The geriatric pain experience in mice: intact cutaneous thresholds but altered responses to tonic and chronic pain.

Older individuals have an elevated risk for chronic pain as half of all individuals over 65 years old have at least one chronic pain condition. Unfortunately, relevant assessment tools and recommendations for chronic pain management targeting older adults are lacking. This study explores changes in response to pain between young (2-3 months old) and geriatric (20-24 months old) ages using mice. Although cutaneous thresholds to brisk stimuli (von Frey and radiant heat assays) were not affected, behavioral responses to tonic stimuli (acetone and capsaicin assays) were more pronounced in geriatric animals. After nerve injury, geriatric mice present an altered neuropathic pain profile with hypersensitivity to mechanical stimuli but not acetone and an impairment in conditioned noxious stimuli avoidance. This altered behavioral response pattern was associated with an abnormal monoaminergic signature in the medial prefrontal cortex, suggesting decreased COMT function. We conclude that young and geriatric mice exhibit different behavioral and physiological responses to the experience of pain, suggesting that knowledge and practices must be adjusted for geriatric populations.

Spinal microglia are required for long-term maintenance of neuropathic pain.

While spinal microglia play a role in early stages of neuropathic pain etiology, whether they are useful targets to reverse chronic pain at late stages remains unknown. Here, we show that microglia activation in the spinal cord persists for >3 months following nerve injury in rodents, beyond involvement of proinflammatory cytokine and chemokine signalling. In this chronic phase, selective depletion of spinal microglia in male rats with the targeted immunotoxin Mac1-saporin and blockade of brain-derived neurotrophic factor-TrkB signalling with intrathecal TrkB Fc chimera, but not cytokine inhibition, almost completely reversed pain hypersensitivity. By contrast, local spinal administration of Mac1-saporin did not affect nociceptive withdrawal threshold in control animals nor did it affect the strength of afferent-evoked synaptic activity in the spinal dorsal horn in normal conditions. These findings show that the long-term, chronic phase of nerve injury-induced pain hypersensitivity is maintained by microglia-neuron interactions. The findings also effectively separate the central signalling pathways underlying the maintenance phase of the pathology from the early and peripheral inflammatory reactions to injury, pointing to different targets for the treatment of acute vs chronic injury-induced pain.

Dynamics of spinal microglia repopulation following an acute depletion.

Our understanding on the function of microglia has been revolutionized in the recent 20 years. However, the process of maintaining microglia homeostasis has not been fully understood. In this study, we dissected the features of spinal microglia repopulation following an acute partial depletion. By injecting intrathecally Mac-1-saporin, a microglia selective immunotoxin, we ablated 50% microglia in the spinal cord of naive mice. Spinal microglia repopulated rapidly and local homeostasis was re-established within 14 days post-depletion. Mac-1-saporin treatment resulted in microglia cell proliferation and circulating monocyte infiltration. The latter is indeed part of an acute, transient inflammatory reaction that follows cell depletion, and was characterized by an increase in the expression of inflammatory molecules and by the breakdown of the blood spinal cord barrier. During this period, microglia formed cell clusters and exhibited a M1-like phenotype. MCP-1/CCR2 signaling was essential in promoting this depletion associated spinal inflammatory reaction. Interestingly, ruling out MCP-1-mediated secondary inflammation, including blocking recruitment of monocyte-derived microglia, did not affect depletion-triggered microglia repopulation. Our results also demonstrated that newly generated microglia kept their responsiveness to peripheral nerve injury and their contribution to injury-associated neuropathic pain was not significantly altered.

Can modulating inflammatory response be a good strategy to treat neuropathic pain?

Neuronal injury not only results in severe alteration in the function of primary sensory neurons and their central projection pathway, but is also associated with a robust immune response at almost every level of the somatosensory system. Evidence from animal studies suggests undoubtedly that bi-directional signalling between the immune system and the nervous system contribute to the development and maintenance of chronic neuropathic pain. Non-neuronal cells, including peripheral immune cells, CNS/PNS glial cells and endothelial cells play important roles in the neuroimmune interaction and subsequent persistent hypersensitivity. Various cytokines and chemokines have been identified as key signalling molecules in the crosstalk. However, majority evidence showing inflammation in neuropathic pain was generated from animal models at acute phase. Whether and to what extent inflammation or non-neuronal cells are involved at chronic stage of neuropathic pain needs to be further explored, and evidence of inflammation in chronic pain from human studies is still largely awaited. Therapeutic agents targeting inflammation provide an exciting prospect. Yet, considering the heterogeneous conditions presented in neuropathic pain, no matter the etiologies, or the pathophysiology during different stages of the disease; and the complexity of the immune response to the damage on the nervous system, it appears that finely tuned strategies of modulating inflammation are essential to warrant an effective treatment for neuropathic pain. We want to reduce pain; we also want to promote tissue repair and functional recovery.

Selectively reducing cytokine/chemokine expressing macrophages in injured nerves impairs the development of neuropathic pain.

It has been well documented that Wallerian degeneration following nerve injury is associated with inflammatory reaction. Such local inflammation contributes to the development of chronic neuropathic pain. Macrophages are one of the major players in the process of either or both degeneration/regeneration and hypersensitivity. To elucidate whether cellular and molecular changes involved in Wallerian degeneration are simultaneously involved in the induction and maintenance of neuropathic pain, and to identify which subpopulation of macrophages can be responsible for the chronic pain following nerve injury, we investigated the peripheral effects of an anti-inflammatory cytokine TGF-ß1 in neuropathic pain. Rat sciatic nerves were partially ligated. Macrophages accumulated in injured sciatic nerves displayed heterogeneity with two distinctive functional phenotypes. While MAC1(+) macrophages were able to express IL-6 and MIP-1α, ED1(+) macrophages were always devoid of signals of inflammatory mediators. Intraneural injection of TGF-ß1 resulted in delayed and attenuated neuropathic pain behaviour. In parallel, we observed that exposure of the nerve to TGF-ß1 dramatically reduced the number of MAC1(+) macrophages. Consequently, the expression of IL-6 and MIP-1α decreased in the injured nerve. Very interestingly, local TGF-ß1 treatment had no effect on the population of ED1(+) phagocytic macrophages. In addition to its effect on selective subsets of macrophages, TGF-ß1 also reduced T-lymphocyte infiltration. Our results revealed the critical roles of cytokine/chemokine secreting MAC1(+) macrophages in the development of neuropathic pain, and highlighted the needs and benefits of targeting specific populations of macrophages in alleviating neuropathic pain without delaying nerve regeneration.

Peripheral nerve injury alters blood-spinal cord barrier functional and molecular integrity through a selective inflammatory pathway.

Peripheral nerve lesion triggers alterations in the spinal microenvironment that contribute to the pathogenesis of neuropathic pain. While neurons and glia have been implicated in these functional changes, it remains largely underexplored whether the blood-spinal cord barrier (BSCB) is also involved. The BSCB is an important component in the CNS homeostasis, and compromised BSCB has been associated with different pathologies affecting the spinal cord. Here, we demonstrated that a remote injury on the peripheral nerve in rats triggered a leakage of the BSCB, which was independent of spinal microglial activation. The increase of BSCB permeability to different size tracers, such as Evans Blue and sodium fluorescein, was restricted to the lumbar spinal cord and prominent for at least 4 weeks after injury. The spinal inflammatory reaction triggered by nerve injury was a key player in modulating BSCB permeability. We identified MCP-1 as an endogenous trigger for the BSCB leakage. BSCB permeability can also be impaired by circulating IL-1ß. In contrast, antiinflammatory cytokines TGF-ß1 and IL-10 were able to shut down the openings of the BSCB following nerve injury. Peripheral nerve injury caused a decrease in tight junction and caveolae-associated proteins. Interestingly, ZO-1 and occludin, but not caveolin-1, were rescued by TGF-ß1. Furthermore, our data provide direct evidence that disrupted BSCB following nerve injury contributed to the influx of inflammatory mediators and the recruitment of spinal blood borne monocytes/macrophages, which played a major role in the development of neuropathic pain. These findings highlight the importance of inflammation in BSCB integrity and in spinal cord homeostasis.

Transforming growth factor-beta1 impairs neuropathic pain through pleiotropic effects.

BACKGROUND: Understanding the underlying mechanisms of neuropathic pain caused by damage to the peripheral nervous system remains challenging and could lead to significantly improved therapies. Disturbance of homeostasis not only occurs at the site of injury but also extends to the spinal cord and brain involving various types of cells. Emerging data implicate neuroimmune interaction in the initiation and maintenance of chronic pain hypersensitivity. RESULTS: In this study, we sought to investigate the effects of TGF-beta1, a potent anti-inflammatory cytokine, in alleviating nerve injury-induced neuropathic pain in rats. By using a well established neuropathic pain animal model (partial ligation of the sciatic nerve), we demonstrated that intrathecal infusion of recombinant TGF-beta1 significantly attenuated nerve injury-induced neuropathic pain. TGF-beta1 treatment not only prevents development of neuropathic pain following nerve injury, but also reverses previously established neuropathic pain conditions. The biological outcomes of TGF-beta1 in this context are attributed to its pleiotropic effects. It inhibits peripheral nerve injury-induced spinal microgliosis, spinal microglial and astrocytic activation, and exhibits a powerful neuroprotective effect by preventing the induction of ATF3+ neurons following nerve ligation, consequently reducing the expression of chemokine MCP-1 in damaged neurons. TGF-beta1 treatment also suppresses nerve injury-induced inflammatory response in the spinal cord, as revealed by a reduction in cytokine expression. CONCLUSION: Our findings revealed that TGF-beta1 is effective in the treatment of neuropathic by targeting both neurons and glial cells. We suggest that therapeutic agents such as TGF-beta1 having multipotent effects on different types of cells could work in synergy to regain homeostasis in local spinal cord microenvironments, therefore contributing to attenuate neuropathic pain.

Characterization of cell proliferation in rat spinal cord following peripheral nerve injury and the relationship with neuropathic pain.

Glial activation is a typical response of the central nervous system to nerve injury. In the current investigation, we characterized the temporal and spatial pattern of glial proliferation, one of the most conspicuous features of glial activation, in relation to nerve injury-induced neuropathic pain. Using bromodeoxyuridine (BrdU) as a mitotic marker, we analyzed cell proliferation in the spinal cord, identified the phenotype of dividing cells, traced their fate, and correlated these phenomena with behavioural assays of the neuropathic pain syndrome. Our results demonstrated that peripheral nerve injury induced an early and transient cell proliferation, on the spinal cord ipsilateral to the nerve lesion which peaked at day 3 post-surgery. The majority of the proliferating cells were Iba-1(+) microglia, together with some NG2(+) oligodendrocyte progenitors, and GFAP(+) astrocytes. These newly generated cells continued to divide over time with the response peaking at day 14 post-injury. Microglia were always the predominant phenotype which made up over 60% of activated microglia derived from this newly generated cell population. There was a close temporal correlation between microglial proliferation in the spinal cord dorsal horn and the abnormal pain responses, suggesting a contribution of the new microglia to the genesis of the neuropathic pain symptoms.

Expression of CCR2 in both resident and bone marrow-derived microglia plays a critical role in neuropathic pain.

Neuropathic pain resulting from damage to or dysfunction of peripheral nerves is not well understood and difficult to treat. Although CNS hyperexcitability is a critical component, recent findings challenge the neuron-centric view of neuropathic pain etiology and pathology. Indeed, glial cells were shown to play an active role in the initiation and maintenance of pain hypersensitivity. However, the origins of these cells and the triggers that induce their activation have yet to be elucidated. Here we show that, after peripheral nerve injury induced by a partial ligation on the sciatic nerve, in addition to activation of microglia resident to the CNS, hematogenous macrophage/monocyte infiltrate the spinal cord, proliferate, and differentiate into microglia. Signaling from chemokine monocyte chemoattractant protein-1 (MCP-1, CCL2) to its receptor CCR2 is critical in the spinal microglial activation. Indeed, intrathecal injection of MCP-1 caused activation of microglia in wild-type but not in CCR2-deficient mice. Furthermore, treatment with an MCP-1 neutralizing antibody prevented bone marrow-derived microglia (BMDM) infiltration into the spinal cord after nerve injury. In addition, using selective knock-out of CCR2 in resident microglia or BMDM, we found that, although total CCR2 knock-out mice did not develop microglial activation or mechanical allodynia, CCR2 expression in either resident microglia or BMDM is sufficient for the development of mechanical allodynia. Thus, to effectively relieve neuropathic pain, both CNS resident microglia and blood-borne macrophages need to be targeted. These findings also open the door for a novel therapeutic strategy: to take advantage of the natural ability of bone marrow-derived cells to infiltrate selectively affected CNS regions by using these cells as vehicle for targeted drug delivery to inhibit hypersensitivity and chronic pain.