Effect Of Bone Mesenchymal Stromal Cells On Neuropathic Pain And Its Mechanism

Background:Neuropathic pain is pain caused by a lesion or disease of the somatosensory nervous system.Neuropathic pain is triggered by lesions to the somatosensory nervous system that alter its structure and function so that pain occurs spontaneously and responses to noxious and innocuous stimuli are pathologically amplified.Importantly,neuropathic pain is mechanistically dissimilar to other chronic pain conditions such as inflammatory pain.Essentially,lesions or diseases of the somatosensory nervous system lead to altered and disordered transmission of sensory signals into the spinal cord and the brain.These maladaptive plasticity leads to persistent changes and,therefore,needs to be considered a disease state of the nervous system in its own right,independent of the etiological factor that triggered it.The mechanisms of neuropathic pain are immensely complex,involving both structural and functional changes throughout the nociceptive pathways,spanning the entire nervous system from the site of peripheral nerve injury to the DRG,spinal cord,and brain.Multiple alterations distributed widely across the nervous system contribute to complex pain phenotypes.These alterations include ectopic generation of action potentials,facilitation and disinhibition of synaptic transmission,loss of synaptic connectivity and formation of new synaptic circuits,and neuroimmune interactions.Diagnosis of neuropathic pain and preparation of animal models are difficult,as pain is a subjective measurement.Patients with neuropathic pain typically experience a distinct set of symptoms,such as spontaneous pain,hyperalgesia(the increased pain perception of noxious stimuli),and mechanical allodynia(pain hypersensitivity to normally innocuous stimuli).Common features of neuropathic pain are the presence of allodynia.Multivariate analyses confirmed that the neuropathic nature of the pain,as indicated by the DN4 score,made a significant contribution to poorer quality of life in terms of both physical and mental scores in these subjects,regardless of pain severity.Peripheral neuropathic pain will probably become more common because of the ageing global population,increased incidence of diabetes mellitus and the increasing rates of cancer and the consequence of chemotherapy.First-line treatment for neuropathic pain includes tricyclic antidepressants,pregabalin and gabapentin,which focus on treating symptoms and do not target the underlying mechanism,thus often provide transient pain relief and only in a minority of patients.Above mentioned etiological approach does not capture the essential feature of neuropathic pain,which is the manifestation of maladaptive plasticity in the nervous system.These treatments are also not specific to the different phases in the development of neuropathic pain.Therapy based on Neuronal Mechanisms are emerging and promise important roles in the treatment of neuropathic pain.Mesenchymal stromal cells(MSCs)are a heterogeneous subset of stromal stem cells that can be isolated from many adult tissues.With increasing evidence for the role of MSCs in direct modulation of the innate immune system,MSC therapy is increasingly being viewed as a novel and promising treatment for many diseases such as neuropathic pain.To date,results have suggested that i.t.delivery of BMSC into thoracic and lumbar spinal cerebrospinal fluid(CSF)features a favorable safety profile as it has higher tissue-specificity,less systemic side effects,and smaller dose requirements as measured by the total number of implanted cells.However,in the context of neuropathic pain,efficacy results of i.t.BMSC have been variable,ranging from a significant improvement in symptoms for several weeks to negligible behavioral changes.These conflicting results might be due in part to the lack of a thorough understanding of how BMSCs act to alleviate neuropathic pain,thereby resulting in sub-optimal experimental designs to investigate its efficacy.Thus,further investigation into BMSCs’ mechanism is needed to fully optimize its analgesic application.Microglia constitute the major immune cells that permanently reside in the central nervous system(CNS)alongside neurons and other glial cells.These resident immune cells are critical for proper brain development,actively maintain brain health throughout the lifespan and rapidly adapt their function to the physiological or pathophysiological needs of the organism.There is increasing evidence that microglial cells are not only responsible for the removal of synapses during development,but also in the normal healthy brain in the framework of neuronal plasticity.Microglial cells are highly active in their presumed resting state,continually surveying their microenvironment with extremely motile processes and protrusions.Microglia are activated by any type of pathologic event or change in brain homeostasis.When activated,they can perform many diverse functions which may be either beneficial or harmful depending on the situation.This activation process is highly diverse and depends on the context and type of the stressor or pathology.Purines are chemical substances that have been regarded as the major intracellular energy sources and drive most of the reactions in the animal body.A growing body of evidence indicates that spinal microglia react and undergo a series of changes that directly in fluence the establishment of neuropathic pain states.P2X4R was the first molecule to be found that increased in expression in spinal microglia after PNI,which play a critical role in neuropathic pain.Importantly,inhibiting the function or expression of P2X4Rs and P2X4R-regulating molecules suppresses the aberrant excitability of dorsal horn neurons and neuropathicpain.These findings indicate that P2X4R-positive microglia are a central player in mechanisms for neuropathic pain.Thus,microglial P2X4Rs are a potential target for treating the chronic pain state.While both BMSCs and microglia are known to impact neuropathic pain,whether the mechanism of BMSC therapy involves microglia regulation remains largely unknown.Researches from our group have demonstrated that TRPV4 in DRG neurons are upregulated following CCD in rats.TRPV4 participates in the peripheral and central sensitization mechanism in neuropathic pain.TRPV4 was involved in ectopic discharge of dorsal root ganglion neurons in rats after CCD surgery.In this context of the DRG’s involvement in the development of NP,the interactions between BMSCs and the expression of TRPV4 in the DRG neuron are worthy further study.Object:1)To observe the therapeutic effect of intrathecal injection of mesenchymal stem cells on neuropathic pain induced by chronic compression of dorsal root ganglion in rats,and to analyze the safety of intrathecal injection of BMSC.2)The migration of BMSC cells in spinal cord was observed and the analgesic mechanism was discussed.3)To investigate the microglial reaction in response to CCD and BMSC and explored interactions between BMSCs and microglia.4)Whether the regulation of P2X4 expression is involved in the therapeutic mechanism of MSC.5)To investigate the interactions between BMSCs and the expression of TRPV4 in the DRG neuron。Statistical analysesAll data are presented as mean ± SEM and differences between groups were examined for statistical significance using generalized linear models repeated measures followed by LSD post hoc test correcting for multiple comparisons,one-way factorial analysis of variance(ANOVA),or Student’s t test.Significant differences are indicated if P values were below 0.05.The above tests were conducted using SPSS software version 17.0(SPSS,Chicago,IL,USA).GraphPad Prism software version 5.00(GraphPad Software,San Diego,CA,USA)was used for data representation.Results:1)Results showed that CCD,but not sham control animals,exhibited mechanical allodynia in the injured limbs starting 4 days post CCD,with CCD animals recording significantly larger paw withdrawals than sham animals.our CCD model was executed with specific and precise surgical methods that reliably produces neuropathic pain and its hallmark symptom of allodynia.2)When compared with CCD mice that did not receive BMSC injections,those treated with i.t.BMSC had significantly attenuated allodynia behaviors one day after treatment.i.t.delivery of BMSC causes a sizeable and transient reduction of neuropathic pain.3)Intrathecal injection of BMSC had no significant adverse effect on normal pain sensation and motor function.No changes in motor performance were observed between pre-and post-i.t.BMSC treatment.4)BMSC-treated rats showed significant fluorescence around the lumbar enlargement at 1-day post-injection,with the maximum fluorescence intensity located at the injection point on the cavosurface of canalis spinalis.There was no evidence of BMSCs in spinal cord parenchyma and DRG post-injection.5)One day after i.t.injection of BMSC-lysate for both time points,tactile allodynia was attenuated compared to pre-injection.BMSC lysates retarded neuropathic pain behavior to a similar extent as seen in i.t.BMSC-treated animals.6)Injection of ATP-stimulated microglia pretreated with BMSC lysate resulted in significantly fewer paw withdrawals.Tactile allodynia in response to ATP-stimulated microglia was partially negated by BMSC lysate.7)After CCD surgery,the expression of iba-1 and OX42,markers of microglial cell activation,both increased.Microglia activation is associated with neuropathic pain following CCD surgery,but i.t.BMSC did not have an impact on mieroglial activation.8)Bone marrow mesenchymal stem cells can reduce the expression of P2X4R protein in microglia in vivo and in vitro.Inhibiting microglial P2X4R is necessary and responsible for i.t.BMSC’s analgesic effect9)Western blot and immunohistochemical results showed that there was no significant difference in TRPV4 expression between CCD group and BMSC group.Intrathecal BMSC does not affect TRPV4 expression on DRG neuronsConclusion:Altogether,our results suggest that i.t.BMSC is an effective and saf’e treatment of neuropathic pain and provides novel evidence that BMSC’s analgesic effects are largely mediated by the release of BMSC-derived factors.Regulation of P2X4R expression is an important mechanism for mesenchymal stem cells to exert analgesic effect.The analgesic mechanism of BMSC is independent of TRPV4 expression in DRG neurons.As future BMSC based therapeutics are developed for neuropathic pain,we believe that combination therapies directed at multiple targets in neuropathic pain might be worthwhile.