Mechanical Allodynia Circuitry in the Dorsal Horn Is Defined by the Nature of the Injury.

The spinal dorsal horn is a major site for the induction and maintenance of mechanical allodynia, but the circuitry that underlies this clinically important form of pain remains unclear. The studies presented here provide strong evidence that the neural circuits conveying mechanical allodynia in the dorsal horn differ by the nature of the injury. Calretinin (CR) neurons in lamina II inner convey mechanical allodynia induced by inflammatory injuries, while protein kinase C gamma (PKCγ) neurons at the lamina II/III border convey mechanical allodynia induced by neuropathic injuries. Cholecystokinin (CCK) neurons located deeper within the dorsal horn (laminae III-IV) are important for both types of injuries. Interestingly, the Maf+ subset of CCK neurons is composed of transient vesicular glutamate transporter 3 (tVGLUT3) neurons, which convey primarily dynamic allodynia. Identification of an etiology-based circuitry for mechanical allodynia in the dorsal horn has important implications for the mechanistic and clinical understanding of this condition.

Time Course of Inflammation in Dorsal Root Ganglia Correlates with Differential Reversibility of Mechanical Allodynia.

Some individuals recover from the pain of nerve trauma within 12 months or less whereas others experience life-long intractable pain. This transition between reversible pain and the establishment of chronic neuropathic pain is poorly understood. We examined the role of persistent inflammation in the dorsal root ganglia (DRG) in the long-term maintenance of mechanical allodynia; an index of neuropathic pain. Male Sprague-Dawley rats underwent chronic constriction injury (CCI), spared nerve injury (SNI) or sham surgery. Both CCI and SNI animals displayed robust mechanical allodynia in the ipsilateral paw at 7 d post-surgery; however, only SNI animals maintained mechanical allodynia at 42 d post-surgery. DRGs were extracted at 7 d or 42 d post-surgery to assess inflammation via rt-qPCR or immunohistochemistry to measure colony stimulating factor 1 (CSF1) expression, satellite glial cell (SGC) activation, presence of Iba1 positive macrophages and interleukin1 ß (IL-1ß) mRNA levels. Whereas DRGs from SNI animals continued to display inflammatory markers at 42 d, those from CCI animals did not. Moreover, the level of allodynia displayed by each individual animal correlated with the extent of DRG inflammation. These data support the hypothesis that the amount of CSF1 immunoreactivity and the persistence of inflammation in ipsilateral DRGs contribute to the difference between transient and persistent mechanical allodynia observed in the CCI and SNI models. We also suggest that feedback loops involving cytokines and neurotransmitters may contribute to increased DRG activity in chronic neuropathic pain. Consequently, targeting persistent CSF1 production and peripheral neuroinflammation may be an effective approach to the management of chronic neuropathic pain.

Long-term actions of interleukin-1ß on K+, Na+ and Ca2+ channel currents in small, IB4-positive dorsal root ganglion neurons; possible relevance to the etiology of neuropathic pain.

Excitation of dorsal root ganglion (DRG) neurons by interleukin 1ß (IL-1ß) is implicated in the onset of neuropathic pain. To understand its mechanism of action, isolectin B4 positive (IB4+) DRG neurons were exposed to 100pM IL-1ß for 5-6d. A reversible increase in action potential (AP) amplitude reflected increased TTX-sensitive sodium current (TTX-S INa). An irreversible increase in AP duration reflected decreased Ca2+- sensitive K+ conductance (BK(Ca) channels). Different processes thus underlie regulation of the two channel types. Since changes in AP shape facilitated Ca2+ influx, this explains how IL-1ß facilitates synaptic transmission in the dorsal horn; thereby provoking pain.

Sensory Neurons of the Dorsal Root Ganglia Become Hyperexcitable in a T-Cell-Mediated MOG-EAE Model of Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system. Patients with MS typically present with visual, motor, and sensory deficits. However, an additional complication of MS in large subset of patients is neuropathic pain. To study the underlying immune-mediated pathophysiology of pain in MS we employed the myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalitis (EAE) model in mice. Since sensory neurons are crucial for nociceptive transduction, we investigated the effect of this disease on sensory neurons of the lumbar dorsal root ganglia (DRG). Here, we report the disease was associated with activation of the complement system and the NLRP3 inflammasome in the DRG. We further observe a transient increase in the number of complement component 5a receptor 1-positive (C5aR1+) immune cells, CD4+ T-cells, and Iba1+ macrophages in the DRG. The absence of any significant change in the levels of mRNA for myelin proteins in the DRG and the sciatic nerve suggests that demyelination in the PNS is not a trigger for the immune response in the DRG. However, we did observe an induction of activating transcription factor 3 (ATF3) at disease onset and chronic disruption of cytoskeletal proteins in the DRG demonstrating neuronal injury in the PNS in response to the disease. Electrophysiological analysis revealed the emergence of hyperexcitability in medium-to-large (≥26 µm) diameter neurons, especially at the onset of MOG-EAE signs. These results provide conclusive evidence of immune activation, neuronal injury, and peripheral sensitization in MOG-EAE, a model classically considered to be centrally mediated.

Acute anti-allodynic action of gabapentin in dorsal horn and primary somatosensory cortex: Correlation of behavioural and physiological data.

Neuropathic pain is a debilitating consequence of neuronal injury or disease. Although first line treatments include the alpha-2-delta (α2δ)-ligands, pregabalin and gabapentin (GBP), the mechanism of their anti-allodynic action is poorly understood. One specific paradox is that GBP relieves signs of neuropathic pain in animal models within 30min of an intraperitoneal (IP) injection yet its actions in vitro on spinal dorsal horn or primary afferent neurons take hours to develop. We found, using confocal Ca2+ imaging, that substantia gelatinosa neurons obtained ex vivo from rats subjected to sciatic chronic constriction injury (CCI) were more excitable than controls. We confirmed that GBP (100 mg/kg) attenuated mechanical allodynia in animals subject to CCI within 30min of IP injection.Substantia gelatinosa neurons obtained ex vivo from these animals no longer displayed CCI-induced increased excitability. Electrophysiological analysis of substantia gelatinosa neurons ex vivo suggest that rapidly developing in vivo anti-allodynic effects of GBP i) are mediated intracellularly, ii) involve actions on the neurotransmitter release machinery and iii) depend on decreased excitatory synaptic drive to excitatory neurons without major actions on inhibitory neurons or on intrinsic neuronal excitability. Experiments using in vivo Ca2+ imaging showed that 100 mg/kg GBP also suppressed the response of the S1 somatosensory cortex of CCI rats, but not that of control rats, to vibrotactile stimulation. Since the level of α2δ1 protein is increased in primary afferent fibres after sciatic CCI, we suggest this dictates the rate of GBP action; rapidly developing actions can only be seen when α2δ1 levels are elevated.

Increased excitability of medium-sized dorsal root ganglion neurons by prolonged interleukin-1ß exposure is K(+) channel dependent and reversible.

KEY POINTS: Neuropathic pain resulting from peripheral nerve injury is initiated and maintained by persistent ectopic activity in primary afferent neurons. Sciatic nerve injury increases the excitability of medium-sized dorsal root ganglion (DRG) neurons. Levels of the inflammatory cytokine interleukin 1ß (IL-1ß) increase and peak after 7 days. Five to six days of exposure of medium sized DRG neurons to 100 pm IL-1ß promotes persistent increases in excitability which abate within 3-4 days of cytokine removal. This is associated with a profound attenuation of K(+) channel currents but only modest increases in function of cyclic nucleotide-sensitive hyperpolarization-activated channels (HCNs) and of voltage-gated Na(+) and Ca(2+) channel currents. It is unlikely, therefore, that direct interaction of IL-1ß with DRG neurons is capable of initiating an enduring phenotypic shift in their electrophysiological properties that follows sciatic nerve injury. The findings also underline the importance of K(+) channel modulation in the actions of inflammatory mediators on peripheral neurons. ABSTRACT: Chronic constriction injury of rat sciatic nerve promotes signs of neuropathic pain. This is associated with an increase in the level of interleukin 1ß (IL-1ß) in primary afferents that peaks at 7 days. This initial cytokine exposure has been proposed to trigger an enduring alteration in neuronal phenotype that underlies chronic hyper-excitability in sensory nerves, which initiates and maintains chronic neuropathic pain. We have shown previously that 5-6 days of exposure of rat dorsal root ganglia (DRGs) to 100 pm IL-1ß increases the excitability of medium-sized neurons. We have now found using whole-cell recording that this increased excitability reverts to control levels within 3-4 days of cytokine removal. The effects of IL-1ß were dominated by changes in K(+) currents. Thus, the amplitudes of A-current, delayed rectifier and Ca(2+) -sensitive K(+) currents were reduced by ∼68%, ∼64% and ∼36%, respectively. Effects of IL-1ß on other cation currents were modest by comparison. There was thus a slight decrease in availability of high voltage-activated Ca(2+) channel current, a small increase in rates of activation of hyperpolarization-activated cyclic nucleotide-gated channel current (IH ), and a shift in the voltage dependence of activation of tetrodotoxin-sensitive sodium current (TTX-S INa ) to more negative potentials. It is unlikely, therefore, that direct interaction of IL-1ß with DRG neurons initiates an enduring phenotypic shift in their electrophysiological properties following sciatic nerve injury. Persistent increases in primary afferent excitability following nerve injury may instead depend on altered K(+) channel function and on the continued presence of slightly elevated levels IL-1ß and other cytokines.