Integration of polygenic and gut metagenomic risk prediction for common diseases.

Multiomics has shown promise in noninvasive risk profiling and early detection of various common diseases. In the present study, in a prospective population-based cohort with ~18 years of e-health record follow-up, we investigated the incremental and combined value of genomic and gut metagenomic risk assessment compared with conventional risk factors for predicting incident coronary artery disease (CAD), type 2 diabetes (T2D), Alzheimer disease and prostate cancer. We found that polygenic risk scores (PRSs) improved prediction over conventional risk factors for all diseases. Gut microbiome scores improved predictive capacity over baseline age for CAD, T2D and prostate cancer. Integrated risk models of PRSs, gut microbiome scores and conventional risk factors achieved the highest predictive performance for all diseases studied compared with models based on conventional risk factors alone. The present study demonstrates that integrated PRSs and gut metagenomic risk models improve the predictive value over conventional risk factors for common chronic diseases.

Role of Gut Microbiota in Statin-Associated New-Onset Diabetes-A Cross-Sectional and Prospective Analysis of the FINRISK 2002 Cohort.

BACKGROUND: Dyslipidemia is treated effectively with statins, but treatment has the potential to induce new-onset type-2 diabetes. Gut microbiota may contribute to this outcome variability. We assessed the associations of gut microbiota diversity and composition with statins. Bacterial associations with statin-associated new-onset type-2 diabetes (T2D) risk were also prospectively evaluated. METHODS: We examined shallow-shotgun-sequenced fecal samples from 5755 individuals in the FINRISK-2002 population cohort with a 17+-year-long register-based follow-up. Alpha-diversity was quantified using Shannon index and beta-diversity with Aitchison distance. Species-specific differential abundances were analyzed using general multivariate regression. Prospective associations were assessed with Cox regression. Applicable results were validated using gradient boosting. RESULTS: Statin use associated with differing taxonomic composition (R2, 0.02%; q=0.02) and 13 differentially abundant species in fully adjusted models (MaAsLin; q<0.05). The strongest positive association was with Clostridium sartagoforme (ß=0.37; SE=0.13; q=0.02) and the strongest negative association with Bacteroides cellulosilyticus (ß=-0.31; SE=0.11; q=0.02). Twenty-five microbial features had significant associations with incident T2D in statin users, of which only Bacteroides vulgatus (HR, 1.286 [1.136-1.457]; q=0.03) was consistent regardless of model adjustment. Finally, higher statin-associated T2D risk was seen with [Ruminococcus] torques (ΔHRstatins, +0.11; q=0.03), Blautia obeum (ΔHRstatins, +0.06; q=0.01), Blautia sp. KLE 1732 (ΔHRstatins, +0.05; q=0.01), and beta-diversity principal component 1 (ΔHRstatin, +0.07; q=0.03) but only when adjusting for demographic covariates. CONCLUSIONS: Statin users have compositionally differing microbiotas from nonusers. The human gut microbiota is associated with incident T2D risk in statin users and possibly has additive effects on statin-associated new-onset T2D risk.

Novel RET agonist for the treatment of experimental neuropathies.

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) alleviate symptoms of experimental neuropathy, protect and stimulate regeneration of sensory neurons in animal models of neuropathic pain, and restore their functional activity. However, clinical development of GFL proteins is complicated by their poor pharmacokinetic properties and multiple effects mediated by several receptors. Previously, we have identified a small molecule that selectively activates the major signal transduction unit of the GFL receptor complex, receptor tyrosine kinase RET, as an alternative to GFLs, for the treatment of neuropathic pain. We then introduced a series of chemical changes to improve the biological activity of these compounds and tested an optimized compound named BT44 in a panel of biological assays. BT44 efficiently and selectively stimulated the GFL receptor RET and activated the intracellular mitogene-activated protein kinase/extracellular signal-regulated kinase pathway in immortalized cells. In cultured sensory neurons, BT44 stimulated neurite outgrowth with an efficacy comparable to that of GFLs. BT44 alleviated mechanical hypersensitivity in surgery- and diabetes-induced rat models of neuropathic pain. In addition, BT44 normalized, to a certain degree, the expression of nociception-related neuronal markers which were altered by spinal nerve ligation, the neuropathy model used in this study. Our results suggest that the GFL mimetic BT44 is a promising new lead for the development of novel disease-modifying agents for the treatment of neuropathy and neuropathic pain.

Genetic variation in P2RX7 and pain tolerance.

P2X7 is a nonselective cation channel activated by extracellular ATP. P2X7 activation contributes to the proinflammatory response to injury or bacterial invasion and mediates apoptosis. Recently, P2X7 function has been linked to chronic inflammatory and neuropathic pain. P2X7 may contribute to pain modulation both by effects on peripheral tissue injury underlying clinical pain states, and through alterations in central nervous system processing, as suggested by animal models. To further test its role in pain sensitivity, we examined whether variation within the P2RX7 gene, which encodes the P2X7 receptor, was associated with experimentally induced pain in human patients. Experimental pain was assessed in Tromsø 6, a longitudinal and cross-sectional population-based study (N = 3016), and the BrePainGen cohort, consisting of patients who underwent breast cancer surgery (N = 831). For both cohorts, experimental pain intensity and tolerance were assessed with the cold-pressor test. In addition, multisite chronic pain was assessed in Tromsø 6 and pain intensity 1 week after surgery was assessed in BrePainGen. We tested whether the single-nucleotide polymorphism rs7958311, previously implicated in clinical pain, was associated with experimental and clinical pain phenotypes. In addition, we examined effects of single-nucleotide polymorphisms rs208294 and rs208296, for which previous results have been equivocal. Rs7958311 was associated with experimental pain intensity in the meta-analysis of both cohorts. Significant associations were also found for multisite pain and postoperative pain. Our results strengthen the existing evidence and suggest that P2X7 and genetic variation in the P2RX7-gene may be involved in the modulation of human pain sensitivity.

A Novel Small Molecule GDNF Receptor RET Agonist, BT13, Promotes Neurite Growth from Sensory Neurons in Vitro and Attenuates Experimental Neuropathy in the Rat.

Neuropathic pain caused by nerve damage is a common and severe class of chronic pain. Disease-modifying clinical therapies are needed as current treatments typically provide only symptomatic relief; show varying clinical efficacy; and most have significant adverse effects. One approach is targeting either neurotrophic factors or their receptors that normalize sensory neuron function and stimulate regeneration after nerve damage. Two candidate targets are glial cell line-derived neurotrophic factor (GDNF) and artemin (ARTN), as these GDNF family ligands (GFLs) show efficacy in animal models of neuropathic pain (Boucher et al., 2000; Gardell et al., 2003; Wang et al., 2008, 2014). As these protein ligands have poor drug-like properties and are expensive to produce for clinical use, we screened 18,400 drug-like compounds to develop small molecules that act similarly to GFLs (GDNF mimetics). This screening identified BT13 as a compound that selectively targeted GFL receptor RET to activate downstream signaling cascades. BT13 was similar to NGF and ARTN in selectively promoting neurite outgrowth from the peptidergic class of adult sensory neurons in culture, but was opposite to ARTN in causing neurite elongation without affecting initiation. When administered after spinal nerve ligation in a rat model of neuropathic pain, 20 and 25 mg/kg of BT13 decreased mechanical hypersensitivity and normalized expression of sensory neuron markers in dorsal root ganglia. In control rats, BT13 had no effect on baseline mechanical or thermal sensitivity, motor coordination, or weight gain. Thus, small molecule BT13 selectively activates RET and offers opportunities for developing novel disease-modifying medications to treat neuropathic pain.

Do Diuretics have Antinociceptive Actions: Studies of Spironolactone, Eplerenone, Furosemide and Chlorothiazide, Individually and with Oxycodone and Morphine.

Spironolactone, eplerenone, chlorothiazide and furosemide are diuretics that have been suggested to have antinociceptive properties, for example via mineralocorticoid receptor antagonism. In co-administration, diuretics might enhance the antinociceptive effect of opioids via pharmacodynamic and pharmacokinetic mechanisms. Effects of spironolactone (100 mg/kg, i.p.), eplerenone (100 mg/kg, i.p.), chlorothiazide (50 mg/kg, i.p.) and furosemide (100 mg/kg, i.p.) were studied on acute oxycodone (0.75 mg/kg, s.c.)- and morphine (3 mg/kg, s.c.)-induced antinociception using tail-flick and hot plate tests in male Sprague Dawley rats. The diuretics were administered 30 min. before the opioids, and behavioural tests were performed 30 and 90 min. after the opioids. Concentrations of oxycodone, morphine and their major metabolites in plasma and brain were quantified by mass spectrometry. In the hot plate test at 30 and 90 min., spironolactone significantly enhanced the antinociceptive effect (% of maximum possible effect) of oxycodone from 10% to 78% and from 0% to 50%, respectively, and that of morphine from 12% to 73% and from 4% to 83%, respectively. The brain oxycodone and morphine concentrations were significantly increased at 30 min. (oxycodone, 46%) and at 90 min. (morphine, 190%). We did not detect any independent antinociceptive effects with the diuretics. Eplerenone and chlorothiazide did not enhance the antinociceptive effect of either opioid. The results suggest that spironolactone enhances the antinociceptive effect of both oxycodone and morphine by increasing their concentrations in the central nervous system.

Structural and functional interactions between six-transmembrane µ-opioid receptors and ß2-adrenoreceptors modulate opioid signaling.

The primary molecular target for clinically used opioids is the µ-opioid receptor (MOR). Besides the major seven-transmembrane (7TM) receptors, the MOR gene codes for alternatively spliced six-transmembrane (6TM) isoforms, the biological and clinical significance of which remains unclear. Here, we show that the otherwise exclusively intracellular localized 6TM-MOR translocates to the plasma membrane upon coexpression with ß2-adrenergic receptors (ß2-ARs) through an interaction with the fifth and sixth helices of ß2-AR. Coexpression of the two receptors in BE(2)-C neuroblastoma cells potentiates calcium responses to a 6TM-MOR ligand, and this calcium response is completely blocked by a selective ß2-antagonist in BE(2)-C cells, and in trigeminal and dorsal root ganglia. Co-administration of 6TM-MOR and ß2-AR ligands leads to substantial analgesic synergy and completely reverses opioid-induced hyperalgesia in rodent behavioral models. Together, our results provide evidence that the heterodimerization of 6TM-MOR with ß2-AR underlies a molecular mechanism for 6TM cellular signaling, presenting a unique functional responses to opioids. This signaling pathway may contribute to the hyperalgesic effects of opioids that can be efficiently blocked by ß2-AR antagonists, providing a new avenue for opioid therapy.

Pain in 1,000 women treated for breast cancer: a prospective study of pain sensitivity and postoperative pain.

BACKGROUND: This article describes the methods and results of the early part (experimental pain tests and postoperative analgesia) of a study that assesses genetic and other factors related to acute pain and persistent pain after treatment of breast cancer in a prospective cohort of 1,000 women. METHODS: One thousand consenting patients were recruited to the study. Before surgery (breast resection or mastectomy with axillary surgery), the patients filled in questionnaires about health, life style, depression (Beck Depression Inventory), and anxiety (State-Trait Anxiety Inventory). They were also exposed to experimental tests measuring heat (43° and 48°C, 5 s) and cold (2-4°C) pain intensity and tolerance. Anesthesia was standardized with propofol and remifentanil, and postoperative analgesia was optimized with i.v. oxycodone. RESULTS: The patients showed significant interindividual variation in heat and cold pain sensitivity and cold pain tolerance. There was a strong correlation between the experimental pain measures across the tests. Presence of chronic pain, the number of previous operations, and particularly state anxiety were related to increased pain sensitivity. Previous smoking correlated with decreased heat pain sensitivity. These factors explained 4-5% of the total variance in pain sensitivity in these tests. Oxycodone consumption during 20 h was significantly higher in patients who had axillary clearance. Oxycodone consumption had only a weak correlation with the experimental pain measures. CONCLUSIONS: Contact heat and cold pressure tests identify variability in pain sensitivity which is modified by factors such as anxiety, chronic pain, previous surgery, and smoking. High levels of anxiety are connected to increased pain sensitivity in experimental and acute postoperative pain.

Effect of catechol-o-methyltransferase-gene (COMT) variants on experimental and acute postoperative pain in 1,000 women undergoing surgery for breast cancer.

BACKGROUND: Catechol-O-methyltransferase (COMT) metabolizes catecholamines in different tissues. Polymorphisms in COMT gene can attenuate COMT activity and increase sensitivity to pain. Human studies exploring the effect of COMT polymorphisms on pain sensitivity have mostly included small, heterogeneous samples and have ignored several important single nucleotide polymorphisms (SNPs). This study examines the effect of COMT polymorphisms on experimental and postoperative pain phenotypes in a large ethnically homogeneous female patient cohort. METHODS: Intensity of cold (+2-4°C) and heat (+48°C) pain and tolerance to cold pain were assessed in 1,000 patients scheduled for breast cancer surgery. Acute postoperative pain and oxycodone requirements were recorded. Twenty-two COMT SNPs were genotyped and their association with six pain phenotypes analyzed with linear regression. RESULTS: There was no association between any of the tested pain phenotypes and SNP rs4680. The strongest association signals were seen between rs165774 and heat pain intensity as well as rs887200 and cold pain intensity. In both cases, minor allele carriers reported less pain. Neither of these results remained significant after strict multiple testing corrections. When analyzed further, the effect of rs887200 was, however, shown to be significant and consistent throughout the cold pressure test. No evidence of association between the SNPs and postoperative oxycodone consumption was found. CONCLUSIONS: SNPs rs887200 and rs165774 located in the untranslated regions of the gene had the strongest effects on pain sensitivity. Their effect on pain is described here for the first time. These results should be confirmed in further studies and the potential functional mechanisms of the variants studied.

Transient proteolytic modification of mesenchymal stromal cells increases lung clearance rate and targeting to injured tissue.

Systemic infusion of therapeutic cells would be the most practical and least invasive method of administration in many cellular therapies. One of the main obstacles especially in intravenous delivery of cells is a massive cell retention in the lungs, which impairs homing to the target tissue and may decrease the therapeutic outcome. In this study we showed that an alternative cell detachment of mesenchymal stromal/stem cells (MSCs) with pronase instead of trypsin significantly accelerated the lung clearance of the cells and, importantly, increased their targeting to an area of injury. Cell detachment with pronase transiently altered the MSC surface protein profile without compromising cell viability, multipotent cell characteristics, or immunomodulative and angiogenic potential. The transient modification of the cell surface protein profile was sufficient to produce effective changes in cell rolling behavior in vitro and, importantly, in the in vivo biodistribution of the cells in mouse, rat, and porcine models. In conclusion, pronase detachment could be used as a method to improve the MSC lung clearance and targeting in vivo. This may have a major impact on the bioavailability of MSCs in future therapeutic regimes.

Intrathecal atipamezole augments the antinociceptive effect of morphine in rats.

BACKGROUND: Opioid analgesics are effective in the treatment of chronic pain, but they have serious adverse effects such as development of tolerance and dependence. Adrenergic α(2) agonists and µ-opioid receptor agonists show synergistic potentiation and cross-tolerance in spinal analgesia, whereas α(2)-adrenergic antagonists have shown pronociceptive effects. However, at ultralow doses, spinal α(2)-adrenergic antagonists have been reported to paradoxically enhance opioid antinociception. New data have suggested a functional µ-opioid-α(2)-adrenoceptor complex, which may help in interpreting the paradoxical effect of the α(2)-adrenergic antagonists. In the present study we assessed the effects of low doses of atipamezole, a nonselective α(2)-adrenergic antagonist, on both systemic and spinal morphine antinociception and tolerance. METHODS: Antinociception was assessed in male Sprague-Dawley rats using hotplate, tail-flick, and paw pressure tests. Spinal or systemic opioid tolerance was induced for 4 days. The effects of both intrathecal and subcutaneous atipamezole on acute morphine-induced antinociception and established morphine tolerance were studied. RESULTS: Systemic or spinal atipamezole itself did not produce antinociception at the doses studied (subcutaneous 0.03, 0.3, 3 µg/kg or intrathecal 0.1, 1, 10 ng). The combined administration of spinal morphine and 1 ng of atipamezole increased the antinociceptive effect of acute spinal morphine 30 minutes after the administration of test drugs in the tail-flick test. Furthermore, 10 ng of intrathecal atipamezole attenuated established morphine tolerance 30 minutes after the administration of test drugs in the tail-flick test. However, subcutaneous atipamezole had no significant effect on systemic morphine antinociception, and it did not attenuate morphine tolerance. CONCLUSIONS: Spinal coadministration of low doses of atipamezole augmented the antinociceptive effect of morphine in naïve and tolerant rats. Heterodimerization of µ-opioid- and α(2A)-adrenoceptors with consequent changes in function and interaction could explain these results. This also suggests an interesting explanation for the variability in opioid response and tolerance in patients experiencing stress or having an increased noradrenergic tone due to other causes, e.g., drugs.

Nitecapone reduces development and symptoms of neuropathic pain after spinal nerve ligation in rats.

Neuropathic pain is caused by damage or malfunctioning of the nervous system. It is fairly common and more resistant to treatment than other types of pain. Since nitecapone, an inhibitor of catechol-O-methyl-transferase (COMT), has decreased neuropathic symptoms in diabetic rats, we studied its effects in another model of neuropathic pain, the spinal nerve ligation (SNL) model. Spinal nerves L5-6 were ligated in male Wistar rats under anaesthesia to produce the SNL model of neuropathic pain. Nitecapone (30 mg/kg, i.p.) or vehicle was administered once daily starting either 1h before or 2 days after surgery and continued for 14-19 days. Threshold for mechanical allodynia was measured with the digital von Frey test and responses to cold stimuli with the acetone test, before surgery and every other day after it 1h before drug administration. Mechanical and cold allodynia developed in all study groups. Both nitecapone treatments significantly reduced mechanical allodynia and withdrawal thresholds were 80-95% higher compared with the control group. In the acetone test, both nitecapone groups also showed less signs of cold allodynia than the control groups. In nitecapone-naïve animals a single dose of nitecapone also reduced mechanical allodynia on the 14th day after the surgery. Nitecapone reduced the symptoms of neuropathic pain after the SNL, which is in line with the earlier study. Our results suggest that nitecapone and other COMT inhibitors should be studied further in the treatment of neuropathic pain.

Catechol-O-methyltransferase and pain.

In animals, different types of COMT inhibitors, irrespective of their brain penetration, are pro-nociceptive in several models of acute and inflammatory pain. Similarly, COMT knock-out mice are more sensitive to nociceptive stimuli, whereas in mice over-expressing a high activity COMT variant nociceptive sensitivity is decreased. COMT knock-out mice also show altered response to opioids and stress-induced analgesia. In different rat models of neuropathic pain, the action of nitecapone is opposite: it is antinociceptive and antiallodynic. Complex actions of low COMT activity may be caused by enhanced adrenergic and dopaminergic activities that play different and even contrasting roles at different parts of the nociceptive system. Also compensatory changes in other neurotransmitters may occur. Pro-nociceptive effects seem to be caused by increased activation of peripheral adrenergic ß(2)- and ß(3) -receptors. Other properties of COMT inhibitors, like scavenging of oxygen and nitrogen radicals, may be important in antiallodynic effects found in neuropathic pain models. Increased number of µ-opioid receptors in certain brain areas may be responsible of enhanced opioid effects associated with a low COMT activity. In human pain studies, a low COMT activity is often associated with increased pain sensitivity in experimental pain models and with increased pre- and postoperative pain in acute clinical situations. As a rule, a simultaneous occurrence of several SNPs within the haplotype, causing low COMT activity, is more often associated with pain than any single SNP alone. In experimental pain studies, all negative findings resulted from concentrating solely on SNP rs4680 (Val158Met). Virtually all studies assessing haplotypes were able to confirm an association of a low COMT and increased pain. In chronic clinical pain, the effect of COMT polymorphisms depends on the pain conditions. Hence, in neuropathic and cancer pains, COMT activity is meaningless but in some chronic musculoskeletal pain conditions and migraine or headache low COMT activity appears to increase incidence and symptoms. A low COMT activity also increases availability of opioid receptors and may enhance opioid analgesia and adverse effects at least in cancer pains.

Effect of S-COMT deficiency on behavior and extracellular brain dopamine concentrations in mice.

INTRODUCTION: Catechol-O-methyltransferase (COMT) has soluble (S-COMT) and membrane bound (MB-COMT) isoforms. Our aims were to assess the behavioral phenotype of S-COMT mutant mice and to clarify the role of MB-COMT in dopamine metabolism in different brain areas. METHODS: Behavioral phenotype of the S-COMT mutant mice was assessed using a test battery designed to describe anxiety phenotype, spontaneous locomotor activity, sensorymotor gating, social behavior, and pain sensitivity. Microdialysis was used to explore the effect of S-COMT deficiency on extracellular dopamine under an L: -dopa load (carbidopa /L: -dopa 30/10 mg/kg i.p.). RESULTS: In behavioral tests, mature adult S-COMT mutants that only possessed MB-COMT exhibited enhanced acoustic startle without alterations in sensorimotor gating. They also showed barbering of vibrissae and nonaggressive social dominance, suggesting a change in their social interactions. In addition, S-COMT deficiency slightly and sex-dependently affected spinal pain reflex and the effect of morphine on hot-plate latency. In microdialysis studies under L: -dopa load, S-COMT mutants of both sexes had higher accumbal dopamine levels, but male S-COMT mutant mice showed paradoxically lower prefrontal cortical dopamine concentrations than wild-type animals. S-COMT deficiency induced the accumulation of 3,4-dihydroxyphenylacetic acid in all brain areas, which was accentuated after L: -dopa loading. The lack of S-COMT decreased extracellular homovanillic acid levels. However, after L: -dopa loading, homovanillic acid concentrations in the prefrontal cortex of S-COMT mutants were similar to those of wild-type mice. CONCLUSION: A lack of S-COMT has a notable, albeit small, brain-area and sex-dependent effect on the O-methylation of dopamine and 3,4-dihydroxyphenylacetic acid in the mouse brain. It also induces subtle changes in mouse social interaction behaviors and nociception.

Modulation of morphine-induced antinociception in acute and chronic opioid treatment by ibudilast.

BACKGROUND: Opioid analgesics are effective in relieving chronic pain, but they have serious adverse effects, including development of tolerance and dependence. Ibudilast, an inhibitor of glial activation and cyclic nucleotide phosphodiesterases, has shown potential in the treatment of neuropathic pain and opioid withdrawal. Because glial cell activation could also be involved in the development of opioid tolerance in rats, the authors studied the antinociceptive effects of ibudilast and morphine in different models of coadministration. METHODS: Antinociception was assessed using male Sprague- Dawley rats in hot plate and tail-flick tests. The effects of ibudilast on acute morphine-induced antinociception, induction of morphine tolerance, and established morphine tolerance were studied. RESULTS: Systemic ibudilast produced modest dose-related antinociception and decreased locomotor activity at the studied doses of 2.5-22.5 mg/kg. The highest tested dose of 22.5 mg/kg produced 52% of the maximum possible effect in the tail-flick test. It had an additive antinociceptive effect when combined with systemic morphine. Coadministration of ibudilast with morphine did not attenuate the development of morphine tolerance. However, in morphine-tolerant rats, ibudilast partly restored morphine-induced antinociception. CONCLUSIONS: Ibudilast produces modest antinociception, and it is effective in restoring but not in preventing morphine tolerance. The mechanisms of the effects of ibudilast should be better understood before it is considered for clinical use.

Stress-induced analgesia and morphine responses are changed in catechol-O-methyltransferase-deficient male mice.

Catechol-O-methyltransferase (COMT) polymorphisms modulate pain and opioid analgesia in human beings. It is not clear how the effects of COMT are mediated and only few relevant animal studies have been performed. Here, we used old male Comt gene knock-out mice as an animal model to study the effects of COMT deficiency on nociception that was assessed by the hot plate and tail flick tests. Stress-induced analgesia was achieved by forced swim. Morphine antinociception was measured after 10 mg/kg of morphine subcutaneously. Morphine tolerance was produced with subcutaneous morphine pellets and withdrawal provoked with subcutaneous naloxone. In the hot plate test, morphine-induced antinociception was significantly greater in the COMT knock-out mice, compared to the wild-type mice. This may be due to increased availability of opioid receptors as suggested by previous human studies. In the tail flick test, opioid-mediated stress-induced analgesia was absent and morphine-induced analgesia was decreased in COMT knock-out mice. In the hot plate test, stress-induced analgesia developed to all mice regardless of the COMT genotype. There were no differences between the genotypes in the baseline nociceptive thresholds, morphine tolerance and withdrawal. Our findings show, for the first time, the importance of COMT activity in stress- and morphine-induced analgesia in mice. COMT activity seems to take part in the modulation of nociception not only in the brain, as suggested earlier, but also at the spinal/peripheral level.