L5 DRG Research Articles

Assessing spontaneous sensory neuron activity using in vivo calcium imaging.

Heightened spontaneous activity in sensory neurons is often reported in individuals living with chronic pain. It is possible to study this activity in rodents using electrophysiology, but these experiments require great skill and can be prone to bias. Here, we have examined whether in vivo calcium imaging with GCaMP6s can be used as an alternative approach. We show that spontaneously active calcium transients can be visualised in the fourth lumbar dorsal root ganglion (L4 DRG) through in vivo imaging in a mouse model of inflammatory pain. Application of lidocaine to the nerve, between the inflamed site and the DRG, silenced spontaneous firing and revealed the true baseline level of calcium for spontaneously active neurons. We used these data to train a machine learning algorithm to predict when a neuron is spontaneously active. We show that our algorithm is accurate in 2 different models of pain: intraplantar complete Freund adjuvant and antigen-induced arthritis, with accuracies of 90.0% ±1.2 and 85.9% ±2.1, respectively, assessed against visual inspection by an experienced observer. The algorithm can also detect neuronal activity in imaging experiments generated in a different laboratory using a different microscope configuration (accuracy = 94.0% ±2.2). We conclude that in vivo calcium imaging can be used to assess spontaneous activity in sensory neurons and provide a Google Colaboratory Notebook to allow anyone easy access to our novel analysis tool, for the assessment of spontaneous neuronal activity in their own imaging setups.

MR thermometry imaging for low intensity focused ultrasound modulation of spinal nervous tissue

ObjectivesPreviously in rodent and swine models, we have shown that external low intensity focused ultrasound (liFUS) can be used to modulate pain responses. To ensure no adverse heating events occur with liFUS modulation in a non-invasive manner, we perform initial work in swine to show that magnetic resonance thermometry imaging (MRTI) is capable of measuring <2.0 °C changes at the L5 DRG. Further, we show that our device can be constructed in an MR-compatible fashion to minimize artifact. MethodsThree MRTI techniques (referenceless, corrected proton resonance frequency shift (PRFS), and PRFS) were applied to assess accuracy of detecting thermal changes at the L5 DRG in unheated euthanized swine. A region of interest (ROI) that includes the L5 DRG was delineated, within which MRTI temperature changes were spatially averaged (ground truth 0 °C). In separate experiments with phantoms, B0 field-inhomogeneity, RF transmit (B1+) and fast gradient echo (fSPGR) magnitude images were acquired to downselect liFUS device materials that produce the least MRI artifacts. ResultsReferenceless, corrected PRFS, and PRFS MRTI resulted in temperature measurements of 0.8 ± 1.1 °C, 1.1 ± 1.3 °C and 5.2 ± 5 °C, respectively. Both materials caused B0 perturbation but minimal B1+ and MRTI artifacts. The presence of imaging artifacts did not preclude thermal imaging of the region. SignificanceWe provide preliminary data suggesting that referenceless MRTI can adequately detect small thermal changes at the DRG that may occur with neuromodulation, which is one of the first steps in creating a table of safe parameters for liFUS therapy in humans.

The timing point of exercise intervention regulates neuropathic pain-related molecules in the ipsilateral dorsal root ganglion neurons after sciatic nerve injury.

The purpose of this study was to determine whether the timing of tread-mill exercise application can control expression levels of neuropathic pain- and regeneration-related proteins in the ipsilateral lumbar 4 (L4) to 6 (L6) dorsal root ganglion cells (DRG) after sciatic nerve injury (SNI). The experimental rats were randomly divided into five groups: the normal control, SNI+sedentary (IS), exercise+SNI (EI), SNI+exercise (IE), exercise+SNI+exercise (EIE) groups. The rats in exercise groups per-formed treadmill exercise at a speed of 8 m/min for 30 min once a day during 14 days before and/or after SNI. For investigating the expression of specific neuropathic pain and regeneration-related proteins in DRG, we prepared L4 to L6 DRG in the ipsilateral side. In the quantitative analysis, growth associated protein 43 (GAP-43) and brain-derived neurotrophic factor levels were further increased in the ipsilateral DRG at all treadmill exercise groups than those in IS group. In the histological findings, GAP-43 was qualitatively increased IE and EIE groups than IS group at DRG. Wnt3a and β-catenin were dramatically downregulated in EIE and IE groups than IS groups. In addition, nuclear factor kappa-light-chain-enhancer of activated B cells and tumor necrosis factor-α were significantly decreased in IE and EIE groups than IS group in the ipsilateral DRG. Our findings suggested novel information that regular low-intensity exercise before and/or after SNI might be a therapeutic and preventive approaches for relieving neuropathic pain and improving axonal elongation after peripheral nerve injury.

The Effect of Clinically Controllable Factors on Neural Activation During Dorsal Root Ganglion Stimulation

ObjectiveDorsal root ganglion stimulation (DRGS) is an effective therapy for chronic pain, though its mechanisms of action are unknown. Currently, we do not understand how clinically controllable parameters (e.g., electrode position, stimulus pulse width) affect the direct neural response to DRGS. Therefore, the goal of this study was to utilize a computational modeling approach to characterize how varying clinically controllable parameters changed neural activation profiles during DRGS. Materials and MethodsWe coupled a finite element model of a human L5 DRG to multicompartment models of primary sensory neurons (i.e., Aα-, Aβ-, Aδ-, and C-neurons). We calculated the stimulation amplitudes necessary to elicit one or more action potentials in each neuron, and examined how neural activation profiles were affected by varying clinically controllable parameters. ResultsIn general, DRGS predominantly activated large myelinated Aα- and Aβ-neurons. Shifting the electrode more than 2 mm away from the ganglion abolished most DRGS-induced neural activation. Increasing the stimulus pulse width to 500 μs or greater increased the number of activated Aδ-neurons, while shorter pulse widths typically only activated Aα- and Aβ-neurons. Placing a cathode near a nerve root, or an anode near the ganglion body, maximized Aβ-mechanoreceptor activation. Guarded active contact configurations did not activate more Aβ-mechanoreceptors than conventional bipolar configurations. ConclusionsOur results suggest that DRGS applied with stimulation parameters within typical clinical ranges predominantly activates Aβ-mechanoreceptors. In general, varying clinically controllable parameters affects the number of Aβ-mechanoreceptors activated, although longer pulse widths can increase Aδ-neuron activation. Our data support several Neuromodulation Appropriateness Consensus Committee guidelines on the clinical implementation of DRGS.

Changes in peripheral HCN2 channels during persistent inflammation

ABSTRACT Nociceptor sensitization following nerve injury or inflammation leads to chronic pain. An increase in the nociceptor hyperpolarization-activated current, Ih, is observed in many models of pathological pain. Pharmacological blockade of Ih prevents the mechanical and thermal hypersensitivity that occurs during pathological pain. Alterations in the Hyperpolarization-activated Cyclic Nucleotide-gated ion channel 2 (HCN2) mediate Ih-dependent thermal and mechanical hyperalgesia. Limited knowledge exists regarding the nature of these changes during chronic inflammatory pain. Modifications in HCN2 expression and post-translational SUMOylation have been observed in the Complete Freund’s Adjuvant (CFA) model of chronic inflammatory pain. Intra-plantar injection of CFA into the rat hindpaw induces unilateral hyperalgesia that is sustained for up to 14 days following injection. The hindpaw is innervated by primary afferents in lumbar DRG, L4-6. Adjustments in HCN2 expression and SUMOylation have been well-documented for L5 DRG during the first 7 days of CFA-induced inflammation. Here, we examine bilateral L4 and L6 DRG at day 1 and day 3 post-CFA. Using L4 and L6 DRG cryosections, HCN2 expression and SUMOylation were measured with immunohistochemistry and proximity ligation assays, respectively. Our findings indicate that intra-plantar injection of CFA elicited a bilateral increase in HCN2 expression in L4 and L6 DRG at day 1, but not day 3, and enhanced HCN2 SUMOylation in ipsilateral L6 DRG at day 1 and day 3. Changes in HCN2 expression and SUMOylation were transient over this time course. Our study suggests that HCN2 is regulated by multiple mechanisms during CFA-induced inflammation.

Metabolic Imaging of Pain Matrix Using 18F Fluoro-deoxyglucose Positron Emission Tomography/Computed Tomography for Patients Undergoing L2 Dorsal Root Ganglion Stimulation for Low Back Pain

Nociceptive signals from lumbar intervertebral discs ascend in the sympathetic chain via the L2 dorsal root ganglion (L2 DRG), a potential target for discogenic low back pain in neuromodulation. Positron Emission Tomography/Computed Tomography (PET-CT) measures functional changes in the brain metabolic activity, identified by the changes in the regional cerebral blood flow (rCBF) as determined by the changes of F-18 Fluoro-deoxyglucose (18 F FDG) tracer within brain tissues. Nine patients were recruited to explore the changes in PET-CT imaging at baseline and four-weeks post implantation of bilateral L2 DRG neurostimulation leads and implantable pulse generator (IPG). PET-CT scans were performed 30 min following an IV injection of 250±10% MBq of 18 F FDG tracer. Fifteen frames were acquired in 15 min. PET list-mode raw data were reconstructed and normalized appropriately to a brain anatomical atlas. Nine patients were recruited to the study, where PET-CT imaging data for five patients were analyzed. The right and left insular cortex, primary and secondary somato-sensory cortices, prefrontal cortex, anterior cingulate cortex, thalamus, amygdala, hippocampus and the midline periaqueductal areas, were assessed for any changes in the metabolic activity. A total of 85 pain matrix regions were delineated SUV (standardized uptake value)MAX , SUV MEAN ± SD, and SUVPEAK were calculated for each of these regions of the brain and were compared pre- and post-L2 DRG stimulation. Sixty-one of the 85 matrices showed an increase in metabolic activity whereas 24 matrices showed a reduction in metabolic activity. This is the first ever study reporting the changes in cerebral metabolic activity and multi-frame static brain 18 F FDG PET imaging after L2 DRG stimulation for discogenic low back pain. Predominantly an increased metabolic activity in nociceptive brain matrices are seen with an increased in F18 F FDG uptake following L2 DRG stimulation.

Dorsal Root Ganglion Stimulation in Experimental Painful Diabetic Polyneuropathy: Delayed Wash-Out of Pain Relief After Low-Frequency (1Hz) Stimulation

Up until now there is little data about the pain relieving effect of different frequency settings in DRGS. The aim of this study was to compare the pain relieving effect of DRGS at low-, mid-, and high-frequencies and Sham-DRGS in an animal model of painful diabetic neuropathy (PDPN). Diabetes mellitus was induced by an intraperitoneal injection of streptozotocin in 8-week-old female Sprague-Dawley rats (n = 24; glucose ≥15 mmol/L: n = 20; mechanical hypersensitivity: n = 15). Five weeks later, a DRGS device was implanted at the L5 DRG. Ten animals were included for stimulation, alternating 30 minutes of low (1 Hz)-, mid (20 Hz)-, and high (1000 Hz)-frequencies and Sham-DRGS during four days, with a pulse width of 0.2 msec (average amplitude: 0.19 ± 0.01 mA), using a randomized cross-over design. The effect on mechanical hypersensitivity of the hind paw to von Frey filaments was evaluated. All DRGS frequencies resulted in a complete reversal of mechanical hypersensitivity and "a clinically relevant reduction" was achieved in 70-80% of animals. No significant differences in maximal pain relieving effect were found between the different frequency treatments (p = 0.24). Animals stimulated at 1000 and 20 Hz returned to baseline mechanical hypersensitivity values 15 and 30 min after stimulation cessation, respectively, while animals stimulated at 1 Hz did not. These results show that DRGS is equally effective when applied at low-, mid-, and high-frequency in an animal model of PDPN. However, low-frequency-(1 Hz)-DRGS resulted in a delayed wash-out effect, which suggests that this is the most optimal frequency for pain therapy in PDPN as compared to mid- and high-frequency.

Nonlinear Relation Between Burst Dorsal Root Ganglion Stimulation Amplitude and Behavioral Outcome in an Experimental Model of Painful Diabetic Peripheral Neuropathy

Background and objectiveDorsal root ganglion stimulation (DRGS) has recently emerged as a neuromodulation modality in the treatment of chronic neuropathic pain. The objective of this study was to compare the efficacy of different Burst‐DRGS amplitudes in an experimental model of painful diabetic peripheral neuropathy (PDPN).MethodsDiabetes mellitus was induced in female Sprague–Dawley rats by intraperitoneal injection of streptozotocin (STZ, n = 28). Animals were tested for mechanical hypersensitivity (von Frey paw withdrawal test) before, and four weeks after STZ injection. PDPN rats (n = 13) were implanted with a unilateral bipolar electrode at the L5 DRG. Animals received Burst‐DRGS at 0%, 10%, 33%, 50%, 66%, and 80% of motor threshold (MT) in a randomized crossover design on post‐implantation days 2–7 (n = 9). Mechanical hypersensitivity was assessed before stimulation onset, 15 and 30 min during stimulation, and 15 and 30 min after stimulation.ResultsBurst‐DRGS at amplitudes of 33%, 50%, 66%, and 80% MT resulted in significant attenuation of STZ‐induced mechanical hypersensitivity at 15 and 30 min during stimulation, as well as 15 min after cessation of stimulation. No effect on mechanical hypersensitivity was observed for Burst‐DRGS at 0% MT and 10% MT. Optimal pain relief and highest responder rates were achieved with Burst‐DRGS at 50–66% MT, with an estimated optimum at 52% MT.ConclusionOur findings indicate a nonlinear relationship between Burst‐DRGS amplitude and behavioral outcome, with an estimated optimal amplitude of 52% MT. Further optimization and analysis of DRGS driven by insights into the underlying mechanisms related to the various stimulation paradigms is warranted.

Dorsal Root Ganglion Stimulation in Experimental Painful Diabetic Peripheral Neuropathy: Burst vs. Conventional Stimulation Paradigm

ObjectivesPainful diabetic peripheral neuropathy (PDPN) is a long‐term complication of diabetes mellitus (DM). Dorsal Root Ganglion Stimulation (DRGS) has recently emerged as a neuromodulation modality in the treatment of chronic neuropathic pain. The objective of this study was to compare the effect of burst DRGS (Burst‐DRGS) and conventional DRGS (Con‐DRGS) in an experimental model of PDPN.Materials and MethodsDM was induced in female Sprague–Dawley rats by intraperitoneal injection of streptozotocin (STZ, n = 48). Animals were tested for mechanical hypersensitivity (50% hind paw withdrawal threshold on Von Frey test) before, and 4 weeks after STZ injection. PDPN rats were then implanted with a unilateral bipolar lead at the L5 DRG (n = 22) and were stimulated for 30 min at days 2 and 3 postimplantation. Animals received Con‐DRGS and Burst‐DRGS in a randomized crossover design (n = 10), or received Sham‐DRGS (n = 7) for 30 min, and were tested for mechanical hypersensitivity at baseline, 15 and 30 min during DRGS, and 15 and 30 min following DRGS. Five animals were withdrawn from the study due to electrode‐related technical problems.ResultsCon‐DRGS and Burst‐DRGS normalized STZ‐induced mechanical hypersensitivity at 15 and 30 min during stimulation. A significant difference in terms of mechanical hypersensitivity was observed between both of the stimulated groups and the Sham‐DRGS group at 15 and 30 min during stimulation. Interestingly, Burst‐DRGS showed signs of a residual effect at 15 min after cessation of stimulation, while this was not the case for Con‐DRGS.ConclusionsUnder the conditions tested, Con‐DRGS and Burst‐DRGS are equally effective in attenuating STZ‐induced mechanical hypersensitivity in an animal model of PDPN. Burst‐DRGS showed signs of a residual effect at 15 min after cessation of stimulation, which requires further investigation.

Lumbar spinal cord neurons putatively involved in ejaculation are sexually dimorphic in early postnatal mice.

A crucial role in ejaculation is thought to be played by a population of lumbar spino-thalamic neurons (LSt), which express galanin and other neuropeptides. In rats, these neurons are activated with ejaculation and their lesion selectively abolishes ejaculation but not other mating behaviors. Consistently with their role, in adult rats and humans, LSt neurons are sexually dimorphic, being more numerous in males. Here we examined whether sexual dimorphism arises early in development, using a transgenic mouse line in which the expression of fluorescent protein is driven by the galanin promoter. We focused on postnatal day 4, shortly after a transient perinatal androgen surge in males that could play an organizational role in LSt development. We found a population of brightly fluorescent neurons organized in bilateral columns dorsolateral to the central canal in segments L1-L5, the expected location of the LSt group. Their number was close to that of adult preparations and significantly greater in male than in female siblings (+19%; CI95% : +13% to +27%; p < .01). This was not due to a generalized higher galanin expression in the male since fluorescent L4 DRG neurons, innervating the hindlimbs and lower back, were not significantly dimorphic (-4%; CI95% : -10% to +8%; p = .92). Unexpectedly, we found in cervical segments a population of fluorescent neurons having a location relative to the central canal similar to the LSt. Thus, the LSt group is sexually dimorphic soon after birth. However, it is possible that only a subset of its neurons participate in the control of ejaculation.

(225) Extrinsic Colon Afferents Influence Enteric Neuron Activity and Colon Motility by Engaging Spinal Reflexes

Abdominal pain and bowel dysmotility are hallmark symptoms of functional bowel disorders, yet it is unclear how pain and dysmotility are correlated mechanistically. Extrinsic colon afferents (EPANs) provide sensory input to the CNS, and visceral pain is thought to be due to hypersensitivity of these afferents. The enteric nervous system (ENS) acts autonomously to control motility reflexes, but extrinsic nerve pathways coordinate activity between distant regions of the GI tract, and importantly, allow the central nervous system (CNS) to regulate GI functioning. To test whether EPANs can directly influence ENS activity, we used an ex vivo colon-pelvic nerve-L6 DRG preparation, in which the roots were cut from the spinal cord, that allowed selective activation of EPANs via dorsal root stimulation and simultaneous calcium imaging (GCaMP6s) of myenteric neurons in the colon. Electrical stimulation of the dorsal root activated the majority of L6 DRG neurons, but produced no GCaMP responses in myenteric neurons of the colon in any of the visual fields tested (n=8). By contrast, ventral root stimulation led to GCaMP responses in ∼20% of myenteric neurons, followed by smooth muscle contraction, as indicated by movement of the imaging field. Interestingly, when the spinal cord was left intact, dorsal root stimulation produced responses in 12% of myenteric neurons (n=5), suggesting the presence of a sensory-parasympathetic spinal reflex. To elucidate this reflex in vivo, we used optogenetic stimulation to produce visceromotor responses and then processed spinal cord tissue for the immediate early gene, c-fos. There were significantly greater numbers of parasympathetic preganglionic neurons (PPNs) with c-fos staining in lumbosacral spinal segments from TRPV1-ChR2 mice (n=3) compared to controls (n=3), and TRPV1 central terminals were observed in close apposition to PPN. Overall, our data suggests that colon afferents influence ENS activity and colon motility by engaging parasympathetic pathways through a spinal reflex.

Blockade of anoctamin-1 in injured and uninjured nerves reduces neuropathic pain

The aim of this study was to determine the participation of anoctamin-1 in 2 models of neuropathic pain in rats (L5/L6 spinal nerve ligation [SNL] and L5 spinal nerve transection [SNT]). SNL and SNT diminished withdrawal threshold in rats. Moreover, SNL up-regulated anoctamin-1 protein expression in injured L5 and uninjured L4 DRG whereas that it enhanced activating transcription factor 3 (ATF-3) and caspase-3 expression only in injured L5 DRG. In marked contrast, SNT enhanced ATF-3 and caspase-3, but not anoctamin-1, expression in injured L5 DRG but it did not modify anoctamin-1, ATF-3 nor caspase-3 expression in uninjured L4 DRG. Accordingly, repeated (3 times) intrathecal injection of the anoctamin-1 blocker T16Ainh-A01 (0.1–1 µg) or MONNA (1–10 µg) partially reverted SNL-induced mechanical allodynia in a dose-dependent manner. In contrast, anoctamin-1 blockers only produced a modest effect in SNT-induced mechanical allodynia. Interestingly, intrathecal injection of T16Ainh-A01 (1 µg) or MONNA (10 µg) prevented SNL-induced up-regulation of anoctamin-1, ATF-3 and caspase-3 in injured L5 DRG. Repeated intrathecal injection of T16Ainh-A01 or MONNA also reduced SNT-induced up-regulation of ATF-3 in injured L5 DRG. In contrast, T16Ainh-A01 and MONNA did not affect SNT-induced up-regulation of caspase-3 expression in L5 DRG. Likewise, gabapentin (100 µg) diminished SNL-induced up-regulation of anoctamin-1, ATF-3 and caspase-3 expression in injured L5 DRG. These data suggest that spinal anoctamin-1 in injured and uninjured DRG participates in the maintenance of neuropathic pain in rats. Our data also indicate that expression of anoctamin-1 in DRG is differentially regulated depending on the neuropathic pain model.

Effect of Intravesical Liposome-Based Nerve Growth Factor Antisense Therapy on Bladder Overactivity and Nociception in a Rat Model of Cystitis Induced by Hydrogen Peroxide.

The aim of this study was to evaluate whether liposome-based local suppression of nerve growth factor (NGF) in the bladder has effects on bladder hypersensitivity in a rat cystitis model induced by intravesical instillation of hydrogen peroxide (HP). HP (1.5%) was intravesically administered to adult female Sprague-Dawley rats. Liposomes complexed with NGF antisense oligonucleotide (OND) labeled with TYE563 fluorescent tag were intravesically instilled on day 2. Red fluorescence from the TYE 563 tag was observed with fluorescent microscopy on day 3. Four separate groups of rats were used in the following experiments: (a) sham-liposome group, (b) sham-OND group, (c) cystitis-liposome group, and (d) cystitis-OND group. Saline or 1.5% HP was intravesically administered on day 0. Empty liposomes or liposomes-antisense OND were instilled into the bladder on day 2. The following experiments were conducted to evaluate the effect of NGF antisense treatment on day 7: (a) continuous cystometry was performed in an awake condition; (b) pain behavior induced by instillation of resiniferatoxin into the bladder, including licking behavior (lower abdominal licking) and freezing behavior (motionless head-turning toward lower abdomen), was observed; (c) immunohistochemical staining of the bladder and L6 DRG for NGF was performed; (d) the expression of several genes in the bladder was analyzed by reverse transcription polymerase chain reaction (RT-PCR); and (e) after Fast Blue was injected into the bladder wall, Fast Blue-positive or -negative cells in DRG neurons were separately collected by using a laser-capture microdissection method 7 days later. RT-PCR was performed to evaluate gene expressions in captured neuronal cells. The expression of TYE563 was identified only in the urothelial layer. In cystometric investigation, intercontraction intervals (ICI) were significantly (p = 0.001) shorter in the cystitis-liposome group in comparison to the sham-liposome group. ICI was significantly (p = 0.007) longer in the cystitis-OND group compared to the cystitis-liposome group. Comparisons of the sham-liposome and the sham-OND groups showed no significant difference in ICI (p = 0.56). Licking events did not significantly differ among the four groups. In contrast, the cystitis-liposome group showed significantly more freezing events than the sham-liposome group did (p = 0.002). A significant reduction in the number of freezing events was observed in the cystitis-OND group compared to the cystitis-liposome group (p = 0.04). Immunofluorescence staining demonstrated that NGF expression in the mucosa (p = 0.02) and L6 DRG (p = 0.01) was significantly higher in the cystitis-liposome group than it was in the sham-liposome group. The expression of NGF was significantly lower in the mucosa (p = 0.002) and L6 DRG (p = 0.01) in the cystitis-OND group compared to the cystitis-liposome group. RT-PCR showed that the expression of NGF and TRPV1 mRNA in the mucosa was significantly higher in the cystitis-liposome group than it was in the sham-liposome group (p = 0.001 and 0.03, respectively). On the other hand, these gene expressions were significantly lower in the cystitis-OND group than they were in the cystitis-liposome group (p = 0.007 and 0.02, respectively). The cystitis-liposome group showed significantly higher expression of TRPA1, P2X3, and BDNF mRNA in labeled bladder afferent neurons than the sham-liposome group did (p = 0.03, 0.01, and 0.001, respectively). These gene expressions were significantly lower in the cystitis-OND group compared to the cystitis-liposome group (p = 0.04, 0.006, and 0.03, respectively). The study indicated that intravesical application of liposome-NGF antisense OND significantly improved bladder hypersensitivity induced by chemical cystitis in rats. Intravesical treatment with liposome-OND conjugates could be a novel local therapy of hypersensitive bladder disorders such as bladder pain syndrome/interstitial cystitis.

Localized Sympathectomy Reduces Mechanical Hypersensitivity by Restoring Normal Immune Homeostasis in Rat Models of Inflammatory Pain.

Some forms of chronic pain are maintained or enhanced by activity in the sympathetic nervous system (SNS), but attempts to model this have yielded conflicting findings. The SNS has both pro- and anti-inflammatory effects on immunity, confounding the interpretation of experiments using global sympathectomy methods. We performed a "microsympathectomy" by cutting the ipsilateral gray rami where they entered the spinal nerves near the L4 and L5 DRG. This led to profound sustained reductions in pain behaviors induced by local DRG inflammation (a rat model of low back pain) and by a peripheral paw inflammation model. Effects of microsympathectomy were evident within one day, making it unlikely that blocking sympathetic sprouting in the local DRGs or hindpaw was the sole mechanism. Prior microsympathectomy greatly reduced hyperexcitability of sensory neurons induced by local DRG inflammation observed 4 d later. Microsympathectomy reduced local inflammation and macrophage density in the affected tissues (as indicated by paw swelling and histochemical staining). Cytokine profiling in locally inflamed DRG showed increases in pro-inflammatory Type 1 cytokines and decreases in the Type 2 cytokines present at baseline, changes that were mitigated by microsympathectomy. Microsympathectomy was also effective in reducing established pain behaviors in the local DRG inflammation model. We conclude that the effect of sympathetic fibers in the L4/L5 gray rami in these models is pro-inflammatory. This raises the possibility that therapeutic interventions targeting gray rami might be useful in some chronic inflammatory pain conditions. Sympathetic blockade is used for many pain conditions, but preclinical studies show both pro- and anti-nociceptive effects. The sympathetic nervous system also has both pro- and anti-inflammatory effects on immune tissues and cells. We examined effects of a very localized sympathectomy. By cutting the gray rami to the spinal nerves near the lumbar sensory ganglia, we avoided widespread sympathetic denervation. This procedure profoundly reduced mechanical pain behaviors induced by a back pain model and a model of peripheral inflammatory pain. One possible mechanism was reduction of inflammation in the sympathetically denervated regions. This raises the possibility that therapeutic interventions targeting gray rami might be useful in some inflammatory conditions.

Nerve injury induced activation of fast-conducting high threshold mechanoreceptors predicts non-reflexive pain related behavior

The role of specific subsets of peripheral nerves in pain related behavior remains unclear. To better understand the contribution of differential activation of fast-conducting, high-threshold mechanoreceptor (AHTMR) input, we hypothesized that neuronal activation would be distinct with nerve injury, and that nociceptive input would predictt behavior in the freely exploring animal. A series of surfaces was used to deliver mechanical input to the hindpaws of rats upon voluntary movement and exploration. Neuronal activation increased as apex surface decreased (0.2, 0.6, 1.0 and 1.5mm) using in vivo recording in L4 DRG neurons, and this relationship was enhanced following partial ligation of L5 (pSNL). In behaving animals, apex size was correlated to time spent on each surface following pSNL, but not with sham. Morphine normalized the discriminatory behavior following pSNL. These data indicate that noxious mechanical activation of AHTMR upon normal movement predicts behavior using paradigms that do not rely on reflexive withdrawal responses suggesting that AHTMR activation and central nervous system input contribute to higher order pain behavior after nerve injury beyond the immediate early pain input long attributed to these neurons.

Overexpression of the monocyte chemokine CCL2 in dorsal root ganglion neurons causes a conditioning-like increase in neurite outgrowth and does so via a STAT3 dependent mechanism

Neuroinflammation plays a critical role in the regeneration of peripheral nerves following axotomy. An injury to the sciatic nerve leads to significant macrophage accumulation in the L5 DRG, an effect not seen when the dorsal root is injured. We recently demonstrated that this accumulation around axotomized cell bodies is necessary for a peripheral conditioning lesion response to occur. Here we asked whether overexpression of the monocyte chemokine CCL2 specifically in DRG neurons of uninjured mice is sufficient to cause macrophage accumulation and to enhance regeneration or whether other injury-derived signals are required. AAV5-EF1α-CCL2 was injected intrathecally, and this injection led to a time-dependent increase in CCL2 mRNA expression and macrophage accumulation in L5 DRG, with a maximal response at 3 weeks post-injection. These changes led to a conditioning-like increase in neurite outgrowth in DRG explant and dissociated cell cultures. This increase in regeneration was dependent upon CCL2 acting through its primary receptor CCR2. When CCL2 was overexpressed in CCR2-/- mice, macrophage accumulation and enhanced regeneration were not observed. To address the mechanism by which CCL2 overexpression enhances regeneration, we tested for elevated expression of regeneration-associated genes in these animals. Surprisingly, we found that CCL2 overexpression led to a selective increase in LIF mRNA and neuronal phosphorylated STAT3 (pSTAT3) in L5 DRGs, with no change in expression seen in other RAGs such as GAP-43. Blockade of STAT3 phosphorylation by each of two different inhibitors prevented the increase in neurite outgrowth. Thus, CCL2 overexpression is sufficient to induce macrophage accumulation in uninjured L5 DRGs and increase the regenerative capacity of DRG neurons via a STAT3-dependent mechanism.

Chronic stress and peripheral pain: Evidence for distinct, region-specific changes in visceral and somatosensory pain regulatory pathways

Chronic stress alters the hypothalamic–pituitary–adrenal (HPA) axis and enhances visceral and somatosensory pain perception. It is unresolved whether chronic stress has distinct effects on visceral and somatosensory pain regulatory pathways. Previous studies reported that stress-induced visceral hyperalgesia is associated with reciprocal alterations of endovanilloid and endocannabinoid pain pathways in DRG neurons innervating the pelvic viscera. In this study, we compared somatosensory and visceral hyperalgesia with respect to differential responses of peripheral pain regulatory pathways in a rat model of chronic, intermittent stress. We found that chronic stress induced reciprocal changes in the endocannabinoid 2-AG (increased) and endocannabinoid degradation enzymes COX-2 and FAAH (decreased), associated with down-regulation of CB1 and up-regulation of TRPV1 receptors in L6–S2 DRG but not L4–L5 DRG neurons. In contrast, sodium channels Nav1.7 and Nav1.8 were up-regulated in L4–L5 but not L6–S2 DRGs in stressed rats, which was reproduced in control DRGs treated with corticosterone in vitro. The reciprocal changes of CB1, TRPV1 and sodium channels were cell-specific and observed in the sub-population of nociceptive neurons. Behavioral assessment showed that visceral hyperalgesia persisted, whereas somatosensory hyperalgesia and enhanced expression of Nav1.7 and Nav1.8 sodium channels in L4–L5 DRGs normalized 3days after completion of the stress phase. These data indicate that chronic stress induces visceral and somatosensory hyperalgesia that involves differential changes in endovanilloid and endocannabinoid pathways, and sodium channels in DRGs innervating the pelvic viscera and lower extremities. These results suggest that chronic stress-induced visceral and lower extremity somatosensory hyperalgesia can be treated selectively at different levels of the spinal cord.

Su1871 Effects of DA-9701, a Prokinetic Agent on Gastric Motor Function in Guinea Pig; In Vivo and In Vitro Study

ity was induced in twenty adult male Sprague-Dawley rats by butyrate enemas in the distal colon, and 10 control rats served as vehicle only. After butyrate enema, the rats were divided into 2 groups and were given respectively DA 9701 3mg/kg or vehicle. The rats were sacrificed at 1 and 2 days after treatment, and colonic damage score and tissue myeloperoxidase activity were evaluated. In lumbosacral L1 and L2 DRG, expression level of BDNF, ASIC 1a, ASIC 2b, IL-1β, and TNF-α were assessed using quantitative real-time PCR, ELISA, and western blot. Results: In L1 DRG, the levels of BDNF protein were increased at both 1 and 2 days of colitis (2.2to 3.2-fold, p < 0.05). There were no significant changes in the BDNF protein level in L2 DRG at both days of colitis. In addition, level of ASIC 1a (2.5to 4.3fold, p < 0.05) and 2b (2.7to 3.6-fold, p < 0.05) protein expression in L2 DRG significantly higher after administration. RT PCR results showed significant increases in the level of BDNF mRNA in L1 DRG (2.1to 2.3-fold, p < 0.05) at 1 and 2 days of colitis. ASIC 1a and 2b mRNA expression was significantly increased in L1 level. There was no change in IL-1β and TNF-α expression in colitis group. In butyrate-treated colitis model, administration of DA 9701 led to significantly decreased level of BDNF, ASIC 1a, and ASIC 2b protein and mRNA expression in both L1 and L2 DRG levels compared with control groups. Conclusion: Our data suggest that peripheral BDNF and ASIC1a/2b concomitantly contribute to the development of butyrate-induced colon hypersensitivity. In addition, DA 9701 can negatively modulate the activity of ASICs and BDNF in lumbosacral DRG.

Su1870 Entacapone, A Peripherally Restricted COMT Inhibitor, Regulates Gastrointestinal Motility and Pain Sensitivity in Mice

ity was induced in twenty adult male Sprague-Dawley rats by butyrate enemas in the distal colon, and 10 control rats served as vehicle only. After butyrate enema, the rats were divided into 2 groups and were given respectively DA 9701 3mg/kg or vehicle. The rats were sacrificed at 1 and 2 days after treatment, and colonic damage score and tissue myeloperoxidase activity were evaluated. In lumbosacral L1 and L2 DRG, expression level of BDNF, ASIC 1a, ASIC 2b, IL-1β, and TNF-α were assessed using quantitative real-time PCR, ELISA, and western blot. Results: In L1 DRG, the levels of BDNF protein were increased at both 1 and 2 days of colitis (2.2to 3.2-fold, p < 0.05). There were no significant changes in the BDNF protein level in L2 DRG at both days of colitis. In addition, level of ASIC 1a (2.5to 4.3fold, p < 0.05) and 2b (2.7to 3.6-fold, p < 0.05) protein expression in L2 DRG significantly higher after administration. RT PCR results showed significant increases in the level of BDNF mRNA in L1 DRG (2.1to 2.3-fold, p < 0.05) at 1 and 2 days of colitis. ASIC 1a and 2b mRNA expression was significantly increased in L1 level. There was no change in IL-1β and TNF-α expression in colitis group. In butyrate-treated colitis model, administration of DA 9701 led to significantly decreased level of BDNF, ASIC 1a, and ASIC 2b protein and mRNA expression in both L1 and L2 DRG levels compared with control groups. Conclusion: Our data suggest that peripheral BDNF and ASIC1a/2b concomitantly contribute to the development of butyrate-induced colon hypersensitivity. In addition, DA 9701 can negatively modulate the activity of ASICs and BDNF in lumbosacral DRG.

Tu1871 Excretion of Breath Hydrogen Is Reduced by Inhibition of Sulfate Reduction by Molybdate in Rats

G A A b st ra ct s increased in the DRG during colitis contains CREB binding sequence in its promoter thus is responsive to CREB activity. AIMS: 1) To examine the phosphorylation of CREB and expression of CGRP by BDNF in vivo and in vitro; and 2) To examine whether the PLC/ calcium pathway is involved in BDNF-induced CREB phosphorylation.METHODS: Colonic inflammation was induced in rats by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS: 1.5 mL/kg of 60 mg/mL solution in 50% EtOH). Control animals received 50% EtOH. Rats were killed on day 3 or 7 following the induction of colitis. CREB phosphorylation was examined by western blot and immunohistochemistry with a specific antibody recognizing CREB phospho-serine 133. The pair-matched DRG explants culture were incubated with BDNF (10 ng/mL) for various time points to examine the effects of BDNF signaling on CREB phosphorylation. Endogenous BDNF was blocked by BDNF neutralizing antibody (36 μg/ kg, i.v.). CGRP expression was examined by real-time PCR. RESULTS: Colitis increased CREB phosphorylation level in the L1 DRG at 3 days and 7 days post TNBS treatment examined by both western blot and immunohistochemistry. CREB phosphorylation in the DRG was expressed in small-to-medium sized sensory neurons. Anti-BDNF treatment of the colitic animals reduced colitis-induced CREB phosphorylation in the L1 DRG. Incubation of DRG explants with BDNF also increased CREB phosphorylation level which was blocked by inhibition of the phospholipase C (PLC) pathway with U-73122. Blockade of endogenous BDNF with BDNF neutralizing antibody also reversed colitis-induced up-regulation of CGRP transcripts in the L1 DRG. CONCLUSION: BDNF regulates CREB activity and CGRP expression through the PLC/calcium pathway in the DRG during colitis. The BDNF-PLC/ calcium-CREB-CGRP axis is an important component in visceral hypersensitivity during colitis.