Andrographolide relieved pathological pain generated by spared nerve injury model in mice.
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ABSTRACT: Andrographolide (Andro), found in large quantities in Andrographis paniculata Nees (Acanthaceae), is anti-inflammatory, especially in the central nervous system (CNS) glia.The objective of this study is to test Andro's ability to reduce allodynia in a spared nerve injury model.Male 30?g BalbC mice were divided into four groups: (1) Sham-operated control (Sham-group); (2) nerve injured and treated with saline (Saline-group); (3) nerve injured and treated with Andro (Andro-group); (4) nerve injured and treated with non-steroidal anti-inflammatory drugs (NSAIDS) (NSAIDS-group). Andro or NSAIDS (diclofenac salt) were injected intraperitoneally at 5?mg/kg body weight daily. Mechanical allodynia was assessed by von Frey tests at 3, 7, and 14?d. For immunohistochemical analysis, samples were collected at 7?d.The threshold for inducing allodynia increased and the response percentage reduced in the Andro-group when compared with the Saline-group, as well as when compared with NSAIDS groups throughout 3-14?d. The ratio of threshold for OP-Andro/OP-saline and for OP-Andro/OP-NSAIDS groups was 20.42 and 11.67 at 14?d, respectively. The ratio of response percentage for OP-Andro/OP-saline and for OP-Andro/OP-NSAIDS was 0.32 and 0.39 at 14?d, respectively. Interleukin-1 (IL-1) immunostaining in the spinal cord was reduced in the Andro-group. Astrocytic activities were not significantly reduced in the Andro-group compared with the Saline-group at 7?d post-operation (PO) Conclusions: Andro reduced mechanical allodynia more than NSAIDS at the same concentration, and the observed behaviour was associated with a reduction in inflammatory cytokine produced in the spinal cord.
<h4>Context</h4>Andrographolide (Andro), found in large quantities in Andrographis paniculata Nees (Acanthaceae), is anti-inflammatory, especially in the central nervous system (CNS) glia.<h4>Objective</h4>The objective of this study is to test Andro's ability to reduce allodynia in a spared nerve injury model.<h4>Material and methods</h4>Male 30 g BalbC mice were divided into four groups: (1) Sham-operated control (Sham-group); (2) nerve injured and treated with saline (Saline-group); (3) nerve ...[more]
Project description:Neuropathic pain is a pathological condition induced by a lesion or disease affecting the somatosensory system, with symptoms like allodynia and hyperalgesia. It has a multifaceted pathogenesis as it implicates several molecular signaling pathways involving peripheral and central nervous systems. Affective and cognitive dysfunctions have been reported as comorbidities of neuropathic pain states, supporting the notion that pain and mood disorders share some common pathogenetic mechanisms. The understanding of these pathophysiological mechanisms requires the development of animal models mimicking, as far as possible, clinical neuropathic pain symptoms. Among them, the Spared Nerve Injury (SNI) model has been largely characterized in terms of behavioral and functional alterations. This model is associated with changes in neuronal firing activity at spinal and supraspinal levels, and induces late neuropsychiatric disorders (such as anxious-like and depressive-like behaviors, and cognitive impairments) comparable to an advanced phase of neuropathy. The goal of this review is to summarize current findings in preclinical research, employing the SNI model as a tool for identifying pathophysiological mechanisms of neuropathic pain and testing pharmacological agent.
Project description:Exosomes are 30-150 nm extracellular vesicles mediating intercellular communication. Disease states can alter exosome composition affecting the message carried and thereby, its functional impact. The objective of this study was to identify proteins present in these vesicles in a mouse model of neuropathic pain induced by spared nerve injury (SNI). Small extracellular vesicles (sEVs) were purified from serum four weeks after SNI surgery and the protein composition was determined using tandem mass spectrometry and cytokine array. Proteomic analysis detected 274 gene products within sEVs. Of these, 24 were unique to SNI model, 100 to sham surgery control and five to naïve control samples. In addition to commonly expressed sEVs proteins, multiple members of serpin and complement family were detected in sEVs. Cytokine profiling using a membrane-based antibody array showed significant upregulation of complement component 5a (C5a) and Intercellular Adhesion Molecule 1 (ICAM-1) in sEVs from SNI model compared to sham control. We observed a differential distribution of C5a and ICAM-1 within sEVs and serum between sham and SNI, indicating changes from local or paracrine to long distance signaling under neuropathic pain. Our studies suggest critical roles for cargo sorting of vesicular proteins in mediating signaling mechanisms underlying neuropathic pain. SIGNIFICANCE: Approximately 100 million U.S. adults are burdened by chronic pain. Neuropathic pain resulting from injury or dysfunction of the nervous system is challenging to treat. Unlike acute pain that resolves over time, chronic pain persists resulting in changes in the peripheral and central nervous system. The transport of biomolecular cargo comprised of proteins and RNAs by small extracellular vesicles (sEVs) including exosomes has been proposed to be a fundamental mode of intercellular communication. To obtain insights on the role of exosome-mediated information transfer in the context of neuropathic pain, we investigated alterations in protein composition of sEVs in a mouse model of neuropathic pain induced by spared nerve injury (SNI). Our studies using mass spectrometry and cytokine array show that sEVs from SNI model harbor unique proteins. We observed an upregulation of C5a and ICAM-1 in exosomes from SNI model compared to control. There was a differential distribution of C5a and ICAM-1 within exosomes and serum, between control and SNI suggesting a switch from local to long distance signaling. Our studies suggest critical roles for cargo sorting of vesicular proteins in mediating signaling under neuropathic pain.
Project description:AbstractThe increased presence of senescent cells in different neurological diseases suggests the contribution of senescence in the pathophysiology of neurodegenerative disorders. Microglia can adapt to any type of disturbance of the homeostasis of the central nervous system, and its altered activity can lead to permanent and unresolvable damage. The aim of this work was to characterize the behavioural phenotype of spared nerve injury mice and then associate it with senescence-related mechanisms. In this work, we investigated the timing of the onset of anxiety, depression, or memory decline associated with peripheral neuropathic pain and their correlation with the presence of microglial cellular senescence. Spared nerve injury mice showed a persistent pain hypersensitivity from 3 days after surgery. Twenty-eight days after nerve injury, they also developed anxiety, depression, and cognitive impairment. The appearance of these symptoms was coincident to a significant increase of senescence markers, such as β-galactosidase and senescent-associated secretory phenotype, at the microglial level in the spinal cord and hippocampus of spared nerve injury animals. These markers were unaltered at previous time points. In murine immortalized microglial cells (BV2) stimulated with LPS 500 ng/mL for 10 days (4 hours/day) every other day, we observed an increase of β-galactosidase and senescent-associated secretory phenotype appearance, a reduction of cell viability, and an increase of senescence-associated heterochromatin foci. Therefore, present findings could represent an important step to a better understanding of the pathophysiological cellular mechanisms in comorbidities related to neuropathic pain states.
Project description:AbstractFollowing surgical repair after peripheral nerve injury, neuropathic pain diminishes in most patients but can persist in a small proportion of cases, the mechanism of which remains poorly understood. Based on the spared nerve injury (SNI), we developed a rat nerve repair (NR) model, where a delayed reconstruction of the SNI-injured nerves resulted in alleviating chronic pain-like behavior only in a subpopulation of rats. Multiple behavioral measures were assayed over 11-week presurgery and postsurgery periods (tactile allodynia, pain prick responses, sucrose preference, motor coordination, and cold allodynia) in SNI (n = 10), sham (n = 8), and NR (n = 12) rats. All rats also underwent resting-state functional magnetic resonance imaging under anesthesia at multiple time points postsurgery, and at 10 weeks, histology and retrograde labeling were used to calculate peripheral reinnervation. Behavioral measures indicated that at approximately 5 weeks postsurgery, the NR group separated to pain persisting (NR persisting, n = 5) and recovering (NR recovering, n = 7) groups. Counts of afferent nerves and dorsal root ganglion cells were not different between NR groups. Therefore, NR group differences could not be explained by peripheral reorganization. By contrast, large brain functional connectivity differences were observed between NR groups, where corticolimbic reorganization paralleled with pain recovery (repeated-measures analysis of variance, false discovery rate, P < 0.05), and functional connectivity between accumbens and medial frontal cortex was related both to tactile allodynia (nociception) and to sucrose preference (anhedonia) in the NR group. Our study highlights the importance of brain circuitry in the reversal of neuropathic pain as a natural pain-relieving mechanism. Further studies regarding the therapeutic potentials of these processes are warranted.
Project description:The medial prefrontal cortex (mPFC) modulates a range of behaviors, including responses to noxious stimuli. While various pain modalities alter mPFC function, our understanding of changes to specific cell types underlying pain-induced mPFC dysfunction remains incomplete. Proper activity of cortical GABAergic interneurons is essential for normal circuit function. We find that nerve injury increases excitability of layer 5 parvalbumin-expressing neurons in the prelimbic (PL) region of the mPFC from male, but not female, mice. Conversely, nerve injury dampens excitability in somatostatin-expressing neurons in layer 2/3 of the PL region; however, effects are differential between males and females. Nerve injury slightly increases the frequency of spontaneous excitatory post-synaptic currents (sEPSCs) in layer 5 parvalbumin-expressing neurons in males but reduces frequency of sEPSCs in layer 2/3 somatostatin-expressing neurons in females. Our findings provide key insight into how nerve injury drives maladaptive and sex-specific alterations to GABAergic circuits in cortical regions implicated in chronic pain.
Project description:Low treatment efficacy represents an important unmet need in neuropathic pain patients and there is an urgent need to develop a more effective pharmacotherapy. An increasing number of patients choose complementary medicine to relieve pain. Lavender essential oil (LEO) is approved by the European Medicines Agency as herbal medicine to relieve anxiety and stress. However, the capability of LEO to relieve other nervous system disorders such as neuropathic pain has never been established. Our work aimed to evaluate the antineuropathic properties of lavender on a spared nerve injury (SNI) model of neuropathic pain in mice. An acute oral administration of LEO (100 mg/kg) alleviated SNI-induced mechanical allodynia, evaluated in the von Frey test, with an intensity comparable to the reference drug pregabalin. Investigations into the mechanism of action showed that LEO markedly decreased the phosphorylation of ERK1, ERK2, and JNK1, and decreased the levels of iNOS in the spinal cord; involvement of the endocannabinoid system was also detected using in vitro inhibition of the FAAH and MALG enzymes as well as in vivo experiments with the CB1 antagonist. Conversely, no effect on P38 phosphorylation and NF-kB activation was detected. These antihyperalgesic effects appeared at the same dose able to induce antidepressant-like, anxiolytic-like, and anorexic effects. In addition, gavage with LEO did not significantly alter animals' gross behavior, motor coordination, or locomotor activity, nor impaired memory functions. Oral administration of LEO could represent a therapeutic approach in the management of neuropathic pain states.
Project description:The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is one of the main sources of interleukin-1β (IL-1β) and is involved in several inflammatory-related pathologies. To date, its relationship with pain has not been studied in depth. The aim of our study was to elucidate the role of NLRP3 inflammasome and IL-1β production on neuropathic pain. Results showed that basal pain sensitivity is unaltered in NLRP3-/- mice as well as responses to formalin test. Spared nerve injury (SNI) surgery induced the development of mechanical allodynia and thermal hyperalgesia in a similar way in both genotypes and did not modify mRNA levels of the NLRP3 inflammasome components in the spinal cord. Intrathecal lipopolysaccharide (LPS) injection increases apoptosis-associated speck like protein (ASC), caspase-1 and IL-1β expression in both wildtype and NLRP3-/- mice. Those data suggest that NLRP3 is not involved in neuropathic pain and also that other sources of IL-1β are implicated in neuroinflammatory responses induced by LPS.
Project description:Histone deacetylase inhibitors (HDACi) that modulate epigenetic regulation and are approved for treating rare cancers have, in disease models, also been shown to mitigate neurological conditions, including chronic pain. They are of interest as non-opioid treatments, but achieving long-term efficacy with limited dosing has remained elusive. Here we utilize a triple combination formulation (TCF) comprised of a pan-HDACi vorinostat (Vo at its FDA-approved daily dose of 50mg/Kg), the caging agent 2-hydroxypropyl-β-cyclodextrin (HPBCD) and polyethylene glycol (PEG) known to boost plasma and brain exposure and efficacy of Vo in mice and rats, of various ages, spared nerve injury (SNI) model of chronic neuropathic pain. Administration of the TCF (but not HPBCD and PEG) decreased mechanical allodynia for 4 weeks without antagonizing weight, anxiety, or mobility. This was achieved at less than 1% of the total dose of Vo approved for 4 weeks of tumor treatment and associated with decreased levels of major inflammatory markers and microglia in ipsilateral (but not contralateral) spinal cord regions. A single TCF injection was sufficient for 3-4 weeks of efficacy: this was mirrored in repeat injections, specific for the injured paw and not seen on sham treatment. Pharmacodynamics in an SNI mouse model suggested pain relief was sustained for days to weeks after Vo elimination. Doubling Vo in a single TCF injection proved effectiveness was limited to male rats, where the response amplitude tripled and remained effective for > 2 months, an efficacy that outperforms all currently available chronic pain pharmacotherapies. Together, these data suggest that through pharmacological modulation of Vo, the TCF enables single-dose effectiveness with extended action, reduces long-term HDACi dosage, and presents excellent potential to develop as a non-opioid treatment option for chronic pain.
Project description:BackgroundNeuropathic pain impairs quality of life, is widely prevalent, and incurs significant costs. Current pharmacological therapies have poor/no efficacy and significant adverse effects; safe and effective alternatives are needed. Hyperpolarisation-activated cyclic nucleotide-regulated (HCN) channels are causally implicated in some forms of peripherally mediated neuropathic pain. Whilst 2,6-substituted phenols, such as 2,6-di-tert-butylphenol (26DTB-P), selectively inhibit HCN1 gating and are antihyperalgesic, the development of therapeutically tolerable, HCN-selective antihyperalgesics based on their inverse agonist activity requires that such drugs spare the cardiac isoforms and do not cross the blood-brain barrier.MethodsIn silico molecular dynamics simulation, in vitro electrophysiology, and in vivo rat spared nerve injury methods were used to test whether 'hindered' variants of 26DTB-P (wherein a hydrophilic 'anchor' is attached in the para-position of 26DTB-P via an acyl chain 'tether') had the desired properties.ResultsMolecular dynamics simulation showed that membrane penetration of hindered 26DTB-Ps is controlled by a tethered diol anchor without elimination of head group rotational freedom. In vitro and in vivo analysis showed that BP4L-18:1:1, a variant wherein a diol anchor is attached to 26DTB-P via an 18-carbon tether, is an HCN1 inverse agonist and an orally available antihyperalgesic. With a CNS multiparameter optimisation score of 2.25, a >100-fold lower drug load in the brain vs blood, and an absence of adverse cardiovascular or CNS effects, BP4L-18:1:1 was shown to be poorly CNS penetrant and cardiac sparing.ConclusionsThese findings provide a proof-of-concept demonstration that anchor-tethered drugs are a new chemotype for treatment of disorders involving membrane targets.
Project description:Epigenetic changes in the spinal cord play a key role in the initiation and maintenance of nerve injury-induced neuropathic pain. N6-methyladenosine (m6A) is one of the most abundant internal RNA modifications and plays an essential function in gene regulation in many diseases. However, the global m6A modification status of mRNA in the spinal cord at different stages after neuropathic pain is unknown. In this study, we established a neuropathic pain model in mice by preserving the complete sural nerve and only damaging the common peroneal nerve. High-throughput methylated RNA immunoprecipitation sequencing results showed that after spared nerve injury, there were 55 m6A methylated and differentially expressed genes in the spinal cord. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway results showed that m6A modification triggered inflammatory responses and apoptotic processes in the early stages after spared nerve injury. Over time, the differential gene function at postoperative day 7 was enriched in "positive regulation of neurogenesis" and "positive regulation of neural precursor cell proliferation." These functions suggested that altered synaptic morphological plasticity was a turning point in neuropathic pain formation and maintenance. Results at postoperative day 14 suggested that the persistence of neuropathic pain might be from lipid metabolic processes, such as "very-low-density lipoprotein particle clearance," "negative regulation of cholesterol transport" and "membrane lipid catabolic process." We detected the expression of m6A enzymes and found elevated mRNA expression of Ythdf2 and Ythdf3 after spared nerve injury modeling. We speculate that m6A reader enzymes also have an important role in neuropathic pain. These results provide a global landscape of mRNA m6A modifications in the spinal cord in the spared nerve injury model at different stages after injury.
