Accepted

Radiotherapy-related pain is a common adverse reaction with a high incidence among cancer patients undergoing radiotherapy and remarkably reduces the quality of life. However, the mechanisms of ionizing radiation (IR)-induced pain are largely unknown. In present study, mice were treated with 20 Gy X-ray to establish IR-induced pain model. X-ray evoked a prolonged mechanical, heat and cold allodynia in mice. Transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) were significantly upregulated in lumbar dorsal root ganglion (DRG). The mechanical and heat allodynia could be transiently reverted by intrathecal injection of TRPV1 antagonist capsazepine and TRPA1 antagonist HC-030031. Additionally, the phosphorylated ERK and JNK in pain neural pathway were induced by X-ray treatment. Our findings indicated activation of TRPA1 and TRPV1 is essential for the development of X-ray-induced allodynia. Furthermore, our findings suggest targeting on TRPV1 and TRPA1 may be promising prevention strategies for X-ray-induced allodynia in clinical practice.

In mice, spared nerve injury replicates symptoms of human neuropathic pain and induces upregulation of many genes in somatosensory neurons. Here we used single cell transcriptomics to probe the effects of partial infraorbital transection of the trigeminal nerve at the cellular level. Uninjured neurons were unaffected by transection of major nerve branches, segregating into many different classes. In marked contrast, axotomy rapidly transformed damaged neurons into just two new and closely-related classes where almost all original identity was lost. Remarkably, sensory neurons also adopted this transcriptomic state following various minor peripheral injuries. By genetically marking injured neurons, we showed that the injury-induced transformation was reversible, with damaged cells slowly reacquiring normal gene expression profiles. Thus, our data expose transcriptomic plasticity, previously thought of as a driver of chronic pain, as a programed response to many types of injury and a potential mechanism for regulating sensation during wound healing.

The spinal dorsal horn is the first relay structure coding for pain transmission and modulation. Previous anatomical and electrophysiological studies have examined spinal dorsal horn circuitry, functional studies of circuit connections and network activity. Further work is required to understand spinal cord sensory information processing that underlies pathological neuropathic pain states. Our previous studies suggest that peripheral nerve injury enhances presynaptic excitatory input onto spinal superficial dorsal horn neurons, which in turn contributes to pathologic nociception. The potential changes in local postsynaptic circuits in the dorsal horn that lead to pathologically heightened behavioral responses to pain remain largely unexplored. We combined whole cell electrophysiological recordings with laser scanning photostimulation (LSPS) to test whether the spinal nerve ligation (SNL) mouse model of neuropathic pain leads to alterations in the functional connectivity of spinal cord circuits including lamina II excitatory interneurons. Here we show that SNL enhances excitation and decreases inhibition to lamina II excitatory interneurons along with their increased glutamate-evoked excitability. The enhanced excitatory postsynaptic input and connectivity evoked by SNL eventually return to normal levels concurrently with the resolution of the neuropathic pain states. The physiological pattern highly correlates with mouse pain behaviors following SNL, supporting a neurophysiological mechanism of central sensitization and neuropathic pain that is functionally localized to the spinal dorsal horn. Together, these data support that SNL induces functional changes in synaptic input and connectivity to lamina II excitatory interneurons that code for pain perception, and thus provide new insights into the mechanism and locus of pain hypersensitivity. Neuropathic pain presumably results from alterations in neuronal circuits that process nociception. This form of pain is often maladaptive. The contribution of circuit connections and detailed local spinal cord circuits underlying neuropathic pain are not well understood. Here, we apply laser-scanning photostimulation (LSPS) combined with whole cell recordings to investigate local circuit connectivity onto the lamina II interneurons during and after recovery following spinal nerve ligation that causes pathological neuropathic pain. The present study sheds light on local circuit organization in spinal dorsal horn and shows that reciprocal changes occur in local excitatory interneurons during both peak and after the gradual normalization of neuropathic pain. This elucidates nociceptive processing changes during and after neuropathic pain conditions and suggests new treatments.

The study reported here investigated the role of the central vascular endothelial growth factor-A (VEGF-A) pathway in the development of trigeminal neuropathic pain following nerve injury. A Sprague-Dawley rat model of trigeminal neuropathic pain was produced using malpositioned dental implants. The left mandibular second molar was extracted under anesthesia and replaced with a miniature dental implant to induce injury to the inferior alveolar nerve. The inferior alveolar nerve injury produced a significant upregulation of astrocytic VEGF-A expression in the medullary dorsal horn. The nerve injury-induced mechanical allodynia was inhibited by an intracisternal infusion of VEGF-A164 antibody. Although both VEGF-A Receptor 1 (VEGF-A R1; colocalized with the blood-brain barrier) and VEGF-A Receptor 2 (VEGF-A R2; colocalized with astrocytes) participated in the development of trigeminal neuropathic pain following nerve injury, only the intracisternal infusion of a VEGF-A R1 antibody, and not that of a VEGF-A R2 antibody, inhibited the increased blood-brain barrier (BBB) permeability produced by nerve injury. Finally, we confirmed the participation of the central VEGF-A pathway in the development of trigeminal neuropathic pain by reducing VEGF-A expression using VEGF-A164 siRNA. This suppression of VEGF-A produced significant prolonged anti-allodynic effects. These results suggest that the central VEGF-A pathway plays a key role in the development of trigeminal neuropathic pain following nerve injury through two separate pathways: VEGF-A R1 and VEGF-A R2. Hence, a blockade of the central VEGF-A pathway provides a new therapeutic avenue for the treatment of trigeminal neuropathic pain.

Identification of genetic variants that influence susceptibility to pain is key to identifying molecular mechanisms and targets for effective and safe therapeutic alternatives to opioids. To identify genes and variants associated with persistent pain, we measured late-phase response to formalin injection in 275 male and female Diversity Outbred mice genotyped for over 70,000 single nucleotide polymorphisms. One quantitative trait locus reached genome-wide significance on chromosome 1 with a support interval of 3.1 Mb. This locus, Nociq4 (nociceptive sensitivity quantitative trait locus 4; MGI: 5661503), harbors the well-known pain gene Trpa1 (transient receptor potential cation channel, subfamily A, member 1). Trpa1 is a cation channel known to play an important role in acute and chronic pain in both humans and mice. Analysis of Diversity Outbred founder strain allele effects revealed a significant effect of the CAST/EiJ allele at Trpa1, with CAST/EiJ carrier mice showing an early, but not late, response to formalin relative to carriers of the 7 other inbred founder alleles (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ). We characterized possible functional consequences of sequence variants in Trpa1 by assessing channel conductance, TRPA1-TRPV1 interactions, and isoform expression. The phenotypic differences observed in CAST/EiJ relative to C57BL/6J carriers were best explained by Trpa1 isoform expression differences, implicating a splice junction variant as the causal functional variant. This study demonstrates the utility of advanced, high-precision genetic mapping populations in resolving specific molecular mechanisms of variation in pain sensitivity.

We investigated the calcium dynamics of dorsal root ganglion (DRG) neurons using transgenic mice to target expression of the genetically encoded calcium indicator (GECI), GCaMP6s, to a subset of neurons containing parvalbumin (PV), a calcium-binding protein present in proprioceptors and low-threshold mechanoreceptors. This study provides the first analysis of GECI calcium transient parameters from large-diameter DRG neurons. Our approach generated calcium transients of consistent shape and time-course, with quantifiable characteristics. Four parameters of calcium transients were determined to vary independently from each other and thus are likely influenced by different calcium-regulating mechanisms: peak amplitude, rise time (RT), decay time, and recovery time. Pooled analysis of 188 neurons demonstrated unimodal distributions, providing evidence that PV+ DRG neurons regulate calcium similarly as a population despite their differences in size, electrical properties, and functional sensitivities. Calcium transients increased in size with elevated extracellular calcium, longer trains of action potentials, and higher stimulation frequencies. RT and decay time increased with the addition of the selective sarco/endoplasmic reticulum calcium ATPases (SERCA) blocker, thapsigargin (TG), while peak amplitude and recovery time remained the same. When elevating bath pH to 8.8 to block plasma-membrane calcium ATPases (PMCA), all measured parameters significantly increased. These results illustrate that GECI calcium transients provide sufficient resolution to detect changes in electrical activity and intracellular calcium concentration, as well as discern information about the activity of specific subclasses of calcium regulatory mechanisms.