Animal Studies

Heme oxygenase 1 (HO-1) and carbon monoxide were shown to normalize oxidative stress and inflammatory reactions induced by neuropathic pain in the central nervous system, but their effects in the locus coeruleus (LC) of animals with peripheral inflammation and their interaction with nitric oxide are unknown. In wild-type (WT) and knockout mice for neuronal (NOS1-KO) or inducible (NOS2-KO) nitric oxide synthases with inflammatory pain induced by complete Freund's adjuvant (CFA), we assessed: 1) antinociceptive actions of cobalt protoporphyrin IX (CoPP), an HO-1 inducer; 2) effects of CoPP and tricarbonyldichlororuthenium(II)dimer (CORM-2), a carbon monoxide-liberating compound, on the expression of HO-1, NOS1, NOS2, CD11b/c, GFAP,and mitogen-activated protein kinases (MAPK)in the LC. CoPP reduced inflammatory pain in different time-dependent manners in WT and KO mice. Peripheral inflammation activated astroglia in the LC of all genotypes and increased the levels of NOS1 and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK 1/2) in WT mice. CoPP and CORM-2 enhanced HO-1 and inhibited astroglial activationin all genotypes. Both treatments blocked NOS1 overexpression,and CoPP normalized ERK 1/2 activation. This study reveals an interaction between HO-1 and NOS1/NOS2 during peripheral inflammation andshows that CoPP and CORM-2 improved HO-1 expression and modulated the inflammatory and/or plasticity changes caused by peripheral inflammation in the LC.

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 purpose of this study was to determine effects and mechanisms of sacral nerve stimulation (SNS) on visceral hypersensitivity in rodent models of colonic hypersensitivity. SNS was performed with different sets of parameters for 30 minutes in six regular rats. Visceral sensitivity was assessed by the measurement of electromyogram (EMG) and abdominal withdrawal reflex (AWR) before and after SNS. Real/sham SNS with optimized parameters was performed in eight restraint stress-induced visceral hypersensitivity rats and 10 neonatal acetic acid treated colonic hypersensitivity rats, acute effect of SNS was assessed by comparing EMG and heart rate variability (HRV). Neonatal acetic acid treated rats were treated by SNS (n=10) or sham-SNS (n=10) daily for 7 days for the assessment of the chronic effect of SNS. (1)When the stimulation amplitude was reduced from 90% of motor threshold (MT) to 65% or 40% MT, SNS with certain parameters showed an inhibitory effect on AWR. The best stimulation parameters for SNS was "14Hz, 330μs, and 40% MT". (2)SNS significantly reduced visceral hypersensitivity and improved autonomic function in restraint stress-induced rats. The inhibitory effect was blocked by naloxone.(3)Acute and Chronic SNS significantly reduced visceral hypersensitivity and improved autonomic function in acetic acid treated rats. SNS with reduced stimulation strength may be used to treat colonic hypersensitivity and the best stimulation parameters seem to be"14Hz, 330μs and 40% MT". SNS with optimized parameters improved visceral hypersensitivity in rodent models of colonic hypersensitivity mediated via the autonomic and opioid mechanisms.

Neuropathic pain is a debilitating condition caused by the abnormal processing of somatosensory input. Synaptic inhibition in the spinal dorsal horn plays a key role in that processing. Mechanical allodynia – the misperception of light touch as painful – occurs when inhibition is compromised. Disinhibition is due primarily to chloride dysregulation caused by hypofunction of the potassium-chloride co-transporter KCC2. Here we show, in rats, that excitatory neurons are disproportionately affected. This is not because chloride is differentially dysregulated in excitatory and inhibitory neurons, but, rather, because excitatory neurons rely more heavily on inhibition to counterbalance strong excitation. Receptive fields in both cell types have a center-surround organization but disinhibition unmasks more excitatory input to excitatory neurons. Differences in intrinsic excitability also affect how chloride dysregulation affects spiking. These results deepen understanding of how excitation and inhibition are normally balanced in the spinal dorsal horn, and how their imbalance disrupts somatosensory processing.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have been implicated in the trafficking of postsynaptic glutamate receptors, including N-methyl-D-aspartate (NMDA)-subtype glutamate receptors (NMDARs) that are critical for nociceptive plasticity and behavioral sensitization. However, the components of SNAREs complex involved in spinal nociceptive processing remain largely unknown. Here we found that SNAP25, syntaxin4, VAMP2 and Munc18-1 were localized at postsynaptic sites and formed the complex in the superficial lamina of spinal cord dorsal horn of rats. The complex formation between these SNAREs components were accelerated after intraplantar injection of complete Freund's adjuvant (CFA), pharmacological removal of GABAergic inhibition or activation of NMDAR in intact rats. The increased SNAP25/syntaxin4/VAMP2/Munc18-1 interaction facilitated the surface delivery and synaptic accumulation of NMDAR during inflammatory pain. Disruption of the molecular interaction between SNAP25 with its SNARE partners by using a blocking peptide derived from the C-terminus of SNAP25 effectively repressed the surface and synaptic accumulation of GluN2B-containing NMDARs in CFA-injected rats. This peptide also alleviated inflammatory mechanical allodynia and thermal hypersensitivity. These data suggested that SNAREs complex assembly in spinal cord dorsal horn was involved in the inflammatory pain hypersensitivity through promoting NMDAR synaptic trafficking.

The underlying mechanism of interstitial cystitis/bladder pain syndrome (IC/BPS) is not well understood and evaluation of current therapeutic interventions has not identified any generally effective treatments. Physical activity has shown beneficial effects on individuals suffering from chronic pain. Anxiety-prone rats exposed to water avoidance stress (WAS) develop urinary frequency and lower bladder sensory thresholds with high face and construct validity for the study of IC/BPS. The aim of this study was to evaluate the role of chronic voluntary exercise on urinary frequency, voiding function, and hyperalgesia in animals exposed to WAS.

Chronic constriction injury of the sciatic nerve is frequently considered as a cause of chronic neuropathic pain. Marked activation of microglia in the posterior horn (PH) has been well established with regard to this pain. However, microglial activation in the anterior horn (AH) is also strongly induced in this process. Therefore, in this study, we compared the differential activation modes of microglia in the AH and PH of the lumbar cord 7 days after chronic constriction injury of the left sciatic nerve in Wistar rats. Microglia in both the ipsilateral AH and PH demonstrated increased immunoreactivity of the microglial markers Iba1 and CD11b. Moreover, abundant CD68 phagosomes were observed in the cytoplasm. Microglia in the AH displayed elongated somata with tightly surrounding motoneurons, whereas cells in the PH displayed a rather ameboid morphology and were attached to myelin sheaths rather than to neurons. Microglia in the AH strongly expressed NG2 chondroitin sulfate proteoglycan. Despite the tight attachment to neurons in the AH, a reduction in synaptic proteins was not evident, suggesting engagement of the activated microglia in synaptic stripping. Myelin basic protein immunoreactivity was observed in the phagosomes of activated microglia in the PH, suggesting the phagocytic removal of myelin. CCI caused both motor deficit and hyperalgesia that were evaluated by applying BBB locomotor rating scale and von Frey test, respectively. Motor defict was the most evident at postoperative day1, and that became less significant thereafter. By contrast, hyperalgesia was not severe at day 1 but it became worse at least by day 7. Collectively, the activation modes of microglia were different between the AH and PH, which may be associated with the difference in the course of motor and sensory symptoms.