Modulation of muscle pain is not somatotopically restricted : an experimental model using concurrent hypertonic-normal saline infusions in humans

We have previously shown that during muscle pain induced by infusion of hypertonic saline (HS), concurrent application of vibration and gentle brushing to overlying and adjacent skin regions increases the overall pain. In the current study, we focused on muscle-muscle interactions and tested whether HS-induced muscle pain can be modulated by innocuous/sub-perceptual stimulation of adjacent, contralateral, and remote muscles. Psychophysical observations were made in 23 healthy participants. HS (5%) was infused into a forearm muscle (flexor carpi ulnaris) to produce a stable baseline pain. In separate experiments, in each of the three test locations (n = 10 per site)—ipsilateral hand (abductor digiti minimi), contralateral forearm (flexor carpi ulnaris), and contralateral leg (tibialis anterior)—50 μl of 0.9% normal saline (NS) was infused (in triplicate) before, during, and upon cessation of HS-induced muscle pain in the forearm. In the absence of background pain, the infusion of NS was imperceptible to all participants. In the presence of HS-induced pain in the forearm, the concurrent infusion of NS into the ipsilateral hand, contralateral forearm, and contralateral leg increased the overall pain by 16, 12, and 15%, respectively. These effects were significant, reproducible, and time-locked to NS infusions. Further, the NS-evoked increase in pain was almost always ascribed to the forearm where HS was infused with no discernible percept attributed to the sites of NS infusion. Based on these observations, we conclude that intramuscular infusion of HS results in muscle hyperalgesia to sub-perceptual stimulation of muscle afferents in a somatotopically unrestricted manner, indicating the involvement of a central (likely supra-spinal) mechanism

Histological and top-down proteomic analyses of the visual pathway in the cuprizone demyelination model

A change in visual perception is a frequent early symptom of multiple sclerosis (MS), the pathoaetiology of which remains unclear. Following a slow demyelination process caused by 12 weeks of low-dose (0.1%) cuprizone (CPZ) consumption, histology and proteomics were used to investigate components of the visual pathway in young adult mice. Histological investigation did not identify demyelination or gliosis in the optic tracts, pretectal nuclei, superior colliculi, lateral geniculate nuclei or visual cortices. However, top-down proteomic assessment of the optic nerve/tract revealed a significant change in the abundance of 34 spots in high-resolution two-dimensional (2D) gels. Subsequent liquid chromatography-tandem mass spectrometry (LC-TMS) analysis identified alterations in 75 proteoforms. Literature mining revealed the relevance of these proteoforms in terms of proteins previously implicated in animal models, eye diseases and human MS. Importantly, 24 proteoforms were not previously described in any animal models of MS, eye diseases or MS itself. Bioinformatic analysis indicated involvement of these proteoforms in cytoskeleton organization, metabolic dysregulation, protein aggregation and axonal support. Collectively, these results indicate that continuous CPZ-feeding, which evokes a slow demyelination, results in proteomic changes that precede any clear histological changes in the visual pathway and that these proteoforms may be potential early markers of degenerative demyelinating conditions

CD8 T-cell recruitment into the central nervous system of cuprizone-fed mice : relevance to modelling the etiology of Multiple Sclerosis

Cuprizone (CPZ)-feeding in mice induces atrophy of peripheral immune organs (thymus and spleen) and suppresses T-cell levels, thereby limiting its use as a model for studying the effects of the immune system in demyelinating diseases such as Multiple Sclerosis (MS). To investigate whether castration (Cx) can protect the peripheral immune organs from CPZ-induced atrophy and enable T-cell recruitment into the central nervous system (CNS) following a breach of the blood-brain barrier (BBB), three related studies were carried out. In Study 1, Cx prevented the dose-dependent reductions (0.1% < 0.2% CPZ) in thymic and splenic weight, size of the thymic medulla and splenic white pulp, and CD4 and CD8 (CD4/8) levels remained comparable to gonadally intact (Gi) control males. Importantly, 0.1% and 0.2% CPZ were equipotent at inducing central demyelination and glial activation. In Study 2, combining Cx with 0.1% CPZ-feeding and BBB disruption with pertussis toxin (PT) enhanced CD8+ T-cell recruitment into the CNS. The increased CD8+ T-cell level observed in the parenchyma of the cerebrum, cerebellum, brainstem and spinal cord were confirmed by flow cytometry and western blot analyses of CNS tissue. In Study 3, PT+0.1% CPZ-feeding to Gi female mice resulted in similar effects on the peripheral immune organs, CNS demyelination, and gliosis comparable to Gi males, indicating that testosterone levels alone were not responsible for the immune response seen in Study 2. The combination of Cx+0.1% CPZ-feeding+PT indicates that CPZ-induced demyelination can trigger an “inside-out” immune response when the peripheral immune system is spared and may provide a better model to study the initiating events in demyelinating conditions such as MS

Proteomics of Multiple Sclerosis : inherent issues in defining the pathoetiology and identifying (early) biomarkers

Multiple Sclerosis (MS) is a demyelinating disease of the human central nervous system having an unconfirmed pathoetiology. Although animal models are used to mimic the pathology and clinical symptoms, no single model successfully replicates the full complexity of MS from its initial clinical identification through disease progression. Most importantly, a lack of preclinical biomarkers is hampering the earliest possible diagnosis and treatment. Notably, the development of rationally targeted therapeutics enabling pre‐emptive treatment to halt the disease is also delayed without such biomarkers. Using literature mining and bioinformatic analyses, this review assessed the available proteomic studies of MS patients and animal models to discern (1) whether the models effectively mimic MS; and (2) whether reasonable biomarker candidates have been identified. The implication and necessity of assessing proteoforms and the critical importance of this to identifying rational biomarkers are discussed. Moreover, the challenges of using different proteomic analytical approaches and biological samples are also addressed

Suppression of the peripheral immune system limits the central immune response following cuprizone-feeding : relevance to modelling Multiple Sclerosis

Cuprizone (CPZ) preferentially affects oligodendrocytes (OLG), resulting in demyelination. To investigate whether central oligodendrocytosis and gliosis triggered an adaptive immune response, the impact of combining a standard (0.2%) or low (0.1%) dose of ingested CPZ with disruption of the blood brain barrier (BBB), using pertussis toxin (PT), was assessed in mice. 0.2% CPZ(±PT) for 5 weeks produced oligodendrocytosis, demyelination and gliosis plus marked splenic atrophy (37%) and reduced levels of CD4 (44%) and CD8 (61%). Conversely, 0.1% CPZ(±PT) produced a similar oligodendrocytosis, demyelination and gliosis but a smaller reduction in splenic CD4 (11%) and CD8 (14%) levels and no splenic atrophy. Long-term feeding of 0.1% CPZ(±PT) for 12 weeks produced similar reductions in CD4 (27%) and CD8 (43%), as well as splenic atrophy (33%), as seen with 0.2% CPZ(±PT) for 5 weeks. Collectively, these results suggest that 0.1% CPZ for 5 weeks may be a more promising model to study the ‘insideout’ theory of Multiple Sclerosis (MS). However, neither CD4 nor CD8 were detected in the brain in CPZ±PT groups, indicating that CPZ-mediated suppression of peripheral immune organs is a major impediment to studying the ‘inside-out’ role of the adaptive immune system in this model over long time periods. Notably, CPZ(±PT)-feeding induced changes in the brain proteome related to the suppression of immune function, cellular metabolism, synaptic function and cellular structure/organization, indicating that demyelinating conditions, such as MS, can be initiated in the absence of adaptive immune system involvement

Minocycline treatment reduces mass and force output from fast-twitch mouse muscles and inhibits myosin production in C2C12 myotubes

Minocycline, a tetracycline-class of antibiotic, has been tested with mixed effectiveness on neuromuscular disorders such as amyotrophic lateral sclerosis, autoimmune neuritis and muscular dystrophy. The independent effect of minocycline on skeletal muscle force production and signalling remain poorly understood. Our aim here is to investigate the effects of minocycline on muscle mass, force production, myosin heavy chain abundance and protein synthesis. Mice were injected with minocycline (40 mg/kg i.p.) daily for 5 days and sacrificed at day six. Fast-twitch EDL, TA muscles and slow-twitch soleus muscles were dissected out, the TA muscle was snap-frozen and the remaining muscles were attached to force transducer whilst maintained in an organ bath. In C2C12 myotubes, minocycline was applied to the media at a final concentration of 10 µg/mL for 48 h. In minocycline treated mice absolute maximal force was lower in fast-twitch EDL while in slow-twitch soleus there was an increase in the time to peak and relaxation of the twitch. There was no effect of minocycline treatment on the other contractile parameters measured in isolated fast- and slow-twitch muscles. In C2C12 cultured cells, minocycline treatment significantly reduced both myosin heavy chain content and protein synthesis without visible changes to myotube morphology. In the TA muscle there was no significant changes in myosin heavy chain content. These results indicate that high dose minocycline treatment can cause a reduction in maximal isometric force production and mass in fast-twitch EDL and impair protein synthesis during myogenesis in C2C12 cultured cells. These findings have important implications for future studies investigating the efficacy of minocycline treatment in neuromuscular or other muscle-atrophy inducing conditions

Anti-cancer potential of synergistic phytochemical combinations is influenced by the genetic profile of prostate cancer cell lines

Introduction: Despite strong epidemiological evidence that dietary factors modulate cancer risk, cancer control through dietary intervention has been a largely intractable goal for over sixty years. The effect of tumour genotype on synergy is largely unexplored. Methods: The effect of seven dietary phytochemicals, quercetin (0–100 μM), curcumin (0–80 μM), genistein, indole-3-carbinol (I3C), equol, resveratrol and epigallocatechin gallate (EGCG) (each 0–200 μM), alone and in all paired combinations om cell viability of the androgen-responsive, pTEN-null (LNCaP), androgen-independent, pTEN-null (PC-3) or androgen-independent, pTEN-positive (DU145) prostate cancer (PCa) cell lines was determined using a high throughput alamarBlue® assay. Synergy, additivity and antagonism were modelled using Bliss additivism and highest single agent equations. Patterns of maximum synergy were identified by polygonogram analysis. Network pharmacology approaches were used to identify interactions with known PCa protein targets. Results: Synergy was observed with all combinations. In LNCaP and PC-3 cells, I3C mediated maximum synergy with five phytochemicals, while genistein was maximally synergistic with EGCG. In contrast, DU145 cells showed resveratrol-mediated maximum synergy with equol, EGCG and genistein, with I3C mediating maximum synergy with only quercetin and curcumin. Knockdown of pTEN expression in DU145 cells abrogated the synergistic effect of resveratrol without affecting the synergy profile of I3C and quercetin. Discussion: Our study identifies patterns of synergy that are dependent on tumour cell genotype and are independent of androgen signaling but are dependent on pTEN. Despite evident cell-type specificity in both maximally-synergistic combinations and the pathways that phytochemicals modulate, these combinations interact with similar prostate cancer protein targets. Here, we identify an approach that, when coupled with advanced data analysis methods, may suggest optimal dietary phytochemical combinations for individual consumption based on tumour molecular profile

An investigation into the peripheral substrates involved in the tactile modulation of cutaneous pain with emphasis on the C-tactile fibres

We recently demonstrated the emergence of touch-evoked pain (allodynia) during innocuous tactile stimulation of the skin overlying a painful muscle. This effect appeared to depend on a class of low-threshold unmyelinated mechanoafferents, termed C-tactile fibres (CT). In this study, we investigated the peripheral neurocircuitry of allodynia when pain originates in the skin. Psychophysical observations were carried out in 28 healthy subjects. Cutaneous pain was induced by infusing hypertonic saline (HS: 5 %) into the hairy skin overlying tibialis anterior muscle. An innocuous tactile stimulus (sinusoidal vibration: 200 Hz-200 μm) was concurrently applied to the hairy skin ~90 mm distal to the HS-infusion site. The contribution of different fibre classes to allodynia was determined by employing conduction blocks of myelinated (sciatic nerve compression) and unmyelinated (intradermal anaesthesia, Xylocaine 0.25 %) fibres. In absence of background nociceptive input, vibration was reported as non-painful. During cutaneous pain, vibration evoked a significant and reproducible increase in the overall pain intensity (allodynia). The blockade of myelinated fibres abolished the vibration sense, but the vibration-evoked allodynia persisted. Conversely, the blockade of unmyelinated cutaneous fibres abolished the allodynia (while the myelinated fibres were conducting or not). On the basis of these findings, in addition to our earlier work, we conclude that the allodynic effect of CT-fibre activation is not limited to nociceptive input arising from the muscle, but can be equally realized when pain originates in the skin. These results denote a broader role of CTs in pain modulation

Mechanical allodynia in human glabrous skin mediated by low-threshold cutaneous mechanoreceptors with unmyelinated fibres

We recently showed that C-tactile fibres (CTs) in human hairy skin (anterior leg) mediate crossover between innocuous touch and noxious touch, i.e. mechanical allodynia. Although there is no evidence for existence of a phenotypically identical class of CTs in human glabrous skin, the 'qualia' of affective stimuli are comparable across skin types. In 42 healthy subjects, muscle pain was induced by infusing hypertonic saline (5%) into flexor carpi ulnaris muscle. Concurrently, sinusoidal vibration (200 H z–200 μm) was applied to glabrous skin of little finger. The neural substrate of allodynia was determined by employing conduction blocks of myelinated (ulnar nerve compression) and unmyelinated (low-dose intra-dermal anaesthesia) fibres. In order to compare the expression of allodynia across spinal segments and skin types, vibration was also applied to glabrous skin of index finger and hairy skin of dorsal forearm. In addition, high-precision brushing stimuli were applied at speeds of 1.0 and 3.0 cm s−1 to digital glabrous skin with absent myelinated fibres. During muscle pain, vibration caused a significant and reproducible increase in pain (allodynia). This effect persisted during blockade of myelinated fibres, but was abolished by inactivation of unmyelinated cutaneous fibres. The vibrationevoked effects were found to be comparable across spinal segments and skin types. Furthermore, brushing produced a near-identical expression of C-fibre-mediated allodynia. Prior to induction and upon cessation of muscle pain, vibration and brushing were reported as non-painful. Based on these results, we postulate that a functional homologue of the CTs (hairy skin) mediates allodynia in human glabrous skin

C-tactile fibers contribute to cutaneous allodynia after eccentric exercise

We recently showed that during acute muscle pain, C-tactile (CT) fibers mediate allodynia in healthy human subjects. In this study, we pursued the following questions: Do CTs contribute to allodynia observed in delayed onset muscle soreness (DOMS)? Is CT-mediated allodynia reproducible in a clinical pain state? In 30 healthy subjects, DOMS was induced in anterior compartment muscles of the leg by repeated eccentric contractions. DOMS was confirmed by mapping the emergence of tender points (decreased pressure pain thresholds). Furthermore, we measured pressure pain thresholds in a clinical subject who presented with activity-triggered heel pain but no resting pain. Cutaneous vibration (sinusoidal; 200 Hz–200 μm)—an otherwise innocuous stimulus—was applied to anterolateral leg before exercise, during DOMS, and following recovery from DOMS. The peripheral origin of allodynia was determined by employing conduction blocks of unmyelinated (intradermal anesthesia) and myelinated (nerve compression) fibers. In DOMS state, there was no resting pain, but vibration reproducibly evoked pain (allodynia). The blockade of cutaneous C fibers abolished this effect, whereas it persisted during blockade of myelinated fibers. In the clinical subject, without exposure to eccentric exercise, vibration (and brushing) produced a cognate expression of CT-mediated allodynia. These observations attest to a broader role of CTs in pain processing