Author response: Neuronal complexity is attenuated in preclinical models of migraine and restored by HDAC6 inhibition (2021)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Migraine is the sixth most prevalent disease worldwide but the mechanisms that underlie migraine chronicity are poorly understood. Cytoskeletal flexibility is fundamental to neuronal-plasticity and is dependent on dynamic microtubules. Histone-deacetylase-6 (HDAC6) decreases microtubule dynamics by deacetylating its primary substrate, α-tubulin. We use validated mouse models of migraine to show that HDAC6-inhibition is a promising migraine treatment and reveal an undiscovered cytoarchitectural basis for migraine chronicity. The human migraine trigger, nitroglycerin, produced chronic migraine-associated pain and decreased neurite growth in headache-processing regions, which were reversed by HDAC6 inhibition. Cortical spreading depression (CSD), a physiological correlate of migraine aura, also decreased cortical neurite growth, while HDAC6-inhibitor restored neuronal complexity and decreased CSD. Importantly, a calcitonin gene-related peptide receptor antagonist also restored blunted neuronal complexity induced by nitroglycerin. Our results demonstrate that disruptions in neuronal cytoarchitecture are a feature of chronic migraine, and effective migraine therapies might include agents that restore microtubule/neuronal plasticity. eLife digest Migraines are a common brain disorder that affects 14% of the world's population. For many people the main symptom of a migraine is a painful headache, often on one side of the head. Other symptoms include increased sensitivity to light or sound, disturbed vision, and feeling sick. These sensory disturbances are called aura and they often occur before the headache begins. One particularly debilitating subset of migraines are chronic migraines, in which patients experience more than 15 headache days per month. Migraine therapies are often only partially effective or poorly tolerated, making it important to develop new drugs for this condition, but unfortunately, little is known about the molecular causes of migraines. To bridge this gap, Bertels et al. used two different approaches to cause migraine-like symptoms in mice. One approach consisted on giving mice nitroglycerin, which dilates blood vessels, produces hypersensitivity to touch, and causes photophobia in both humans and mice. In the second approach, mice underwent surgery and potassium chloride was applied onto the dura, a thick membrane that surrounds the brain. This produces cortical spreading depression, an event that is linked to migraine auras and involves a wave of electric changes in brain cells that slowly propagates across the brain, silencing brain electrical activity for several minutes. Using these approaches, Bertels et al. studied whether causing chronic migraine-like symptoms in mice is associated with changes in the structures of neurons, focusing on the effects of migraines on microtubules. Microtubules are cylindrical protein structures formed by the assembly of smaller protein units. In most cells, microtubules assemble and disassemble depending on what the cell needs. Neurons need stable microtubules to establish connections with other neurons. The experiments showed that provoking chronic migraines in mice led to a reduction in the numbers of connections between different neurons. Additionally, Bertels et al. found that inhibiting HDAC6 (a protein that destabilizes microtubules) reverses the structural changes in neurons caused by migraines and decreases migraine symptoms. The same effects are seen when a known migraine treatment strategy, known as CGRP receptor blockade, is applied. These results suggest that chronic migraines may involve decreased neural complexity, and that the restoration of this complexity by HDAC6 inhibitors could be a potential therapeutic strategy for migraine. Introduction Migraine is an extremely common neurological disorder that is estimated to affect 14% of the global population, making it the third most prevalent disease worldwide (Global Burden of Disease Study 2013 Collaborators, 2015; GBD 2016 Disease and Injury Incidence and Prevalence Collaborators, 2017). One particularly debilitating subset of migraine patients are those with chronic migraine, which is defined as having more than 15 headache days a month (Headache Classification Committee of the International Headache Society (IHS), 2013). Despite its high prevalence, migraine therapies are often only partially effective or are poorly tolerated, creating a need for better pharmacotherapies (Ford et al., 2017). Recent clinical success of antibodies and antagonists against calcitonin gene-related peptide (CGRP) and CGRP receptor demonstrate the effectiveness of targeted migraine therapeutics. While there has been more research into understanding the molecular mechanisms of migraine, there remains much to be discovered. Neuroplastic changes play an important role in a variety of chronic neuropsychiatric conditions (Descalzi et al., 2015), and epigenetic alterations through histone deacetylases (HDACs) are frequently investigated. HDACs are best characterized for their ability to deacetylate histones, promoting chromatin condensation and altered gene expression (Krishnan et al., 2014). Intriguingly, some HDACs can also deacetylate non-histone targets, including proteins involved in cytoarchitecture and dynamic cellular structure. Due to its cytoplasmic retention signal, HDAC6 is primarily expressed in the cytosol (Valenzuela-Fernández et al., 2008), and one of its primary targets for deacetylation is α-tubulin (Valenzuela-Fernández et al., 2008; d'Ydewalle et al., 2012). α- and β-tubulin form heterodimers that make up microtubules, which are a major component of the cytoskeleton, and regulate intracellular transport, cell morphology, motility, and organelle distribution (Janke and Bulinski, 2011; Janke and Montagnac, 2017). Microtubules undergo multiple cycles of polymerization and depolymerization, creating a state of dynamic instability (Janke and Bulinski, 2011). Tubulin displays a variety of post-translational modifications, including α-tubulin acetylation, which occurs endogenously through α-tubulin N-acetyltransferase I (αTAT1), and it is correspondingly deacetylated by HDAC6 (Janke and Montagnac, 2017). Tubulin acetylation is associated with increased flexibility and stability of microtubules (Xu et al., 2017). In contrast, deacetylated microtubules are more fragile and prone to breakage or increased cycling between assembly and disassembly (Xu et al., 2017). Microtubules are important for cellular response to injury and play a role in neurite branching (Gallo, 2011); and microtubule dynamics influence neuronal signaling and mediate axonal transport (Covington et al., 2009; Braun et al., 2011; Schappi et al., 2014; Jin et al., 2017; Van Helleputte et al., 2018). Importantly, changes in cellular structure such as alterations in dendritic spine density, have been implicated in disease chronicity (Jochems et al., 2015; Forrest et al., 2018; Singh et al., 2018; Nestler and Lüscher, 2019). The aims of this study were to determine if altered neuronal cytoarchitecture facilitates the chronic migraine state and whether modifying this by inhibition of HDAC6 would be effective in mouse models of migraine. We observed decreased neuronal complexity in headache-processing brain regions in the nitroglycerin (NTG) model of chronic migraine-associated pain. We further demonstrated that treatment with HDAC6 inhibitor reversed these cytoarchitectural changes and correspondingly decreased cephalic allodynia. These studies were extended to a mechanistically distinct model of migraine, cortical spreading depression (CSD), which is thought to be the electrophysiological correlate of migraine aura. Again, we observed decreased neuronal complexity in migraine related sites, which was reversed by a HDAC6 inhibitor. To investigate the translational implication, we also tested the effect of olcegepant, a CGRP receptor inhibitor, and found that it alleviated chronic allodynia induced by NTG, and restored cytoarchitectural changes associated with chronic migraine-associated pain. These results suggest a novel mechanism for migraine pathophysiology and establish HDAC6 as a novel therapeutic target for this disorder. Results Exposure to chronic NTG induces cytoarchitectural changes in key pain processing regions Changes in the structural plasticity of neurons have been observed in a number of neuropsychiatric disorders and can serve as a marker of disease chronicity (Forrest et al., 2018; Nestler and Lüscher, 2019). To investigate if this was also the case in migraine, we treated male and female C57BL/6J mice every other day for 9 days with NTG or vehicle and tested for periorbital mechanical responses on days 1, 5, and 9 (Figure 1A). NTG induced severe and sustained cephalic allodynia as measured by von Frey hair stimulation of the periorbital region as compared to vehicle animals on the same day (Figure 1B). Mice were sacrificed on day 10, 24 hr after the final NTG/VEH treatment, and neuronal size and arborization were examined through a Golgi staining procedure in a key cephalic pain processing region, the trigeminal nucleus caudalis (TNC) (Figure 1C; Goadsby et al., 2017). We observed a dramatic decrease in neuronal complexity after NTG treatment (Figure 1D). Neurons of chronic NTG-treated mice had significantly fewer branch points (Figure 1E), and shorter neurites resulting in decreased overall length of the neurons (Figure 1F). Further examination of the complexity of the neurons using Sholl Analysis, showed a significant decrease in the number of intersections following NTG treatment (Figure 1G–I). In addition to the TNC we also determined if other brain regions related to central pain processing were affected by chronic NTG treatment. We examined the somatosensory cortex (SCx) and periaqueductal gray (PAG) of these mice and found similar results, where neurons from NTG-treated mice had fewer branch points, were shorter in length, and had fewer intersections (Figure 1—figure supplement 1A–D and E–H, respectively). To ensure that this effect was associated with migraine-pain processing and not a non-specific effect of NTG we also analyzed neuronal complexity in the nucleus accumbens shell (NAc), a region more commonly associated with reward, and found no alteration in number of branches, total neuron length, or Sholl analysis for cells in this region (Figure 1—figure supplement 1I–L). Furthermore, we also examined the dorsal horn of the lumbar and cervical spinal cord, important sites for pain processing in the peripheral nervous system. No differences in NTG versus vehicle controls in number of branches, total neuron length, or Sholl analysis were observed (Figure 1—figure supplement 1M–T). These results suggest that decreased neuronal complexity may be a feature that maintains the chronic migraine state; a previously undiscovered phenomenon. Figure 1 with 1 supplement see all Download asset Open asset The NTG model of chronic migraine produces cytoarchitectural changes in a cephalic pain processing region. (A) Schematic of testing schedule, M and F C57Bl/6J mice were treated with vehicle or nitroglycerin (10 mg/kg, IP; NTG) every other day for 9 days. (B) Periorbital mechanical thresholds were accessed prior to Vehicle/NTG administration on days 1, 5, and 9. NTG produced severe cephalic allodynia p<0.0001 effect of drug F = 44.62, time F = 19.16, and interaction F = 16.28, two-way RM ANOVA and Holm-Sidak post hoc analysis. ****p<0.0001, relative to vehicle on day 1; n = 8/group. (C) Representative image taken of Golgi stained TNC at 4x (left) and 20x (right). Chevrons indicate neurons traced from the selected image and demonstrate type of neurons selected from this region. (D) Representative tracing of neurons from mice treated with chronic vehicle (left) or NTG (right) demonstrating reduced neural processes after chronic NTG treatment. (E) The number of branch points/neuron was decreased following chronic NTG. Unpaired t-test. ****p<0.0001, t = 4.029, df = 70, 95% CI (−2.866,–0.9678). (F) Total neuron length was also compromised after NTG treatment. Unpaired t-test. **p=0.0066, t = 2.798, df = 70, 95% CI (−93.04,–15.60). (G) Representative Sholl plots of vehicle (left) and NTG (right) treated mice. (H) Sholl analysis broken up by 0.377 µm/20 pixel distances from the center of the cell showing differences between groups. (I) Sholl analysis revealed a significant decrease in total intersections after chronic NTG treatment. Unpaired t-test. *p=0.0193, t = 2.396, df = 70, 95% CI (−9.825,–0.8977) n = 6 mice/group, six neurons per mouse. Figure 1—source data 1 Data Figure 1B. https://cdn.elifesciences.org/articles/63076/elife-63076-fig1-data1-v2.xlsx Download elife-63076-fig1-data1-v2.xlsx Figure 1—source data 2 Data Figure 1E. https://cdn.elifesciences.org/articles/63076/elife-63076-fig1-data2-v2.xlsx Download elife-63076-fig1-data2-v2.xlsx Figure 1—source data 3 Data Figure 1F. https://cdn.elifesciences.org/articles/63076/elife-63076-fig1-data3-v2.xlsx Download elife-63076-fig1-data3-v2.xlsx Figure 1—source data 4 Data Figure 1H. https://cdn.elifesciences.org/articles/63076/elife-63076-fig1-data4-v2.xlsx Download elife-63076-fig1-data4-v2.xlsx Figure 1—source data 5 Data Figure 1I. https://cdn.elifesciences.org/articles/63076/elife-63076-fig1-data5-v2.xlsx Download elife-63076-fig1-data5-v2.xlsx HDAC6 inhibition increases acetylated α-tubulin and neuronal cytoarchitectural complexity We hypothesized that if we could restore migraine-compromised cytoarchitectural complexity, this might also relieve cephalic pain. Recent studies demonstrate that increased tubulin acetylation facilitates microtubule flexibility and prevents microtubule breakage (Janke and Montagnac, 2017; Portran et al., 2017; Xu et al., 2017). Thus, we hypothesized that inhibiting HDAC6 to promote microtubule stability may restore the neuronal complexity observed following chronic NTG. We tested the selective HDAC6 inhibitor, ACY-738, in the chronic NTG model. Mice were treated with NTG or VEH for 9 days. On day 10, mice were injected with ACY-738, after 4 hr, tissue was processed and analyzed by western blot. ACY-738 treatment resulted in a significant increase in the ratio of acetylated α-tubulin to total tubulin within the trigeminal ganglia (TG), TNC, SCx, NAc, and spinal cord (Figure 2—figure supplement 1). A separate group of mice were analyzed by neuronal tracing in the TNC, 4 hr post-ACY-738 (Figure 2A). Again, chronic NTG treatment caused a decrease in branch points (Figure 2B), combined neurite length (Figure 2C), and number of intersections (Figure 2D–F). In contrast, treatment with ACY-738 led to a significant increase in these measures in both chronic vehicle and NTG groups (Figure 2A–F). Figure 2 with 1 supplement see all Download asset Open asset Treatment with HDAC6 inhibitor restores blunted neuronal complexity. (A) Representative neuron tracing of mice that were chronically treated with vehicle or NTG (10 mg/kg, IP) every other day for 9 days and on day 10 were injected with ACY-738 (50 mg/kg, IP) or vehicle (5% DMSO in 0.9% NaCl, IP) and sacrificed 4 hr later for Golgi staining. (B) The number of branch points/neuron were significantly decreased following NTG treatment (NTG-Vehicle); while ACY-738 treated mice showed increased branching regardless of pretreatment. p=0.0061, F = 7.891, 95% CI (0.4864, 2.841) effect of chronic treatment, p=0.0002, F = 14.61, 95% CI (−3.441,–1.086) drug treatment, p=0.0044, F = 8.537, 95% CI (−5.815,–1.106) and interaction, two-way ANOVA and Holm-Sidak post hoc analysis. ***p=0.0002, t = 4.851 effect of ACY-738; ++++p<0.0001, t = 4.947 effect of NTG. (C) NTG also decreased total length/neuron, while ACY-738 treatment increased it. p<0.0001, F = 109.8, 95% CI (−667.8,–456.1) effect drug treatment, p=0.0004, F = 12.82, 95% CI (−595.8,–172.3) and interaction, two-way ANOVA and Holm-Sidak post hoc analysis. ****p<0.0001, t = 10.06 effect of ACY-738; ++p=0.0019, t = 3.358 effect of NTG. (D) Representative Sholl analysis image of neuronal complexity in the four groups. (E) Sholl analysis broken up by 0.377 µm/20 pixel distances reveal differences between NTG and ACY-738 treatment. (F) Chronic NTG results in significantly fewer total interactions relative to vehicle treatment. ACY-738 increases total interactions in both vehicle and NTG groups. p<0.0001, F = 59.38, 95% CI (−27.60,–15.96) effect drug treatment, and p=0.0073, F = 8.537, 95% CI (−28.16,–4.874) interaction, two-way ANOVA and Holm-Sidak post hoc analysis. ****p<0.001, t = 3.342 effect of ACY-738; +p=0.0403, t = 2.477 effect of NTG. For all analysis n = 6 mice/group, 6–8 neurons per mouse. Figure 2—source data 1 Data Figure 2B. https://cdn.elifesciences.org/articles/63076/elife-63076-fig2-data1-v2.xlsx Download elife-63076-fig2-data1-v2.xlsx Figure 2—source data 2 Data Figure 2C. https://cdn.elifesciences.org/articles/63076/elife-63076-fig2-data2-v2.xlsx Download elife-63076-fig2-data2-v2.xlsx Figure 2—source data 3 Data Figure 2E. https://cdn.elifesciences.org/articles/63076/elife-63076-fig2-data3-v2.xlsx Download elife-63076-fig2-data3-v2.xlsx Figure 2—source data 4 Data Figure 2F. https://cdn.elifesciences.org/articles/63076/elife-63076-fig2-data4-v2.xlsx Download elife-63076-fig2-data4-v2.xlsx HDAC6 inhibition reverses NTG-induced allodynia and restores neuronal complexity in a time dependent manner We next determined if this restored neuronal complexity would affect behavioral outcomes. Mice were treated with chronic intermittent NTG or vehicle for 9 days. On day 10, baseline cephalic allodynia was observed in mice treated chronically with NTG but not vehicle (Figure 3A, baselines). Mice were then injected with ACY-738 or vehicle and tested 4, 24, or 48 hr later. ACY-738 significantly reversed cephalic allodynia in NTG treated mice for up to 24 hr post-injection. Mechanical responses in ACY-738 treated animals, not treated with NTG were unaffected (Figure 3A, VEH-ACY), suggesting that augmentation of neuronal complexity by HDAC6 inhibitor in a pain-free animal does not alter endogenous pain processing. Interestingly, the half-life of ACY-738 is only 12 min (Jochems et al., 2014), thus short-term inhibition of HDAC6 still produced long-lasting behavioral and cytoarchitectural changes. Figure 3 Download asset Open asset ACY-738 reverses established allodynia in a time-dependent manner with neuronal alterations. (A) C57Bl/6J mice underwent chronic intermittent NTG/Veh treatment for 9 days, on day 10 basal mechanical thresholds were assessed, and mice were subsequently injected with ACY-738 (50 mg/kg IP) or Vehicle and tested 4, 24, and 48 hr later. Chronic NTG treatment caused severe cephalic allodynia (Baselines); which was significantly inhibited by ACY-738 at 4 hr and 24 hr post-injection. p<0.0001, F = 209.8 chronic treatment, p=0.0002, F = 16.85 drug, p=0.0046, F = 5.174 time, and interaction p<0.0001 F = 11.19, three-way RM ANOVA, Holm- Sidak post hoc analysis, ***p<0.001, t = 7.006 NTG-Veh as compared to Veh-Veh at baseline, ***p<0.001 t = 9.126 NTG-Veh as compared to Veh-Veh at 4 hr time point, ***p<0.001 t = 6.736 NTG-Veh as compared to Veh-Veh at 24 hr timepoint; +++p<0.001, t = 7.993 NTG-ACY compared to the NTG-Veh treated mice at 4 hr timepoint, +++p<0.001, t = 6.069; n = 12 mice/group. (B) Representative trigeminal nucleus caudalis neurons from mice treated with VEH-ACY (top) or NTG-ACY (bottom) depicting alterations in cytoarchitecture over the 48 hr time course. In the trigeminal nucleus caudalis, the (C) number of branch points/neuron were significantly decreased following NTG treatment (NTG-Vehicle) an effect not observed following treatment with ACY (NTG-ACY); p<0.0001, F = 17.46, 95% CI (1.024, 2.864) effect of chronic treatment, p=0.0664, F = 3.424, 95% CI (−1.781, 0.05890) drug treatment, p=0.0183, F = 5.701, 95% CI (−4.062,–0.3822) and interaction, two-way ANOVA and Holm-Sidak post hoc analysis. ****p<0.0001, t = 4.643 NTG-Veh compared to Veh-Veh; +p=0.0128, t = 2.997 NTG-Veh compared to NTG-ACY. (D) NTG also decreased total length/neuron, while ACY-738 treatment restored length in chronic NTG-treated animals. p=0.0955, F = 2.817, 95% CI (−226.4, 18.49) effect drug treatment, and p=0.0062, F = 7.717, 95% CI (−588.9,–99.19) interaction, two-way ANOVA and Holm-Sidak post hoc analysis. *p=0.0217, t = 2.897 NTG-Veh compared to Veh-Veh; +p=0.0119, t = 3.151 NTG-Veh compared to NTG-ACY. (E) Chronic NTG resulted in significantly fewer total interactions relative to vehicle treatment, an effect not observed in ACY-738-treated groups. p=0.1278, F = 2.347, 95% CI (−8.780, 1.113) effect drug treatment, p=0.2641, F = 1.257, 95% CI chronic treatment, and F = 95% CI interaction, two-way ANOVA and Holm-Sidak post hoc analysis. t = Veh-Veh compared to t = NTG-Veh compared to NTG-ACY. For all n = 6 mice/group, six neurons per mouse. 48 hr, the effects of ACY-738 were no Chronic NTG treated groups showed decreased (F) ****p<0.001, F = 95% CI effect of chronic NTG two-way (G) neurite length was also in NTG treated groups. ****p<0.0001, F = 95% CI effect of chronic NTG. (H) Chronic NTG groups also showed decreased interactions following Sholl analysis at the 48 hr ***p=0.0002, F = 95% CI effect of chronic NTG two-way Figure data 1 Data Figure Download Figure data 2 Data Figure Download Figure data 3 Data Figure Download Figure data 4 Data Figure Download Figure data 5 Data Figure Download Figure data 6 Data Figure Download Figure data Data Figure Download To further the between neuronal complexity and we examined the cytoarchitecture of neurons following treatment with ACY-738 at the 24 and 48 hr time points (Figure A separate of animals were treated with NTG/Veh for 9 days and on day 10 and were sacrificed 24 or 48 hr for neuronal analysis. 24 hr NTG-Veh animals to show significantly fewer branch points as compared to the Veh-Veh controls (Figure In contrast, the NTG-ACY group had a significantly number of compared to Interestingly, at 24 hr we no observed increased neuronal complexity in the as was observed at the 4 hr time (Figure results were observed for total neuronal length (Figure and interactions (Figure These data further the between cytoarchitectural complexity and decreased as at both 4 and 24 hr ACY-738 has 48 hr post-ACY-738 we observed that compared to the the number of branch points were significantly in both the and NTG-ACY groups (Figure and similar results were observed for total neuronal length (Figure the NTG-Veh group showed significantly fewer there was a in the NTG-ACY group (Figure these data show that the effects of with the at which it also restores neuronal complexity. We that this behavioral effect resulted from changes in HDAC6 inhibition. We tested two the inhibitor A Figure and a novel inhibitor, et al., 2017; Figure significantly reversed chronic NTG-induced for a much shorter than In contrast, when we tested the and 2 selective inhibitor, we not in NTG-induced chronic allodynia relative to vehicle controls (Figure These data further that chronic migraine-associated pain can be by and that this effect is not to acetylation of in the cell Figure 4 with 1 supplement see all Download asset Open asset but not inhibitor chronic migraine-associated and ACY-738 can of NTG induced allodynia. and female C57BL/6J mice were treated with chronic intermittent NTG (10 mg/kg IP) or Vehicle for 9 days. On day 10, mice were subsequently tested for baseline responses and then injected with were for NTG-treated mice demonstrating chronic allodynia. groups of mice were tested for (A) Mice were treated with the inhibitor A 2 mg/kg IP) or Vehicle DMSO in M IP) and subsequently tested 2 hr significantly inhibited chronic cephalic allodynia. p<0.0001, F = effect of chronic treatment, F = effect of drug, F = time, and F = interaction, ANOVA and Holm-Sidak post hoc analysis. ****p<0.0001, t = NTG-Veh relative to Veh-Veh at baseline ****p<0.0001, t = relative to Veh-Veh at baseline, t = NTG-Veh compared to Veh-Veh 2 hr time ++++p<0.0001, t = relative to at 2 hr time n = (B) Mice were treated with the novel inhibitor, at 1 or 10 mg/kg or vehicle and 0.9% Mice were subsequently tested in the region 4, and 24 hr at the 10 mg/kg had a significant effect at the 2 and 4 hr time point, and the 1 mg/kg had a significant effect only at the 4 hr time compared to the NTG-Veh of produced effect 24 hr after treatment compared to RM ANOVA and Holm-Sidak post hoc analysis, F = effect of treatment, F = drug, p<0.0001, F = time and p=0.0002, F = p<0.0001, F = effect of treatment, F = drug, F = time and p=0.0002, F = interaction, ****p<0.0001, t = NTG-Veh relative to Veh-Veh at baseline, ****p<0.0001, t = NTG-Veh relative to Veh-Veh at 2 hr time point, ****p<0.0001, t = NTG-Veh relative to Veh-Veh at 4 hr time point, ****p<0.0001, t = NTG-Veh relative to Veh-Veh at 24 hr time ++++p<0.0001, t = high relative to at 2 hr point, ++++p<0.0001, t = high relative to at 4 hr time point, ++++p<0.0001, t = relative to at 4 hr time n = 8/group. (C) chronic NTG/VEH treatment, mice were treated with the I inhibitor, mg/kg IP) or Vehicle and 0.9% NaCl, IP) and had mechanical thresholds taken at 2 and 24 hr to NTG-induced pain. and NTG-Veh treated mice both were significantly different than the Vehicle groups at both 2 and 24 hr time points, ANOVA and Holm-Sidak post hoc analysis p<0.0001, F = effect of chronic treatment, = compared to Veh-Veh at = Veh-Veh compared to at 2 hr time point, = Veh-Veh compared to at 24 hr time treated mice at same time n = (D) Mice were treated with ACY-738 which of basal allodynia. F = effect of NTG/Veh treatment, F = effect of p=0.0073, F = effect of F = effect of interaction of NTG/Veh and t = NTG-Veh day 5 compared to Veh-Veh day 1, t = NTG-Veh day 5 compared to Veh-Veh day 9 n = Figure data 1 Data Figure Download Figure data 2 Data Figure Download Figure data 3 Data Figure Download Figure data 4 Data Figure