2.1 Animals

Male Sprague–Dawley (SD) rats, aged 2–3 months and weighing 200–250 g, were housed in a standard 12-h light–dark cycle with access to water and food ad libitum. All procedures were approved by the Animal Ethics Committee of West China Hospital of Sichuan University and conducted in accordance with the Animal Research: Reporting of in vivo experiments (ARRIVE) guidelines.

2.2 SNI Model

We used the SNI model to investigate the mechanisms underlying neuropathic pain following peripheral nerve injury. The SNI model was adopted from Decosterd and Woolf [18]. Briefly, rats were anesthetized with 2% isoflurane and exposed the branches of left sciatic nerve. Subsequently, the tibial and common peroneal nerves were ligated with 5-0 silk sutures and cut, while sparing the sural nerve intact. Notably, the sciatic nerve on the contralateral side on the right was only exposed without injuring any branches.

2.3 Behavioral Tests

To investigate the pain induced by SNI, we employed behavioral tests to assess mechanical and thermal hyperalgesia. We used the Von Frey test, a widely accepted method for measuring pain in laboratory animals, to assess mechanical hyperalgesia [19]. Prior to testing, rats were acclimatized for 15 min. Then, a series of Von Frey filaments (0.4, 0.6, 1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0, 26.0 g) were applied to the plantar surface of their hind paws according to the up-and-down method described by Dixon [20]. Finally, we calculated the 50% mechanical withdrawal threshold (in grams) using Dixon's formula [21] to quantify reflexive responses following SNI-induced pain.

In addition, we evaluated thermal hyperalgesia in SNI rats using a plantar device (Woodland Hills IITC Life Science, USA). Briefly, rats were placed on a plantar device, and a heat stimulus was applied to the plantar surface of the rat's paw, causing the animal to withdraw its paw as a response. The time it took for the rat to withdraw its paw (latency) was recorded, with each paw tested three times with an interval of 10 min. To prevent tissue damage, we set a cut-off time of 30 s for each test. This allowed us to assess thermal hyperalgesia by measuring the reduced latency to withdrawal in response to heat stimulation, which indicates an increased sensitivity to heat stimuli, a characteristic of neuropathic pain.

2.4 Spinal Cord Slice Preparation and Whole Cell Patch-Clamp Recording

After anesthetized with 2% isoflurane, rats were transcardially perfused with cutting solution (in mM: 230 Sucrose, 26 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 0.5 CaCl₂, 10 MgSO₄, 10 D-Glucose; pH 7.4, 290–305 mOsm/L, 95% O₂ and 5% CO₂). The spinal lumbar enlargements were removed immediately, fixed in agarose gel (3%), and coronally sectioned by a vibratome (VT1000 A, Leica) in ice-cold artificial cerebrospinal fluid (aCSF) (in mM: 125 NaCl, 3 KCl, 26 NaHCO₃, 1.25 NaH₂PO₄, 2 CaCl₂, 1 MgCl₂, 10 D-Glucose; pH 7.4, 290–305 mOsm/L, 95% O₂, and 5% CO₂). The spinal cord slices (350 μm thick) were incubated in preoxygenated aCSF at 35°C for 60 min and then maintained at room temperature. After incubation, the spinal cord slices were mounted in a recording chamber (perfused with preoxygenated aCSF at ~2 mL/min).

The whole cell patch-clamp recording was performed on the neurons in lamina I–II of spinal lumbar enlargement. Electrophysiological recordings were sampled at 20 kHz and filtered at 10 kHz at room temperature using a Sutter DIPA amplifier linked to a computer running Sutterpatch software (Sutter, USA). Current-clamp mode was used to record neural intrinsic excitability with a pipette internal solution comprised as follows (in mM): 135 K-gluconate, 0.5 CaCl₂, 2 MgCl₂, 5 EDTA, 5 HEPES, and 5 Mg-ATP (PH = 7.3 adjusted with KOH). To record the current threshold for evoking action potential (rheobase) and the neuronal firing rate and behavior, the currents were delivered into the neurons from −80 to 120 pA (10 pA step) and from −80 to 320 pA (20 pA step), respectively. As previously described [22, 23], the neuronal firing behaviors were divided into five patterns here, including no firing, delayed firing, phasic firing, tonic firing, and depolarizing block. Resting membrane potential (RMP) was recorded under I = 0 condition prior to current injection. Voltage-clamp mode (holding at −70 mV) was applied for spontaneous excitatory postsynaptic current (sEPSC) recording with a pipette containing modified internal solution (the above plus 1 μM bicuculline and 1 μM strychnine). After filled with intracellular solution, the impedance of the patch pipettes was 4–7 MΩ. The series resistance did not exceed 20 MΩ.

2.5 In Vivo Extracellular Recording of Spinal WDR Neurons

WDR neurons, located at lamina III–VI of SDH, are associated with the transmission of nociceptive information [24], which respond to mechanical and thermal stimuli from various types of Aδ and C fibers [25, 26]. With in vivo extracellular recording, we detected the activity of WDR neurons during NP following SNI. The experimental procedures referred to previous studies [27, 28]. Briefly, the anesthetized animals were fixed in a stereotaxic apparatus (RWD, China). T13 to L1 laminectomy was performed to expose L3–L5 spinal cord and remove the dura. Bipolar silver electrodes were placed on and transcutaneously electrically stimulated the plantar skin innervated by sciatic nerves [28]. Platinum recording electrodes with an impedance of 2–5 MΩ were positioned by stereotaxic apparatus and placed in L3–L5 SDH (approximately 1 mm lateral to the midline). The extracellular electrical activity of WDR neurons was recorded at a depth of 250–1000 μm (that means the lamina III–VI), and the same position was recorded three times with an interval of 10 min. The stimulation intensity was 1.5× the C-fiber response threshold. These electrical signals were recorded and amplified using MP150 system (BIOPAC, USA). The field potential and C-fiber evoked response of WDR neurons were extracted by AcqKnowledge software (BIOPAC, USA). Here, the C-fiber evoked response was located between 300 and 800 ms of each generated signal.

2.6 Statistical Analysis

Data were expressed as the mean ± standard error of the mean (SEM). At first, Kolmogorov–Smirnov test (K-S test) and Levene's test were used to evaluate normal distribution and homogeneity of variance of data, respectively. If the data followed a normal distribution and the variance was homogeneous, Student t-test or one-way analysis of variance (ANOVA; followed by LSD or Tamhane's T2 multiple comparisons tests) were then performed to analyze the data from two or three and above completely randomized design groups, respectively. Paired t-test was used to analyze data from two paired samples (e.g., contralateral and ipsilateral). Two-way ANOVA (followed by a Bonferroni post hoc test) was applied to the repeated data, such as mechanical hyperalgesia threshold, thermal hyperalgesia threshold, body weight, and so on. If not, the data were analyzed via a non-parametric equivalent. The exact analysis of each data is presented in the corresponding figure legend. P value of < 0.05 was considered to be statistically significant. All statistical analyses were performed using IBM SPSS Statistics V19 package (Armonk, NY, USA).

Additional methods have been included in the Data.

3.1 SNI Induces Downregulation of Vof16 in Spinal Dorsal Horn

We first developed an NP model by SNI in SD rats (Figure 1A). The behavioral results showed that SNI induced mechanical and thermal hyperalgesia, and the symptoms developed 3 days after SNI and lasted for at least 3 weeks (Figure 1B,C). This illustrated that SNI model successfully imitated the nociceptive behaviors of NP. Through reverse transcription and quantitative polymerase chain reaction (RT-qPCR), we found that SNI resulted in the downregulation of Vof16 in SDH of lumbar enlargement, which emerged 3 days after SNI and maintained in a low state up to 21 days (Figures 1D–G and S1A). Additionally, fluorescence in situ hybridization (FISH) of Vof16 was performed on spinal lumbar enlargement samples on day 7 and day 14 after SNI. The FISH results were congruent with the data from RT-qPCR (Figure 1H,I). Apart from SDH, the expression of Vof16 in thenar, prefrontal cortex, hippocampus, heart, liver, spleen, and lung tissues was examined on day 14 after SNI. Strangely, except for a slight decrease in thenar innervated by sciatic nerves, there was no change in Vof16 expression in the other tissues after SNI (Figure S1B–H). Because of the close relation between Vof16 and motor function [14, 15] and the existence of pain-motor protective neuroreflex [29, 30], the decline of Vof16 in thenar (directly mediating the hind limb retraction or foot lifting) might not be accidental, but an adaptive change. To sum up, the above results corroborated that SNI induced downregulation of Vof16 in SDH, indicating that Vof16 might be linked with hyperalgesia caused by SNI.

3.2 Disrupting Vof16 Enhances the Intrinsic Excitability of Spinal Neurons, Although It Does Not Induce Hyperalgesia in Non-SNI State

To uncover the role of Vof16, we constructed Vof16 knockout (Vof16 ko) rats by CRISPR-Cas9 technique (Figures 2A,B and S2A,B). Morphologically, disrupting Vof16 did not hamper the physical and neurological development of SD rats (Figure S2C–H). Above all, the disruption of Vof16 also could not induce hyperalgesia independently in the normal circumstances (i.e., in non-SNI state; Figure 2C–E).

The electrophysiological characteristics of spinal neurons in lamina I–II of lumbar enlargement slices from wild-type (WT) and Vof16 ko rats were further recorded by whole-cell patch clamp (Figure 2F). The intrinsic hallmarks of neurons, including RMP, rheobase, and firing rate of action potential, were evaluated by whole-cell current-clamp recordings. Although there was no significant difference in the RMP and rheobase between WT and Vof16 ko neurons, the disruption of Vof16 led to a descending trend in them (Figure 2G,H). Remarkably, disrupting Vof16 improved the firing rate of action potential (p = 0.031, a 240-pA current injection, Figure 2I,J). The synaptic characteristics of neurons were gauged by whole-cell voltage-clamp recording of sEPSC. Even though the difference in amplitude and frequency of sEPSC was not statistically significant between Vof16 ko neurons and WT neurons, both of them showed an increasing trend in Vof16 ko neurons (Figure 2K–N).

Taken together, the disruption of Vof16 in non-SNI condition could slightly enhance the intrinsic excitability of spinal neurons. However, this spontaneous increase in neuronal excitability did not induce hyperalgesia in behavior.

3.3 Vof16 Alters the Firing Patterns of Neurons in Spinal Dorsal Horn

As mentioned above, the firing patterns of neurons consisted of five modes: no firing, delayed firing, phasic firing, tonic firing, and depolarizing block (see Figure 3A). Under distant current stimulation, the firing patterns between Vof16 ko neurons and WT neurons showed significant difference. When responding to small current injection, a 40-pA current injection was more likely to elicit neuronal firing in Vof16 ko neurons (with 80% of no firing) compared to that in WT neurons (with 90% of no firing; Figure 3B,C). In response to moderate current injection, an 80-pA current injection could trigger 20% of tonic firing in Vof16 ko neurons, but only 10% of tonic firing in WT neurons (Figure 3D,E). Once larger currents (e.g.,120-pA or 160-pA) were input, Vof16 ko neurons were more inclined to depolarizing block (with 20% and 50% in 120-pA and 160-pA injection, respectively) in comparison with WT neurons (with 0% and 10% in 120-pA and 160-pA injection, respectively; Figure 3F–H). However, we noted that, under different current injections, the incidence of delayed firing and phasic firing varied between WT and Vof16 ko neurons (Figure 3I,J). Generally, these findings here extrapolated that Vof16 ko neurons showed a higher likelihood of excitability compared to WT neurons.

3.4 Disrupting Vof16 Aggravates Hyperalgesia and Spinal Neuronal Hyperexcitability in SNI State

Next, we investigated the effect of blockading the expression of Vof16 in NP following spared nerve injury. To address this issue, we developed an SNI model in both WT and Vof16 ko rats (Figure 4A). As expected, we confirmed that disrupting the expression of Vof16 in SNI state further increased the mechanical and thermal hyperalgesia of rats compared to WT rats (Figure 4B,C). Additionally, the RMP underwent depolarization (Figure 4D), the rheobase substantially declined (Figure 4E), and the firing rate of action potential significantly increased (Figure 4F,G) in Vof16 ko neurons from lamina I to II of the lumbar enlargement comparing with WT neurons in SNI state. As for sEPSC, the amplitude and frequency were boosted in Vof16 ko neurons of lamina I–II (Figure 4H–K). Furthermore, we investigated the excitability and C-fiber response of WDR neurons in laminae III–VI of WT and Vof16 ko rats by in vivo extracellular recording (Figure 4L). As shown in Figure 4M–O, SNI resulted in elevated neuronal excitability and C-fiber response in both WT and Vof16 ko WDR neurons (Figure 4M–O). Similarly, the neuronal excitability and C-fiber response were higher in Vof16 ko WDR neurons than that in WT WDR neurons in SNI state but not non-SNI state (Figure 4M–O). These results confirmed that the downregulation of Vof16 indeed aggravated hyperalgesia and spinal neuronal hyperexcitability in SNI state.

Previous studies indicated glial cell activation as one of the major features of central sensitization and a contributing factor to hyperalgesia in NP [31, 32]. From this precedent, we also intended to evaluate whether glial cells in the lumbar enlargement regions of the spinal cord were activated as well. Our data revealed that disrupting Vof16 showed no impact on glial cell activation (Figure S3).

3.5 Conditional Overexpression of Vof16 in Spinal Neurons Alleviates Hyperalgesia and Neuronal Hyperexcitability Induced by SNI

As described above, Vof16 only changed the excitability of neurons, but held no effect on glial cell activation (Figure S3). Therefore, we proposed that neuronal hyperexcitability induced when Vof16 was suppressed during NP might lead to hyperalgesia formation. To further determine the role of Vof16 in neuronal hyperexcitability and NP, we then constructed adeno-associated virus vector to conditionally overexpress Vof16 gene in spinal neurons (AAV-Vof16; CON323 is the empty vector), and intraspinally injected it into SDH of lumbar enlargement (Figure 5A). Delightedly, intraspinal injection of AAV vectors with a 33G Hamilton needle at a slow and uniform speed (0.2 μL/min) did not cause visible damage to spinal cord (Figure 5B), or dysfunction in motor and sensory in rats after injection for 4 weeks (Figure 5C,D). Immunofluorescence staining demonstrated that the green fluorescent protein (GFP) carried by AAV vectors was only expressed in spinal neurons, but not in astrocytes and microglia (Figure 5E,F), suggesting that the AAV vectors held a favorable neuronal targeted specificity. As shown in Figure 5G, the percentage of AAV-positive neurons to total neurons around the injection sites was about 30%. Additionally, as expected, the expression of Vof16 in SDH of lumbar enlargement was substantially elevated in AAV-Vof16 rats but not CON323 rats after AAV injection for 4 weeks, and this status persisted at least 7 weeks after injection (equivalent to the end of the experiment; Figure 5H). These data indicated that the constructed AAV-Vof16 vectors could effectively upregulate the expression of Vof16 in spinal neurons.

Furthermore, the effect of conditionally overexpressing Vof16 on NP induced by SNI was investigated in this study. At first, we excluded the influence of AAV empty vector on the experimental results. As the data were shown, whether in SNI or non-SNI states, the empty vector (CON323) did not affect not only mechanical and thermal hyperalgesia of WT rats (Figure 5I,J) but also the intrinsic and synaptic characteristics of neurons in lamina I–II of spinal lumbar enlargement (Figures 6A–H and S4). Then, the effect of upregulated Vof16 on hyperalgesia and neuronal excitability was explored. In non-SNI state, even though conditional overexpression of Vof16 made no impact on mechanical and thermal pain (Figure 5I, upper and 5J, left), it could shrink the intrinsic neuronal excitability (Figure S4). Remarkably, conditional overexpression of Vof16 in spinal neurons attenuated mechanical and thermal hyperalgesia of WT rats in SNI state (Figure 5I,J). In the whole-cell current patch recording, compared to WT neurons, conditional overexpression of Vof16 impelled spinal neurons to be hyperpolarized (Figure 6A). Moreover, in the spinal neurons with conditional overexpression of Vof16, the rheobase increased significantly (Figure 6B), while the firing rate of action potential was opposite (Figure 6C,D). In the whole-cell voltage recording, the amplitude and frequency of sEPSC in Vof16-overexpressed neurons also dwindled significantly in comparison to WT neurons under SNI condition (Figure 6E–H). Additionally, the results of in vivo extracellular recording demonstrated that conditional overexpression of Vof16 abated the field potential and C-fiber response of WDR neurons in spinal lumbar enlargement in SNI but not non-SNI states (Figure 6I–K). These results suggested that conditional overexpression of Vof16 in spinal neurons could relieve hyperalgesia and neuronal hyperexcitability induced by SNI.