Investigating Mechanisms in Nociceptors Driving Ongoing Activity and Ongoing Pain

Ongoing (apparently spontaneous) pain at rest is a major complaint of patients suffering from many forms of acute and chronic pain, including acute and persistent postsurgical pain. Accumulating evidence suggests ongoing activity in nociceptors is a major driver of ongoing pain. Ongoing activity can be generated in sensory neurons in the absence of sensory generator potentials if one or more of three neurophysiological alterations occur – prolonged depolarization of resting membrane potential (RMP), hyperpolarization of action potential (AP) threshold, and/or increased amplitude of depolarizing spontaneous fluctuations of membrane potential (DSFs) to bridge the gap between RMP and AP threshold. Cellular signaling pathways that increase DSF amplitude and promote ongoing activity acutely in nociceptors were unknown for any neuromodulator, as were the neurophysiological alterations that underly ongoing activity in nociceptors after deep tissue incision. The work presented in this dissertation sought to identify a cellular signaling pathway and injury- and inflammation-related neuromodulator that induces alterations that drive hyperexcitability, such as enhanced DSFs, and thereby potentiates ongoing activity in nociceptors, as well as to determine the alterations that contribute to nociceptor hyperactivity associated with postsurgical pain. A combination of whole-cell patch clamp electrophysiology, pharmacology, and high content microscopy was used to define the effects of low concentrations of the injury-related proinflammatory mediator serotonin and the major pathway by which it exerts its effects. This study shows that serotonin enhances DSFs, hyperpolarizes AP threshold, and thereby potentiates OA in isolated rat DRG neurons at a concentration of 100 nM, and serotonin exerts these effects on nonpeptidergic nociceptors via the 5-HT4 receptor and downstream cAMP signaling via PKA and EPAC. Furthermore, whole-cell patch clamp recordings of primary DRG neurons, analysis of spontaneous pain behavior, and RNA sequencing were used to reveal an unexpected persistence of hyperexcitability in isolated DRG neurons and potentially associated differential gene expression, both of which were found after the behavioral expression of postsurgical pain had resolved. In addition to elucidating cell signaling mechanisms that can contribute to acute ongoing activity in nociceptors, these studies set the stage for future investigations addressing important questions about nociceptor contributions to pain

The mechanisms of GABAergic signalling in the peripheral pain pathway

Peripheral pain pathway plays a crucial role in how pain is perceived and felt. The dorsal root ganglia (DRG) which house the primary sensory neurons have become the focus of many emerging pain studies due to its potential as a functional structure in controlling pain transmission, and not only for producing proteins and providing nutrients essential for neuron survival. The major inhibitory neurotransmitter in the nervous system, GABA has been shown to play a significant role in this regard. Within the present study, the mechanism of GABA release within DRG neurons was investigated by studying the expression of vesicular GABA transporter (VGAT) in the DRG neurons. VGAT was highly expressed in the DRG neuron somata. The VGAT-positive neurons also expressed markers of subpopulations of DRG neurons, including those involved in nociception. The availability of VGAT luminal (VGAT-C) and cytoplasmic (VGAT-N) domains were utilised to investigate the mechanism of GABA release in a live DRG neuron culture. This mechanism involves the recycling process of vesicles following their exocytosis. Imaging of the internalization of VGAT-C domain during vesicle recycling indicates GABA is released via exocytosis and has both, tonic and activity-dependent components. Using the in vivo electrophysiological recordings, neuronal firing in the spinal nerve and dorsal branches of the peripheral nerve (before and after the DRG, respectively), was investigated. These data revealed existence of a ‘filter’ in the DRG that decreased the frequency of the neuronal firing passing through the DRG. This filtering effect was overcome by bicuculline, a GABAA receptor antagonist indicating the role of GABAA receptor in peripheral pain pathway. This role of GABAA receptor was also supported by the decrease in GABAA receptor activation in the presence of bicuculline in DRG neurons co-cultured with HEK293 cells. In sum, in the DRG, GABA is liberated into the interneuronal space via Ca2+-dependent vesicular exocytosis, which in turn acts on GABAA receptors. This GABAergic signalling is responsible for filtering the action potentials from the periphery to the central terminals in the spinal cord. These findings identify and further characterize peripheral ‘gate’ within the somatosensory system

A feasibility study examining the utility of sacral dermatomal evoked potentials in assessing urogenital dysfunction

Background: Tarlov cysts are cerebrospinal fluid-filled sacs that form in multiple numbers at the location of dorsal root ganglia in the sacral spinal cord region. Tarlov cysts have been known for over seventy years and are often considered benign findings in MRI reports. A group of predominantly female patients suffer from one or more symptoms, such as lower back pain, lower limb pain, sensory disturbance in the perineum, urinary incontinence, constipation, and sexual dysfunction, for no apparent reasons. All these patients have one common feature - Tarlov cysts in their sacral spinal segment. Most of the traditional diagnostic tests are normal in this group. Since all patients with Tarlov cysts do not suffer from these symptoms, traditionally, their presence is often ignored, and patients with urogenital symptoms are investigated for urological, gynaecological, gastroenterological, or spinal causes. Many tests cause more delays in treatment and create economic and psychological burdens on these patients. Studies have shown that patients can get relief from their symptoms after removing Tarlov cysts, which spurred interest in understanding symptomatic Tarlov cysts. It was hypothesised that some Tarlov cysts can interfere with sacral nerve root functions, causing urogenital dysfunction. Aim: To provide objective evidence for sacral root dysfunction in patients with symptomatic Tarlov cysts. Methods: Prospective, cross-sectional observational studies were conducted in three cohorts. In a healthy group (cohort1) study, 20 healthy volunteers (14 women and 6 men) whose age + SD (women: 39+16.6, men:35+10.6), height in cm (women: 162+8.8, men:176+8.1) and BMI (women: 25.9+6.6, men:24.5+5.5) were recruited to generate normative values for the sacral S2, S3 and S4 dermatomal Somatosensory Evoked Potential (dSEP). Regression equations were generated for the cortical latency, amplitude and inter-side differences using independent age, height and BMI parameters. In the spinal cord injury (cohort 2) study, 20 volunteers (13 women and 7 men) had cauda equina-level lesions (18) or thoracic-level lesions (2) confirmed by MRI. Three of the volunteers had an acute onset of symptoms, and the rest had an onset of symptoms ranging from less than a year to over a decade. All volunteers had sensory deficits on their buttocks, the back of their thighs, or in their perineum. The S2, S3 and S4 dSEPs were tested in this group, and the sensitivity and specificity of the sacral dSEPs were established using the Receiver Operating Curve. In the symptomatic Tarlov cyst (cohort 3) study, 20 volunteers (18 women and 2 men) with one or more Tarlov cysts at their sacral spinal cord segment shown in their MRI were recruited. These volunteers were suffering from lower urinary tract symptoms (70%), perineal pain (65%), Persistent Genital Arousal Disorder - PGAD (20%) and paraesthesia in the perineum (15%). Tibial Somatosensory Evoked Potentials (SEP) and Pudendal SEP were also recorded on all volunteers in all three cohorts. The study was approved by the Manchester Metropolitan University Research Ethics and Governance Team (EthOS Reference Number: 46173). Volunteers in all three cohorts were compensated according to the Health Research Authority recommendations (21/NE/0194). Results: Inter-side cortical latency difference is the most useful parameter when considering sacral root dysfunctions. S2 dSEPs have 75% / 70%, S3 dSEPs have 85% / 85%, and S4 dSEPs have 90% / 85% sensitivity/specificity in detecting unilateral sacral root abnormalities

Estudio retrospectivo de pacientes con cervicobraquialgia que se someten a RMN y a EMG

Estudio retrospectivo de 184 pacientes con cervicobraquialgia en el contexto clínico de hernia discal y/o espondilosis, y sospecha de radiculopatía cervical. Se analizaron los hallazgos RMN y EMG. Las hernias y osteofitos más prevalentes aparecieron en el C5-C6, seguido de C6-C7. Los elementos de espondilosis presentaron relación con el envejecimiento (p<0,01). La alineación vertebral sagital más frecuente fue la rectificación cervical. La raíz con mayor número de hallazgos neurógenos fue C7, seguida de C8-T1. El desbalance anatómico entre el nivel de las lesiones en la RMN y los hallazgos en la EMG sugiere que las raicillas se dañan cranealmente al agujero de conjunción, dentro del canal vertebral

Mapping the Functional Expression Profile of Kv7 Channels in Somatosensory Neurons of Different Sensory Modalities

Pain is a regular and unavoidable experience that dramatically impacts people’s quality of life. Current treatments are often inadequate, expensive or invasive, thus, despite advances, the clinical need is still left unmet and new therapeutic targets are needed. Kv7 (‘M’) potassium channels may represent such a target as these impose strong control over neuronal excitability. Functional M channels are present in peripheral somatosensory neurons but there is currently no consensus as to what the functionally dominant subunit is. Here we combine measures of gene and protein expression with functional patch clamp analysis and behavioural testing to extensively study Kv7 in the peripheral sensory system. We demonstrated that all but Kv7.1 are expressed in rat sensory neurons and that Kv7.2, Kv7.3 and Kv7.5 are abundantly expressed. Kv7.2 showed a bias to small-diameter neurons and Kv7.5 toward larger neurons with significant overlap between the various subunits in rats and mice. Effects of Kv7.2/Kv7.3 selective potentiator, ICA-27243, and Kcnq2 knockdown suggest Kv7.2 is the predominant subunit controlling M current in TRPV1 positive nociceptors. ICA-27243 also significantly attenuated acute and inflammatory pain with comparable efficacy to retigabine. Kcnq2 knockdown significantly reduced the M current and rheobase in TRPV1 positive neurons and significantly increased the excitability of these neurons. This translated to a reduction in thermal and mechanical pain thresholds and an increased pain response to acute painful stimuli when Kcnq2 was knocked down in vivo. There were changes in some parameters associated with neuronal excitability and pain behaviour with Kcnq5 knockdown. These findings identified Kv7.2 as the functionally dominant Kv7 subunit in nociceptors and for response to painful stimuli