200 μL total volume per mouse) for seven days during the second week of DOX administration.

Techniques: Activity Assay