Physiological actions of a humanized P2X4 scFv on peripheral and central neurons in male mice with neuropathic pain

Highlights

• •A single intraperitoneal dose of the hP2X4R scFv reversed mechanical hypersensitivity in male mice suffering from chronic neuropathic pain.
• •hP2X4R scFv altered excitability of spinal dorsal horn and ventrolateral periaqueductal gray Fos+ neurons involved in chronic pain.
• •hP2X4R scFv decreased the excitability of large diameter DRG and TG neurons from neuropathic mice, but had no effect on neurons from naïve mice.
• •This study supports hP2X4R scFv as a promising non-opioid biologic for chronic pain treatment.

Abstract

Neuropathic pain remains a challenging clinical condition due to its resistance to conventional analgesics. The purinergic P2X4 receptor (P2X4R), an ATP-gated ion channel, is upregulated in sensory neurons and glial cells following nerve injury and is pivotal in chronic pain pathogenesis. This study evaluates the therapeutic potential of a novel humanized single-chain variable fragment antibody (hP2X4R scFv) targeting P2X4R in male mice models of neuropathic pain. Using spared nerve injury (SNI) and foramen rotundum inflammatory compression of the trigeminal infraorbital nerve (FRICT-ION) models, we demonstrate that a single intraperitoneal dose of hP2X4R scFv significantly reverses mechanical hypersensitivity for up to four weeks. Electrophysiological recordings from FosTRAP mice revealed that hP2X4R scFv reduced the excitability of Fos+ neurons in the spinal dorsal horn and ventrolateral periaqueductal gray (vlPAG), key regions involved in pain processing. In vitro, patch-clamp studies further showed that hP2X4R scFv selectively decreased action potential firing in larger diameter dorsal root ganglion (DRG) and trigeminal ganglion (TG) neurons from SNI and FRICT-ION mice, respectively, without affecting naïve neurons. These findings suggest that hP2X4R scFv modulates both central and peripheral neuronal excitability associated with chronic pain. The specificity and long-lasting efficacy of hP2X4R scFv highlights its promise as a non-opioid therapeutic candidate for neuropathic pain management.

1. Introduction

Neuropathic pain is one of the most debilitating forms of chronic pain, affecting 7–10 % of the general population (van Hecke et al., 2014). This type of pain does not respond well to nonsteroidal anti-inflammatories and opioids. Therefore, it is essential to explore the underlying mechanisms of neuropathic pain to inform the development of new and more effective treatments. P2X purine receptors, ATP-gated ion channels that are permeable to cations (Burnstock, 2000), play a critical role in pain signaling. Among these, the P2X4 receptor (P2X4R) has been shown to have a significant impact on the development of neuropathic pain. (Tsuda et al., 2013, Zhang et al., 2020, Inoue, 2019).

P2X4R, primarily located on the plasma membrane, responds to ATP (Bo et al., 2003). P2X4R is expressed in the PNS and CNS tissues (Burnstock, 2000, Teixeira et al., 2019, Kuroda et al., 2012, Nakai et al., 2010, Deng et al., 2018, Wang et al., 2020, Ulmann et al., 2008), and under normal conditions, it has a limited role in pain perception (Ulmann et al., 2010). However, following nerve or tissue injury, P2X4R expression in PNS and CNS increases, leading to heightened excitability of sensory neurons, which cause abnormal activity, perpetuating chronic pain states (Teixeira et al., 2019, Kuroda et al., 2012, Nakai et al., 2010, Deng et al., 2018, Wang et al., 2020, Ulmann et al., 2008, Bernier et al., 2018). It has been reported that mice lacking P2X4R do not exhibit mechanical hyperalgesia after nerve injury (Inoue, 2019, Inoue, 2009). Notably, research has shown that male, but not female, rats exhibit increased P2X4R expression after nerve damage, suggesting potential sex-based differences in pain processing mechanisms (Mapplebeck et al., 2016, Lopes et al., 2017, Paige et al., 2018). While upregulation of P2X4R plays a role in neuropathic pain, basal P2X4R is also known to contribute to synaptic plasticity and neuronal excitability in central circuits, which are known players in neuropathic pain (Wildner et al., 2024). Therefore, it is unclear that upregulation of P2X4R alone is the sole mechanism of its contribution to neuropathic pain and functional mechanisms need to be further studied.

Single-chain variable fragment (scFv) antibodies have been used as treatment for a variety of conditions, including cancer and autoimmune disorders (Gezehagn Kussia and Tessema, 2024). We created a panel of scFvs targeting an extracellular peptide sequence of P2X4R through cell-free ribosome display for recombinant antibody selection, as previously described (Westlund et al., 2021a, Westlund et al., 2021b). These scFv antibodies demonstrate binding activity similar to monoclonal antibodies but offer stronger affinity and improved tissue penetrability due to their approximately 30% smaller size. Our previous work with a murine P2X4R scFv demonstrated effectiveness in reducing pain-related behaviors in male, but not female, mice with chronic orofacial pain (Westlund et al., 2021b). More recently we have developed a humanized P2X4 scFv (hP2X4 scFv) which is also effective only in male, but not female mice with neuropathic pain (Kunamneni and Westlund, 2025). Our previous studies did not show an effect of hP2X4 scFv on total expression of hP2X4R in cultured cells after prolonged treatment; however, hP2X4 scFv was able to reduce ATP-evoked currents HEK cells overexpressing P2X4R showing a reduction in function of the receptor (Zurek et al., 2025).

In this study, we assess the physiological mechanisms of a hP2X4 scFv in alleviating neuropathic pain in male mice, specifically those with spared nerve injury (spared nerve injury – (SNI)) and trigeminal nerve injury (foramen rotundum inflammatory compression of the trigeminal infraorbital nerve (FRICT-ION)). We evaluated its effects on neuronal excitability in vitro and ex vivo using cells derived from the peripheral and central nervous system. Central actions of hP2X4 scFv were studied through ex vivo slice patch-clamp recordings of Fos+ neurons of the spinal dorsal horn and ventrolateral periaqueductal grey (vlPAG) from FosTRAP (targeted recombination in active populations) SNI and FRICT-ION mice treated with scFv. These critical regions of the spinal cord and brain are essential for chronic pain processing (Heinricher et al., 2009, Alles and Smith, 2018) and help elucidate the mechanisms of our humanized scFv. Additionally, we examined changes in the electrophysiological properties and excitability of dissociated DRG neurons from SNI mice and TG neurons from FRICT-ION mice in response to hP2X4 scFv in vitro treatment compared to controls. Our findings demonstrate that the hP2X4 scFv is effective in modulating chronic pain-associated neuronal excitability and highlights P2X4R as potential as a target for therapeutic development.

2. Methods

2.1. Animals

All animal research was conducted with the approval of the University of New Mexico Institutional Animal Care and Use Committee. Our facilities are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and hold all necessary federal certifications: Animal Welfare Assurance #D16-00,228 (A3350-01) and USDA Registration # 85-R-0014.

All rodents were housed in a well-ventilated room maintained at 20–22 °C, with a reversed 10/14  h dark/light cycle to facilitate testing during their active phase. They were acclimated for one week prior to commencing the studies. They had unrestricted access to food and water throughout the experimental period, receiving a standard chow diet with low soybean content.

Male BALB/cAnNHsd mice (5–6 weeks old; Jackson Laboratories) and male FosTRAP (Fos2iCreERT2/J crossed with B6-Tg Ai14tdTOMfl mice (also 5–6 weeks old) underwent surgical procedures to induce models of neuropathic pain models, specifically SNI and foramen rotundum inflammatory compression of the trigeminal infraorbital nerve (FRICT-ION). All surgeries were conducted under sterile conditions using a surgical microscope, and mice were anesthetized with 2 to 5 % isoflurane.

2.2. Generation of humanized scFv

The humanized P2X4 heavy-chain single variable fragment (hP2X4 scFv) was generated as described and characterized previously (Kunamneni and Westlund, 2025, Zurek et al., 2025). A parental mouse scFv targeting P2X4R was previously characterized and described (Westlund et al., 2021b, High and Kunamneni, 2025).

2.3. Surgical induction of the neuropathic pain models

2.3.1. Spared nerve injury model

Male BALB/cAnNHsd mice aged 5–6 weeks (obtained from Jackson Laboratories) and male Fos2iCreERT2/J x B6-Tg Ai14tdTOMfl mice of the same age were used to establish the SNI model of neuropathic pain (Decosterd and Woolf, 2000). In brief, the tibial and common peroneal nerves were ligated and cut distal to the ligation site, while the sural nerve remained intact. In the sham surgery group, the nerve was exposed without any disturbance. Mechanical hypersensitivity in the left hind paw was assessed weekly using the “up and down” von Frey method (Chaplan et al., 1994).

2.3.2. Trigeminal neuropathic pain model

Male BALB/cAnNHsd mice (aged 5–6 weeks; from Jackson Laboratories) and male Fos2iCreERT2/J x B6-Tg Ai14tdTOMfl mice of the same age were tested in a FRICT-ION-induced neuropathic pain model. The acronym FRICT-ION refers to a chronic model that causes compression and chemical irritation of the trigeminal nerve. Our lab established the FRICT-ION trigeminal neuropathic pain model by introducing 3 mm of chromic catgut suture (4-0) through a tiny scalpel incision in the oral buccal/cheek crease along the trigeminal maxillary nerve branch (V2) as it enters the foramen rotundum of the skull (Montera and Westlund, 2020). In the sham surgery group, mice received the oral buccal/cheek incision, but the nerve remained intact. All animals in the FRICT-ION group developed mechanical hypersensitivity on the snout within one week, which was tested as previously described (Montera and Westlund, 2020).

Mechanical sensitivity on the left hind paw (SNI) and snout (FRICT-ION) was assessed prior to nerve injury to establish baseline thresholds and weekly after nerve injury using the “up and down” von Frey approach (Chaplan et al., 1994). Briefly, graded thin nylon von Frey filaments with defined bending forces (tensile strength) were applied to either the left hind paw in the SNI induced models (Goins et al., 2022) or the lateral snout/buccal area in the FRICT-ION induced models (Montera and Westlund, 2020). Five applications of several mid-range von Frey filaments were applied once every three to four seconds. A positive response for hypersensitivity was measured by reflexive withdrawal from mechanical stimulation. The mechanical hypersensitivity threshold was determined by systematically selecting the next filament based on the mouse’s response to the stimulation, with repeated trials to confirm positive responses. Gram force was calculated from the fiber strength using an arithmetic technique. A reduced sensitivity threshold, or “hypersensitivity,” was indicated by responses to a lower gram force than that observed in the control group.

2.5. Preparation of ex vivo spinal cord slices from FosTRAP mice

Male Fos2iCreERT2/J x B6-Tg Ai14tdTOMfl SNI FosTRAP mice (TRAP2) (DeNardo et al., 2019) underwent acute spinal cord slicing. After 3 weeks of SNI, the mice were given a single intraperitoneal (i.p.) injection of either hP2X4 scFv (4.0 mg/kg) or a vehicle. We employed the light touch-TRAP technique to activate and “trap” Fos+ nociceptive neurons in the spinal cord, as described by Corder et al. (2019). After administration of the hP2X4 scFv or vehicle for two weeks, a von Frey filament (no.08) was lightly applied to the lateral aspect of the left hind paw (sural nerve innervation receptive field) with enough force to cause a slight bend of the filament for up to 1 s before being retracted. The filament stimulus was applied once every 30 s over 10 min. Following 30 min of stimulation, the mice received a single i.p. injection of tamoxifen 50 mg/kg. Two weeks later, the animals were deeply anesthetized (with 3 % isoflurane) before being decapitated for spinal cord dissection. Following anesthesia, cardiac arrest and loss of ocular reflexes, the spinal cord was extracted via laminectomy and placed in a solution with a partly frozen 252 mM sucrose base containing (in mM) 2.5 KCl, 2 MgCl₂, 2 CaCl₂, 1.2 NaH₂PO₄, 26 NaHCO₃, 10 D-glucose, and 5 Kynurenic Acid, with a pH of 7.4. This solution was constantly aerated with carbogen (95 % O₂, 5 % CO₂). The L4-L6 area of the spinal cord was attached using cyanoacrylate adhesive to a trapezoid block made from 4 % agar. Slices measuring 300–350 μm in thickness were produced with a Leica VT1000S vibratome (Leica Biosystems, Germany). The sections were placed in approximately 200 ml of artificial cerebrospinal fluid (aCSF) with the following concentrations (in mM): 113 NaCl, 3.0 KCl, 25 NaHCO₃, 1.0 NaH2PO4, 2.0 CaCl₂, 2.0 MgCl₂, and 11 D-glucose, pH 7.4. This solution was continuously bubbled with carbogen (95 % O2, 5 % CO₂) at 37 °C for 30 min before recording. It was then kept at room temperature (22–24 °C) for the remainder of the experimental day. Infrared-differential interference contrast (IR-DIC) was used to identify the substantia-gelatinosa for recordings due to its translucent appearance (Alles et al., 2017).

2.6. Preparation of ex vivo brain slices from FosTRAP mice

Male Fos2iCreERT2/J x B6-Tg Ai14tdTOMfl FRICT-ION FosTRAP mice (“TRAP2”) (DeNardo et al., 2019) underwent acute brain slicing. The mice received a single i.p. injection of either hP2X4 scFv (4.0 mg/kg) or a vehicle following 3 weeks of the FRICT-ION. After 2 weeks of receiving hP2X4 scFv or vehicle, a von Frey filament (no.08) was lightly applied to the snout to activate and trap Fos+ nociceptive neurons in the brain, as described in section 2.6 (Corder et al., 2019). After 30 min of stimulation, mice were given a single i.p. injection of tamoxifen (50 mg/kg). Two weeks later, the animals were sacrificed, and brains harvested for slicing. The brain was then placed in a partially frozen sucrose solution, as described in section 2.5. Brain slices, ranging from 300 to 350 μm in thickness, were prepared using a Leica VT1000S vibratome and incubated in warm and carbogen-bubbled aCSF for 30 min before the recording (Ting et al., 2018). The slices were then transferred to room temperature aCSF with carbogen bubbling before examining the ventrolateral periaqueductal gray (vlPAG) region under 4X magnification. Cells were randomly selected for recording and distributed along the rostral/caudal axis of the periaqueductal gray (PAG).

2.7. Cell cultures

Two to four weeks following injury, DRG and TG cell culture assays were performed using SNI and FRICT-ION mice, respectively.

2.7.1. Dorsal root ganglion culture

Male BALB/cAnNHsd SNI mice were deeply anesthetized with 3 % isoflurane and then euthanized by decapitation prior to dissecting the dorsal root ganglia (DRG) for primary cultures intended for electrophysiological studies. The bilateral L3-L5 DRGs were carefully removed and placed in ice-cold Hank’s Balanced Salt Solution (HBSS) without Ca²⁺/Mg²⁺ (Stem Cell Technologies, Cat# 37250). The ganglia were mechanically disrupted and cut into smaller fragments using a scalpel.

Subsequently, the ganglia were digested for 20 min in an enzymatic solution composed of HBSS, papain (1 mg protein/mL, Worthington, Cat# LS003126), and L-Cysteine (5.5 mM, Sigma, Cat# C7352-25 g). This was followed by another 20-minute digestion in a solution containing HBSS, dispase II (4 mg/mL, Sigma, Cat# D4693-1 g), and collagenase type 2 (6 mg/mL, Worthington, Cat# LS004176). Both enzymatic digestions were carried out at 37 °C with gentle agitation every 10 min. The digested ganglia were suspended in 10% Fetal Bovine Serum (Gibco, Cat# 26140–079) and 2 % Penicillin-Streptomycin (Gibco, Cat# 15070–063) in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Cat# 11995–065). Flame-polished Pasteur pipettes were used to triturate the suspension until it could flow easily through the pipette. Cells were then plated on 12 mm glass coverslips and coated with 20 µg/mL Poly-D-Lysine (Gibco, Cat# A38904-01) and 16 µg/mL Laminin (Sigma, Cat# L2020) in HBSS without Ca²⁺/Mg²⁺ (Malin et al., 2007). Following cell plating, cells were incubated at 37 °C and 5 % CO₂ until use.

2.7.2. Trigeminal ganglion culture

Male BALB/cAnNHsd FRICT-ION mice were deeply anesthetized with 3% isoflurane and then euthanized by decapitation prior dissecting the trigeminal ganglia (TG) for primary cultures intended for electrophysiological studies. The skull was opened, and the brain was extracted to reveal the TG. The TG were carefully extracted from the connective tissue and dura mater at the cranium's base. The ganglia were put in a 5 cm culture dish containing ice-cold Hank's Balanced Salt Solution without Ca²⁺/Mg²⁺ (HBSS) (Stem Cell Technologies, Cat# 37250). Using a scalpel, the ganglia were mechanically disrupted and cut into smaller fragments. These pieces were transferred to a 1.5 ml tube prefilled with ice-cold HBSS and centrifuged for 1 min at 300 x g to pellet the tissue. The supernatant was aspirated, and the ganglia underwent a 20 min digestion period at 37 °C in an enzymatic solution comprised of HBSS, papain (1 mg protein/mL, Worthington, Cat# LS003126), and L-Cysteine (5.5 mM, Sigma, Cat# C7352-25g) with gentle agitation at 10 min. After digestion, the papain digested tissue suspension was centrifuged for 1 min at 200 × g, and the supernatant was removed. A new digestion solution consisting of HBSS, collagenase type 2 (6 mg/mL, Worthington, Cat# LS004176) and dispase II (4 mg/mL, Sigma, cat# D4693) was added. This mixture was again digested for 20 min at 37 °C and gentle agitation at the 10-minute mark. The digested tissue suspension spun down for 4 min at 400 × g. After removing the supernatant, the pellet was resuspended in 0.5 ml warm complete Leibovitz’s L15 medium which contained L-15 (Quality Biological, cat# 112-029-101), 5 % fetal bovine serum (Gibco, cat# 26140-079), 2 % 1 M HEPES (Sigma, cat# H0887), and 1 % Penicillin-Streptomycin (Gibco, cat# 15070–063). The suspension was triturated 10 times with a 200 µl pipette tip to ensure thorough digested, resulting in a uniformly cloudy suspension. The digested tissue suspension was then carefully layered on top of a Percoll gradient (12.5 % Percoll (Sigma, cat# P4937) layered on top of 28% Percoll) and centrifuged at 1300 x g for 10 min. Care was taken not to disturb the lower layers of the spun-down gradient, and 4.5 ml of supernatant was aspirated from the top to eliminate most of the debris. Following this, 4.5 ml warm complete L-15 medium was added to the remaining solution in the tube. Centrifugation was performed again for 6 min at a speed of 1000 x g. Once the supernatant was discarded, the pellet was resuspended in 0.5 ml of warm complete DMEM (Gibco, cat# 11995-065), which contained 10 % fetal bovine serum and 2 % penicillin–streptomycin. A volume of 125 µl of the mouse TG cell suspension was added to each 12 mm coverslip, which had been pre-coated with poly-d-lysine (20 µg/ml, Gibco, Cat# A38904-01) and coated with laminin (16 µg/ml, Sigma, Cat# L2020). The TG cells were allowed to attach for 40–60 min before the wells were gently flooded with enough complete DMEM media to fill each well (1–2 mL for a 12-well plate) (Westlund et al., 2021a). Following cell plating, the cells were incubated at 37 °C and 5% CO₂ until used.

2.8. Whole-cell patch-clamp electrophysiology

Whole-cell patch-clamp electrophysiology was performed as previously described (Goins et al., 2022, Ehsanian et al., 2021, Zurek et al., 2024, Zurek et al., 2024, Goyal et al., 2025). Neurons were identified using differential interference contrast optics linked to an IR-2000 digital camera (Dage MTI, Indiana City, MI). Cell size was assessed with Dage MTI camera software or ImageJ (NIH, Bethesda, MD) and calibrated using a micro-scale calibration slide. Current clamp recordings were carried out with a Multiclamp 700B (Molecular Devices, San Jose, CA). Signals were acquired using a Digidata 1550B converter (Molecular Devices, San Jose, CA) and recorded with Clampex 11 software (Molecular Devices, San Jose, CA). Patch pipettes were prepared using a Zeitz puller (Werner Zeitz, Martinsried, Germany) from borosilicate thick glass (GC150F, Sutter Instruments, Novato, CA). Bridge equilibrium was maintained in all recordings. Cell capacitance was calculated using the whole-cell capacitance compensation circuit in the Multiclamp 700B (Molecular Devices). Cells were excluded from further analysis if they did not fire action potentials (APs), had a resting membrane potential (RMP) of greater than −35 mV or exhibited an access resistance exceeding 15 MΩ. For recordings from DRG and TG cultures, the intracellular solution consisted of 125 K-gluconate, 6 KCl, 10 HEPES, 10 EGTA, 2 Mg-ATP, with pH adjusted to 7.3 using KOH, with an osmolarity range of 290–310 mOsm. The electrode resistance ranged from 3 to 6 MΩ. Electrophysiological measurements were performed 16 to 24 h after the cells were placed in the culture dish, following a 1-hour exposure to either the vehicle or hP2X4 scFv (4.5 μg/ml) prior to recording. For spinal cord and brain (vlPAG) slice recordings, pipette resistance ranged from 6 to 9 MΩ. The internal solution consisted of 130 potassium gluconate, 1 MgCl₂, 2 CaCl₂, 10 HEPES, 10 EGTA, 4 mg-ATP, and 0.3Na-GTP, with pH adjusted to 7.3 using KOH, with an osmolarity range of 290–310 mOsm. Junction potential was not adjusted for in these recordings. During recording, cultured cells or slices were continuously perfused with carbogen-bubbled aCSF at 37 °C.

2.9. Electrophysiology analysis methods

Current clamp recordings began with 25 ms (ms) of the cell at rest, followed by a 500 ms current pulse that increased in 10pA increments from −100 pA in increments until reaching inactivation, or a maximum of 4nA. There was a 500 ms recovery between each current clamp step. Electrophysiological analysis was conducted using Easy Electrophysiology, as previously described (Zurek et al., 2024, Zurek et al., 2024, Goyal et al., 2025). Rheobase was defined as the current injection that elicited neuronal firing, excluding any rebound firing or spontaneous activity. The minimum possible value for rheobase is 10 pA. Cells were continually stimulated in a stepwise manner until they reached rheobase, after which they were further activated until inactivation. Inactivation was defined as the point where the cell ceased firing or exhibited a reduced number of APs after reaching maximum firing up to 4 nA.

RMP was calculated as an average of at least 10 sweeps and was not corrected for junction potential. The hyperpolarizing −100 pA step was used to determine input resistance (Rin). Cells that fired more than one AP during any current injection at or above rheobase were classified as multi-firing. APs resulting from spontaneous activity were excluded when determining multi-firing cells. Spontaneous activity was assessed with either no current injection or enough current applied to maintain the membrane voltage at −45 mV during a 30 s recording. Neurons that generated one or more APs during the 30 s recording were classified as having spontaneous activity. Rebound firing was defined as AP firing occurring during the 500 ms recovery period following any hyperpolarizing current injections.

Firing frequency plots were created considering only the multi-firing cells. The number of APs generated above rheobase was plotted and analyzed in GraphPad Prism v10.0.2 at each current level. Cell diameters were used to characterize cells by size: small cells measured less than 25 µm in diameter, while large cell diameters were 25 µm or larger.

2.10. Statistical and data analysis

Electrophysiological analysis was conducted using Easy Electrophysiology v.2.5.1 (London, UK) and Clampfit 11.2 (Molecular Devices, San Jose, CA). Statistical analysis was conducted with GraphPad Prism v10.0.2 (Boston, MA). Error bars denote the mean ± standard error of the mean (SEM) unless specified otherwise. A p-value of less than 0.05 was considered statistically significant. Statistical tests are shown in the figure legends.

3. Results

3.1. Humanized hP2X4 scFv reduced pain-like behavior and excitability of lamina II spinal dorsal horn neurons from SNI FosTRAP mice

Administration of a single dose of hP2X4 scFv (4 mg/kg, i.p.) restored mechanical sensitivity to naïve levels in the SNI mice (Fig. 1a). After three weeks of ongoing mechanical hypersensitivity, hP2X4 scFv effectively reduced mechanical hypersensitivity, with effects persisting for at least 4 weeks, compared to the control group (n = 4 mice in each group, p < 0.0001; two-way ANOVA followed by Tukey’s multiple comparisons test). Given that the spinal cord laminae II is a key intermediate node in chronic pain signaling (Alles and Smith, 2018, Furue et al., 2004, Basbaum, 1999), we examined the excitability of laminae II Fos+ neurons in ex vivo spinal cord slices from hP2X4 scFv treated FosTRAP SNI male mice exhibiting chronic pain like behavior as described previously.

Fig. 1.

Humanized P2X4 scFv reversed pain like behavior and reduced excitability of lamina II spinal dorsal horn neurons from SNI FosTRAP mice. (a) A single dose of hP2X4 scFv given 3 weeks after induction of SNI model reverses mechanical hypersensitivity in ipsilateral hind paw to von Frey stimulation in mice through to the end of the seven-week study time course (n = 4 mice per group, p < 0.0001; two-way ANOVA followed by Tukey’s multiple comparisons test). (b) Photo of laminae II, spinal cord slice with patch pipette (dotted lines indicate lamina II) and Fos+ neuron (circled with blue dotted line). Electrophysiology recordings of laminae II Fos+ substantia-gelatinosa neurons in ex-vivo spinal cord slices from hP2X4 scFv intraperitoneal-injected FosTRAP SNI male mice (n = 4 per group). Significant difference was found in the percentage of cells that displayed (c) multi-firing property and (d) spontaneous activity with holding current to bring potential to −45 mV following hP2X4 scFv treatment (Vehicle treated cells n = 5 and hP2X4 scFv treated cells n = 5, p < 0.0001; Fisher’s exact test) compared to vehicle control. (e) No significant effect was observed on the rebound firing following hP2X4 scFv treatment. Example traces illustrating multi/rebound firing and SA in vehicle cell. (f) Rheobase (minimum current to elicit firing) (g) resting membrane potential and (h) input resistance were not significantly changed following hP2X4 scFv treatment. (i) No significant effect was observed on action potential firing frequency (F-I) during the current step protocol (10 pA stepwise increase of 500 ms current injection window) as current injection increased from rheobase. F-I analysis is restricted to multi-firing cells, thus vehicle treated cells n = 5 and hP2X4 scFv treated cells n = 4. Example traces of multi-firing SC neurons on current step protocol, vehicle blue and hP2X4 scFv red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Current clamp recordings were obtained from L4-L6 spinal cord lamina II Fos+ neurons. Using whole-cell patch-clamp electrophysiology, we observed a significant reduction in the percentage of cells that displayed multi- firing when treated with hP2X4 scFv compared to vehicle controls 80 % vs 100 %, p < 0.0001; Fisher’s exact test) (Fig. 1c). Additionally, we noted a reduction in the incidence of spontaneous activity (holding potential = −45 mV) of Fos+ dorsal horn neurons from hP2X4 scFv-treated mice compared to controls (0 % vs 40 %, p < 0.0001; Fisher’s exact test) (Fig. 1d). No significant differences in rebound firing were observed following hP2X4 scFv treatment compared to controls (Fig. 1e). Our results indicated that intrinsic properties of rheobase, resting membrane potential (RMP), input resistance, and action potential (AP) firing frequency (frequency-current (F-I) plot) did not significantly change after hP2X4 scFv treatment (Fig. 1f–i).

3.2. Humanized P2X4 scFv reduced pain-like behavior and altered excitability of ventrolateral periaqueductal neurons from FRICT-ION FosTRAP mice

A single dose of hP2X4 scFv (4 mg/kg, i.p.) restored mechanical sensitivity to naïve levels in the FRICT-ION mice (Fig. 2a). After three weeks of ongoing mechanical hypersensitivity, the use of hP2X4 scFv effectively decreased mechanical hypersensitivity, and these effects persisted for at least 4 weeks, compared to the control group (n = 4 mice in each group, p < 0.0001; two-way ANOVA followed by Tukey’s multiple comparisons test).

Fig. 2.

Humanized P2X4 scFv reversed pain-like behavior and altered excitability of ventrolateral periaqueductal gray (vlPAG) neurons from FRICT-ION FosTRAP mice. (a) A single dose of hP2X4 scFv given 3 weeks after induction of FRICT-ION model reverses mechanical hypersensitivity in snout to von Frey stimulation in mice through to the end of the seven-week study time course (n = 4 mice per group, p < 0.0001; two-way ANOVA followed by Tukey’s multiple comparisons test). (b) Photo of vlPAG, brain slice with patch pipette (dotted lines indicate vlPAG) and Fos+ vlPAG neurons (circled with blue dotted line). Electrophysiology recordings of Fos+ ventrolateral periaqueductal gray (vlPAG) neurons in ex-vivo brain slices from hP2X4 scFv intraperitoneal-injected FosTRAP FRICT-ION male mice (n = 4 per group). No significant effect was observed on (c) multi-firing property and (d) spontaneous activity (holding at −45 mV) following hP2X4 scFv treatment. (e) Significant difference was found in the percentage of cells that displayed rebound firing following hP2X4 scFv treatment (Vehicle treated cells n = 20 and hP2X4 scFv treated cells n = 16, p < 0.001; Fisher’s exact test) compared to vehicle control. Example traces illustrating multi/rebound firing and SA in vehicle cell. (f) Rheobase (minimum current to elicit firing) (g) resting membrane potential and (h) input resistance were not significantly changed following hP2X4 scFv treatment. (i) No significant effect was observed on action potential firing frequency (F-I) during the current step protocol (10 pA stepwise increase of 500 ms current injection window) as current injection increased from rheobase. F-I analysis is restricted to multi-firing cells, thus vehicle treated cells n = 14 and hP2X4 treated cells n = 16. Example traces of multi-firing vlPAG neurons on current step protocol, vehicle blue and hP2X4 scFv red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Since vlPAGs play a key role in chronic pain (Snyder et al., 2018), we examined Fos+ vlPAG in ex vivo slices from male FosTRAP FRICT-ION mice that had received i.p. hP2X4 scFv and exhibited chronic pain-like behavior as described above. Using ex vivo slice electrophysiology, we did not observe significant changes in the percentage of cells exhibiting multi-firing properties or spontaneous activity when holding current to bring the membrane potential to −45 mV (Fig. 2c-d). We found a significant increase in the percentage of cells that displayed rebound firing following hP2X4 scFv treatment compared to vehicle control mice (87 % vs 65 %, p < 0.001; Fisher’s exact test) (Fig. 2e). Further, intrinsic properties, such as rheobase, RMP, input resistance, and AP firing frequency (F-I), were not significantly altered following hP2X4 scFv treatment (Fig. 2f–i).

3.3. Humanized P2X4 scFv reduced larger-diameter DRG neuron excitability from SNI, but not naïve, mice

We assessed the direct impact of hP2X4 scFv on the excitability of DRG neurons using whole-cell patch-clamp electrophysiology recordings. These recordings were conducted on dissociated DRG neurons that were plated 16–20 h prior. The neurons were exposed to 4.5 µg/ml hP2X4 scFv or vehicle in culture media for one hour before recording from naïve and SNI mice.

Using current clamp, we recorded small (<25 µm) and large (>25 µm) diameter neurons in both naïve and SNI mice. We observed no significant changes in rheobase or RMP showed no significant changes in small and large neurons from both naïve and SNI mice after hP2X4 scFv in vitro treatment compared to vehicle controls (Fig. 3A(a–d), 3B(g–j). Additionally, there were no significant effects on firing frequency (F-I) in small and large diameter neurons of naïve mice or in small diameter neurons of SNI mice after hP2X4 scFv in vitro treatment (Fig. 3A(e-f) & 3B(k)).

Fig. 3.

Humanized P2X4 scFv reduces large DRG neuron excitability obtained from SNI mice. Current clamp results from small (<25 μm) and large (>25 μm) diameter dorsal root ganglion neurons from naïve and SNI mice (8–12 weeks old male BALB/c, n = 6 each group) treated in vitro with hP2X4 scFv for 1 h at 4.5 µg/ml prior to recording. Results for all cells compared for treated vs vehicle. hP2X4 scFv acts directly on large diameter (>25 μm) sensory neurons to decrease excitability in SNI mice. A(a-d) No significant effect on rheobase (minimum current to elicite firing) and resting membrane potential (RMP) observed in the small (Naïve + Veh cells n = 16, Naive + hP2X4 scFv cells n = 17) and large (Naïve + Veh cells n = 20, Naive + hP2X4 scFv cells n = 18) neurons of naïve mice. B(g-j) Similarly, there were no significant changes on rheobase and RMP seen in the small (SNI + Veh cells n = 19, SNI + hP2X4 scFv cells n = 20) and large (SNI + Veh cells n = 21, SNI + hP2X4 scFv cells n = 21) neurons of SNI mice. A(e-f) & B(k) No significant effect on firing frequency (F-I) seen in the small (Naïve + Veh cells n = 13, Naïve + hP2X4 scFv cells n = 12) and large (Naïve + Veh cells n = 13, Naive + hP2X4 scFv cells n = 15) neurons of naïve mice as well as small (SNI + Veh cells n = 13, SNI + hP2X4 scFv cells n = 12) neurons of SNI mice during the current step protocol (10 pA stepwise increase of 500 ms current injection window) as current injection increased from rheobase. F-I analysis is restricted to multi-firing cells. B(l) There was a significant decrease in firing frequency (F-I) found in the large neurons of SNI mice (SNI + Veh cells n = 15 and SNI + hP2X4 scFv cells n = 19, *p < 0.05; two-way repeated measures ANOVA followed by Sidak multiple comparison test) compared to vehicle control. Example traces of multi-firing DRG neurons current step protocol, vehicle blue, hP2X4 scFv red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

However, in SNI mice, there was a significant decrease in action potential firing frequency in large DRG neurons treated with hP2X4 scFv compared to vehicle control (n = 6 mice per group, *p < 0.05; two-way repeated measures ANOVA followed by Sidak’s multiple comparison test) (Fig. 3B(i)). In summary, hP2X4 scFv was only effective in reducing the excitability of larger-diameter DRG neurons from SNI mice and had no effect on the excitability of DRG neurons from naïve mice.

3.4. Humanized P2X4 scFv reduces larger-diameter TG neuron excitability from FRICT-ION, but not naïve, mice

To assess the direct impact of hP2X4 scFv on TG neuron excitability, we conducted patch-clamp recordings on cultured TG neurons 16–20 h after plating. Cells were treated with 4.5 µg/ml hP2X4 scFv/vehicle for one hour before recording from naïve and FRICT-ION mice. Current clamp recordings were performed on small (<25 µm) and large (>25 µm) diameter neurons of both naïve and FRICT-ION mice.

There was no significant alteration in rheobase and RMP in either small or large diameter neurons of naïve and FRICT-ION mice after hP2X4 scFv treatment compared to vehicle control (Fig. 4A(a-d) & 4B(g-j)). Additionally, there were no significant changes in firing frequency (F-I) in small or large diameter neurons of naïve as well as small diameter neurons of FRICT-ION mice after hP2X4 scFv in vitro treatment (Fig. 4A(e-f) & 4B(k)). A significant reduction in the AP firing frequency was observed in hP2X4 scFv treated large TG neurons of FRICT-ION mice when compared to vehicle controls (n = 6 each group, p**<0.01; two-way repeated measures ANOVA followed by Sidak multiple comparison test) (Fig. 4B(i)). In summary, hP2X4 scFv effectively reduced the excitability of larger-diameter TG neurons from FRICT-ION mice, but had no effect on the excitability of TG neurons from naïve mice.

Fig. 4.

Humanized P2X4 scFv reduces large TG neuron excitability obtained from FRICT-ION mice. Current clamp results from small (<25 μm) and large (>25 μm) diameter TG neurons from naïve and FRICT-ION mice (8–12 weeks old male BALB/c, n = 6 each group) treated in vitro with hP2X4 scFv for 1 h at 4.5 µg/ml prior to recording. Results for all cells compared for treated vs vehicle. hP2X4 scFv significantly decreased excitability of cultured large diameter TG neurons in FRICT-ION mice. A(a–d) No significant effect on rheobase and resting membrane potential observed in the small (Naïve + Veh cells n = 14, Naive + hP2X4 scFv cells n = 15) and large (Naïve + Veh cells n = 22, Naive + hP2X4 scFv cells n = 22) neurons of naïve mice. B(g–j) Similarly, there were no significant changes on rheobase and RMP seen in the small (FRICT-ION + Veh cells n = 14, FRICT-ION + hP2X4 scFv cells n = 18) and large (FRICT-ION + Veh cells n = 28, FRICT-ION + hP2X4 scFv cells n = 25) neurons of FRICT-ION mice. A(e–f) & B(k) No significant effect on firing frequency (F-I) seen in the small (Naïve + Veh cells n = 14, Naïve + hP2X4 scFv cells n = 13) and large (Naïve + Veh cells n = 14, Naive + hP2X4 scFv cells n = 14) neurons of naïve mice as well as small (FRICT-ION + Veh cells n = 8, FRICT-ION + hP2X4 scFv cells n = 5) neurons of FRICT-ION mice during the current step protocol (10 pA stepwise increase of 500 ms current injection window) as current injection increased from rheobase. F-I analysis is restricted to multi-firing cells. B(l) There was a significant decrease in firing frequency (F-I) found in the large neurons of FRICT-ION mice (FRICT-ION + Veh cells n = 13 and FRICT-ION + hP2X4 scFv cells n = 22, **p < 0.01; two-way repeated measures ANOVA followed by Sidak multiple comparison test) compared to vehicle control. Example traces of multi-firing TG neurons current step protocol, vehicle blue, hP2X4 scFv red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Discussion

This study examined the behavioral and electrophysiological effects a single i.p. dose of hP2X4 scFv on peripheral and central neurons in male mice with SNI and FRICT-ION models of chronic pain. We confirmed that a single dose of hP2X4 scFv increases the withdrawal threshold for mechanical allodynia in FosTRAP male mice with SNI and FRICT-ION chronic pain models. Our data indicates that hP2X4 scFv reduces neuronal excitability within lamina II neurons in SNI mice by decreasing the prevalence of multi-firing and spontaneous activity. This effect correlates with an overall decrease in the excitability of large (>25 μm) DRG neurons in SNI mice. These findings suggest that hP2X4 scFv may inform the development of new therapeutic interventions for chronic pain.

The SNI and FRICT-ION models produce mechanical hypersensitivity of the hind paw of SNI mice and in the snout of FRICT-ION mice (Alles et al., 2017, Goins et al., 2022, Kunamneni et al., 2023, Westlund et al., 2021b). Our behavioral data show that a single administration of hP2X4 scFv effectively restores mechanical sensitivity to normal levels in both chronic pain models (Fig. 1, Fig. 2). These results are consistent with our previously published findings (Kunamneni and Westlund, 2025). This reduction occurs within one week, with a full return of mechanical hypersensitivity behavioral responses to naïve baseline within two weeks. This highlights the crucial role of P2X4 receptors in pain modulation (Inoue, 2019, Tsuda et al., 2003, Zhang et al., 2020).

In our investigation of the central effects of hP2X4 scFv, we observed a reduction in the excitability of Fos+ neurons in lamina II of the spinal dorsal horn in SNI FosTRAP mice. The spinal dorsal horn serves as a relay station for nociceptive inputs received from peripheral DRG neurons. Increased excitability of spinal dorsal horn neurons is associated with neuropathic pain (Alles and Smith, 2018, Furue et al., 2004, Basbaum, 1999). Furthermore, decreased excitability of lamina II dorsal horn neurons has been linked to reduced pain behaviors following the administration of analgesics, such as gabapentin (Alles et al., 2017) or the knockout of sodium channels involved in nociception, like Na_v1.7 (Alles et al., 2020). Several studies have indicated that the excitability of the spinal dorsal horn plays a key role in the mechanism of action of P2X4R (Mapplebeck et al., 2018). Our results align with these findings demonstrating that P2X4R plays a role in sensitization of these neurons in chronic pain.

The actions of hP2X4 scFv on vlPAG neurons result in an increase in the incidence of rebound firing of Fos+ neurons in the vlPAG of FRICT-ION FosTRAP mice. The vlPAG is a critical hub for processing nociceptive information, and has been strongly implicated in chronic pain states (Heinricher et al., 2009). Rebound firing is associated with T-type voltage-gated calcium channel activity, which has been linked to trigeminal neuropathic pain (Montera et al., 2020, Alaklabi et al., 2023, Gambeta et al., 2022). In addition, it has been shown that the activity of neurons that project from the vlPAG to the LC is reduced in neuropathic pain (Yu et al., 2024); therefore, it is possible that the activity of these neurons is increased by hP2X4 scFv by increasing the incidence of rebound firing. While it is known that P2X4R can modulate the activity of T-type channels, such as Ca_V3.1 (Cazade et al., 2014), these studies did not examine the cross-talk within a native system. Our studies indicate that hP2X4 scFv affects rebound firing, possibly via T-type channels, in vlPAG neurons, which attenuate trigeminal neuropathic pain. Further research using brain slice preparations is necessary to explore this possible mechanism.

Peripheral sensitization plays a crucial role in initiating neuropathic pain (Liu et al., 2022). Our findings in SNI mice demonstrate that in vitro treatment with hP2X4 scFv results in results in decreased excitability of large (>25 µm), but not small (<25 µm) DRG neurons. Similarly, in TG, our findings indicate that hP2X4 scFv in vitro treatment also results decrease the excitability of large (>25 µm) TG, but has no effect on small (<25 µm) TG in FRICT-ION mice. These results emphasize the specificity of hP2X4 scFv in targeting larger-diameter peripheral sensory neurons under injury conditions. Large-diameter neurons are essential in chronic pain behaviors, including spontaneous pain and tactile allodynia (Viana et al., 2002, Nitzan-Luques et al., 2011, MacDonald et al., 2021). It has also been shown that P2X4R is primarily expressed on larger-diameter DRG neurons, whereas smaller-diameter neurons mainly express the P2X3 receptor (Chen et al., 2022). Additionally, responses to extracellular ATP in trigeminal satellite glial cells during inflammation are largely due to the contribution of P2X4R receptors (Khakh et al., 2001, Khakh et al., 1999, Kushnir et al., 2011). The present study demonstrates the therapeutic potential of hP2X4 scFv in modulating peripheral pain pathways.

Overall, this study highlights the effectiveness of hP2X4 scFv as a potential targeted non-opioid therapy for chronic pain. Most importantly, we reveal the electrophysiological mechanisms underlying reversal of chronic pain-related behavioral effects of a single dose of hP2X4 scFv.

Funding sources

We would like to acknowledge funding from Veterans Affairs BLRD Merit Review Award 1I01 BX005937-01 (KNW, SRAA, AK), Department of Defense Chronic Pain Management Research Program, Investigator-Initiated Research Award # W81XWH-20-1-0930 (KNW, SRAA, AK) and the Research Endowment Fund of the Department of Anesthesiology and Critical Care Medicine, University of New Mexico Health Sciences Center.

CRediT authorship contribution statement

Sachin Goyal: Writing – review & editing, Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Ian Adams: Writing – review & editing, Software, Methodology, Investigation, Formal analysis, Data curation. Marena Montera: Writing – review & editing, Software, Methodology, Investigation, Formal analysis, Data curation. Nesia A. Zurek: . Shivali Goyal: Writing – review & editing, Methodology. Adinarayana Kunamneni: Writing – review & editing, Visualization, Resources, Methodology, Investigation, Funding acquisition, Conceptualization. Karin N. Westlund: Writing – review & editing, Writing – original draft, Visualization, Supervision, Software, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Sascha R.A. Alles: Writing – review & editing, Writing – original draft, Visualization, Supervision, Software, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The humanized P2X4 hscFv is protected under provisional patent World Intellectual Property Organization (WIPO) International Publication WO 2023/114962 A1 filed by UNM Rainforest Innovations, Inventors KNW and AK.

Data availability

Data will be made available on request.