Jun-Ming Zhang

Cytokines, Inflammation, and Pain

Cytokines are small secreted proteins released by cells have a specific effect on the interactions and communications between cells. Cytokine is a general name; other names include lymphokine (cytokines made by lymphocytes), monokine (cytokines made by monocytes), chemokine (cytokines with chemotactic activities), and interleukin (cytokines made by one leukocyte and acting on other leukocytes). Cytokines may act on the cells that secrete them (autocrine action), on nearby cells (paracrine action), or in some instances on distant cells (endocrine action). There are both pro-inflammatory cytokines and anti-inflammatory cytokines. There is significant evidence showing that certain cytokines/chemokines are involved in not only the initiation but also the persistence of pathologic pain by directly activating nociceptive sensory neurons. Certain inflammatory cytokines are also involved in nerve-injury/inflammation-induced central sensitization, and are related to the development of contralateral hyperalgesia/allodynia. The discussion presented in this chapter describes several key pro-inflammatory cytokines/chemokines and anti-inflammatory cytokines, their relation with pathological pain in animals and human patients, and possible underlying mechanisms.

Neuropathic pain: Early spontaneous afferent activity is the trigger

&NA; Intractable neuropathic pain often results from nerve injury. One immediate event in damaged nerve is a sustained increase in spontaneous afferent activity, which has a well‐established role in ongoing pain. Using two rat models of neuropathic pain, the CCI and SNI models, we show that local, temporary nerve blockade of this afferent activity permanently inhibits the subsequent development of both thermal hyperalgesia and mechanical allodynia. Timing is critical—the nerve blockade must last at least 3–5 days and is effective if started immediately after nerve injury, but not if started at 10 days after injury when neuropathic pain is already established. Effective nerve blockade also prevents subsequent development of spontaneous afferent activity measured electrophysiologically. Similar results were obtained in both pain models, and with two blockade methods (200 mg of a depot form bupivacaine at the injury site, or perfusion of the injured nerve just proximal to the injury site with TTX). These results indicate that early spontaneous afferent fiber activity is the key trigger for the development of pain behaviors, and suggest that spontaneous activity may be required for many of the later changes in the sensory neurons, spinal cord, and brain observed in neuropathic pain models. Many pre‐clinical and clinical studies of pre‐emptive analgesia have used much shorter duration of blockade, or have not started immediately after the injury. Our results suggest that effective pre‐emptive analgesia can be achieved only when nerve block is administered early after injury and lasts several days.

A comparison of chronic pain behavior following local application of tumor necrosis factor α to the normal and mechanically compressed lumbar ganglia in the rat

&NA; To study the role of inflammatory cytokines in the initiation and persistence of radiculopathy as seen in humans, tumor necrosis factor &agr; (TNF‐&agr;) was administered either to normal, uninjured L5 dorsal root ganglia (DRG) of rats via a hole drilled through the transverse process, or to chronically compressed L5 DRG via a hollow stainless steel rod inserted into the intervertebral foramen. In other experiments, a mixture of soluble TNF receptors (sTNF‐Rs: sTNF‐RI±sTNF‐RII) was locally delivered to the chronically or acutely compressed DRG to neutralize the activity of endogenous TNF‐&agr;. Behavioral tests of mechanical allodynia were performed before and after TNF‐&agr; administration. Infusion of the normal DRG with TNF‐&agr; at a rate of 1 &mgr;l/h for 7 days induced ipsilateral mechanical allodynia (i.e. decreased mechanical withdrawal threshold) that lasted about 2 weeks. Infusion of the compressed DRG did not alter compression‐induced allodynia within the first operative week but substantially enhanced the ipsilateral allodynia after the first postoperative week. Neutralizing the activity of endogenous TNF‐&agr; of the compressed DRG with sTNF‐Rs reduced allodynia for 3 days, but was subsequently without effect. Similar results were obtained when sTNF‐Rs were chronically administrated at the acutely compressed ganglion. Results demonstrated that exogenous TNF‐&agr; causes pain and mechanical allodynia when deposited at the normal DRG, and further enhances the ongoing allodynia when administrated at the compressed DRG. Results also suggest that endogenous TNF‐&agr; contributes to the early development of mechanical allodynia in rats with chronic DRG compression.

Peripheral Nociception Associated With Surgical Incision Elicits Remote Nonischemic Cardioprotection Via Neurogenic Activation of Protein Kinase C Signaling

Background— Although remote ischemic stimuli have been shown to elicit cardioprotection against ischemia/reperfusion injury, there is little known about the effects of nonischemic stimuli. We previously described a remote cardioprotective effect of nonischemic surgical trauma (abdominal incision) called remote preconditioning of trauma (RPCT). In the present study, we elucidate mechanisms underlying this phenomenon. Methods and Results— We used a murine model of myocardial infarction to evaluate ischemia/reperfusion injury, and either abdominal surgical incision, or application of topical capsaicin, to elicit cardioprotection. We show that the cardioprotective effect of RPCT is initiated by skin nociception, and requires neurogenic signaling involving spinal nerves and activation of cardiac sensory and sympathetic nerves. Our results demonstrate bradykinin-dependent activation and repression, respectively, of PKCϵ and PKC&dgr; in myocardium after RPCT, and we show involvement of the KATP channels in cardioprotection. Finally, we show that topical application of capsaicin, which selectively activates C sensory fibers in the skin, mimics the cardioprotective effect of RPCT against myocardial infarction. Conclusions— Nontraumatic nociceptive preconditioning represents a novel therapeutic strategy for cardioprotection with great potential clinical utility.

Robust Increase of Cutaneous Sensitivity, Cytokine Production and Sympathetic Sprouting in Rats with Localized Inflammatory Irritation of the Spinal Ganglia

We investigated the role and mechanisms of inflammatory responses within the dorsal root ganglion (DRG) in the development of chemogenic pathological pain. DRG inflammation was induced by a single deposit of the immune activator zymosan in incomplete Freunds adjuvant in the epidural space near the L5 DRG via a small hole drilled through the transverse process. After a single zymosan injection, rats developed bilateral mechanical hyperalgesia and allodynia which began by day 1 after surgery, peaked at days 3-7, and lasted up to 28 days. The number of macrophages in ipsilateral and contralateral DRGs increased significantly, lasting over 14 days. Robust glial activation was observed in inflamed ganglia. Cytokine profile analysis using a multiplexing protein array system showed that, in normal DRG, all but interleukin (IL)-5, IL-10 and granulocyte-macrophage colony stimulating factor (GM-CSF) were detectable with concentrations of up to 180 pg/mg protein. Local inflammatory irritation selectively increased IL-1beta, IL-6, IL-18, monocyte chemoattractant protein-1 (MCP-1), and growth-related oncogene (GRO/KC) up to 17-fold, and decreased IL-2 and IL-12 (p70) up to threefold. Inflaming the DRG also remarkably increased the incidence of spontaneous activity of A- and C-fibers recorded in the dorsal root. Many of the spontaneously active A-fibers exhibited a short-bursting discharge pattern. Changes in cytokines and spontaneous activity correlated with the time course of pain behaviors, especially light stroke-evoked tactile allodynia. Finally, local inflammation induced extensive sprouting of sympathetic fibers, extending from vascular processes within the inflamed DRG. These results demonstrate the feasibility of inducing chronic localized inflammatory responses in the DRG in the absence of traumatic nerve damage, and highlight the possible contribution of several inflammatory cytokines/chemokines to the generation of spontaneous activity and development and persistence of chemogenic pathologic pain.

Abnormal spontaneous activity and responses to norepinephrine in dissociated dorsal root ganglion cells after chronic nerve constriction

&NA; As part of ongoing studies of the cellular mechanisms of sympathetically maintained pain, we investigated the effects of a peripheral nerve injury on the responses of dissociated dorsal root ganglion (DRG) cells to norepinephrine (NE). Whole‐cell patch‐clamp recordings under current clamp were obtained from L4 and L5 DRG cells from adult rats in acute culture 11–25 days after a loose or tight ligation of the sciatic nerve. Only small to medium‐sized cells from normal (uninjured) nerves and from loosely ligated nerves were tested with NE. One of 15 cells obtained from uninjured nerves responded to NE (500 &mgr;M, the highest dose, elicited a small depolarization without action potentials). In contrast, many cells from injured nerves responded to NE with a membrane depolarization, accompanied in some cases by the generation of action potentials. Fifty‐two percent responded to 500 &mgr;M, while a significantly lower percentage responded to the lower doses of 100 &mgr;M (26%) and 10 &mgr;M (14%). Cells responsive to NE also responded to capsaicin. Spontaneous activity was observed in 14% and 21% of cells from loosely and tightly ligated nerves, respectively, but none of the cells from uninjured nerves. We conclude that the abnormal electrogenesis and responsiveness to NE in certain nerve‐injured primary sensory neurons are due at least in part to changes in the membrane properties of the soma.

Exercise-induced gene expression in soleus muscle is dependent on time after spinal cord injury in rats.

Cycling exercise attenuates atrophy in hindlimb muscles and causes changes in spinal cord properties after spinal cord injury in rats. We hypothesized that exercising soleus muscle expresses genes that are potentially beneficial to the injured spinal cord. Rats underwent spinal cord injury at T10 and were exercised on a motor‐driven bicycle. Soleus muscle and lumbar spinal cord tissue were used for messenger RNA (mRNA) analysis. Gene expression of brain‐derived neurotrophic factor (BDNF) and glial cell line‐derived neurotrophic factor (GDNF) was elevated 11‐ and 14‐fold, respectively, in soleus muscle after one bout of exercise performed 5 days after spinal cord transection. Also, c‐fos and heat shock protein‐27 (HSP27) mRNA abundance were increased 11‐ and 7‐fold, respectively. When exercise was started 2 days after the injury, the changes in gene expression were not observed. By contrast, at 2 but not at 5 days after transection, expression of the HSP27 gene was elevated sixfold in the lumbar spinal cord, independent of exercise. Electromyographic activity in soleus muscles was also decreased at 2 days, indicating that the spinal cord was less permissive to exercise at this early time. Long‐term exercise for 4 weeks attenuated muscle atrophy equally well in rats started at 2 days or 5 days after injury. We conclude that BDNF and GDNF released from exercising muscle may be involved in exercise‐induced plasticity of the spinal cord. Furthermore, the data suggest that the lumbar spinal cord undergoes time‐dependent changes that temporarily impede the ability of the muscle to respond to exercise. Muscle Nerve 29: 73–81, 2004

An in vitro study of ectopic discharge generation and adrenergic sensitivity in the intact, nerve-injured rat dorsal root ganglion.

Abstract A chronic, loose constriction of the sciatic nerve in rat produces behavioral signs of spontaneous pain and cutaneous hyperalgesia (Bennett and Xie, Pain, 33 (1988) 87–107) as well as an abnormal spontaneous activity and adrenergic sensitivity of certain dorsal root ganglion (DRG) cells with axons in the injured nerve (Kajander et al., Neurosci. Lett., 138 (1992) 225–228; Xie et al., J. Neurophysiol., 73 (1995)1811–1820) The present study investigated whether the spontaneous activity and adrenergic sensitivity were intrinsic properties of injured DRG cells and manifested in vitro, i.e., not dependent on intact blood circulation and an intact, functioning sympathetic nervous system. Two weeks after a loose constriction of the sciatic nerve, the L4 or L5 DRG with its ligated nerve and dorsal root attached was removed from the rat and placed in a chamber. Extracellular recordings were made from teased dorsal root fibers. Spontaneous activity (>0.05 imp/s in 3 min) originating within or close to the DRG was often found in C‐, A&dgr;‐ and A&bgr;‐fibers from nerve‐injured rats, but was rare in fibers with peripheral axons from uninjured nerve. The incidence of various patterns of spontaneous discharge was similar to that previously recorded in vivo. Nineteen of 30 C‐fibers, four of five A&dgr;‐ and three of seven A&bgr;‐fibers from injured nerve responded to different doses of norepinephrine (NE) applied topically to the DRG. Five of seven C‐ and one of two A&bgr;‐fibers from injured nerve responded to clonidine, a more selective &agr;2 adrenergic agonist. The thresholds ranged from 500 to 10 &mgr;M, the lowest dose delivered. None of the fibers from uninjured nerve responded to NE or clonidine (500 &mgr;M). Since the experiments were carried out in vitro in the intact DRG, the existence of spontaneous activity in DRG cells in nerve‐injured rats was independent of any blood borne chemicals, such as norepinephrine. We hypothesize that abnormal activity and adrenergic sensitivity in injured DRG neurons are due to an intrinsic alteration of the cell body membrane.

Early Blockade of Injured Primary Sensory Afferents Reduces Glial Cell Activation in Two Rat Neuropathic Pain Models

Satellite glial cells in the dorsal root ganglion (DRG), like the better-studied glia cells in the spinal cord, react to peripheral nerve injury or inflammation by activation, proliferation, and release of messengers that contribute importantly to pathological pain. It is not known how information about nerve injury or peripheral inflammation is conveyed to the satellite glial cells. Abnormal spontaneous activity of sensory neurons, observed in the very early phase of many pain models, is one plausible mechanism by which injured sensory neurons could activate neighboring satellite glial cells. We tested effects of locally inhibiting sensory neuron activity with sodium channel blockers on satellite glial cell activation in a rat spinal nerve ligation (SNL) model. SNL caused extensive satellite glial cell activation (as defined by glial fibrillary acidic protein [GFAP] immunoreactivity) which peaked on day 1 and was still observed on day 10. Perfusion of the axotomized DRG with the Na channel blocker tetrodotoxin (TTX) significantly reduced this activation at all time points. Similar findings were made with a more distal injury (spared nerve injury model), using a different sodium channel blocker (bupivacaine depot) at the injury site. Local DRG perfusion with TTX also reduced levels of nerve growth factor (NGF) in the SNL model on day 3 (when activated glia are an important source of NGF), without affecting the initial drop of NGF on day 1 (which has been attributed to loss of transport from target tissues). Local perfusion in the SNL model also significantly reduced microglia activation (OX-42 immunoreactivity) on day 3 and astrocyte activation (GFAP immunoreactivity) on day 10 in the corresponding dorsal spinal cord. The results indicate that early spontaneous activity in injured sensory neurons may play important roles in glia activation and pathological pain.

Human acupuncture points mapped in rats are associated with excitable muscle/skin–nerve complexes with enriched nerve endings

As part of our ongoing investigation into the neurological mechanisms of acupuncture, we have tried to correlate the distribution of afferent nerve endings with acupuncture points (AP) in the rat hind limbs. In vivo extracellular microfilament recordings of Aalpha/Abeta/Adelta fibers were taken from peripheral nerves to search for units with nerve endings or receptive fields (RF) in the skin or the muscles. The location of the RFs for each identified unit was marked on scaled diagrams of the hind limb. Noxious antidromic stimulation-induced Evans blue extravasation was used to map the RFs of C-fibers in the skin or muscles. Results indicate that, for both A- and C-fibers, the distribution of RFs was closely associated with the APs. In the skin, the RFs concentrate either at the sites of APs or along the orbit of meridian channels. Similarly, the majority of sarcous sensory receptors are located at the APs in the muscle. Results from our studies strongly suggest that APs in humans may be excitable muscle/skin-nerve complexes with high density of nerve endings.