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Brain-derived TNFα mediates neuropathic pain
Brain Research 841 Ž1999. 70–77 www.elsevier.comrlocaterbres
Research report
Brain-derived TNFa mediates neuropathic pain Tracey A. Ignatowski a,b , William C. Covey a , Paul R. Knight b , Charles M. Severin c , Tracey J. Nickola a , Robert N. Spengler a,b,) a
Department of Pathology, State UniÕersity of New York at Buffalo, 204 Farber Hall, 3435 Main Street, Buffalo, NY, 14214, USA b Department of Anesthesiology, State UniÕersity of New York at Buffalo, 3435 Main Street, Buffalo, NY, 14214, USA c Department of Anatomy, State UniÕersity of New York at Buffalo, 3435 Main Street, Buffalo, NY, 14214, USA Accepted 22 June 1999
Abstract Neuropathic pain is a chronic pain state that develops a central component following acute nerve injury. However, the pathogenic mechanisms involved in the expression of this central component are not completely understood. We have investigated the role of brain-associated TNF in the evolution of hyperalgesia in the chronic constriction injury ŽCCI. model of neuropathic pain. Thermal nociceptive threshold has been assessed in rats Žmale, Sprague–Dawley. that have undergone loose, chromic gut ligature placement around the sciatic nerve. Total levels of TNF in regions of the brain, spinal cord and plasma have been assayed ŽWEHI-13VAR bioassay.. Bioactive TNF levels are elevated in the hippocampus. During the period of injury, hippocampal noradrenergic neurotransmission demonstrates a decrease in stimulated norepinephrine ŽNE. release, concomitant with elevated hippocampal TNF levels. Continuous intracerebroventricular Ži.c.v.. microinfusion of TNF-antibodies ŽAbs. starting at four days, but not six days, following ligature placement completely abolishes the hyperalgesic response characteristic of this model, as assessed by the 588C hot-plate test. Antibody infusion does not decrease spinal cord or plasma levels of TNF. Continuous i.c.v. microinfusion of rrTNFa exacerbates the hyperalgesic response by ligatured animals, and induces a hyperalgesic response in animals not receiving ligatures. Likewise, field-stimulated hippocampal adrenergic neurotransmission is decreased upon continuous i.c.v. microinfusion of TNF. These results indicate an important role of brain-derived TNF, both in the pathology of neuropathic pain, as well as in fundamental pain perception. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Pain; TNF; Norepinephrine; a 2 -Adrenergic receptor; Microinfusion; Hippocampus
1. Introduction Chronic pain syndromes often develop a central nervous system ŽCNS.-mediated component that plays a role in the cognitive experience of pain and associated mood changes. Chronic persistent pain that has developed a central component is a difficult therapeutic challenge. Neuroplastic changes at several sites in the spinal cord and brain have been implicated w8,16x. Neuropathic pain develops from intense, prolonged noxious stimulation, or from nerve injury, and leads to long-term functional changes in the CNS, resulting in amplification and persistence of the pain. Two common symptoms of neuropathic pain include hy) Corresponding author. [email protected]
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peralgesia Žan enhanced response to normally painful stimuli. and allodynia Ža painful response to a normally innocuous stimuli.. Drugs that appear to modify adrenergic receptors in the CNS Že.g., clonidine, amitriptyline. are efficacious, to varying degrees, in preventing or alleviating these symptoms w4,6x. Elevated levels of systemic proinflammatory cytokines occur in association with increases in pain perception, as well as during changes in general mood status w18,30,31x. During systemic illness Že.g., rheumatoid arthritis and microbial infections., following activation of the proinflammatory cytokine cascade, patients experience mood changes, such as general malaise and somnolence, along with the appearance of nondescript pain symptoms, such as polyarthralgias, myalgias, and hypesthesia w9x. When TNF is administered intravenously for 24 h to human volun-
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teers, lethargy and general malaise are consistently produced w18x. While systemically administered cytokines may affect responses in the CNS, how this corresponds to regional TNF levels achieved, as well as sites of cytokine action are not known. A considerable amount of evidence supports a pronociceptive role for TNF during chronic pain w11,30x. Findings from our laboratory have led us to hypothesize that proinflammatory cytokines, such as TNF, synthesized in neurons within distinct regions of the CNS are involved in promoting neuroplastic changes of noradrenergic neurons involved in the perception of pain w12,13x. Watkins et al. w32x have proposed that hyperalgesia is produced by the peripheral, local production of proinflammatory cytokines which can activate vagal afferents mediating signals of pain perception to the brain. These signals to the brain result, via descending circuitry, in alterations at the level of the spinal cord that are implicated in various forms of hyperalgesia w32x. An intense afferent barrage appears necessary to elicit the signals that establish persistent pain. A number of observations support the role of the central noradrenergic nervous system in pain perception. The locus coeruleus ŽLC. is the largest noradrenergic nucleus within the brain w26x, and the noradrenergic neuronal cell bodies there represent the primary source of norepinephrine ŽNE. in the CNS w21x. Norepinephrine is a principal neurotransmitter associated with the modulation of nociception and analgesia w21x. Electrical stimulation of the LC has been demonstrated to produce analgesia w17x. The hippocampus, a region of the brain rich in noradrenergic nerve terminals, associated with pain perception w16x, receives its NE innervation exclusively from the LC. The LC and the adjacent subcoeruleus ŽSC. also represent the major source of noradrenergic nerve terminals to the spinal cord w19x. Finally, drugs such as the a 2-adrenergic agonist clonidine act at noradrenergic synapses and are useful in the management of chronic pain w6x. We have postulated that proinflammatory cytokine production by neurons within specific regions of the brain at the time of pain-elicited afferent barrage plays an important role in the pathogenesis of ‘‘central sensitization’’. Biologically active TNF and TNF mRNA can be detected in noradrenergic regions of the rat brain w12,13x. Additionally, noradrenergic neuronal immunohistochemical staining for TNF has been demonstrated w14x. Administration of IL-1b via intracerebroventricular Ži.c.v.. injection produces hyperalgesia, although intrathecal injection does not, indicating that the brain and not the spinal cord is the site of cytokine action w30x. In the present study, we report on the role of brain-derived TNF in the pathogenesis of hyperalgesia using a chronic constriction injury ŽCCI. of the rat sciatic nerve, developed by Bennett and Xie w2x. In order to examine this interaction, we assessed TNF levels in select nuclei of the brain and infused TNF as well as TNF antibody ŽAb. into the right lateral cerebral ventricle. The effect of this cy-
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tokine or antibody administration on hyperalgesia has been evaluated.
2. Materials and methods 2.1. CCI peripheral neuropathic pain model Experimental animal care has been approved in accordance with IACUC institutional guidelines. Rats Žmale, Sprague–Dawley, 250–350 g. are anesthetized with ketamine Ž60 mgrkg. and xylazine Ž3 mgrkg. i.p. prior to operation. The sciatic nerve is exposed unilaterally, and four ligatures Ž4.0 chromic gut. are loosely placed around the nerve, 1 mm apart proximal to the trifurcation. In sham procedures, the nerve is exposed and freed of adherent tissuermuscle, but no ligatures are placed. 2.2. Thermal nociception measurements Thermal nociceptive threshold is measured in each hind paw using a 588C hot plate test. Changes in thresholds Žseconds. are expressed as either the percentage of the Maximal Possible Effect Ž% MPE. for antihyperalgesic effects, or the percentage of the Maximal Possible Response Ž% MPR. for increased hyperalgesic response, using the equation: wŽExperimental latencyy Mean basal latency.rŽMaximal latencyy Mean basal latency.x = 100. Pre-ligation hind paw withdrawal latencies Žmean basal latencies. are subtracted from those taken post-ligation Žexperimental latencies. in order to generate a ‘‘difference score’’, which is used as an index of hyperalgesia. Normal latency withdrawal values pre-CCI were 6.0–10.0 s Ž15 s being the maximal latency accepted.. Cutaneous hind-paw temperature measurement was also recorded. Animals are held and the plantar hind paw is gently pressed with a BT-1 surface microprobe thermocouple sensor connected to a digital thermometer ŽModel TH-5, Physitemp, Clifton, NJ.. Stable temperature recordings were obtained within 30–60 s and were measured to the nearest 0.18C. The change in cutaneous temperature was calculated as the difference of averaged pre-CCI recordings from each postCCI recording. Normal cutaneous hind paw skin temperatures averaged 28.38C for the contralateral and 28.28C for the ipsilateral hind paw. All measurements were recorded between 0700–0800 h. All surgeries were performed between 0800–1200 h. 2.3. TNF bioassay Total biologically-active TNF was assayed before or after specific noxious stimuli andror pharmacological interventions for TNF activity using the WEHI-13VAR bioassay w24x. The selectivity of WEHI fibroblast cells for TNF-induced lysing by TNF from tissue has been well established by this laboratory and others w15,24x.
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2.4. IntracerebroÕentricular microinfusion Mini osmotic pumps Ž0.5 ulrh, 14 days, Alza, Palo Alto, CA. and brain infusion cannulae ŽAlza, Palo Alto, CA. are assembled according to the manufacturer’s guidelines. The vehicle used for delivery of the infused compounds is aCSF ŽNaCl, 216.5 mM; KCl, 3 mM; CaCl 2 P 2H 2 O, 1.4 mM; MgCl 2 P 6H 2 O, 0.8 mM; Na 2 HPO4 P 7H 2 O, 0.8 mM; and NaH 2 PO4 P H 2 O, 0.2 mM.. Gentamycin Ž0.1 mgrml solution; Sigma, St. Louis, MO. is added to the aCSF to prevent bacterial growth. Rat albumin Žfraction V, 1 mgrml; Sigma, St. Louis, MO. was added to stabilize the infused compound and to prevent proteins and charged molecules from binding non-specifically. Rats Žmale, Sprague–Dawley, 250–350 g., anesthetized with ketamine Ž60 mgrkg. and xylazine Ž3 mgrkg, i.p.., are secured on a stereotaxic platform. With bregma used as the zero point, the following are the stereotaxic coordinates: A–P, y0.92 mm; lateral, 1.6 mm; vertical, 3.5 mm, for trephining the skull prior to cannulation of the right lateral cerebral ventricle. Compounds to be infused include the following: polyclonal rabbit anti-mouse TNFa ŽGenzyme, Cambridge, MA. and normal rabbit serum ŽSigma, St. Louis, MO.; monoclonal hamster anti-murine TNFa and Armenian hamster IgG isotype control Ab ŽGenzyme, Cambridge, MA.; recombinant rat TNFa ŽR & D Systems, Minneapolis, MN.; as well as aCSF alone. The dosage for each infused compound is based on the EC 50 value for inhibition of 3–10 pgrml TNF in the bioassay.
The viability of each Ab, its control, and rrTNFa were confirmed in the bioassay following 14 days of incubation in aCSF at 378C.
3. Results 3.1. CCI induction of hyperalgesia and hyperthermia The CCI model of neuropathic pain consists of a central and peripheral component w2x. Rats undergo unilateral ligature placement and are evaluated every other day. Hyperalgesia is measured by changes in paw-withdrawal latency on a 588C hot plate w3x. Mean baseline response latency over all experimental conditions was 7.9 " 0.2 seconds. Heat-evoked hind paw withdrawal latencies are consistently reduced in CCI animals as compared to sham operated rats ŽFig. 1.. Hyperalgesia, quantified by a difference in paw-withdrawal latency Žseconds., and expressed as the percentage of the Maximal Possible Response Ž% MPR., begins to escalate on day two post-surgery and reaches a maximum on day 10. Unoperated animals tested at the same times do not differ from sham animals, with scores remaining close to the zero score Ždata not shown.. Alterations of sympathetic activity, assessed as changes in skin temperature, have been observed in neuropathic pain models w29x. Changes in hind paw skin temperature are expressed as the temperature difference in degree centigrade Ž8C. from the average of three recordings taken on
Fig. 1. Assessment of hyperalgesia in the CCI model of neuropathic pain. Data are presented as the percent Maximal Possible Response wŽpost-CCI pre-CCI values.rŽ15 - pre-CCI values.x = 1004 of the latency-to-hind paw withdrawal using the 588C hot plate test. The effect of rrTNFa i.c.v. infusion on the CCI-induced nociceptive response is demonstrated. Each point is expressed as the negative score for mean " S.E.M. with the number of rats indicated in parentheses. Statistical significance evaluated with ANOVA. Statistical significance different from sham animals: U p - 0.05, UU p - 0.01. Significantly different from CCI rats, ap - 0.02, aap - 0.01. CCI s chronic constriction injury.
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each of two days prior to and on the day of ligature placement. Hyperthermia, indicative of decreased sympathetic activity Žvasodilatation., occurs during the initial 14 days following CCI Ždata not shown.. Interestingly, this response is noted in both the ipsilateral and contralateral hind paws of CCI rats, a hallmark for a central origin. 3.2. Brain-associated TNF leÕels Regional total TNF levels increase in a time-dependent fashion, coincident with the development of hyperalgesia in the CCI neuropathic pain model. At day six Ženhanced development of hyperalgesia., TNF levels increase in the hippocampus Ž6.5 " 2.2 pgr100 mg tissue at day 4 postCCI as compared to 19.6 " 2.1 pgr100 mg tissue at day 6 post-CCI, p - 0.02, Student’s t-test.. These data clearly demonstrate that an increase occurs in total TNF levels in a region of the brain associated with pain cognition w16x, with a time course indicative of TNF playing a primary role in neuropathic pain. 3.3. Norepinephrine (NE) oÕerflow during field stimulation of hippocampal brain slices We have previously demonstrated that coincident with tricyclic antidepressant ŽTCA.-induced loss in neuron-associated TNF there is a significant increase in field-stimulated release of 3 H-NE from hippocampal brain slices w12,13x. While this class of drugs ŽTCA. is used to treat chronic pain syndromes, we have presently investigated the effect chronic pain has on 3 H-NE overflow from hippocampal brain slices obtained from CCI rats. 3 H-NE
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ŽL-wring-2,5,6,-3 Hx-; NEN, spec. act. 59.1 Cirmmol. release, after electrical field stimulation, is measured directly in a series of hippocampal brain slices w12x. The release of preloaded 3 H-labeled neurotransmitter during electrical field stimulation is an established method for studying neurotransmitter release w25x. The hippocampus is studied because this region is implicated in pain perception and mood states w16x, and drugs efficacious in the treatment of pain have been demonstrated to elicit neuroplastic changes associated with hippocampal functioning w12,13x. Electrical field-stimulated release of NE is frequency-dependent ŽFig. 2.. Hippocampal slices obtained from CCI rats at eight days post-surgery demonstrate a significant decrease in NE release at both the 1 Hz and 4 Hz frequencies of stimulation. Transmitter release from rat hippocampal slices is a regulated event w25x. At the higher frequency of stimulation a greater expression of regulatory mechanisms governing further NE release may be expected. While TNF obviously plays a pivotal role in the adrenergic neuroplastic change and also in the hyperalgesia, factors such as the afferent barrage are also involved in neuropathic pain. Furthermore, the changes in NE release are coincident with the development of hyperalgesia. Similar results occur when TNF is continuously microinfused into the right lateral cerebral ventricle of an untreated rat ŽFig. 2b.. When hippocampal slices from rrTNFa-administered rats are field-stimulated, the fractional release is significantly decreased at 1 Hz ŽS1. and at 4 Hz ŽS2.. The S2rS1 ratio is also significantly decreased indicating a more striking decrease of release at the higher electrical frequency. Consequently, during both chronic pain and continuous microinfusion of TNF into the brain, noradrenergic neu-
Fig. 2. Stimulated neuronal release of 3 H-NE. Neuronal release was studied in hippocampal slices by applying field stimulations consisting of trains of square wave pulses Ž26 V, 2 ms duration. at either 1 Hz or 4 Hz for two minute periods. Each slice was positioned between two nylon mesh disks and placed within each of six chambers connected to two platinum wire electrodes designed to stimulate adrenergic nerve endings Žfield stimulation.. Ža. Stimulated hippocampal NE release from CCI rats at day-8 post-surgery. Žb. Stimulated hippocampal NE release from control animals continuously microinfused with rrTNFa. The data are expressed as the percent release of the total 3 H pool in the tissue at the time of stimulation in excess of spontaneous efflux. Each bar represents the mean " S.E.M. from 11 separate determinations. Statistical significance different from control was reached at U p - 0.05, UU p - 0.01, or UUU p - 0.001 using a paired Student’s t-test.
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ronal activity is altered resulting in decreased NE release in the brain. 3.4. Effects of i.c.Õ. microinfusion of TNF-Ab on hyperalgesic responses to CCI An increase in TNF generation in the hippocampus is associated with an increased intensity of neuropathic pain perception and a change in adrenergic responses ŽFig. 2., resulting in a decrease in NE release. In order to further test this hypothesis, we have infused either polyclonal or monoclonal TNF-Abs into the right lateral cerebral ventricle of CCI rats w33x. Antibody infusion is performed both four and six days post-ligature placement, corresponding to the time periods just preceding and during the appearance of elevated levels of the cytokine in the brain. Continuous microinfusion of polyclonal TNF-Abs ŽpTNF-Ab, 0.5 mlrh. ŽFig. 3., and with lesser efficacy monoclonal TNFAbs ŽmTNF-Abs. Ždata not presented., into the brains of rats starting at four days post-ligature placement abolishes the hyperalgesic response characteristic of this model. These rats also demonstrate a hypoalgesic response following Ab infusion. These observations indicate an important role for TNF both in the pathogenesis of neuropathic pain, as well as in pain perception. The effect of TNF-Ab on pain is dependent upon the time administered post nerve injury. Microinfusion of Ab at six days post-ligature place-
ment does not reduce the hyperalgesic response ŽFig. 3.. Infusion of the vehicle ŽaCSF. or normal rabbit serum has no effect on this response when compared to rats receiving ligature placement only. Infusion with IgG Ab as a control for monoclonal TNF-Ab demonstrates a small decrease in the nociceptive response early during the infusion period Ždata not shown.. We speculate that this decrease in response may be due to non-specific immunoglobulininduced modification of constitutive TNF production. This phenomenon can be demonstrated in mononuclear cells w10,27x. The concentrations of TNF-Ab used for microinfusion are pharmacological doses of 17 times a 1r100,000 dilution for the pTNF-Ab and 17 times a 1r6000 dilution for the mTNF-Ab, based on the ability of each type of Ab to completely block the EC 50 concentration of mrTNFa in the WEHI bioassay. This concentration of TNF-Ab reaches concentrations appropriate to block TNF in neighboring brain regions due to complete CSF turnover every one to two hours in the rat brain, such that the half-life of most CSF solutes is 0.75 h w33x. The ability of TNF-Ab administered by this approach to alter systemic TNF levels was assessed by assaying for plasma levels of TNF. The levels of plasma TNF did not differ in CCI rats infused with TNF-Abs as compared to those infused with aCSF alone ŽaCSF s 2.28 " 0.91 pgrml; pTNF-Ab s 3.63 " 1.16 pgrml; mTNF-Abs 5.20 " 3.49 pgrml; NS.. The levels
Fig. 3. Nociceptive threshold for latency-to-hind paw withdrawal in the CCI model of neuropathic pain. Results are presented as the negative of the percent MPE using the 588C hot plate test. Note: Infusion of pTNF-Ab beginning 6 days post-CCI placement had no effect on the hyperalgesic response characteristic of this neuropathic pain model, while infusion 4 days following CCI ablated the hyperalgesic response. CCI s chronic constriction injury. Each point represents mean " S.E.M. with the number of determinations indicated in parentheses. Significantly different from CCI rats, U p - 0.05, ŽANOVA.. Significantly different from CCI rats, aCSF-infused CCI rats, and Rb serum-infused rats, UU p - 0.05 ŽANOVA..
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Fig. 4. Effect of rrTNFa i.c.v. microinfusion on pain perception in control animals. rrTNFa-infused control rats demonstrate a subsequent decrease in nociceptive threshold similar to that shown by CCI rats. Each point represents the negative score for mean " S.E.M. from the number of rats in parentheses.
of TNF in the cervical and thoraco-lumbar regions of the spinal cord also did not differ in animals infused with aCSF alone or with TNF-Abs Ždata not shown.. These findings indicate that the effects of microinfusion of TNFAb into the brain are most likely due to the blockade of TNF activity in neighboring brain regions of the cerebral right lateral ventricle. 3.5. Effect of intracerebroÕentricular microinfusion of rrTNFa on pain perception Whether TNF is a causative factor contributing to the onset of the development of neuropathic pain symptoms was examined by the infusion of rrTNF into the brains of CCI rats as well as into unoperated rats. Right lateral cerebral ventricle infusions consisted of rats receiving either 30 ng rrTNFar24 h or 1000 ng rrTNFar24 h at a rate of 0.5 mlrh for 14 days. These concentrations were chosen in order to achieve appropriate levels of the cytokine in brain tissue Žwithin the synapse.. We have previously demonstrated that a TNF concentration of 1 to 30 ngrml is needed to affect in vitro neurotransmission in the hippocampus w12,13x. Infusion of 30 ng rrTNFar24 h for 14 days does not effect the hyperalgesic response nor does it affect the total levels of TNF in the rat brain, spinal cord, sciatic nerve, or plasma Ždata not shown.. However, i.c.v. infusion of 1000 ng rrTNFar24 h beginning on day four post-CCI enhances CCI-induced hyperalgesia. Differences are apparent at day 14 post-CCI Žday 10 post-infusion. and remain so
throughout the duration of the infusion ŽFig. 1.. Interestingly, the increases in TNF tissue levels, expressed as percent of control rat values, are highest in regions of the brain anatomically closest to the site of infusion Žhippocampus, 60,000% control.. However, there are no detectable increases in TNF levels, as compared to controls, in the region containing the lumbar spinal cord nor in the systemic circulation Žplasma, data not shown., indicating that TNF is acting at higher brain centers to induce an increase in pain perception. Infusion of 1000 ng rrTNFar24 h into unoperated control animals induces a hyperalgesic response equal to that experienced by a CCI animal ŽFig. 4.. While the initial hyperalgesic response at day two post-infusion of rrTNF appears to be due to the increase in total TNF levels in the CNS, the initial hyperalgesic response expressed by the CCI animal is probably due to immediate changes in TNF levels at the nerve terminal. Infusion of the vehicle alone, aCSF, does not modify the nociceptive threshold as assessed by the hot plate test Ždata not shown..
4. Discussion We have examined levels of biologically-active TNF during experimental neuropathic pain in a region of the brain involved in cognition and implicated in pain perception w16x. Furthermore, we have also examined levels of biologically-active TNF in select regions of the brain and spinal cord in rats microinfused with TNF into the right
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lateral cerebral ventricle while experiencing experimental neuropathic pain. The temporal increases in the levels of this cytokine and the physical manifestations of persistent pain occur concomitantly. The development of the physical manifestations of persistent pain, as well as neuroplastic changes were determined by quantitating hyperalgesia, as well as changes in NE release. These experiments were performed in order to determine the requirement for TNF in the development of hyperalgesia. While changes in levels of TNF in the spinal cord as well as at the peripheral site of nerve injury during neuropathic pain has been well-documented w7,23,28x, an effect on its levels in the brain is original. Therefore, the changes in hippocampal TNF levels along with the involvement of brain-derived TNF in pain shows that the cognitive perception of pain must play an integral role in hyperalgesia. Intracerebroventricular infusion of TNF-Abs into the right lateral cerebral ventricle abolishes hyperalgesia, a hallmark of neuropathic pain. Conversely, infusion of rrTNFa not only enhances CCI-induced hyperalgesia, but also induces hyperalgesia in unoperated rats. The lack of changes in TNF levels in the lumbar spinal cord and in the systemic circulation following i.c.v. infusion strongly indicates that the action of TNF occurs at brain centers that modulate pain perception. Our previous work demonstrates that TNF regulates NE release from adrenergic nerve terminals w12,13x. TCA administration Ždrug class used to treat chronic pain. to unmanipulated rats produces a depletion in neuron-associated TNF expression in regions of the brain with adrenergic activity w14x. Furthermore, chronic administration of the TCA enhances adrenergic neurotransmission at the same time that it transforms the response elicited by the a 2-adrenergic receptor w12x. During chronic pain the fractional release of NE is decreased ŽFig. 2a.. Therefore, TCAs may exert their effects on nociception through similar mechanisms as in depression. The majority of investigators examining central adrenergic receptor participation in centrally-mediated pain have focused on adrenergic receptor function in the spinal cord. However, these results also suggest that supraspinal sites are involved. Prominent innervation of the spinal cord by descending noradrenergic neurons indicates that NE mediates many of the neuronal processes in the spinal cord w5x. Of particular interest, the activity of noradrenergic neurons in the supraspinal regions ŽLC, SC. can be decreased by central administration of a 2-adrenergic agonists w1x. Therefore, modifying the activity of LC neurons may alter the activity of their axon terminals in the spinal cord, thus modulating the descending noradrenergic inhibition of nociception in the spinal cord. However, we have addressed how changes in adrenergic activity occur in the hippocampus due to changes in TNF production and release in the LC and hippocampus w12,13x. The present findings complement this previous work, demonstrating how these changes would play a primary role in neuropathic pain.
The use of anti-TNF agents as possible therapies for conditions associated with nerve injury, such as those seen in rheumatoid arthritis w22x, has gained interest. However, because of the redundant and overlapping actions of many of the cytokines, interpretation of data derived from studies undertaken to inhibit cytokine activity is usually not straightforward. Clinically, this has led to many of the failures associated with therapies that are based on experimental data used to evaluate cytokine cascades in disease Že.g., strategies to improve the outcome of sepsis.. This may be particularly important when the stimulus is multifactorial Žcentralized pain, depression.. The most obvious and direct example of this is the overlapping responses to IL-1 and TNF. Both of these initiation cytokines have very similar actions in inducing subsequent cytokines that may play a causative role in adrenergic adaptational changes. In addition, many of the biological responses of IL-1 and TNF are additive or synergistic. Therefore, it should be noted that a time-dependence in the efficacy of TNF-Ab infusion regarding nociceptive threshold withdrawal latencies was noted in our studies ŽFig. 3.. This finding demonstrates an induction of the cytokine cascade, whereby following the initial increase in central TNF levels, TNFAbs are ineffective in preventing ensuing pain perception. Similarly, the finding that IL-1ra is able to block TNF-induced hyperalgesia, when TNF was injected either i.p. w31x or i.c.v. w20x, suggests that TNF induces or enhances the release of IL-1 which, in turn, produces hyperalgesia. However, the present studies do indeed demonstrate a pivotal role for the proinflammatory cytokine, TNF, in the mediation of centrally mediated chronic pain syndromes.
Acknowledgements This work was supported by PVA grant a RFA008 ŽRNS. and NIMH grant a MH53946 ŽRNS..
References w1x G.K. Aghajanian, C.P. VanderMaelen, Alpha 2 -adrenoceptor mediated hyperpolarization of locus coeruleus neurons: intracellular studies in vivo, Science 215 Ž1982. 1394–1396. w2x G.J. Bennett, Y.-K. Xie, A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain 33 Ž1988. 87–107. w3x M. Bianchi, P. Sacerdote, P. Ricciardi-Castagnoli, P. Mantegazza, A. Panerai, Central effects of tumor necrosis factor alpha and interleukin-1 alpha on nociceptive thresholds and spontaneous locomotor activity, Neuroscience Letters 148 Ž1992. 76–80. w4x H.M. Bryson, M.I. Wilde, Amitriptyline: a review of its pharmacological properties and therapeutic use in chronic pain states, Drugs and Aging 8 Ž1996. 459–476. w5x F.M. Clark, H.K. Proudfit, The projection of locus coeruleus neurons to the spinal cord in the rat determined by anterograde tracing combined with immunocytochemistry, Brain Res. 538 Ž1991. 231– 245. w6x E.E. Codd, J.B. Press, R.B. Raffa, Alpha-2 adrenoceptors vs. imida-
T.A. Ignatowski et al.r Brain Research 841 (1999) 70–77
w7x
w8x
w9x
w10x
w11x
w12x
w13x
w14x
w15x
w16x w17x
w18x
w19x
w20x
zoline receptors: implications for alpha-2 mediated analgesia and other non-cardiovascular therapeutic uses, wReviewx, Life Sciences 56 Ž1995. 63–74. J.A. Deleo, R.W. Colburn, A.J. Rickman, Cytokine and growth factor immunohistochemical spinal profiles in two animal models of mononeuropathy, Brain Res. 759 Ž1997. 50–57. R. Dubner, M.A. Ruda, Activity-dependent neuronal plasticity following tissue injury and inflammation, Trends Neurol. Sci. 15 Ž1992. 96–103. S.H. Ferreira, The role of interleukins and nitric oxide in the mediation of inflammatory pain and its control by peripherial analgesics, wReviewx, Drugs 46 Ž1. Ž1993. 1–9, Suppl. J.L. Foreback, D.G. Remick, E. Crockett-Torabi, P.A. Ward, Cytokine responses of human blood monocytes stimulated with Igs, Inflammation 21 Ž1997. 501–517. H. Fukuoka, M. Kawatani, T. Hisamitsu, C. Takeshige, Cutaneous hyperalgesia induced by peripheral injection of interleukin-1 beta in the rat, Brain Res. 657 Ž1994. 133–140. T.A. Ignatowski, R.N. Spengler, Tumor necrosis factor-a: presynaptic sensitivity is modified after antidepressant drug administration, Brain Res. 665 Ž1994. 293–299. T.A. Ignatowski, R.C. Chou, R.N. Spengler, Changes in noradrenergic sensitivity to tumor necrosis factor-a in brains of rats administered clonidine, J. Neuroimmunol. 70 Ž1996. 55–63. T.A. Ignatowski, B.K. Noble, J.R. Wright, J.L. Gorfien, R.R. Heffner, R.N. Spengler, Neuronal-associated tumor necrosis factor ŽTNFa .: its role in noradrenergic functioning and modification of its expression following antidepressant drug administration, J. Neuroimmunol. 79 Ž1997. 84–90. K.S.A. Khabar, S. Siddiqui, J.A. Armstrong, WEHI-13VAR: a stable and sensitive variant of WEHI 164 clone 13 fibrosarcoma for tumor necrosis factor bioassay, Immunol. Lett. 46 Ž1995. 107–110. S. Khanna, J.G. Sinclair, Noxious stimuli produce prolonged changes in the CA1 region of the rat hippocampus, Pain 39 Ž1989. 337–343. D. Margalit, M. Segal, A pharmacologic study of analgesia produced by stimulation of the nucleus locus coeruleus, Psychopharmacology 62 Ž1979. 169–173. A. Mittelman, C. Puccio, E. Gafney, N. Coombe, B. Singh, D. Wood, P. Nadler, T. Ahmed, Z. Arlin, A phase I pharmacokinetic study of recombinant human TNF administered by a 5-day continuous infusion, Investigational New Drugs 10 Ž1992. 183–190. R.Y. Moore, F.E. Bloom, Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems, Annu. Rev. Neuroscience 2 Ž1979. 113–168. T. Oka, Y. Wakugawa, M. Hosoi, K. Oka, T. Hori, Intracerebroven-
w21x
w22x
w23x
w24x
w25x
w26x
w27x
w28x w29x
w30x
w31x
w32x
w33x
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tricular injection of tumor necrosis factor-alpha induces thermal hyperalgesia in rats, Neuroimmunomodulation 3 Ž1996. 135–140. H.K. Proudfit, Pharmacological evidence for the modulation of nociception by noradrenergic neurons, Prog. Brain Res. 77 Ž1998. 357–370. E.C. Rankin, E.H. Choy, D. Kassimos, G.H. Kingsley, A.M. Sopwith, D.A. Isenberg, G.S. Panayi, The therapeutic effects of an engineered human anti-tumor necrosis factor alpha antibody ŽCDP571. in rheumatoid arthritis, Br. J. Rheumatol. 34 Ž1995. 334–342. C. Sommer, M. Marziniak, R.R. Myers, The effect of thalidomide treatment on vascular pathology and hyperalgesia caused by chronic constriction injury of rat nerve, Pain 74 Ž1998. 83–91. R.N. Spengler, S.W. Chensue, D.A. Giacherio, N. Blenk, S.L. Kunkel, Endogenous norepinephrine regulates tumor necrosis factor-alpha production from macrophages in vitro, J. Immunol. 152 Ž1994. 3024–3031. K. Starke, M. Gothert, H. Kilbinger, Modulation of neurotransmitter release by presynaptic autoreceptors, Physiol. Rev. 69 Ž1989. 864– 989. L.W. Swanson, B.K. Hartman, The central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-beta-hydroxylase as a marker, J. Comp. Neurol. 163 Ž1975. 467–505. Y. Ueda, K.H. Lee, S. Ito, K. Saito, N. Niesen, J.R. Brentjens, R.O.B. Gans, In vivo neutralization of tumor necrosis factor attenuates the generalized Shwartzman reaction in the rabbit, J. Endotoxin Res. 3 Ž1996. 67–75. R. Wagner, R.R. Myers, Endoneurial injection of TNF-alpha produces neuropathic pain behaviors, NeuroReport 7 Ž1996. 2897–2901. S. Wakisaka, K.C. Kajander, G.J. Bennett, Abnormal skin temperature and abnormal sympathetic vasomotor innervation in an experimental painful peripheral neuropathy, Pain 46 Ž1991. 299–313. L.R. Watkins, E.P. Wiertelak, L.E. Goehler, K.P. Smith, D. Martin, S.F. Maier, Characterization of cytokine-induced hyperalgesia, Brain Res. 654 Ž1994. 15–26. L.R. Watkins, L.E. Goehler, J. Relton, M.T. Brewer, S.F. Maier, Mechanisms of tumor necrosis factor-alpha ŽTNF-alpha. hyperalgesia, Brain Res. 692 Ž1995. 244–250. L.R. Watkins, S.F. Maier, L.E. Goehler, Immune activation: the role of proinflammatory cytokines in inflammation, illness responses and pathological pain states, Pain 63 Ž1995. 289–302. J.D. White, M.W. Schwartz, Using osmotic minipumps for intracranial delivery of amino acids and peptides, Methods Neurosci. 21 Ž1994. 187–200.
Research report
Brain-derived TNFa mediates neuropathic pain Tracey A. Ignatowski a,b , William C. Covey a , Paul R. Knight b , Charles M. Severin c , Tracey J. Nickola a , Robert N. Spengler a,b,) a
Department of Pathology, State UniÕersity of New York at Buffalo, 204 Farber Hall, 3435 Main Street, Buffalo, NY, 14214, USA b Department of Anesthesiology, State UniÕersity of New York at Buffalo, 3435 Main Street, Buffalo, NY, 14214, USA c Department of Anatomy, State UniÕersity of New York at Buffalo, 3435 Main Street, Buffalo, NY, 14214, USA Accepted 22 June 1999
Abstract Neuropathic pain is a chronic pain state that develops a central component following acute nerve injury. However, the pathogenic mechanisms involved in the expression of this central component are not completely understood. We have investigated the role of brain-associated TNF in the evolution of hyperalgesia in the chronic constriction injury ŽCCI. model of neuropathic pain. Thermal nociceptive threshold has been assessed in rats Žmale, Sprague–Dawley. that have undergone loose, chromic gut ligature placement around the sciatic nerve. Total levels of TNF in regions of the brain, spinal cord and plasma have been assayed ŽWEHI-13VAR bioassay.. Bioactive TNF levels are elevated in the hippocampus. During the period of injury, hippocampal noradrenergic neurotransmission demonstrates a decrease in stimulated norepinephrine ŽNE. release, concomitant with elevated hippocampal TNF levels. Continuous intracerebroventricular Ži.c.v.. microinfusion of TNF-antibodies ŽAbs. starting at four days, but not six days, following ligature placement completely abolishes the hyperalgesic response characteristic of this model, as assessed by the 588C hot-plate test. Antibody infusion does not decrease spinal cord or plasma levels of TNF. Continuous i.c.v. microinfusion of rrTNFa exacerbates the hyperalgesic response by ligatured animals, and induces a hyperalgesic response in animals not receiving ligatures. Likewise, field-stimulated hippocampal adrenergic neurotransmission is decreased upon continuous i.c.v. microinfusion of TNF. These results indicate an important role of brain-derived TNF, both in the pathology of neuropathic pain, as well as in fundamental pain perception. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Pain; TNF; Norepinephrine; a 2 -Adrenergic receptor; Microinfusion; Hippocampus
1. Introduction Chronic pain syndromes often develop a central nervous system ŽCNS.-mediated component that plays a role in the cognitive experience of pain and associated mood changes. Chronic persistent pain that has developed a central component is a difficult therapeutic challenge. Neuroplastic changes at several sites in the spinal cord and brain have been implicated w8,16x. Neuropathic pain develops from intense, prolonged noxious stimulation, or from nerve injury, and leads to long-term functional changes in the CNS, resulting in amplification and persistence of the pain. Two common symptoms of neuropathic pain include hy) Corresponding author. [email protected]
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peralgesia Žan enhanced response to normally painful stimuli. and allodynia Ža painful response to a normally innocuous stimuli.. Drugs that appear to modify adrenergic receptors in the CNS Že.g., clonidine, amitriptyline. are efficacious, to varying degrees, in preventing or alleviating these symptoms w4,6x. Elevated levels of systemic proinflammatory cytokines occur in association with increases in pain perception, as well as during changes in general mood status w18,30,31x. During systemic illness Že.g., rheumatoid arthritis and microbial infections., following activation of the proinflammatory cytokine cascade, patients experience mood changes, such as general malaise and somnolence, along with the appearance of nondescript pain symptoms, such as polyarthralgias, myalgias, and hypesthesia w9x. When TNF is administered intravenously for 24 h to human volun-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 7 8 2 - 5
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teers, lethargy and general malaise are consistently produced w18x. While systemically administered cytokines may affect responses in the CNS, how this corresponds to regional TNF levels achieved, as well as sites of cytokine action are not known. A considerable amount of evidence supports a pronociceptive role for TNF during chronic pain w11,30x. Findings from our laboratory have led us to hypothesize that proinflammatory cytokines, such as TNF, synthesized in neurons within distinct regions of the CNS are involved in promoting neuroplastic changes of noradrenergic neurons involved in the perception of pain w12,13x. Watkins et al. w32x have proposed that hyperalgesia is produced by the peripheral, local production of proinflammatory cytokines which can activate vagal afferents mediating signals of pain perception to the brain. These signals to the brain result, via descending circuitry, in alterations at the level of the spinal cord that are implicated in various forms of hyperalgesia w32x. An intense afferent barrage appears necessary to elicit the signals that establish persistent pain. A number of observations support the role of the central noradrenergic nervous system in pain perception. The locus coeruleus ŽLC. is the largest noradrenergic nucleus within the brain w26x, and the noradrenergic neuronal cell bodies there represent the primary source of norepinephrine ŽNE. in the CNS w21x. Norepinephrine is a principal neurotransmitter associated with the modulation of nociception and analgesia w21x. Electrical stimulation of the LC has been demonstrated to produce analgesia w17x. The hippocampus, a region of the brain rich in noradrenergic nerve terminals, associated with pain perception w16x, receives its NE innervation exclusively from the LC. The LC and the adjacent subcoeruleus ŽSC. also represent the major source of noradrenergic nerve terminals to the spinal cord w19x. Finally, drugs such as the a 2-adrenergic agonist clonidine act at noradrenergic synapses and are useful in the management of chronic pain w6x. We have postulated that proinflammatory cytokine production by neurons within specific regions of the brain at the time of pain-elicited afferent barrage plays an important role in the pathogenesis of ‘‘central sensitization’’. Biologically active TNF and TNF mRNA can be detected in noradrenergic regions of the rat brain w12,13x. Additionally, noradrenergic neuronal immunohistochemical staining for TNF has been demonstrated w14x. Administration of IL-1b via intracerebroventricular Ži.c.v.. injection produces hyperalgesia, although intrathecal injection does not, indicating that the brain and not the spinal cord is the site of cytokine action w30x. In the present study, we report on the role of brain-derived TNF in the pathogenesis of hyperalgesia using a chronic constriction injury ŽCCI. of the rat sciatic nerve, developed by Bennett and Xie w2x. In order to examine this interaction, we assessed TNF levels in select nuclei of the brain and infused TNF as well as TNF antibody ŽAb. into the right lateral cerebral ventricle. The effect of this cy-
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tokine or antibody administration on hyperalgesia has been evaluated.
2. Materials and methods 2.1. CCI peripheral neuropathic pain model Experimental animal care has been approved in accordance with IACUC institutional guidelines. Rats Žmale, Sprague–Dawley, 250–350 g. are anesthetized with ketamine Ž60 mgrkg. and xylazine Ž3 mgrkg. i.p. prior to operation. The sciatic nerve is exposed unilaterally, and four ligatures Ž4.0 chromic gut. are loosely placed around the nerve, 1 mm apart proximal to the trifurcation. In sham procedures, the nerve is exposed and freed of adherent tissuermuscle, but no ligatures are placed. 2.2. Thermal nociception measurements Thermal nociceptive threshold is measured in each hind paw using a 588C hot plate test. Changes in thresholds Žseconds. are expressed as either the percentage of the Maximal Possible Effect Ž% MPE. for antihyperalgesic effects, or the percentage of the Maximal Possible Response Ž% MPR. for increased hyperalgesic response, using the equation: wŽExperimental latencyy Mean basal latency.rŽMaximal latencyy Mean basal latency.x = 100. Pre-ligation hind paw withdrawal latencies Žmean basal latencies. are subtracted from those taken post-ligation Žexperimental latencies. in order to generate a ‘‘difference score’’, which is used as an index of hyperalgesia. Normal latency withdrawal values pre-CCI were 6.0–10.0 s Ž15 s being the maximal latency accepted.. Cutaneous hind-paw temperature measurement was also recorded. Animals are held and the plantar hind paw is gently pressed with a BT-1 surface microprobe thermocouple sensor connected to a digital thermometer ŽModel TH-5, Physitemp, Clifton, NJ.. Stable temperature recordings were obtained within 30–60 s and were measured to the nearest 0.18C. The change in cutaneous temperature was calculated as the difference of averaged pre-CCI recordings from each postCCI recording. Normal cutaneous hind paw skin temperatures averaged 28.38C for the contralateral and 28.28C for the ipsilateral hind paw. All measurements were recorded between 0700–0800 h. All surgeries were performed between 0800–1200 h. 2.3. TNF bioassay Total biologically-active TNF was assayed before or after specific noxious stimuli andror pharmacological interventions for TNF activity using the WEHI-13VAR bioassay w24x. The selectivity of WEHI fibroblast cells for TNF-induced lysing by TNF from tissue has been well established by this laboratory and others w15,24x.
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2.4. IntracerebroÕentricular microinfusion Mini osmotic pumps Ž0.5 ulrh, 14 days, Alza, Palo Alto, CA. and brain infusion cannulae ŽAlza, Palo Alto, CA. are assembled according to the manufacturer’s guidelines. The vehicle used for delivery of the infused compounds is aCSF ŽNaCl, 216.5 mM; KCl, 3 mM; CaCl 2 P 2H 2 O, 1.4 mM; MgCl 2 P 6H 2 O, 0.8 mM; Na 2 HPO4 P 7H 2 O, 0.8 mM; and NaH 2 PO4 P H 2 O, 0.2 mM.. Gentamycin Ž0.1 mgrml solution; Sigma, St. Louis, MO. is added to the aCSF to prevent bacterial growth. Rat albumin Žfraction V, 1 mgrml; Sigma, St. Louis, MO. was added to stabilize the infused compound and to prevent proteins and charged molecules from binding non-specifically. Rats Žmale, Sprague–Dawley, 250–350 g., anesthetized with ketamine Ž60 mgrkg. and xylazine Ž3 mgrkg, i.p.., are secured on a stereotaxic platform. With bregma used as the zero point, the following are the stereotaxic coordinates: A–P, y0.92 mm; lateral, 1.6 mm; vertical, 3.5 mm, for trephining the skull prior to cannulation of the right lateral cerebral ventricle. Compounds to be infused include the following: polyclonal rabbit anti-mouse TNFa ŽGenzyme, Cambridge, MA. and normal rabbit serum ŽSigma, St. Louis, MO.; monoclonal hamster anti-murine TNFa and Armenian hamster IgG isotype control Ab ŽGenzyme, Cambridge, MA.; recombinant rat TNFa ŽR & D Systems, Minneapolis, MN.; as well as aCSF alone. The dosage for each infused compound is based on the EC 50 value for inhibition of 3–10 pgrml TNF in the bioassay.
The viability of each Ab, its control, and rrTNFa were confirmed in the bioassay following 14 days of incubation in aCSF at 378C.
3. Results 3.1. CCI induction of hyperalgesia and hyperthermia The CCI model of neuropathic pain consists of a central and peripheral component w2x. Rats undergo unilateral ligature placement and are evaluated every other day. Hyperalgesia is measured by changes in paw-withdrawal latency on a 588C hot plate w3x. Mean baseline response latency over all experimental conditions was 7.9 " 0.2 seconds. Heat-evoked hind paw withdrawal latencies are consistently reduced in CCI animals as compared to sham operated rats ŽFig. 1.. Hyperalgesia, quantified by a difference in paw-withdrawal latency Žseconds., and expressed as the percentage of the Maximal Possible Response Ž% MPR., begins to escalate on day two post-surgery and reaches a maximum on day 10. Unoperated animals tested at the same times do not differ from sham animals, with scores remaining close to the zero score Ždata not shown.. Alterations of sympathetic activity, assessed as changes in skin temperature, have been observed in neuropathic pain models w29x. Changes in hind paw skin temperature are expressed as the temperature difference in degree centigrade Ž8C. from the average of three recordings taken on
Fig. 1. Assessment of hyperalgesia in the CCI model of neuropathic pain. Data are presented as the percent Maximal Possible Response wŽpost-CCI pre-CCI values.rŽ15 - pre-CCI values.x = 1004 of the latency-to-hind paw withdrawal using the 588C hot plate test. The effect of rrTNFa i.c.v. infusion on the CCI-induced nociceptive response is demonstrated. Each point is expressed as the negative score for mean " S.E.M. with the number of rats indicated in parentheses. Statistical significance evaluated with ANOVA. Statistical significance different from sham animals: U p - 0.05, UU p - 0.01. Significantly different from CCI rats, ap - 0.02, aap - 0.01. CCI s chronic constriction injury.
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each of two days prior to and on the day of ligature placement. Hyperthermia, indicative of decreased sympathetic activity Žvasodilatation., occurs during the initial 14 days following CCI Ždata not shown.. Interestingly, this response is noted in both the ipsilateral and contralateral hind paws of CCI rats, a hallmark for a central origin. 3.2. Brain-associated TNF leÕels Regional total TNF levels increase in a time-dependent fashion, coincident with the development of hyperalgesia in the CCI neuropathic pain model. At day six Ženhanced development of hyperalgesia., TNF levels increase in the hippocampus Ž6.5 " 2.2 pgr100 mg tissue at day 4 postCCI as compared to 19.6 " 2.1 pgr100 mg tissue at day 6 post-CCI, p - 0.02, Student’s t-test.. These data clearly demonstrate that an increase occurs in total TNF levels in a region of the brain associated with pain cognition w16x, with a time course indicative of TNF playing a primary role in neuropathic pain. 3.3. Norepinephrine (NE) oÕerflow during field stimulation of hippocampal brain slices We have previously demonstrated that coincident with tricyclic antidepressant ŽTCA.-induced loss in neuron-associated TNF there is a significant increase in field-stimulated release of 3 H-NE from hippocampal brain slices w12,13x. While this class of drugs ŽTCA. is used to treat chronic pain syndromes, we have presently investigated the effect chronic pain has on 3 H-NE overflow from hippocampal brain slices obtained from CCI rats. 3 H-NE
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ŽL-wring-2,5,6,-3 Hx-; NEN, spec. act. 59.1 Cirmmol. release, after electrical field stimulation, is measured directly in a series of hippocampal brain slices w12x. The release of preloaded 3 H-labeled neurotransmitter during electrical field stimulation is an established method for studying neurotransmitter release w25x. The hippocampus is studied because this region is implicated in pain perception and mood states w16x, and drugs efficacious in the treatment of pain have been demonstrated to elicit neuroplastic changes associated with hippocampal functioning w12,13x. Electrical field-stimulated release of NE is frequency-dependent ŽFig. 2.. Hippocampal slices obtained from CCI rats at eight days post-surgery demonstrate a significant decrease in NE release at both the 1 Hz and 4 Hz frequencies of stimulation. Transmitter release from rat hippocampal slices is a regulated event w25x. At the higher frequency of stimulation a greater expression of regulatory mechanisms governing further NE release may be expected. While TNF obviously plays a pivotal role in the adrenergic neuroplastic change and also in the hyperalgesia, factors such as the afferent barrage are also involved in neuropathic pain. Furthermore, the changes in NE release are coincident with the development of hyperalgesia. Similar results occur when TNF is continuously microinfused into the right lateral cerebral ventricle of an untreated rat ŽFig. 2b.. When hippocampal slices from rrTNFa-administered rats are field-stimulated, the fractional release is significantly decreased at 1 Hz ŽS1. and at 4 Hz ŽS2.. The S2rS1 ratio is also significantly decreased indicating a more striking decrease of release at the higher electrical frequency. Consequently, during both chronic pain and continuous microinfusion of TNF into the brain, noradrenergic neu-
Fig. 2. Stimulated neuronal release of 3 H-NE. Neuronal release was studied in hippocampal slices by applying field stimulations consisting of trains of square wave pulses Ž26 V, 2 ms duration. at either 1 Hz or 4 Hz for two minute periods. Each slice was positioned between two nylon mesh disks and placed within each of six chambers connected to two platinum wire electrodes designed to stimulate adrenergic nerve endings Žfield stimulation.. Ža. Stimulated hippocampal NE release from CCI rats at day-8 post-surgery. Žb. Stimulated hippocampal NE release from control animals continuously microinfused with rrTNFa. The data are expressed as the percent release of the total 3 H pool in the tissue at the time of stimulation in excess of spontaneous efflux. Each bar represents the mean " S.E.M. from 11 separate determinations. Statistical significance different from control was reached at U p - 0.05, UU p - 0.01, or UUU p - 0.001 using a paired Student’s t-test.
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ronal activity is altered resulting in decreased NE release in the brain. 3.4. Effects of i.c.Õ. microinfusion of TNF-Ab on hyperalgesic responses to CCI An increase in TNF generation in the hippocampus is associated with an increased intensity of neuropathic pain perception and a change in adrenergic responses ŽFig. 2., resulting in a decrease in NE release. In order to further test this hypothesis, we have infused either polyclonal or monoclonal TNF-Abs into the right lateral cerebral ventricle of CCI rats w33x. Antibody infusion is performed both four and six days post-ligature placement, corresponding to the time periods just preceding and during the appearance of elevated levels of the cytokine in the brain. Continuous microinfusion of polyclonal TNF-Abs ŽpTNF-Ab, 0.5 mlrh. ŽFig. 3., and with lesser efficacy monoclonal TNFAbs ŽmTNF-Abs. Ždata not presented., into the brains of rats starting at four days post-ligature placement abolishes the hyperalgesic response characteristic of this model. These rats also demonstrate a hypoalgesic response following Ab infusion. These observations indicate an important role for TNF both in the pathogenesis of neuropathic pain, as well as in pain perception. The effect of TNF-Ab on pain is dependent upon the time administered post nerve injury. Microinfusion of Ab at six days post-ligature place-
ment does not reduce the hyperalgesic response ŽFig. 3.. Infusion of the vehicle ŽaCSF. or normal rabbit serum has no effect on this response when compared to rats receiving ligature placement only. Infusion with IgG Ab as a control for monoclonal TNF-Ab demonstrates a small decrease in the nociceptive response early during the infusion period Ždata not shown.. We speculate that this decrease in response may be due to non-specific immunoglobulininduced modification of constitutive TNF production. This phenomenon can be demonstrated in mononuclear cells w10,27x. The concentrations of TNF-Ab used for microinfusion are pharmacological doses of 17 times a 1r100,000 dilution for the pTNF-Ab and 17 times a 1r6000 dilution for the mTNF-Ab, based on the ability of each type of Ab to completely block the EC 50 concentration of mrTNFa in the WEHI bioassay. This concentration of TNF-Ab reaches concentrations appropriate to block TNF in neighboring brain regions due to complete CSF turnover every one to two hours in the rat brain, such that the half-life of most CSF solutes is 0.75 h w33x. The ability of TNF-Ab administered by this approach to alter systemic TNF levels was assessed by assaying for plasma levels of TNF. The levels of plasma TNF did not differ in CCI rats infused with TNF-Abs as compared to those infused with aCSF alone ŽaCSF s 2.28 " 0.91 pgrml; pTNF-Ab s 3.63 " 1.16 pgrml; mTNF-Abs 5.20 " 3.49 pgrml; NS.. The levels
Fig. 3. Nociceptive threshold for latency-to-hind paw withdrawal in the CCI model of neuropathic pain. Results are presented as the negative of the percent MPE using the 588C hot plate test. Note: Infusion of pTNF-Ab beginning 6 days post-CCI placement had no effect on the hyperalgesic response characteristic of this neuropathic pain model, while infusion 4 days following CCI ablated the hyperalgesic response. CCI s chronic constriction injury. Each point represents mean " S.E.M. with the number of determinations indicated in parentheses. Significantly different from CCI rats, U p - 0.05, ŽANOVA.. Significantly different from CCI rats, aCSF-infused CCI rats, and Rb serum-infused rats, UU p - 0.05 ŽANOVA..
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Fig. 4. Effect of rrTNFa i.c.v. microinfusion on pain perception in control animals. rrTNFa-infused control rats demonstrate a subsequent decrease in nociceptive threshold similar to that shown by CCI rats. Each point represents the negative score for mean " S.E.M. from the number of rats in parentheses.
of TNF in the cervical and thoraco-lumbar regions of the spinal cord also did not differ in animals infused with aCSF alone or with TNF-Abs Ždata not shown.. These findings indicate that the effects of microinfusion of TNFAb into the brain are most likely due to the blockade of TNF activity in neighboring brain regions of the cerebral right lateral ventricle. 3.5. Effect of intracerebroÕentricular microinfusion of rrTNFa on pain perception Whether TNF is a causative factor contributing to the onset of the development of neuropathic pain symptoms was examined by the infusion of rrTNF into the brains of CCI rats as well as into unoperated rats. Right lateral cerebral ventricle infusions consisted of rats receiving either 30 ng rrTNFar24 h or 1000 ng rrTNFar24 h at a rate of 0.5 mlrh for 14 days. These concentrations were chosen in order to achieve appropriate levels of the cytokine in brain tissue Žwithin the synapse.. We have previously demonstrated that a TNF concentration of 1 to 30 ngrml is needed to affect in vitro neurotransmission in the hippocampus w12,13x. Infusion of 30 ng rrTNFar24 h for 14 days does not effect the hyperalgesic response nor does it affect the total levels of TNF in the rat brain, spinal cord, sciatic nerve, or plasma Ždata not shown.. However, i.c.v. infusion of 1000 ng rrTNFar24 h beginning on day four post-CCI enhances CCI-induced hyperalgesia. Differences are apparent at day 14 post-CCI Žday 10 post-infusion. and remain so
throughout the duration of the infusion ŽFig. 1.. Interestingly, the increases in TNF tissue levels, expressed as percent of control rat values, are highest in regions of the brain anatomically closest to the site of infusion Žhippocampus, 60,000% control.. However, there are no detectable increases in TNF levels, as compared to controls, in the region containing the lumbar spinal cord nor in the systemic circulation Žplasma, data not shown., indicating that TNF is acting at higher brain centers to induce an increase in pain perception. Infusion of 1000 ng rrTNFar24 h into unoperated control animals induces a hyperalgesic response equal to that experienced by a CCI animal ŽFig. 4.. While the initial hyperalgesic response at day two post-infusion of rrTNF appears to be due to the increase in total TNF levels in the CNS, the initial hyperalgesic response expressed by the CCI animal is probably due to immediate changes in TNF levels at the nerve terminal. Infusion of the vehicle alone, aCSF, does not modify the nociceptive threshold as assessed by the hot plate test Ždata not shown..
4. Discussion We have examined levels of biologically-active TNF during experimental neuropathic pain in a region of the brain involved in cognition and implicated in pain perception w16x. Furthermore, we have also examined levels of biologically-active TNF in select regions of the brain and spinal cord in rats microinfused with TNF into the right
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lateral cerebral ventricle while experiencing experimental neuropathic pain. The temporal increases in the levels of this cytokine and the physical manifestations of persistent pain occur concomitantly. The development of the physical manifestations of persistent pain, as well as neuroplastic changes were determined by quantitating hyperalgesia, as well as changes in NE release. These experiments were performed in order to determine the requirement for TNF in the development of hyperalgesia. While changes in levels of TNF in the spinal cord as well as at the peripheral site of nerve injury during neuropathic pain has been well-documented w7,23,28x, an effect on its levels in the brain is original. Therefore, the changes in hippocampal TNF levels along with the involvement of brain-derived TNF in pain shows that the cognitive perception of pain must play an integral role in hyperalgesia. Intracerebroventricular infusion of TNF-Abs into the right lateral cerebral ventricle abolishes hyperalgesia, a hallmark of neuropathic pain. Conversely, infusion of rrTNFa not only enhances CCI-induced hyperalgesia, but also induces hyperalgesia in unoperated rats. The lack of changes in TNF levels in the lumbar spinal cord and in the systemic circulation following i.c.v. infusion strongly indicates that the action of TNF occurs at brain centers that modulate pain perception. Our previous work demonstrates that TNF regulates NE release from adrenergic nerve terminals w12,13x. TCA administration Ždrug class used to treat chronic pain. to unmanipulated rats produces a depletion in neuron-associated TNF expression in regions of the brain with adrenergic activity w14x. Furthermore, chronic administration of the TCA enhances adrenergic neurotransmission at the same time that it transforms the response elicited by the a 2-adrenergic receptor w12x. During chronic pain the fractional release of NE is decreased ŽFig. 2a.. Therefore, TCAs may exert their effects on nociception through similar mechanisms as in depression. The majority of investigators examining central adrenergic receptor participation in centrally-mediated pain have focused on adrenergic receptor function in the spinal cord. However, these results also suggest that supraspinal sites are involved. Prominent innervation of the spinal cord by descending noradrenergic neurons indicates that NE mediates many of the neuronal processes in the spinal cord w5x. Of particular interest, the activity of noradrenergic neurons in the supraspinal regions ŽLC, SC. can be decreased by central administration of a 2-adrenergic agonists w1x. Therefore, modifying the activity of LC neurons may alter the activity of their axon terminals in the spinal cord, thus modulating the descending noradrenergic inhibition of nociception in the spinal cord. However, we have addressed how changes in adrenergic activity occur in the hippocampus due to changes in TNF production and release in the LC and hippocampus w12,13x. The present findings complement this previous work, demonstrating how these changes would play a primary role in neuropathic pain.
The use of anti-TNF agents as possible therapies for conditions associated with nerve injury, such as those seen in rheumatoid arthritis w22x, has gained interest. However, because of the redundant and overlapping actions of many of the cytokines, interpretation of data derived from studies undertaken to inhibit cytokine activity is usually not straightforward. Clinically, this has led to many of the failures associated with therapies that are based on experimental data used to evaluate cytokine cascades in disease Že.g., strategies to improve the outcome of sepsis.. This may be particularly important when the stimulus is multifactorial Žcentralized pain, depression.. The most obvious and direct example of this is the overlapping responses to IL-1 and TNF. Both of these initiation cytokines have very similar actions in inducing subsequent cytokines that may play a causative role in adrenergic adaptational changes. In addition, many of the biological responses of IL-1 and TNF are additive or synergistic. Therefore, it should be noted that a time-dependence in the efficacy of TNF-Ab infusion regarding nociceptive threshold withdrawal latencies was noted in our studies ŽFig. 3.. This finding demonstrates an induction of the cytokine cascade, whereby following the initial increase in central TNF levels, TNFAbs are ineffective in preventing ensuing pain perception. Similarly, the finding that IL-1ra is able to block TNF-induced hyperalgesia, when TNF was injected either i.p. w31x or i.c.v. w20x, suggests that TNF induces or enhances the release of IL-1 which, in turn, produces hyperalgesia. However, the present studies do indeed demonstrate a pivotal role for the proinflammatory cytokine, TNF, in the mediation of centrally mediated chronic pain syndromes.
Acknowledgements This work was supported by PVA grant a RFA008 ŽRNS. and NIMH grant a MH53946 ŽRNS..
References w1x G.K. Aghajanian, C.P. VanderMaelen, Alpha 2 -adrenoceptor mediated hyperpolarization of locus coeruleus neurons: intracellular studies in vivo, Science 215 Ž1982. 1394–1396. w2x G.J. Bennett, Y.-K. Xie, A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain 33 Ž1988. 87–107. w3x M. Bianchi, P. Sacerdote, P. Ricciardi-Castagnoli, P. Mantegazza, A. Panerai, Central effects of tumor necrosis factor alpha and interleukin-1 alpha on nociceptive thresholds and spontaneous locomotor activity, Neuroscience Letters 148 Ž1992. 76–80. w4x H.M. Bryson, M.I. Wilde, Amitriptyline: a review of its pharmacological properties and therapeutic use in chronic pain states, Drugs and Aging 8 Ž1996. 459–476. w5x F.M. Clark, H.K. Proudfit, The projection of locus coeruleus neurons to the spinal cord in the rat determined by anterograde tracing combined with immunocytochemistry, Brain Res. 538 Ž1991. 231– 245. w6x E.E. Codd, J.B. Press, R.B. Raffa, Alpha-2 adrenoceptors vs. imida-
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w7x
w8x
w9x
w10x
w11x
w12x
w13x
w14x
w15x
w16x w17x
w18x
w19x
w20x
zoline receptors: implications for alpha-2 mediated analgesia and other non-cardiovascular therapeutic uses, wReviewx, Life Sciences 56 Ž1995. 63–74. J.A. Deleo, R.W. Colburn, A.J. Rickman, Cytokine and growth factor immunohistochemical spinal profiles in two animal models of mononeuropathy, Brain Res. 759 Ž1997. 50–57. R. Dubner, M.A. Ruda, Activity-dependent neuronal plasticity following tissue injury and inflammation, Trends Neurol. Sci. 15 Ž1992. 96–103. S.H. Ferreira, The role of interleukins and nitric oxide in the mediation of inflammatory pain and its control by peripherial analgesics, wReviewx, Drugs 46 Ž1. Ž1993. 1–9, Suppl. J.L. Foreback, D.G. Remick, E. Crockett-Torabi, P.A. Ward, Cytokine responses of human blood monocytes stimulated with Igs, Inflammation 21 Ž1997. 501–517. H. Fukuoka, M. Kawatani, T. Hisamitsu, C. Takeshige, Cutaneous hyperalgesia induced by peripheral injection of interleukin-1 beta in the rat, Brain Res. 657 Ž1994. 133–140. T.A. Ignatowski, R.N. Spengler, Tumor necrosis factor-a: presynaptic sensitivity is modified after antidepressant drug administration, Brain Res. 665 Ž1994. 293–299. T.A. Ignatowski, R.C. Chou, R.N. Spengler, Changes in noradrenergic sensitivity to tumor necrosis factor-a in brains of rats administered clonidine, J. Neuroimmunol. 70 Ž1996. 55–63. T.A. Ignatowski, B.K. Noble, J.R. Wright, J.L. Gorfien, R.R. Heffner, R.N. Spengler, Neuronal-associated tumor necrosis factor ŽTNFa .: its role in noradrenergic functioning and modification of its expression following antidepressant drug administration, J. Neuroimmunol. 79 Ž1997. 84–90. K.S.A. Khabar, S. Siddiqui, J.A. Armstrong, WEHI-13VAR: a stable and sensitive variant of WEHI 164 clone 13 fibrosarcoma for tumor necrosis factor bioassay, Immunol. Lett. 46 Ž1995. 107–110. S. Khanna, J.G. Sinclair, Noxious stimuli produce prolonged changes in the CA1 region of the rat hippocampus, Pain 39 Ž1989. 337–343. D. Margalit, M. Segal, A pharmacologic study of analgesia produced by stimulation of the nucleus locus coeruleus, Psychopharmacology 62 Ž1979. 169–173. A. Mittelman, C. Puccio, E. Gafney, N. Coombe, B. Singh, D. Wood, P. Nadler, T. Ahmed, Z. Arlin, A phase I pharmacokinetic study of recombinant human TNF administered by a 5-day continuous infusion, Investigational New Drugs 10 Ž1992. 183–190. R.Y. Moore, F.E. Bloom, Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems, Annu. Rev. Neuroscience 2 Ž1979. 113–168. T. Oka, Y. Wakugawa, M. Hosoi, K. Oka, T. Hori, Intracerebroven-
w21x
w22x
w23x
w24x
w25x
w26x
w27x
w28x w29x
w30x
w31x
w32x
w33x
77
tricular injection of tumor necrosis factor-alpha induces thermal hyperalgesia in rats, Neuroimmunomodulation 3 Ž1996. 135–140. H.K. Proudfit, Pharmacological evidence for the modulation of nociception by noradrenergic neurons, Prog. Brain Res. 77 Ž1998. 357–370. E.C. Rankin, E.H. Choy, D. Kassimos, G.H. Kingsley, A.M. Sopwith, D.A. Isenberg, G.S. Panayi, The therapeutic effects of an engineered human anti-tumor necrosis factor alpha antibody ŽCDP571. in rheumatoid arthritis, Br. J. Rheumatol. 34 Ž1995. 334–342. C. Sommer, M. Marziniak, R.R. Myers, The effect of thalidomide treatment on vascular pathology and hyperalgesia caused by chronic constriction injury of rat nerve, Pain 74 Ž1998. 83–91. R.N. Spengler, S.W. Chensue, D.A. Giacherio, N. Blenk, S.L. Kunkel, Endogenous norepinephrine regulates tumor necrosis factor-alpha production from macrophages in vitro, J. Immunol. 152 Ž1994. 3024–3031. K. Starke, M. Gothert, H. Kilbinger, Modulation of neurotransmitter release by presynaptic autoreceptors, Physiol. Rev. 69 Ž1989. 864– 989. L.W. Swanson, B.K. Hartman, The central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-beta-hydroxylase as a marker, J. Comp. Neurol. 163 Ž1975. 467–505. Y. Ueda, K.H. Lee, S. Ito, K. Saito, N. Niesen, J.R. Brentjens, R.O.B. Gans, In vivo neutralization of tumor necrosis factor attenuates the generalized Shwartzman reaction in the rabbit, J. Endotoxin Res. 3 Ž1996. 67–75. R. Wagner, R.R. Myers, Endoneurial injection of TNF-alpha produces neuropathic pain behaviors, NeuroReport 7 Ž1996. 2897–2901. S. Wakisaka, K.C. Kajander, G.J. Bennett, Abnormal skin temperature and abnormal sympathetic vasomotor innervation in an experimental painful peripheral neuropathy, Pain 46 Ž1991. 299–313. L.R. Watkins, E.P. Wiertelak, L.E. Goehler, K.P. Smith, D. Martin, S.F. Maier, Characterization of cytokine-induced hyperalgesia, Brain Res. 654 Ž1994. 15–26. L.R. Watkins, L.E. Goehler, J. Relton, M.T. Brewer, S.F. Maier, Mechanisms of tumor necrosis factor-alpha ŽTNF-alpha. hyperalgesia, Brain Res. 692 Ž1995. 244–250. L.R. Watkins, S.F. Maier, L.E. Goehler, Immune activation: the role of proinflammatory cytokines in inflammation, illness responses and pathological pain states, Pain 63 Ž1995. 289–302. J.D. White, M.W. Schwartz, Using osmotic minipumps for intracranial delivery of amino acids and peptides, Methods Neurosci. 21 Ž1994. 187–200.