Combining fecal 16 S rRNA sequencing and spinal cord metabolomics analysis to explain the modulatory effect of PPARα on neuropathic pain.

BACKGROUND: Existing evidence suggests that the composition of the gut microbiota is associated with neuropathic pain (NP), but the mechanistic link is elusive. Peroxisome proliferator-activated receptor α (PPARα) has been shown to be a pharmacological target for the treatment of metabolic disorders, and its expression is also involved in inflammatory regulation. The aim of this study was to investigate the important modulatory effects of PPARα on gut microbiota and spinal cord metabolites in mice subjected to chronic constriction injury. METHODS: We analyzed fecal microbiota and spinal cord metabolic alterations in mice from the sham, CCI, GW7647 (PPARα agonist) and GW6471 (PPARα antagonist) groups by 16 S rRNA amplicon sequencing and untargeted metabolomics analysis. On this basis, the intestinal microbiota and metabolites that were significantly altered between treatment groups were analyzed in a combined multiomics analysis. We also investigated the effect of PPARα on the polarization fractionation of spinal microglia. RESULTS: PPARα agonist significantly reduce paw withdrawal threshold and paw withdrawal thermal latency, while PPARα antagonist significantly increase paw withdrawal threshold and paw withdrawal thermal latency. 16 S rRNA gene sequencing showed that intraperitoneal injection of GW7647 or GW6471 significantly altered the abundance, homogeneity and composition of the gut microbiome. Analysis of the spinal cord metabolome showed that the levels of spinal cord metabolites were shifted after exposure to GW7647 or GW6471. Alterations in the composition of gut microbiota were significantly associated with the abundance of various spinal cord metabolites. The abundance of Licheniformes showed a significant positive correlation with nicotinamide, benzimidazole, eicosanoids, and pyridine abundance. Immunofluorescence results showed that intraperitoneal injection of GW7647 or GW6471 altered microglial activation and polarization levels. CONCLUSION: Our study shows that PPARα can promote M2-type microglia polarization, as well as alter gut microbiota and metabolites in CCI mice. This study enhances our understanding of the mechanism of PPARα in the treatment of neuropathic pain.

Alterations in the gut microbiota and metabolite profiles in the context of neuropathic pain.

The aim of this study was to explore the relationships among gut microbiota disturbances and serum and spinal cord metabolic disorders in neuropathic pain. 16S rDNA amplicon sequencing and serum and spinal cord metabolomics were used to identify alterations in the microbiota and metabolite profiles in the sham rats and the chronic constriction injury (CCI) model rats. Correlations between the abundances of gut microbiota components at the genus level, the levels of serum metabolites, and pain-related behavioural parameters were analysed. Ingenuity pathway analysis (IPA) was applied to analyse the interaction networks of the differentially expressed serum metabolites. First, we found that the composition of the gut microbiota was different between rats with CCI-induced neuropathic pain and sham controls. At the genus level, the abundances of Helicobacter, Phascolarctobacterium, Christensenella, Blautia, Streptococcus, Rothia and Lactobacillus were significantly increased, whereas the abundances of Ignatzschineria, Butyricimonas, Escherichia, AF12, and Corynebacterium were significantly decreased. Additionally, 72 significantly differentially expressed serum metabolites and 17 significantly differentially expressed spinal cord metabolites were identified between the CCI rats and the sham rats. Finally, correlation analysis showed that changes in the gut microbiota was significantly correlated with changes in serum metabolite levels, suggesting that dysbiosis of the gut microbiota is an important factor in modulating metabolic disturbances in the context of neuropathic pain. In conclusion, our research provides a novel perspective on the potential roles of the gut microbiota and related metabolites in neuropathic pain.

A network approach to investigating the key microbes and stability of gut microbial communities in a mouse neuropathic pain model.

BACKGROUND: Neuropathic pain is an abnormally increased sensitivity to pain, especially from mechanical or thermal stimuli. To date, the current pharmacological treatments for neuropathic pain are still unsatisfactory. The gut microbiota reportedly plays important roles in inducing neuropathic pain, so probiotics have also been used to treat it. However, the underlying questions around the interactions in and stability of the gut microbiota in a spared nerve injury-induced neuropathic pain model and the key microbes (i.e., the microbes that play critical roles) involved have not been answered. We collected 66 fecal samples over 2 weeks (three mice and 11 time points in spared nerve injury-induced neuropathic pain and Sham groups). The 16S rRNA gene was polymerase chain reaction amplified, sequenced on a MiSeq platform, and analyzed using a MOTHUR- UPARSE pipeline. RESULTS: Here we show that spared nerve injury-induced neuropathic pain alters gut microbial diversity in mice. We successfully constructed reliable microbial interaction networks using the Metagenomic Microbial Interaction Simulator (MetaMIS) and analyzed these networks based on 177,147 simulations. Interestingly, at a higher resolution, our results showed that spared nerve injury-induced neuropathic pain altered both the stability of the microbial community and the key microbes in a gut micro-ecosystem. Oscillospira, which was classified as a low-abundance and core microbe, was identified as the key microbe in the Sham group, whereas Staphylococcus, classified as a rare and non-core microbe, was identified as the key microbe in the spared nerve injury-induced neuropathic pain group. CONCLUSIONS: In summary, our results provide novel experimental evidence that spared nerve injury-induced neuropathic pain reshapes gut microbial diversity, and alters the stability and key microbes in the gut.

Gut microbiota regulates neuropathic pain: potential mechanisms and therapeutic strategy.

Neuropathic pain (NP) is a sustained and nonreversible condition characterized by long-term devastating physical and psychological damage. Therefore, it is urgent to identify an effective treatment for NP. Unfortunately, the precise pathogenesis of NP has not been elucidated. Currently, the microbiota-gut-brain axis has drawn increasing attention, and the emerging role of gut microbiota is investigated in numerous diseases including NP. Gut microbiota is considered as a pivotal regulator in immune, neural, endocrine, and metabolic signaling pathways, which participates in forming a complex network to affect the development of NP directly or indirectly. In this review, we conclude the current understanding of preclinical and clinical findings regarding the role of gut microbiota in NP and provide a novel therapeutic method for pain relief by medication and dietary interventions.

Leprosy piRnome: exploring new possibilities for an old disease.

Leprosy, which is caused by the human pathogen Mycobacterium leprae, causes nerve damage, deformity and disability in over 200,000 people every year. Because of the long doubling time of M. leprae (13 days) and the delayed onset of detectable symptoms, which is estimated to be approximately 3-7 years after infection, there is always a large percentage of subclinically infected individuals in the population who will eventually develop the disease, mainly in endemic countries. piRNAs comprise the largest group of small noncoding RNAs found in humans, and they are distinct from microRNAs (miRNAs) and small interfering RNAs (siRNAs). piRNAs function in transposon silencing, epigenetic regulation, and germline development. The functional role of piRNAs and their associated PIWI proteins have started to emerge in the development of human cancers and viral infections, but their relevance to bacterial diseases has not been investigated. The present study reports the piRNome of human skin, revealing that all but one of the piRNAs examined are downregulated in leprosy skin lesions. Considering that one of the best characterized functions of piRNAs in humans is posttranscriptional mRNA silencing, their functions are similar to what we have described for miRNAs, including acting on apoptosis, M. leprae recognition and engulfment, Schwann cell (SC) demyelination, epithelial-mesenchymal transition (EMT), loss of sensation and neuropathic pain. In addition to new findings on leprosy physiopathology, the discovery of relevant piRNAs involved in disease processes in human skin may provide new clues for therapeutic targets, specifically to control nerve damage, a prominent feature of leprosy that has no currently available pharmaceutical treatment.

Chronic aspects of leprosy-neglected but important.

The chronic aspects of leprosy are discussed here. They are a consequence of the peripheral nerve damage that affects many patients during their lifetime with leprosy. The peripheral nerve damage leaves people unable to feel and with weakness in their hands and feet. They are at risk of damaging their hands and feet, causing the disabilities and deformities that characterise late leprosy. More than 200 000 new leprosy patients are diagnosed globally each year. Better data are needed from cohort studies to estimate the number of patients developing nerve damage and modelling studies are needed to estimate the number of patients who develop disabilities. For some of them, this will be a lifelong disability. Nerve damage is caused by inflammation in leprosy-affected nerves. Patients with nerve damage of <6-mo duration need treatment with steroids. About 66% of multibacillary patients will develop nerve damage. Plastic graded monofilaments can be used to detect nerve damage in leprosy and diabetic clinics. Assessing nerve damage and treating patients with steroids in leprosy programmes needs to be strengthened. The World Health Organization has a successful programme for supplying antibiotics for treating leprosy infection to national leprosy programmes. They should take responsibility for providing steroids to national programmes since this is a core part of the treatment for >66% of multibacillary patients. Patients need to be asked about neuropathic pain symptoms and treated if necessary. Treated leprosy patients are at risk of developing ulcers in their feet. Treatment and prevention needs to be improved through health education, providing protective footwear and patient empowerment.

Key role of gut microbiota in anhedonia-like phenotype in rodents with neuropathic pain.

Patients with chronic neuropathic pain frequently suffer from symptoms of anhedonia, which is a core symptom of depression. Accumulating studies suggest that gut microbiota may play a role in depression via gut-microbiota-brain axis. However, it is unknown whether gut microbiota plays a role in neuropathic pain-associated anhedonia. Here, we used a rat model of spared nerve injury (SNI). Hierarchical cluster analysis of sucrose preference test (SPT) results was used to classify the SNI rats with or without anhedonia-like phenotype. The 16S ribosomal RNA sequencing analysis showed abnormal composition of gut microbiota in the anhedonia susceptible compared to sham-operated rats and resilient rats. Furthermore, antibiotics-treated mice showed pain as well as depression-like and anhedonia-like phenotypes, suggesting a role of gut microbiota in these abnormal behaviors. Transplantation of fecal microbiota from anhedonia susceptible rats into antibiotics-treated pseudo-germ-free mice significantly exaggerated pain and depression-like phenotypes, including anhedonia. In contrast, transplantation of fecal microbiota from resilient rats into antibiotics-treated pseudo-germ-free mice significantly improved pain and depression-like phenotypes, including anhedonia. In conclusion, this study suggests that abnormal composition of gut microbiota may contribute to anhedonia susceptibility post SNI surgery, and that gut microbiota also plays a role in the pain as well as depression-like phenotypes. Interestingly, fecal microbiota transplantation from SNI rats with or without anhedonia can alter pain, depression-like and anhedonia-like phenotypes in the pseudo-germ-free mice. Therefore, it is likely that gut microbiota plays a key role in the pain as well as depression-like phenotypes including anhedonia in rodents with neuropathic pain.

Effectiveness and safety of clofazimine and pentoxifylline in type 2 lepra reaction: a double-blind, randomized, controlled study.

BACKGROUND: Type 2 lepra reaction (T2R) is a difficult-to-manage condition in leprosy, and an effective and safe steroid-sparing agent is needed for its management. The World Health Organization proposes clofazimine and recommends pentoxifylline for T2R. Our study was done to compare the effectiveness and safety of clofazimine and pentoxifylline therapy in patients with T2R. METHODS: Twenty patients with T2R were randomized equally. Group A received pentoxifylline 400 mg t.d.s, group B received clofazimine 100 mg t.d.s. for 12 weeks. Both groups received prednisolone 40 mg o.d., tapered over 12 weeks. The effectiveness parameters were days needed for resolution of cutaneous and systemic manifestations, relapses, cutaneous score, systemic score, and average daily prednisolone intake. Safety parameters were spontaneously appearing adverse events and laboratory parameter changes. RESULTS: The cutaneous scores in the clofazimine (P < 0.001) and pentoxifylline groups (P < 0.001) showed a progressive decline in subsequent follow-ups. Individual follow-ups were significantly lower than baseline in both groups (P < 0.05). Systemic scores fared similarly. There were no significant intergroup changes. Average daily prednisolone intake progressively decreased in group B (P < 0.001). Cutaneous and systemic manifestations took a comparable time to resolve. Both drugs were safe. CONCLUSION: Pentoxifylline effectively reduces initial severity; clofazimine provides sustained improvement but acts slowly.

Prevalence and characteristics of neuropathic pain in leprosy patients treated years ago.

The aim of this study was to determine the prevalence of neuropathic pain, now recognized as another late complication of leprosy, and its characteristics among leprosy patients. A cross-sectional study was carried out of people treated for leprosy up to at least 5 years ago in a rural area of Ethiopia. Seventy-four patients were interviewed using the Neuropathic Pain Symptom Inventory (NPSI) questionnaire. In total, 78.9% of the patients were female with a mean age of 42.9. The mean time from initial diagnosis to the time of the study was 28.0 years, and 73.0% of patients were diagnosed over 20 years ago. Fifty-two (70.3%) reported having symptoms suggestive of neuropathic pain and the majority described the pain as burning (88.5%), electric (80.8%), stabbing (76.9%), cutting (76.9%), tingling (65.4%), squeezing (57.7%), and/or pressure (53.8%). The pain caused a severe or moderate impact on daily life in 75% and 57.7% of cases, respectively, and 92.3% suffered from disrupted sleep. Eighty percent of patients with pain (42/52) took some medication for pain relief. Neuropathic pain is common in patients treated for leprosy and in more than half of them, it causes disruption in their daily life and sleep, limiting their quality of life even more.

Tactile allodynia in patients with postherpetic neuralgia: lack of change in skin blood flow upon dynamic stimulation.

Tactile allodynia is a common, troublesome feature of neuropathic pain. Allodynia has been proposed to involve abnormal Abeta-afferent coupling in the dorsal horn resulting in C-fibre activation and increased skin blood flow (SBF). Thus, changes in SBF could provide an objective measure of allodynia. We searched for this mechanism in patients with postherpetic neuralgia (PHN) with varying degrees of cutaneous sensory loss. We mapped the allodynic area in PHN patients using cotton buds and von Frey hairs. Quantitative thermal testing was performed to assess small fibre function in the affected and mirror-image areas. At a subsequent visit the area of allodynia was remapped. Then the SBF in the affected and control areas was quantified before and after allodynic stimulation using laser Doppler imaging and subsequent single point continuous monitoring to detect rapid changes. We enrolled 10 PHN patients (medians: age 77 yrs, duration 20 months, ongoing pain 5). The allodynic area (range 11-546 cm2) was stable across the sessions. Thermal testing showed similar (n=5) or reduced (n=5) warmth and pain sensation in the affected versus control area. Following allodynic stimulation (median evoked pain-5) we saw no changes in SBF using either imaging (repeated measures ANOVA, P=0.73) or single point monitoring. This was the case for all patients regardless of the degree of sensory impairment in the affected dermatome. In conclusion, in a representative population of PHN patients we found no evidence of changes in SBF in response to allodynic stimulation. Hence, SBF measurements are not suitable for assessing allodynia.

[Herpes zoster and post-herpetic neuralgia. Comparison between elderly patients and young adults treated with acyclovir]. / Herpes zoster e nevralgia post-erpetica. Confronto tra anziani e giovani adulti trattati con acyclovir.

Herpes zoster (HZ) is a common skin disease due to a virus identical to that responsible for chickenpox. In a variable number of cases neuritic pain persist after cutaneous healing. Aim of this investigation was to analyze zoster clinical evolution in 102 immunocompetent patients, subdivided by age (< 60 years and > or = 60 years) and sex, after treatment with acyclovir (4 g/die x 10 days). Signs and symptoms of the disease were evaluated, with particular attention to pain and the duration of post-herpetic neuralgia. Vescicular eruption was most frequently found in the thoraco-abdominal region and in the trigeminal one, with no significant differences among the subgroups. Two thirds of the subjects complained of pain and it was prevalent in female sex (84% of cases vs 53%, p < 0.01) but not in any age-class. After 1 months from the episode (and its pharmacological treatment), post-herpetic neuralgia was still present in about 20% of the patients, above all in those > or = 60 years; this last difference reached statistical significance after 6 months (9.7% vs 1.4% for subjects > or = 60 years and < 60 years respectively, p < 0.05). No patient showed any adverse pharmacological effect after treatment. We conclude that acyclovir is well accepted both in young and elderly immune-competent subjects suffering from HZ, but it necessitates further efficacy investigations in sight of its broader utilization.

Treatment of the cutaneous pain of acute herpes zoster with 9% lidocaine (base) in petrolatum/paraffin ointment.

BACKGROUND: Treatment of the pain of acute herpes zoster by local anesthetic injections has drawbacks. Topical percutaneous local anesthesia (TPLA) may offer another strategy of providing regional analgesia in affected patients. OBJECTIVE: We evaluate the analgesic efficacy and safety of 9% (wt/vol) lidocaine (base) in petrolatum/paraffin ointment in patients with acute herpes zoster. METHODS: Ointment was applied to the affected skin of 22 patients. Pain, tenderness, sensitivity to pinprick and cold, and blood lidocaine concentration were measured repeatedly during a 20-hour interval and intermittently thereafter. RESULTS: Mean pain, tenderness, and cutaneous sensation scores were reduced at measurements taken from 4 to 20 hours after ointment application (p < 0.05), but not every patient obtained relief. No patient had local skin irritation or systemic toxic effects related to the local anesthetic. CONCLUSIONS: TPLA is a promising therapy for control of cutaneous pain of acute herpes zoster. Controlled studies should be performed to prove efficacy, determine optimal TPLA formulation, and define dosage limits.

Pharmacologic management of herpes zoster and postherpetic neuralgia.

Herpes zoster is an infection caused by reactivation of dormant varicella-zoster virus. The acute course of herpes zoster is generally benign; however, some patients will experience postherpetic neuralgia characterized by severe, relentless, and at times disabling pain that is often refractory to treatment. While herpes zoster responds to acyclovir, cost-benefit considerations limit the drug's usefulness to only a select group. Postherpetic neuralgia requires a holistic approach, including pharmacologic therapy using several different classes of drugs.

Postherpetic neuralgia. Are C-nociceptors involved in signalling and maintenance of tactile allodynia?

Under normal conditions acute stimulation and sensitization of polymodal nociceptive C-fibres cause pain and, due to afferent axon reflex activation, a local skin vasodilatation, flare reaction and skin temperature increase. Two questions arise: (i) Do sensitized C-nociceptors signal allodynia in chronic postherpetic neuralgia? (ii) If not, does ongoing peripheral nociceptive C-fibre input maintain a central process that accounts for allodynia? Ten patients with postherpetic neuralgia and tactile allodynia and 10 control subjects were studied using a laser Doppler perfusion monitor. Peripheral nociceptive C-fibre function was assessed by quantitative measurement of the axon reflex vasodilatation and flare reaction induced by histamine iontophoresis and compared with non-neural vasodilatation induced by local skin heating. Resting skin temperature, skin resistance and resting skin blood flow were the same in the allodynic area and the contralateral homologous skin area. The histamine responses (vasodilatation and flare) were significantly reduced or nearly abolished in the allodynic area compared with the contralateral side, whereas the temperature-dependent vasodilatation in patients and the histamine responses in healthy controls showed no side differences. C-fibre mediated pain and itch sensations were also decreased in the allodynic area. These findings indicate a considerable impairment of cutaneous nociceptive C-fibre function in the allodynic area. Allodynic stimuli of 20 s did not cause any local blood flow change. Impairment of C-fibre function was positively correlated with intensity of neuropathic pain. We conclude that sensitized nociceptive C-fibres are not involved in signalling allodynia. Changes in CNS processing may occur after zoster infection that strengthen the synaptic ties between central pain signalling pathways and low-threshold mechanoreceptors with A beta-fibres. This altered central processing is not maintained by ongoing cutaneous nociceptive C-fibre input, at least in some patients with postherpetic neuralgia. On the contrary, an anatomical synaptic reorganization depending on afferent C-fibre degeneration seems to be more likely, particularly in advanced stages of postherpetic neuralgia.

A retrospective and an observational study with acyclovir.

Retrospective analysis: This open controlled non-randomized study was carried out to investigate the influence of intravenous acyclovir (ACV) on the incidence of post-herpetic neuralgia (PHN). Twelve women and 11 men (mean age 52 years, range 19-89) received ACV 5 mg/kg every 8 hours) for 10 days (I). Twenty-seven untreated patients (mean age 62 years, range 20-89) were taken as a control group (II). Six to 24 months after the onset of herpes zoster (shingles) the patients were reexamined. The analysis revealed a significantly lower incidence of both general pain and severe pain (P < 0.05, chi 2 = 5.55 and 4.39) for (I) compared to (II). For 21 patients who were treated for a period of 10 days, the significance level was 1% (chi 2 = 7.82 and 8.62). Observational study: Fifteen thousand eight hundred and thirty-one non-hospitalized patients with shingles (mean age 55.2 years) received oral ACV (800 mg five times daily) for 7 days. At the onset of therapy, 15,420 patients (97.6%) reported pain (severe 42.6%, moderate 43.1%, mild 14.3%). The pain during treatment was documented by the patients (n = 5,728) in a diary and transferred to a scoring system (0 = none, 1 = mild, 2 = moderate, 3 = severe). From day 1 to day 7 there was a decrease in the pain score level from 2.3 to 0.9. Three months after the onset of herpes zoster, 2,519 of 14,858 patients (16.95%) reported pain; 311 patients (2.1%) complained of continuous pain, typical for PHN.(ABSTRACT TRUNCATED AT 250 WORDS)