The role of spinal neuroimmune activation in morphine tolerance/hyperalgesia in neuropathic and sham-operated rats.

Hypersensitivity resulting from nerve injury or morphine tolerance/hyperalgesia is predicted to involve similar cellular and molecular mechanisms. One expected but incompletely explored mechanism is the activation of central neuroimmune responses associated with these conditions. To begin to address this, we undertook three separate studies: First, we determined the acute antinociceptive action of morphine, the rate of development of opioid tolerance, and withdrawal-induced hyperalgesia/allodynia in nerve-injured and sham-operated rats using noxious (thermal and mechanical) and non-noxious (mechanical allodynia) behavioral paradigms. Second, we investigated the impact of chronic morphine treatment on spinal glial activation and cytokine expression after L5 spinal nerve transection or sham surgery. Third, we examined the consequences of spinal administration of cytokine inhibitors on the development of morphine tolerance and morphine withdrawal-induced hyperalgesia and allodynia. Results demonstrated that after nerve injury, the antinociceptive effect of acute morphine was significantly decreased, and the rate of development of tolerance and opioid withdrawal-induced hyperalgesia/allodynia was significantly enhanced compared with that after sham surgery. Chronic administration of morphine to sham-operated rats activated spinal glia and upregulated proinflammatory cytokines [interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha]. This neuroimmune activation was further enhanced in nerve-injured rats after chronic morphine treatment. Spinal inhibition of proinflammatory cytokines restored acute morphine antinociception in nerve-injured rats and also significantly reversed the development of morphine tolerance and withdrawal-induced hyperalgesia and allodynia in nerve-injured or sham-operated rats. Targeting central cytokine production and glial activation may improve the effectiveness of morphine and reduce the incidence of morphine withdrawal-induced hyperalgesia and allodynia in neuropathic pain conditions.

Anti-hyperalgesic and morphine-sparing actions of propentofylline following peripheral nerve injury in rats: mechanistic implications of spinal glia and proinflammatory cytokines.

Injury to peripheral nerves often produces non-physiological, long-lasting spontaneous pain, hyperalgesia and allodynia that are refractory to standard treatment and often insensitive to opioids, such as morphine. Recent studies demonstrate spinal glial activation and increased proinflammatory cytokines in animal models of neuropathic pain. When these data are considered together, a unifying hypothesis emerges which implicates a role of central neuroimmune processes in the etiology of neuronal and behavioral hypersensitivity. The present investigation assessed the influence of propentofylline, a glial modulating and anti-inflammatory agent, on the development of L5 spinal nerve transection-induced hyperalgesia and associated enhancement of spinal neuroimmune responses using real-time reverse transcription-polymerase chain reaction, RNase protection assay, enzyme-linked immunosorbent assay, and immunocytochemistry in rats. The results show that chronic propentofylline treatment attenuated the development of hyperalgesia and restored the analgesic activity of acute morphine in neuropathic rats. These findings directly correlated with the ability of propentofylline to inhibit glial activation and enhanced spinal proinflammatory cytokines following peripheral nerve injury. These findings along with our earlier observations of an anti-allodynic activity of propentofylline using the identical animal model of mononeuropathy supports the concept that modulation of glial and neuroimmune activation may be potential therapeutic targets to treat or prevent neuropathic pain. Further, restoration of the analgesic activity of morphine by propentofylline treatment suggests that increased glial activity and proinflammatory cytokine responses may account for the decreased analgesic efficacy of morphine observed in the treatment of neuropathic pain.

Central administration of methotrexate reduces mechanical allodynia in an animal model of radiculopathy/sciatica.

We have recently reported that injury to a lumbar root in a rat model of radiculopathy produces spinal glial activation associated with elevated proinflammatory cytokines. Based on our hypothesis that central neuroinflammatory processes may manifest clinically as radicular pain, we undertook pharmacological intervention using the immunosuppressive agent methotrexate (MTX). The L5 lumbar spinal root (central to the dorsal root ganglia) was exposed unilaterally and loosely constricted with chromic gut. In the prevention (phase I) study, MTX was administered intrathecally (1 mg/kg) and around the spinal root (1 mg/kg) at surgery and at days 2 and 4 postsurgery (group A). Saline injection was employed for the control group (group B). Sham operated animals were administered MTX to determine the potential for behavioral/neural side effects (group C). In the existing pain paradigm (phase II) study, the experiment was extended to day 14 with three additional groups. The same dose and method of delivery of MTX or saline was administered as in phase I in the first week on days 0, 2, and 4 and in the second week on days 7, 9, and 11 postsurgery. To measure the effects of MTX on existing behaviors saline was administered in the first week and MTX during the second (group D; Saline:MTX). The control group received saline during both weeks (group E; Saline:Saline). To examine the possible recurrence of radicular pain after MTX termination, MTX was given in the first week and saline in the second (group F; MTX:Saline). Gait disturbance and mechanical allodynia (using von Frey filaments) were assessed up to day 7 in the prevention study (Phase I) and day 14 in the existing pain paradigm (Phase II). The L5 spinal cord segments were harvested for assessment of immunohistochemical glial activation using the antibodies OX-42 (microglial marker) and glial fibrillary acidic protein (GFAP: astrocytic marker) and for the presence of Major Histocompatibility Complex (MHC) Class II expression. Group C (Sham+MTX) did not demonstrate any evidence of gait disturbance or mechanical allodynia after MTX administration. The rats in group B (Surgery+Saline) demonstrated mechanical allodynia from one day postsurgery to the time of euthanization. When allodynia was assessed using the 12 g von Frey filament, the MTX treated rats in group A showed significantly decreased mechanical allodynia as compared to the saline treated rats (group B) (repeated measured ANOVA, P<0.0001). In the phase II study, the rats in group D (Saline:MTX) and E (Saline:Saline) showed robust allodynia in the first week after the surgery. In the second week, mechanical allodynia significantly decreased in group D, while mechanical allodynia continued in the saline treated group (repeated measured ANOVA, P=0.0121). Allodynia was significantly attenuated in group F (MTX: Saline) as compared to the response in groups D and E at day 7 (one-way ANOVA, P<0.0001) and remained significantly lower as compared to group E up to day 11 postsurgery (one-way ANOVA, P9=0. 0013: P11=0.0048). OX-42 and GFAP expression were elevated in the gray matter of the L5 spinal section in all groups that underwent the root ligature with chromic gut (Groups A, B, D-F). There were no significant differences in glial activation between the groups. However, spinal expression of MHC II was markedly reduced in the MTX treated group as compared with the saline treated group. The exact mechanism of action of MTX in attenuating mechanical allodynia has not yet been elucidated. The present results indicate that MTX administration may offer a new treatment modality for radicular pain with or without disc herniation as well as directing new research into the development of novel immunomodulators for the treatment of chronic neuropathic and radicular pain.

Focal peripheral nerve injury induces leukocyte trafficking into the central nervous system: potential relationship to neuropathic pain.

The present study was undertaken to determine whether leukocytes are recruited into the spinal cord following a peripheral L5 spinal nerve transection that results in mechanical allodynia (increased tactile sensitivity behavior correlates with neuropathic pain). In rats subjected to bone marrow irradiation, donor-specific major histocompatibility complex (MHC) class I (I1-69) positive peripheral immune cells trafficked to the L5 spinal cord in response to an L5 spinal nerve injury. The number of I1-69 positive cell profiles increased over time and correlated with increased mechanical allodynia. At early time points following injury, I1-69 positive immune cells co-regionalized with the expression of the macrophage marker ED2. At later time points following injury, some of the infiltrating immune cells did not co-regionalize with the macrophage marker ED2. At no time did the infiltrating cells co-regionalize with the neuronal marker (NeuN). Both macrophage-like morphology and T cell-like morphology were observed in the I1-69 positive cellular infiltrate. Conversely, animals that underwent sham surgery demonstrated little mechanical allodynia and a minimal number of infiltrating peripheral immune cells. In a separate group of rats, infiltration of CD3+ T-lymphocytes was confirmed at 14 days post-nerve transection. This study demonstrates trafficking of leukocytes into the lumbar spinal cord at time points that correlate with mechanical allodynia suggesting a role of central neuroinflammation in persistent neuropathic pain.

The Role of Cytokines in the Initiation and Maintenance of Chronic Pain.

Recent advancements in the pain field have identified a central nervous system (CNS) neuroimmune response that may act as the driving force for neuronal hypersensitivity, the pathological correlate to chronic pain following peripheral nerve injury. Neuroimmune activation involves the activation of nonneuronal cells such as endothelial and glial cells, which when stimulated leads to enhanced production of a host of inflammatory mediators, such as cytokines. The central production of proinflammatory cytokines, such as interleukin-1beta (IL-1beta), IL-6 and tumor necrosis factor have been found to play a key role in the propagation of persistent pain states. In addition, chemotactic cytokines, chemokines, have also been recently identified in the CNS neuroimmune cascade that ensues after injury to a peripheral nerve. The extravasation of leukocytes from the blood to the site of perceived injury is defined as the neuroinflammatory aspect of this cascade. Chemokines directly control this leukocyte transmigration process. They are synthesized at the site of injury and establish a concentration gradient through which immune cells migrate. Recent studies have demonstrated leukocyte trafficking into the CNS following peripheral nerve or lumbar nerve root injury. With the use of selective cytokine inhibitors and neutralizing antibodies, tactile and thermal hypersensitivity is attenuated in animal models of neuropathy. A further understanding of the role of nonneuronal cells, the physiological mechanisms of CNS cytokines and chemokines, and their relevance to neuro- immune activation and neuroinflammatory processes may lead to the development of novel pharmacological agents for the treatment and prevention of chronic pain. (c) 2002 Prous Science. All rights reserved.

The magnitude of mechanical allodynia in a rodent model of lumbar radiculopathy is dependent on strain and sex.

STUDY DESIGN: This study examined the differences in tactile hypersensitivity across 6 different strains of male mice, and between male and female rats of 3 different strains in a rodent model of low back pain associated with lumbar radiculopathy. OBJECTIVE: We investigated the possibility that differences in tactile allodynia following the same nerve root injury are affected by genotype and sex in rodents. SUMMARY OF BACKGROUND DATA: Low back pain associated with radiculopathy affects countless people throughout the world, encompassing a wide range of individual pain susceptibility. The roles of genetics and sex on differences in nociceptive sensitivities following lumbar nerve root injury have yet to be fully characterized. METHODS: Six strains of mice (BALB/cJ, CBA/J, C57BL/6J, 129P3/J, C3H/HeJ, and C58/J; all males) and male and female Sprague Dawley, Holtzman, and Long-Evans rats underwent a lumbar nerve root injury followed by assessment of tactile allodynia. RESULTS: The most sensitive mouse strains following nerve root injury were: 129P3/J, C58/J, and BALB/cJ; and the less sensitive strains were: C57BL/6J, C3H/HeJ, and CBA/J. Female Sprague Dawley and Long-Evans rats displayed increased hypersensitivity following nerve root injury compared to males. No sex differences were observed in Holtzman rats. CONCLUSIONS: Different mouse strains, and male and female rats that are exposed to identical nerve root injuries have diverse levels of tactile hypersensitivity, supporting the hypothesis that genetic factors and sex play a key role in radicular pain. Our results correlate with data compiled in identical mouse and rat strains after L5-L6 nerve ligation, suggesting that the precise nature of the injury is not relevant to the inheritance of neuropathic symptom sensitivity.

Lumbar nerve root injury induces central nervous system neuroimmune activation and neuroinflammation in the rat: relationship to painful radiculopathy.

STUDY DESIGN: These studies were designed to examine the role of central neuroimmune activation and neuroinflammation in a rat model of lumbar radiculopathy. OBJECTIVES: In the present study the authors investigated the role of neuroimmune activation using immunocytochemistry to detect expression of major histocompatibility complex Class II, cluster determinant 4, intracellular adhesion molecule-1 (ICAM-1), and platelet endothelial cellular adhesion molecule-1 (PECAM-1). The role of central neuroinflammation was investigated using radiation bone marrow chimeric rats. SUMMARY OF BACKGROUND DATA: The pathologic mechanisms resulting in painful lumbar radiculopathy secondary to nerve root injury remain obscure. There is a growing body of evidence that central neuroimmune activation and neuroinflammation may play a key role in the initiation and maintenance of various pain states, including lumbar radiculopathy. METHODS: Male Holtzman rats undergoing mechanical sensitivity testing were divided into two groups: a sham group and a chromic gut suture group. Animals were killed on day 14 post surgery. Male Holtzman rats, used to detect cluster determinant 4, major histocompatibility complex Class II, and CAM spinal expression, were divided into three groups: a normal group, a sham surgery group, and a chromic group. The male Brown Norway rats used to make the radiation bone marrow chimeras were divided into two groups: a sham group and a chromic group. Animals were killed at 1, 3, 7 or 14 days following surgery. RESULTS: Nerve root injury in the rat produced increased spinal major histocompatibility complex Class II, cluster determinant 4, ICAM-1, and PECAM-1 immunoreactivity and increased bilateral sensitivity to tactile stimuli. Leukocyte trafficking into the spinal parenchyma was observed, which increased over time after nerve root injury. CONCLUSIONS: The presence of bilateral mechanical allodynia and spinal neuroimmune changes following nerve root injury supports the hypothesis that central sensitization through activation of immune mediators, coupled with macrophage traffic across the blood-brain barrier, plays a key role in the development and maintenance of radicular pain.