The Journal of Neuroscience. June 1993. 13(6): 2689-2702

Patterns of Increased Brain Activity Indicative of Pain in a Rat Model of Peripheral Mononeuropathy

Jianren Mao, David J. Mayer, and Donald D. Price Department of Anesthesiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298

Regional changes in brain neural activity were examined in haviors in CCI rats and clinical symptoms in neuropathic pain rats with painful peripheral mononeuropathy (chronic con- patients. strictive injury, Ccl) by using the fully quantitative W-2- [Key words: neuropathic pain, P-deoxyglucose, hyperal- deoxyglucose (2-DG) autoradiographic technique to mea- gesia, spontaneous pain, nerve injury, brainstem, cerebral sure local glucose utilization rate. CCI rats used in the cortex, thalamusj experiment exhibited demonstrable thermal hyperalgesia and spontaneous pain behaviors 10 d after sciatic nerve ligation when the 2-DG experiment was carried out. In the absence Peripheral nerve injury sometimesresults in a chronic neuro- of overt peripheral stimulation, reliable increases in 2-DG pathic pain syndrome characterized by hyperalgesia, sponta- metabolic activity were observed in CCI rats as compared neouspain, radiation of pain, and nociceptive responsesto nor- to sham-operated rats within extensive brain regions that mally innocuous stimulation (allodynia) (Bon@ 1979; Thomas, have been implicated in supraspinal nociceptive processing. 1984; Price et al., 1989). Most of these symptoms have been These brain regions included cortical somatosensory areas, recently observed in a rodent model of painful peripheral mon- clngulate cortex, amygdala, ventral posterolateral thalamic oneuropathy induced by loose ligation of the rat’s common nucleus, posterior thalamic nucleus, hypothalamic arcuate sciatic nerve(chronicconstrictive injury, CCI) (Bennett and Xie, nucleus, central gray matter, deep layers of superior collic- 1988). For example, one prominent changeseen in CC1 rats is ulus, pontine reticular nuclei, locus coeruleus, parabrachial the appearanceof ongoing behaviors such as typical hind paw nucleus, gigantocellular reticular nucleus, and paragigan- guarding positions following sciatic nerve ligation, suggesting tocellular nucleus. The increase in 2-DG metabolic activity the presenceof persistent spontaneouspain (Bennett and Xie, was bilateral in most brain regions of CCI rats. However, 1988; Attai et al., 1990; Mao et al., 1992a,b). However, based somatosensory regions within the thalamus and the cerebral on these behaviors alone, it is difficult to determine whether cortex were activated in CCI rats. High levels of 2-DG met- CCI rats truly experience spontaneouspain or these ongoing abolic activity were obsenred within the cortical hind limb behaviors simply occur as a defense against pains evoked by area, ventral posterolateral thalamic nucleus, and posterior mechanical or thermal stimuli from the affected area. Some thalamic nucleus contralateral to the ligated sciatic nerve, evidence indicating the presenceof spontaneouspain in CC1 and these levels were higher than ipsilateral corresponding rats derives from recent W-2-deoxyglucose (2-DG) autoradio- regions in CCI rats. In addition, patterns of increased neural graphic studies that show that spinal cord neural activity (in- activity found in the brain of CCI rats showed some similar- ferred from increased local glucose utilization rate) increases ities and differences to those found in the brain of rats ex- substantially in the absenceof overt peripheral stimulation in posed to acute nociceptton induced by noxious heat or for- CC1 rats 10 d after sciatic nerve ligation (Price et al., 1991; Mao malin stimulation. Thus, these CCLinduced spontaneous et al., 1992a). Consistent with spontaneousincreases in spinal increases in neural activity within extensive brain regions of cord neural activity in CC1 rats, electrophysiological studies CCI rats previously implicated in sensory-discriminative and have indicated increasesin spontaneousneuronal discharges affective-motivational dimensions of pain as well as cen- both in spinothalamic tract neurons (Paleceket al., 1991) and trifugal modulation of pain are likely to reflect brain neural in the ventrobasal thalamic complex (Guilbaud et al., 1990) of processing of spontaneous pain. Implications of increased CC1 rats 2-3 weeksafter peripheral nerve injury. In view of the brain neural activity in mechanisms of neuropathic pain are critical roles of supraspinalstructures in nociceptive processing, discussed with emphasis on correlations between spatial more conclusive evidence for the existenceof spontaneouspain patterns of altered brain neural activity and pain-related be- in CC1rats should be provided if increasedneural activity occurs in brain regions that have been implicated in pain. Therefore, it would be of great interest to examine the spatial Received Aug. 24, 1992; revised Nov. 10, 1992; accepted Jan. 12, 1993. distribution of neural activity throughout the entire brain of We thank Dr. John G. McHaRie and Juan Lu for the technical assistance. We also thank Dr. Robert C. Coghill for his assistance with the 2-DG technique. This CC1 rats, examining brain structures implicated in supraspinal work was supported by U.S. Public Health Service Grant NS-24009. nociceptive processing.The advantage of usinga neural imaging Correspondence should be addressed to Jianren Mao, M.D., Ph.D., Department of Anesthesiology, Medical College of Virginia, P.O. Box 516, Richmond, VA approach such as the 2-DG technique is that it allows simul- 23298. taneous examination of multiple CNS areasaffected by a stim- Copyright 0 1993 Society for Neuroscience 0270-6474/93/132689-14$05.00/O ulus or behavioral condition. As reported previously, increases 2690 Mao et al. l Patterns of Increased Brain Activity Indicative of Pain in spinal cord neural activity in CC1rats exhibit specificpatterns lowski and Marshall, 1983; Di Rocco ct al., 1989; Coghill et al., with respect to the spatial distribution of spontaneously in- 1991; Price et al., 1991; Mao et al., 1992a). creasedneural activity (Price et al., 1991; Mao et al., 1992a). These patterns of increased neural activity can serve as the Materials and Methods guidelines for examining spatial patterns of increasesin neural Subjccfs. Adult male Sprague-Dawley rats (Hilltop) weighing 300-350 activity in specificbrain regionsof CC1rats. For instance,spinal gm were used. Animals were individually housed in cages with water cord regions showing increasedneural activity in theseCC1 rats and food pellets available ad libitum. The animal room was artificially include superficial laminae I-II, deep dorsal horn laminae V- illuminated from 0700 to 1900 hr. All experimental procedures were VI, and the ventral horn lamina VII (Price et al., 199 1; Mao et approved by our Institutional Animal Care and Use Committee. Lum- bar spinal cords of the rats used in the present study have been analyzed al., 1992a),regions highly implicated in spinal cord nociceptivc in our previous 2-DC studies (Price et al., 199 1; Mao et al., 1992a). processingand representing the main origin of ascending spi- Surgical preparation of CC1 rats. Rats in both the ligation and the nothalamic, spinoreticular,and spinomesencephalictracts. These sham operation group (n = 6/group) were anesthetized with 50 mg/kg pathways transmit somatosensoryinformation to many brain pentobarbital. For sciatic nerve ligation, the right sciatic nerve was structures involved in supraspinalnociccptive processing(Wil- exposed and loosely ligated with four ligatures (4-O chromic gut) ac- cording to the method of Bennett and Xie (1988). For sham operation, lis, 1985; Price, 1988). the nerve was exposed as above but not ligated. All animals received Given the bilateral pattern and the regional specificity (lam- intramuscular potassium penicillin (30,000 W/rat) postoperatively. inae I-IV, V-VI, and VII) ofincreased spinalcord neural activity Behavioral assessmcnfs. Thermal hyperalgesia and spontaneous pain in CC1 rats (seeresults in Price et al., 199 1; Mao et al., 1992a), behaviors were examined in both ligated and sham-operated rats on days 0 (baseline), 5, 7, and IO postsurgety. Thermal hyperalgesia to several hypotheses may be made with respect to the possible radiant heat was assessed by using a foot withdrawal test. Foot with- spatial distribution of increasesin brain neural activity. First, drawal latency was defined as the time elapsed from the onset of radiant increasesin neural activity should occur bilaterally in broad heat to the hind paw withdrawal. Since the foot withdrawal latency supraspinal regions of CC1 rats in association with ascending taken from the hind paw directly touching the ground differs from that nociceptive pathways (including the spinothalamic tract), par- slightly elevated from the ground, a condition sometimes seen in the nerve-ligated hind paw of CC1 rats, we tested CC1 rats only during the ticularly those in connection with spinorcticular and spinomes- period when their affected hind paws were in contact with the ground. encephalic tracts, such as brainstem reticular formation and Foot withdrawal latency difference scores (contralateral side minus ip- central gray matter. Second, since at the spinal level maximal silateral side) were used to determine the degree of hyperalgesia as increasesin neural activity occur in somatotopically appropriate described in detail elsewhere (Mao et al., 1992a,b). Spontaneous pain behaviors were evaluated by observing postsurgical hind paw guarding L,-L, spinal laminae I-IV and V-VI (the main origin of the positions including (1) placement of only the medial portion or the heel spinothalamic tract) in CC1rats, thalamic nuclei receiving direct of the ligated hind paw onto ground and (2) lifting of the ligated hind input from thesespinal cord regions, such as the ventral pos- paw as described in our previous experiments (Mao et al., 1992a,b). terolateral thalamic nucleus and posterior thalamic nucleus, “C-2-DC administration. The procedures for 2-DC administration were exactly as those described in our previous 2-DG experiments (Price should exhibit somatotopically organized activation. Likewise, et al., 1991; Mao et al., 1992a). Briefly, on the tenth day after nerve cortical somatosensoryareas that, in turn, receive input from ligation or sham operation, animals were anesthetized with isollurane these thalamic nuclei should be activated as well. Moreover, (4.5% for induction, 1.5% for maintenance in a 70% N,O and 30% 0, greater neural activity should be observed on the side contra- mixture). The femoral vein and artery contralateral to the ligated or lateral to nerve injury than on the ipsilateral side. Third, since sham-operated side were catheterized with PE-50 tubing filled with hcparinized saline. The surgical field was infiltrated repeatedly with a spinothalamic and spinoreticular tracts arc likely to participate local anesthetic, bupivicaine, before and after surgery to minimize con- in both sensoryand affective dimensionsof pain (Willis, 1985; founding nociceptive input. Animals were allowed to recover completely Price, 1988), regions that may participate in neural processing from anesthesia for at least I hr after catheterization to regain a phys- of affective-motivational states related to pain, such as the iologically stable and awake condition. amygdala and the cingulate cortex, also would be expected to Afferent feedback from flexor motor reflexes occurring in CC1 rats could conceivably contribute to the increases in 2-DC metabolic activity show increasesin neural activity following CCI. in CC1 rats. In order to examine this possibility, one-half of the animals In addition, since similarities and difherencesin spatial pat- from each group (ligation and sham operation) were intubated and terns of spinal cord neural activity are observed between acute paralyzed with curare (I .5 mg) under the initial anesthesia induced by pain (Coghill et al., 1991) and persistent neuropathic pain in isoflurane as described in detail elsewhere (Price et al., 1991; Mao et al., 1992a). The initial anesthesia was discontinued after surgery, and this CC1 model (Price et al., 1991; Mao et al., 1992a), accord- rats were artificially ventilated during the experimental period. End- ingly, similarities and differencesmight be seenbetween patterns tidal CO, was monitored, and respiratory rate/volume was adjusted to of brain neural activity in CC1 rats and in rats exposed to for- maintain end-tidal CO, at 4.5%. Anesthesia was discontinued in both malin injection, an acute noxious stimulus (Porro et al., 199 1b). paralyzed and nonparalyzed preparations because anesthesia would sub- General similarities also might be expected between patterns of stantially alter brain sensory and motor activity. In addition, surgical procedures eliminating sensory awareness (e.g., decerebration) would brain neural activity in CC1 rats and human subjects during also substantially alter brain neural activity. CC1 produces an ongoing noxious heat stimulation (Talbot ct al., 1991) in similar func- sensory abnormality in the normally behaving (nonparalyzed) prepa- tional activity mapping studies.The presenceof similarities and ration. However, based on the absence of differences in spinal cord differencesbetween patterns of brain neural activity under acute 2-DG metabolic activity between paralyzed and nonparalyzed rats (Price et al., I99 I; Mao et al., 1992a), the introduction of paralysis does not and chronic pain conditions should provide insights into CNS seem to alter the magnitude of abnormal sensory input produced by mechanismsof postinjury neuropathic pain and possibly other CCI. Moreover, both paralyzed and nonparalyzed animals did not re- chronic pain conditions. These possibilities were investigated ceive any additional noxious stimulation. Thus, both paralyzed and in CC1 rats 10 d after sciatic nerve injury by utilizing the fully nonparalyzcd animals were very likely to receive similar sensory inputs quantitative 2-DG technique, a functional mapping technique related to an ongoing sensory abnormality. Furthermore, paralysis did not appear to produce any additional stress in both sham-operated and that enablessimultaneous examinations of both relative intcn- CC1 animals. Neither heart rates (before vs after: 187.3 * 15.4 vs 185.5 sity of neural activity and its spatial distribution (Kennedy et t 8.9; paired 1 test, P > 0.05) nor blood pressure (systolic/diastolic: al., 1975; Sokoloff et al., 1977; Singer and Mehler, 1980; Koz- 93.7 & 5.9j71.6 + 5.4 vs 91.7 ? 7.1/68.8 f 4.3; paired I test, P z TM Journal of Neurosdnce. June 1993, U(6) 2891

0.05) were changed upon administration ofcurare. Thus, paralysis does Results not appear to exacerbate stress in sham-operated animals or pain and stress in CC1 animals. Behuvioml uhnormalities in CC1 rats The catheterized paralyzed or nonparalyzed animal was comfortably Behavioral assessmentconfirmed that CC1 rats used in the ex- positioned in a well-ventilated, tube-shaped plastic restrainer (intcmal periment had demonstrablethermal hyperalgesia,that is, higher diameter 6.5 cm x internal length 25 cm) with fore and hind paws foot withdrawal latency difference scores,on days 3, 5, and IO extending through holes at the bottom of the restrainer. The use of a restrainer in nonparalyzed rats was to secure the intravascular cannulas after nerve ligation as compared to sham-operatedrats (ANO- implanted for the blood sampling, a procedure necessary for the fully VA, P < 0.01; see results in Mao et al., 1992a). CC1 rats used quantitative 2-DG autoradiography. A restrainer also was used with in this experiment all exhibited foot withdrawal latency differ- paralyzed rats in order to apply the same experimental conditions as ence scoreshigher than 2 sec. Accordingly, these animals had those applied with nonparalyzed rats. All animals were habituated to reliably higher spinal cord 2-DG metabolic activity as compared restraint 1 hr per day for 5 d prior to 2-DG administration. The place- ment of a nonparalyzed rat into a restrainer does not immobilize the to sham-operatedcontrols (Price et al., 1991; Mao et al., 1992a). rat. The rat can still move to a certain degree within the restrainer. In All CC1 rats also showed typical postsurgicalguarding positions fact, rats usually voluntarily enter a tube-shaped restrainer during the such as placement of only the medial portion or the heel of the period of habituation before the 2-DG administration. No signs of ligated hind paw onto ground and frequent lifting of the ligated distress were observed for these rats during restraint as indicated by the observed lack of aggressive behaviors and by the presence of stable hind paw. blood pressure values before and throughout the experiment. Neither noxious nor overt innocuous peripheral stimulation was delivered to Glohul chunges in brain 2-DG metabolic activity in CCI rats animals within each group during the following 45 min blood sampling period. At the beginning of a 45 min blood sampling period, lpC-2-DG Brain 2-DG metabolism in sham-operatedrats rangedfrom 29 (50 PCi) was injected into the femoral vein and 13 scheduled blood * 8 (hypothalamic arcuate nucleus) to 57 f 7 (the posterior samples were drawn from the femoral artery to determine plasma glu- thalamic nucleus)rmol/lOO gm/min (Fig. 1, left column; Table cose and 2-DG radioactive levels for the performance of the fully quan- I). No significant differences in 2-DG metabolic activity be- titative “C-2-DG autoradiography. Blood pressure and heart rates were monitored during the experimental period. End-tidal CO, also was mon- tween sampledbrain regions ipsilateral and contralateral to sur- itored in paralyzed, artificially ventilated rats throughout the experi- gery were found in sham-operatedrats. Thus, shamsurgery had ment. On completion of the blood sampling procedure, rats were killed a negligible effect on LGUR. In contrast to sham-operatedrats, with pentobarbital and perfused with saline and 10% formalin solution. CC1 rats examined IO d after sciatic nerve ligation exhibited The brain and spinal cords were carefully removed, immediately frozen strikingly increasedbrain 2-DG metabolic activity (Fig. I, right in cooled 2-methylbutane (- SOOC),and then stored at - 70°C for later cryostat sectioning and autoradiography. Lumbar spinal cords of these column; Table 1). Overall LGUR pooled from 36 sampledROIs rats have been analyzed previously as described in detail elsewhere was reliably higher in CC1 rats than in sham-operatedrats (li- (Price et al., 1991; Mao et al., 1992a). gation, 64.9 f 13.1, vs sham operation, 41.4 f 6.2; Student’s Autoradiography and image processing. Coronal sections of the brain t test, P < 0.0 1). LGUR in the thalamus and the cerebral cortex (20 pm per section) were made by using a cryostat at -20°C. Autora- diography was carried out by exposing x-ray film (Kodak SB-5) to brain was comparatively higher than that in lower brainstem regions sections and methyl methacrylate standards (Amersham) for 5 d. Au- of sham-operatedrats. Accordingly, reliably higher LGUR also toradiographic images were digitized and analyzed with a commercially was observed at cortical and thalamic levels in comparisonwith available image processing system (MCID, Imaging Research Inc.). lower brainstem regionsin CC1rats (cerebralcortex + thalamus, Thirty-six regions of interest (ROIs) selected from the medulla to the 73.8 + 9.4, vs lower brainstem, 56.1 f 5.8; Student’s t test, P cerebral cortex were sampled by manually outlining the territory ofeach ROI based on the atlas of Paxinos and Watson (1986). The histological < 0.05; Fig. 1, right column; Fig. 2). verification of these regions was made by using dark-field microscopic observations, and microscopic photos were taken when necessary. Four Topographic changes in brain 2-136 metabolic activity in CCI sections were sampled for each ROI per rat. Whenever applicable, ip- rats silateral and contralateral portions of an ROI were sampled separately. Optical density was then converted into local glucose utilization rate Changesin 2-DG metabolic activity occurred within extensive (LGUR; rmol/lOO gm/min) according to the method of Sokoloff et al. brain regions from the medulla to the cerebral cortex of CC1 (1977). rats 10 d following sciatic nerve ligation. However, as indicated Statisticaldata ana/y.ses. Mean individual LGUR was first calculated for each ROI for both ligated or sham-operated rats. Mean group LGUR for specific areasdescribed below, increasesin brain neural ac- of each ROI was then obtained by averaging individual means of each tivity occurred in specific nuclei and were topographically or- ROI from each group. Consistent with the lack of differences seen in ganized. spinal cord 2-DG metabolic activity between paralyzed and nonpara- Me&/la. There was an general elevation of LGUR in CC1 lyzed rats in each group (Price et al., 1991; Mao et al., 1992a), there was also no difference in brain 2-DG metabolic activity (pooled from rats at the medullary level, particularly within both dorsal and all 36 brain regions analyzed) between paralyzed and nonparalyzed rats ventral portions of the medullary reticular formation (Fig. IE, in each UOUD (paralvzed CC1 rats vs nonparalyzed CC1 rats: 67.2 + 9.7 Table 1). As shown in Figure I, reliably higher LGUR was vs 62.6-k 13.2; paralyzed sham rats vsnonparalyzed sham rats: 40.3 observed in CC1 rats within a clearly outlined medullary region, f 4.5 vs 42.5 ? 6.9; Student’s t tests, each P > 0.05). In addition, the parvocellular reticular nucleus, both ipsilateral and contra- LGUR in each sampled brain ROI also was not different between par- alyzed and nonparalyzed rats in both ligation and sham operation groups lateral to the side of nerve ligation as compared to sham-op- (analysis of variance, ANOVA, P > 0.05). All of these indicate a neg- erated rats (P < 0.01; Table I). Reliable increasesin LGUR ligible influence from the flexor afferent feedback on brain 2-DG met- also occurred in bilateral ventral gigantocellular reticular nu- abolic activity. Thus, data from paralyzed and nonparalyzed rats were cleus,lateral paragigantoccllular nucleus,and the entire nucleus pooled to yield group means (the ligation group and the sham-operation raphe magnus(P < 0.01; Table 1). group). Two-way ANOVA was used to examine overall differences in brain LGUR within the vestibular nuclei was not different between LGUR between ligation and sham operation groups as well as differences CC1 rats and sham-operatedrats (P > 0.05; Table l), although in each ROI between groups. Univariate tests were used to further there seemedto be a slight increasewithin bilateral vestibular analyzedifferences between ROIs, if applicable,ipsilateral or contra- nuclei in CC1 rats (Fig. 1E). In addition, 2-DG metabolic ac- lateral to surgical procedures (nerve ligation or sham operation). tivity in caudal and interpolar portions of the spinal trigeminal CP

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BRAIN REGIONS J OWER BRAINSTEM PARVOCELLU. RETl. N PARAGIGAN. N, LAT. PONTINE RETl. N LoaJscoERuLEus IPSILATERAL PARABRACHIAL N CONTRALATERAL SUP. coLLlcuLus THAI Au VENT. POST. LAT. N VENT. POST. MED. N POST. THALA. N VENT. THALA. N, LAT. VENT. THALA. N, MED. CORTEX & SUBCOR. CAUDATE-PUTAMEN AMYGDALA CINGULATE Cx Figure 2. Net increases in 2-DG met- FORE LIMB Cx abolic activity in sampled bilateral brain HIND LIMB Cx regions that were found to have signif- PARIETAL Cx icant elevations in 2-DG activity (see RETROSPLENIAL Cx Table 1). Net increases (ligation group minus sham operation group) in LGUR 0 10 20 30 40 50 (pmol/l 00 gm/min) were calculated for each side (ipsilateral or contralateral) of NET INCREASE IN LGUR a bilateral ROI exhibiting statistical sig- (umoledl OOglmin) nificance between two groups. nuclei remained unchanged in CC1 rats in comparison with Although there appeared to be a slight increasein 2-DG met- corresponding regions of sham-operated rats (P > 0.05; Fig. lE, abolic activity within occipital areasin CC1 rats (Fig. 1C, Table Table 1). l), no significant difference in LGUR was detected within these Pans. LGUR within ventral and caudal pontine reticular nu- areasbetween CC1 rats and sham-operatedrats (P > 0.05; Table clei was reliably higher in CC1 rats than in sham-operatedrats, 1). and similar increasesin LGUR were observed in bilateral locus Thalamus and hypothalamus. At the thalamic and hypotha- coeruleusof CC1 rats (Fig. 1D, Table 1). As shown in Figure 1, lamic level, two apparent changesoccurred with regard to in- the activation of the parabrachial nucleus complex, including creased 2-DG metabolic activity in CC1 rats: (1) LGUR was lateral and medial parabrachial nuclei, was lateralized in CC1 substantially higher within the thalamus and the hypothalamus rats. Thus, although both ipsilateral and contralateral parabra- than that seenin the lower brainstem (Figs. M-E, 2), and (2) chial nuclei showed increasedLGUR in CC1 rats as compared more brain regions were activated as compared to the number to sham-operatedrats, higher LGUR occurred in the contra- of regions showing reliable increases in LGUR within the lower lateral (seenin the left half of a section, the side opposite to the brainstem (Fig. M-E). However, regions exhibiting increased ligated sciatic nerve) parabrachial nuclei as compared to the 2-DG metabolic activity at the thalamic and hypothalamic level correspondingipsilateral (right half of a section) region in CC1 were segregatedinto several distinguishablegroups in CC1 rats rats (P < 0.05, paired t test; Table 1). (Fig. 1B). Midbrain. Reliable increasesin LGUR were observed within 2-DG metabolic activity reliably increased in thalamic so- the dorsolateral portion of the central gray matter, the dorsal matosensory relay nuclei. These nuclei included ventral pos- raphe nucleus, and bilateral reticular tegmental nucleus (P < terolateral thalamic nucleus,ventral posteromedialthalamic nu- 0.05; Fig. 1C, Table 1). Increasesin LGUR within the superior cleus,posterior thalamic nucleus,and ventral medial and ventral colliculus of CC1 rats were heterogeneous;that is, reliable in- lateral thalamic nuclei. While increasesin LGUR were observed creasesin LGUR were seenin deep layers of bilateral superior bilaterally within these nuclei as compared to sham-operated colliculus (P < 0.05) but not in superficial layers of this nucleus rats (P < 0.0 I), higher LGUR occurred on the side contralateral as compared to sham-operatedrats (P > 0.05; Fig. lC, Table to the injured sciatic nerve than on the ipsilateral side, partic- 1). ularly in ventral posterolateral thalamic nucleus, posterior tha- t Fig&z I. Topographic presentation of coronal sections showing local glucose utilization in the brain of a CC1 rat (right column) and a sham- operated rat (left column). Sciatic nerve ligation produced substantial increases in metabolic activity within cortical hind limb (ZZL) and fore limb (FL) area, retrosplenial granular cortex (RG), cingulate cortex (CZ), parietal area (PA), caudate-putamen complex (CP), amygdala (AC), posterior thalamic nucleus (PO), ventral posterolateral thalamic nucleus (VK), ventral posteromedial thalamic nucleus (VZ’M), ventral medial thalamic nucleus (VW, occipital cortex (OC), central gray matter (CC), superior colliculus (SC’), pontine reticular nucleus (PR), parabrachial nuclei (Z’S), parvocellular reticular nucleus (PCR), spinal vestibular nuclei (SV), and lateral paragigantocellular nucleus (LPG) as compared to corresponding regions of sham-operated rats. This increase was more pronounced on the contralateral side (left side of an image) of CC1 rats than on the ipsilateral side. The anteroposterior coordinates (from bregma) of each coronal section (from A to E) are -0.80 mm, -3.14 mm, -7.80 mm, -9.80 mm, and - 11.96 mm, respectively, based on the Paxinos and Watson atlas (Paxinos and Watson, 1986). The color bar represents the calibration of the LGUR (pmol/ 100 gm/min) for these images. 2894 Mao et al. - Patterns of Increased Brain Activity Indicative of Pain

Table 1. Regional changes in LGUR (pmol/lOO gm/min)

Ligation Sham Brain redons Mean -t SE Mean f SE Medulla Spinal trigeminal N, caudal 46 k 5 0 38 + 3 47 f 5 (1) 37 f 5 Spinal trigeminal N, interpolar 48 2 6 0 38 + 4 54 f I (1) 36 k 5 Medullar reticular formation, dorsal 55 + 5 ((3 42 + 6 57 + 6 (1) 42 + 5 Medullar reticular formation, ventral 52 f 4 (Cl 39 k 3 50 + 3 (1) 38 2 5 Lateral reticular N 47 f 5 (Cl 32 + 4 50 f 6 (1) 38 f 5 Vestibular N 53 k 5 ((2 46 t 4 60 k 7 (1) 53 f 5 Parvocellular reticular N 73 f 7** (C) 43 f 5 67 + 3* (1) 45 f 6 Lateral paragigantocellular N 62 k 6** (C) 28 2 4 58 t 6** (1) 33 f 3 Gigantocellular reticular N, ventral 61 f 8** 31+4 Raphe magnus N 56 f 4* 34 f 5

Pons Pontine reticular N, caudal 76 k 9* (C) 49 + 6 69 -c 5** (1) 45 * 4 Pontine reticular N, ventral 56 2 3* 36 f 6 LAXUS coeruleus 58 k 3** (Cl 32 f 4 58 f 11* (1) 30 f 3 Parabrachial N, lateral-medial 61 f I**,t 0 41 f 2 52 f I** (1) 40 f 3 Midbrain Central gray matter Dorsal lateal 58 k 4* (C) 42 f 5 57 * 5* (1) 41 2 5 Ventral lateral 60 f 5 (C) 46 k 6 60 ? 5 (1) 45 f 6 Raphe N, dorsal 62 f 3* 46 k 7 Reticular tegmental N 55 f 5* (C) 36 + 5 56 f 6* (1) 36 + 4 Superior colliculus 57* 1* (0 37 * 3 Deep layers 55 f I* (1) 38 k 3 Upper layers 40 t 2 (C) 38 f 2 42 f 2 (1) 37 + 2 Thalamus and hypothalamus Ventral posterolateral N 82 f lo*+ 63 49 f 6 66 f 9 (1) 49 f 6 Ventral posteromedial N 88 I! 11* CC) 53 f 6 82 + lO* (1) 51 -t6 Posterior thalamic N 94 + 7*+ (C) 57 f 7 82 + 4* (1) 52 f 5 Ventral lateral thalamic N 92 + 11’ (C) 56 f 7 80 + 12* (1) 51 f 5 Ventral medial thalamic N 82 f I**,$ 0 41 -t 3 69 f 2** (1) 42 + 3 Central lateral-central medial N 63 f 2* 41 f 3 Dorsomedial hypothalamic N 62 t 9 62 43 f 6 62 2 8 (1) 42 f 5 Hypothalamic arcuate N 62 f 9* 29 f 8 Lateral geniculate N 42 k I (Cl 39 f 2 42 2 1 (1) 42 + 4 The Journal of Neuroscience. June 1993, X3(6) 2695

Table 1. Continued

Ligation Sham Brainregions Mean -t SE Mean + SE Cortexand subcorticalregions Parietalarea 1 80 f 14* 6-3 46 k 6 76 f 13* (1) 46 f 7 Parietalarea 2 82 k 14* CC) 48 f 6 79 + 12* (1) 48 f 5 Retrosplenialgranular cortex 92 2 5** (C) 42 f 2 89 f 4** (1) 40 f I Hind limb area 82 k 6**.t e-2 40 ?I 5 69 f 6* (1) 39 f 5 Fore limb area 77 f 6** 62 40 f 5 67 + 5* (1) 41 * 5 Amygdala 81 f 5**+ (C) 40 + 5 66 * 5* (1) 40 + 6 Cingulatearea 82 + 14* CC) 46 + 6 78 + 14* (1) 46 + 8 Caudate-putamenN 86 f 5**,t CC) 50 f 4 75 f 5* (1) 47 f 4 Occipitalarea 45 * I CC) 41 f 3 42 + I (1) 39 + 3

Whenever applicable the metabolic rate calculated from contralateral (C)and ipsilateral (I) brain regions is given separately in the table. l , P < 0.05; l *, P < 0.01 (ANOVA); comparisonsmade between ligation and sham groups. t, P < 0.05 (paired r test), comparisons made between contralateral and ipsilateral side of an ROI in CC1 rats.

lamic nucleus, and ventral medial thalamic nucleus (P < 0.05, higher than that of the ipsilateral amygdala in CC1 rats (P < paired t tests;Fig. lB, Table I). In contrast to increasesin LGUR 0.05, paired t test; Fig. lA, Table 1). within the thalamic somatosensoryrelay nuclei, no differences Retrosplenial granular cortex, a cortical area seenin the tha- in 2-M; metabolic activity were observed in the lateral genic- lamic section (Fig. 1B), was highly activated in CC1 rats. 2-DG ulate body, a sensory relay nucleus within the visual system, metabolic activity in this cortical area increasedbilaterally and between CC1 rats and sham-operatedrats (P > 0.05; Table 1). homogeneouslyin CC1 rats as compared to sham-operatedrats Comparatively smaller but significant increasesin 2-DG met- (P < 0.01). Moreover, this area exhibited the highest increase abolic activity were observed within the central medial and in LGUR among 36 sampledbrain regions in CC1 rats (Fig. 2). central lateral nuclei in CC1 rats as compared to sham-operated 2-DG metabolic activity within the bilateral caudate and pu- rats (P < 0.05; Fig. lB, Table 1). Similar increasesin LGUR tamen complex, a portion of the subcortical basalganglia, also occurred within the hypothalamus such as the hypothalamic was increasedin CC1 rats in comparison with sham-operated arcuate nucleus (P < 0.05; Fig. lB, Table 1). rats (P < 0.01; Fig. IA, Table 1). Moreover, significantly higher Cortex and subcortica[ areas. At the cortical and subcortical LGUR was seenin the contralateral caudateand putamencom- level, regions with increased2-DG metabolic activity in CC1 plex than in the corresponding ipsilateral region in CC1 rats (P rats were largely divided into two groups: the cortical somato- < 0.05, Table I). sensoryareas and the limbic structures.With respectto the first group, reliable bilateral increasesin LGUR were observed with- Characteristicsof spatial patterns of increasedbrain Z-DG in parietal cortex and within cortical hind limb and fore limb metabolic activity in CCI rats somatosensoryareas of CC1 rats in comparison with corre- The spatial patterns of strikingly increased2-DG metabolic ac- sponding regions of sham-operated rats (P < 0.01; Fig. 1A). tivity describedabove for CC1 rats were characterized by several Appropriately, peak activity occurred within the hind limb area distinct features(see above for detailed statistical analyses).First, (Fig. 1A). Consistentwith somatotopical activation of thalamic LGUR was increasedbilaterally in most sampledROIs of CC1 somatosensoryprojection nuclei in CC1rats, increasesin LGUR rats as compared to corresponding regions of sham-operated within the cortical hind limb area were also lateralized; that is, rats (Table 1, Fig. 2). Second, consistent with somatotopic pro- rehably higher LGUR was seenin the contralateral hind limb jections of the ascendingspinothalamic tract and its correspond- area as compared to the ipsilateral hind limb area in CC1 rats ing cortical arcas, LGUR was reliably higher within appropriate (P < 0.05, paired t test; Fig. lA, Table I). The activation of contralateral thalamic and cortical regions,including the ventral cortical regionswas largely concentrated within the middle and posterolateralthalamic nucleus, posterior thalamic nucleus,and deep layers of the cortex (Fig. lA,B). cortical hind limb area in CC1 rats (Figs. I, 2). Third, although Two regionsclassifiedas limbic structures,that is, the bilateral there occurred a general elevation in background 2-DG meta- amygdala and cingulate cortex, were significantly activated in bolic activity in CC1 rats, peak increasesin metabolic activity CC1 rats as compared to sham-operatedrats (P < 0.01; Fig. lA, (ligated rats minus sham-operatedrats) were observed, among Table 1). LGUR within the contralateral amygdala was reliably 36 sampledROIs, in the contralateral cortical hind limb area, 2996 Mao et al. - Patterns of Increased Brain Activity lndiitive of Pain amygdala, ventral posterolateral thalamic nucleus, posterior types of neurons and therefore may be an incomplete marker thalamic nucleus, and bilateral retrosplenial granular cortex in of neural activity (Bullitt, 1990). Moreover, the 2-DG technique five out of six CC1 rats (Figs. 1, 2). Finally, LGUR in brain is based on the well-understood mechanism that the increase regions known not to be directly involved in somatosensory in local cerebral glucose utilization is proportional to the in- processing, such as those within the visual pathway (lateral ge- crease in neural activity (Sokoloff et al., 1977). This technique niculate body, occipital cortex) and the vestibular pathway provides a unique means to allow simultaneous examination of (medullary vestibular nuclei), remained unchanged in CC1 rats. both relative intensity of neural activity and its spatial distri- bution within the CNS by measuring LGUR during a relatively long period (3w5 min). Thus, the 2-DG technique is partic- Discussion ularly suitable for mapping CNS neural activity during pro- The present findings that striking increases in 2-DG metabolic longed noxious stimulation (Coghill et al., 1991) or ongoing activity occur in widespread brain regions of CC1 rats provide nociception such as in this CC1 model (Price et al., 199 1; Mao the first functional mapping of supraspinal neural activity re- et al., 1992a). Comparable electrophysiological single-unit ex- sulting from a chronic pain condition. These increases in 2-DG aminations of the spatial distribution of neural activity within metabolic activity occurred in CC1 rats with demonstrable ther- the entire CNS would be extremely time consuming and thereby mal hyperalgesia and spontaneous pain behaviors. The fact that impractical. Nevertheless, the use of different methodologies peak increases in metabolic activity occurred in contralateral should provide better understanding of CNS nociceptive pro- brain regions previously implicated in supraspinal nociccptive cessing than any one technique alone. processing suggests a causal relationship between increases in brain neural activity (as measured by 2-DG metabolic activity) Functional rekrtionships to brain regions implicated in and neuropathic pain behaviors in CC1 rats. These striking in- nociceptive processing creases in neural activity, which occur in the absence of overt Given the chronic course of behavioral and physiological changes peripheral stimulation, provide further evidence that ongoing subsequent to nerve ligation in this CC1 model, it is conceivable pain behaviors reflect persistent spontaneous pain in CC1 rats that a number of potential changes only indirectly related to and are not exclusively defensive behaviors directed toward chronic neuropathic pain may have occurred and have contrib- avoiding pain. This overall conclusion is supported by the fol- uted to regional increases in brain 2-M; metabolic activity in lowing discussion, which deals with methodological consider- CC1 rats. Such changes might occur in brain regions involving ations and anatomical-functional interpretations of brain regions regulation of motor activity, food and water intake, cardiovas- showing increased neural activity. cular activity, and respiratory functions. This may partially ex- plain the bilateral activation ofcertain brain regions in CC1 rats. Methodological considerations However, the extensive activation of brain regions observed in One major limitation of currently applied autoradiographic the present 2-DG experiment most likely reflects CNS process- functional activity mapping techniques, including the I%-2-DG ing of neuropathic pain in CC1 rats for several reasons. First, technique, is their potential inability to distinguish excitatory increases in brain neural activity ofCC1 rats reflect the anatomic from inhibitory activity, since both excitatory and inhibitory and functional continuation of ascending spinal cord somato- events involve energy consumption, and therefore increases in sensory pathways (e.g., spinoreticular, spinomescncephalic, and local glucose utilization (Ackcrmann et al., 1984; Nudo and spinothalamic tracts), whose spinal cord origins (laminae I-IV, Masterton, 1986). However, studies of combined 2-DG mea- V-VI, VII) exhibit substantially elevated neural activity in these surement and electrophysiological recording of primate so- same CCI rats (cf. results in Price et al., 199 1; Mao et al., 1992a). matosensory cortex strongly suggest that neural excitation is a Second, brain regions that are very unlikely to be involved in major contribution to increases in 2-DG metabolic activity (Ju- pain, such as those within the visual pathway (lateral geniculate liano and Whitsel, 1987) and in fact, inhibitory neural events body, occipital cortex) and the vestibular pathway (medullar are shown to decrease local glucose utilization in similar 2-DG vcstibular nuclei), remained unchanged in CC1 rats as compared studies (Sharp et al., 1988). Consistent with these observations, to sham-operated rats. Third, a large body of evidence indicates recent electrophysiological studies have reported increased that the increase in brain neural activity in CC1 rats exhibits background neuronal discharges within the lumbar spinal cord regional specificity with respect to known anatomical and elec- (Palccek et al., 1991) and the thalamic ventrobasal complex trophysiological involvement of these regions in supraspinal (Guilbaud et al., 1990) ofCC1 rats, in which substantial increases nociceptive processing. Nevertheless, a complete resolution of in 2-DG metabolic activity are detected in previous (Price et this interpretational problem awaits the analysis ofbrain activity al., 199 1; Mao et al., 1992a) and present 2-DG experiments. by a variety of noxious stimuli with varying nonspecific se- Thus, increases in 2-DG metabolic activity observed in brain quelae. The relationships between observed increases in neural regions of CC1 rats are more likely to reflect excitatory than activity and evidence for involvement in nociceptive processing inhibitory activity. An additional limitation of this 2-DG tech- or pain modulation will be discussed for individual brain regions nique relates to its lack of cellular resolution; that is, changes in turn. in 2-DG metabolic activity are undistinguishable between neu- Medulla. The activation oflateral paragigantocellular nucleus, ronal perikarya and nerve fibers (Di Rocco ct al., 1989). An gigantoccllular reticular nucleus, nucleus raphe magnus, and alternative functional activity mapping technique utilizing the parvocellular reticular nucleus at the medullary level is consis- c-fos-like protein expression provides such cellular resolution tent with their possible roles in nociception and pain modulation (Hunt et al., 1987). (Basbaum and Fields, 1984; Lovick, 1987; Mayer and Price, Despite its limitations, the 2-DG technique is a well estab- 1989; Zhuo and Gebhart, 199 1; Kicfel et al., I 992). Extracellular lished autoradiographic mapping technique (Sokoloffet al., 1977). recordings made from the gigantocellular reticular nucleus in- It is different from the c-fos method, which can label only certain dicate that neuronal discharges of nociceptive neurons within The Journal of Neurosdence. June 1993, 13(6) 2697 this nucleus reliably increase in response to peripheral noxious dromically identified parabrachial-amygdaloid projection neu- heat (Sotgiu, 1988) or mechanical (pinch) stimulation (Drower rons) were classified as nociceptive-specific neurons, and no and Hammond, 1988) and the electrical activation of this nu- wide-dynamic-range neurons were recorded within this pathway cleus with low-intensity stimulation facilitates spinal cord no- (Bcmard and Besson, 1990). ciceptive transmission as assessed using the tail-flick test (Zhuo The role of this spinopontoamygdaloid pathway in pain pro- and Gebhart, 1990b, 199 1). On the other hand, most of these cessing is not yet clear. It is unlikely that this pathway partici- nuclei are also known to be major origins of descending noci- pates in neural processing of sensoryAiscriminative dimensions ceptive inhibitory pathways (Lovick, 1986b, 1987; Zhuo and of pain since little evidence exists indicating the involvement Gebhart, 1990a,b, 199 1; Cho and Basbaum, 199 I) and to relay of the amygdala in sensory discrimination of pain. This as- descending pain modulatory pathways from upper brain regions cending pathway is, however, likely to be involved in affective- (Lovick, 1986a; Ness and Gebhart, 1986; Mokha et al., 1987; motivational dimensions of pain, particularly in the coordina- Drower and Hammond, 1988; Kiefel et al., 1992). Since the tion ofnociceptive, emotional, and autonomic responses to nox- activation ofdescending inhibition by noxious or environmental ious stimulation (Bernard et al., 1989; Berkley and Scofield, peripheral input has been known to play an important role in 1990; Bernard and Besson, 1990), since the amygdala is known endogenous analgesic systems (Mayer and Price, 1989) the ac- to be a forebrain region implicated in stress, arousal, and fearful tivation of these nuclei could relate to both nociceptive pro- reactions (Werka and Marek, 1990; Tanaka et al., 199 1). On cessing and descending modulation. the other hand, both anatomical and electrophysiological studies It should be noted that both lateral paragigantocellular nu- have indicated an efferent pathway originating from the amyg- cleus and gigantocellular reticular nucleus are also implicated dala, directly or indirectly (via the medial hypothalamus) syn- in the autonomic regulation ofcardiovascular functions (Lovick, apsing within the pcriaqueductal gray, to the spinal cord dorsal I986b, 1987; Van Bockstaele et al., 1989; Aichcr and Randich, horn (Hopkins and Holstege, 1978; Krettek and Price, 1978; 1990). Thus, it will be of interest to determine whether the Beitz, 1982; Watson et al., 1983; Zhang et al., 1991). This ef- activation of these nuclei is related to abnormal sympathetic ferent pathway from the forebrain has been implicated in de- responses in CC1 rats, reflected for example by differences in scending modulation of pain (Basbaum and Fields, 1984; Shaikh skin temperature between ligated and nonligated hind paws in et al., 199 1; Zhanget al., 199 1). It should be noted that structures CC1 rats (Bennett and Ochoa, 199 1). within both the spinopontoamygdaloid pathway and the amyg- Pans and midbrain. The observation of increases in 2-DC daloid efferent pathway were found to be highly activated in metabolic activity within the pontine reticular nucleus, locus our present and previous (Price et al., 199 1; Mao et al., 1992a) coeruleus, dorsal raphe nucleus, reticular tegmental nucleus, and 2-DC studies. Thus, there appears to be a neural loop that central gray matter confirms the previous behavioral and elec- connects the spinal cord dorsal horn (laminae I-II), pontine trophysiological data that these regions participate in central parabrachial nuclei, amygdala, periaqueductal gray, and back nociceptive processing(Basbaum and Fields, 1984; Willis, 1985; to the spinal cord. This proposed neural loop might be important Ness and Gebhart, 1986; Yezierski and Schwartz, 1986; Mayer in efferent modulation ofpain. Interestingly, a recent behavioral and Price, 1989; Muller and Klingberg, 1989; Kiefel et al., 1992). study has indicated important roles of the parabrachial nucleus Of further significance is that higher metabolic activity is seen in development of neuropathic pain behaviors (autotomy) fol- in deep layers than in superficial layers ofthe superior colliculus. lowing sciatic and saphenous nerve sections (Wall et al., 1988). This observation is consistent with electrophysiological studies Destruction ofcell bodies with ibotenic acid in the contralateral showing that wide-dynamic-range and nociceptive-specific neu- parabrachial nuclei accelerates the onset of autotomy in nerve- rons occur in deep but not superficial layers of the superior sectioned rats (Wall et al., 1988). It will be of interest to inves- colliculus (McHaffie et al., 1989). The involvement of this mid- tigate whether such an inhibitory influence from the parabra- brain nucleus in nociceptive processing has also been reported chial nucleus on development of neuropathic pain is mediated in several recent electrophysiological studies (Larson et al., 1987; by this proposed neural loop. McHaffie et al., 1989; Nelson et al., 1989). In view of its possible Thalamus and hypothalamus. The role of thalamic somato- role in sensory-motor integration (McHaffe et al., 1989) the sensory relay nuclei and their adjacent regions in brain noci- superior colliculus may have specific roles in abnormal behav- ceptive processing has been well documented (Willis, 1985; iors in CC1 rats related to orientation to the location of periph- Chung et al., 1986; Guilbaud et al., 1986; Taguchi et al., 1987; eral nociception. Downie et al., 1988; Lenz et al., 1988; Price, 1988; Dostrovsky The activation of the parabrachial nucleus at the pontine level and Guilbaud, 1990). Accordingly, the ventral posterolateral is of particular interest since this pontine nucleus is strategically thalamic nucleus, posterior thalamic nucleus, ventral lateral and located within a recently delineated spinopontoamygdaloid medial thalamic nuclei, and central medial and lateral thalamic pathway (Ma and Peschanski, 1988; Bernard et al., 1989; Hyld- nuclei are shown to be highly activated in the present 2-DG en et al., 1989; Berkley and Scofield, 1990; Bernard and Besson, experiment. Moreover, results from our 2-DC labeling are also 1990). This spinopontoamygdaloid pathway originates from su- consistent with the data derived from a recent electrophysio- perficial spinal laminae I-II and is relayed within the pontine logical study (Guilbaud et al., 1990). In that study, abnormal parabrachial nucleus to the amygdala (Ma and Pcschanski, 1988; neuronal responses within the ventrobasal thalamic complex Bernard et al., 1989; Hylden et al., 1989; Berkley and Scofield, (regions corresponding to major thalamic relay nuclei in our 1990; Bernard and Besson, 1990), a structure that was found to 2-DC labeling) were observed in the same CC1 model used in be highly activated in the present experiment. Projections from the present 2-DC experiment, including increased spontaneous laminae I-II cells to the parabrachial nucleus were found to be neuronal activity, fading of the response with repetitive stim- bilateral and some of them were shown to be collaterals of the ulation, and prolonged afterdischarges (Guilbaud et al., 1990). spinothalamic tract (Hylden et al., 1989). The majority (69%) Consistent with the involvement of the hypothalamic arcuate of neurons within this pathway (recordings made from anti- nucleus in pain modulation (Mao and Yin, 1987; Hamba, 1988; 2899 Mao et al. * Patterns of Increased Brain Activity Indicative of Pain

Sathaye and Bodnar, 1989; Wanget al., 1990), increases in 2-DG ular cortex, which is involved in learning and memory, lends metabolic activity are also seen in this hypothalamic nucleus in further support to the hypothesis that neural mechanismsof CC1 rats. Direct spinohypothalamic projections from both su- postinjury neuropathic pain may be related to CNS neuronal perficial spinal laminae I-11 and the base ofthe dorsal horn have plastic changessimilar to those occurring in learning and mem- been recently reported (Burstein et al., 1987). This direct spi- ory (Mao et al., 1992~4). nohypothalamic pathway may at least partially account for the The activation of the cingulate cortex and amygdala may be activation of hypothalamic nuclei in CC1 rats. The hypothalam- rclatcd to generalarousal, anxiety, and stress(Papez, 1937; Vogt ic arcuate nucleus is the major origin of &endorphinergic neu- et al., 1979; Werka and Marek, 1990; Tanaka et al., 1991), rons in the brain (Bloom et al., 1978) and /3-endorphin is known although these areas have also been implicated in nociceptive to play important roles in pain modulation (Loh et al., 1976). processingand pain modulation (Hylden et al., 1989; Al Rodhan Thus, the activation of the hypothalamic arcuate nucleus may et al., 1990; Werka and Marek, 1990; Joneset al., 1991; Talbot have a descending inhibitory influence on nociccptive process- et al., 1991). However, sincerats usedin the present experiment ing in CC1 rats. On the other hand, since the hypothalamic had been habituated for restraint before 2-DG administration arcuate nucleus has numerous connections with brainstem struc- and did not reccivc overt peripheral stimulation during the ex- tures and functional relationships with the pituitary (Sim and perimental period, and since bilateral elevations of 2-DG met- Joseph, 199 1; Takcshige et al., 199 1a) and is therefore involved abolic activity within theseareas are observed only in CC1 rats in regulation of many physiological reactions including stress but not sham-operatedrats handled in the same manner, the and emotional reactions (Takeshige et al., 199 la,b), its activa- activation of the cingulate cortex and amygdala may primarily tion in CC1 rats may reflect possible roles of this hypothalamic reelect neural processingrelated to pain, particularly the affec- nucleus in the affective-motivational dimension of pain in CC1 tivc-motivational dimension of chronic neuropathic pain. In rats. fact, this feature ofthe bilateral activation ofthe cingulatecortex Cortex and subcortical areas. Regionsactivated at the cortical in CC1 rats is consistent with the clinical observation that the level such as parietal areas and the hind limb somatosensory affective-motivational dimension of persistent pain in human area, areas corresponding to the rat’s primary somatosensory patients, including neuropathic pain patients, can be partially area (SI) (Mountcastle and Darian-Smith, 1968), are consistent relieved after bilateral lesions of this area (Foltz and White, with those thalamic somatosensory relay nuclei showing in- 1962; Hurt and Ballantine, 1973). Thus, it will be of interest to creasedneural activity in CC1 rats. Of particular interest is the determine whether the activation of the cingulate cortex, amyg- fact that peak activity occurred within the hind limb arca of the dala, and retrosplenial granular cortex contributes to the im- somatosensorycortex (Fig. 1). Nociceptive neuronal responses mediate unpleasantnessstage or the secondary stage(related to within thesecortical areas have been reported in previous an- long-term implications and suffering) of the affective dimension imal and human studies (Lamour et al., 1983a; Willis, 1985; of pain (Price and Harkins, 1992). Price, 1988; Talbot et al., 1991). It should be emphasizedthat the activation of cortical areas in CC1 rats presentsa general Comparisons ofpatterns of increased brain activity between laminar organization (Fig. 1). Thus, increasedneural activity acute and persistent pain and implications for mechanisms of occurs mainly within middle and deep layers of the cortex, a neuropathic pain feature consistentwith the heavy concentration ofwide-dynam- Patterns of increasedbrain neural activity following CC1 are ic-range and nociceptive-specific neuronsfound in thesecortical similar in many respectsto those induced by acute noxious heat layers in electrophysiological studies(Lamour et al., 1983a,b). or formalin stimulation (Porro et al., 199I b; Talbot et al., 1991). The caudate and putamen complex is part of the subcortical First, lower brainstem regions activated in CC1 rats overlap basalganglia that is known as a central region involved in co- primarily with those activated by hind paw formalin injection, ordination of motor activity (Mountcastle and Darian-Smith, both including medullar and pontine reticular nuclei, nucleus 1968). Several recent reports, however, have indicated its role raphe magnus,dorsal raphc nucleus, and central gray matter. in pain modulation (Ohno et al., 1987; Li and Xu, 1990; Mul- These regions receive input from spinoreticular and spinome- gaonker and Mascarenhas, 1991; He, 1992). Given the abnor- sencephalicpathways, the spinal cord origins ofwhich are highly mal motor activity resulting from abnormal guarding postures activated in similar 2-DG studies(Porro et al., 199 la; Price et in CC1 rats, the activation of the caudate and putamcn complex al., 1991; Mao et al., 1992a). Second, the increase in 2-DG may be related to pain modulation and/or pain-rclatcd abnor- metabolicactivity is bilateral in most sampledbrainstem regions mal motoric activity. but with greaterincreases within the contralateral side following It is surprising that the greatest increase in 2-DG metabolic either unilateral sciatic nerve injury or unilateral hind paw for- activity occurs within the retrosplenial granular cortex of CC1 malin stimulation, a feature consistent with bilateral increases rats (Fig. 2). The retrosplenial granular cortex is an association in spinal cord 2-DG metabolic activity in both cases(Porro et cortex that has numerousreciprocal connections with the thal- al., 1991 a; Price et al., 1991; Mao et al., 1992a). The bilateral amus, hypothalamus, limbic structures, and other cortical areas activation of brain structures following unilateral sciatic nerve (Thompson and Robertson, 1987; Groen and Wyss, 1990). To ligation is consistent with behavioral observations that me- our knowledge, the role of this cortical area in nociceptivc pro- chanical hyperalgesia occurs in hind paws of CC1 rats both cessinghas not yet been investigated. This areais known to play ipsilateral and contralateral to the side of nerve ligation (Attal important roles in learning and memory in many species et al., 1990). Bilateral activation also occurs in the spinal cord (Thompsonand Robertson, 1987; Groen and Wyss, 1990,1992). ofrats exposedto noxious heat stimulation (Coghill et al., 199l), Of interest is that intracellular changesof spinal cord neurons although much higher contralateral spinal cord neural activity similar to those during mammalian learning and memory pro- is seenin CC1 rats (Mao ct al., 1992a)than in rats stimulated cesseshave been reported in CC1 rats (Mao et al., 1992e). This with noxious heat (Coghill et al., 1991). Third, cortical and striking increasein neural activity within the retrosplenial gran- subcortical arcas with increasedneural activity in CC1 rats are The Journal of Neuroscience, June 1993. 13(6) 2698

generally consistent with those activated by noxious heat stim- also highly activated in CC1 rats. Such a multiple-system acti- ulation in awake human subjects (Jones et al., I99 1; Talbot et vation is consistent with clinical characteristics of chronic neu- al., I99 I) including cortical somatosensory regions, the cingulate ropathic pain syndromes that involve disorders associated with cortex, and thalamic regions. Thus, the same brain regions in- sensory, motor, and autonomic systems (Bon&, 1979; Thomas, volved in CNS nociceptive processing appear to be activated 1984; Price et al., 1989). Similar pathological changes are seen under both acute and chronic pain conditions. in CC1 rats including thermal hyperalgesia, spontaneous pain An important difference in spatial patterns of brain neural behaviors (abnormal guarding positions), and asymmetry of hind activity is that brain regions activated in CC1 rats appear to be paw skin temperature (Bennett and Xie, 1988; Attal et al., 1990). even more extensive than those CNS regions activated during These symptoms are likely to be mediated by activation of formalin or noxious heat stimulation (Porro et al., 199 1b; Talbot multiple brain regions and their anatomic interconnections. et al., 199 1). For example, the activation of the brain is bilateral It is important to point out that sciatic nerve injury in CC1 in many thalamic and cortical regions of CC1 rats including the rats activates not only those brain regions implicated in afferent ventral posterolateral thalamic nucleus, posterior thalamic nu- processing of sensory+liscriminative and affective-motivation- cleus, cortical hind limb area, fore limb area, and parietal areas. al dimensions of pain but also those associated with centrifugal The existence of large and bilateral receptive fields of nocicep- modulation of pain. The latter structures may include the med- tive neurons within spinal cord lamina VII (Menetrey et al., ullary reticular formation, nucleus raphe magnus, parabrachial 1984) may have conceivably contributed to such bilateral ac- nuclei, dorsal raphe nucleus, central gray matter, and hypotha- tivations in CC1 rats. On the other hand, the bilateral activation lamic arcuate nucleus. Such a dual-functional activation of both of brain regions (e.g., the cortical fore limb area) in CC1 rats afferent and efferent nociceptive processing systems in CC1 rats may result from the divergence ofneural responses at both spinal may reflect CNS mechanisms of adaptation to a persistent pain and supraspinal levels. For instance, propriospinal connections condition. Thus, this CCI-induced increase in neural activity have been shown to extend from caudal spinal cord segments across extensive brain regions implicated in both sensory+Iis- up to the cervical enlargement (Yezierski et al., 1980; English criminative and affective-motivational dimensions of pain as et al., 1985), a region receiving fore limb somatosensory input. well as centrifugal modulation of pain may represent patterns Consistent with these anatomical connections, elevations of of supraspinal processing related to chronic pain conditions. spontaneous neural activity are observed across the entire sam- The use of the 2-DG functional activity mapping technique pled spinal cord lumbar segments (L,-L,) with little decrease at provides a three-dimensional view of the entire picture of brain both rostra1 (L,) and caudal (L,) extremes in CC1 rats (Price et nociceptive processes under a persistent pain condition. Such al., 1991; Mao et al., 1992a). Thus, it is likely that changes in an approach is likely to lead to improved understanding of CNS neural activity within the spinal cord may extend well beyond processing related to acute and chronic pain. For instance, al- the primary afferent terminations of the ligated sciatic nerve in though temporal properties of nociceptive neural activity (such CC1 rats and may even include the fore limb area. The diver- as neuronal discharge frequency, temporal summation) may or gence of spinal cord activity may, thereby, account for much of may not differ greatly between acute and chronic pain (Willis, the widespread activity in thalamic and cortical somatosensory 1985; Price, 1988), the spatial distribution of CNS nociceptive regions. processing in the latter appears to be distinguished by greater The activation of brain structures is not, however, restricted divergent activation of multiple brain regions. Furthermore, the to regions receiving input directly from known spinal cord as- unexpected and as yet unexplained activation of certain brain cending nociceptive pathways in CC1 rats. For example, the regions in CC1 rats such as the retrosplenial granular cortex and activation of the ventral posteromedial thalamic nucleus in CC1 caudate-putamen complex suggests that studies of the role of rats is unexpected, since this thalamic region receives trigemi- these brain regions in the neural processing of pain might pro- nothalamic but little, if any, spinothalamic tract input (Yen et vide new insights into the neurobiology of neuropathic pain and al., I99 1). Since no reliable increases in metabolic activity were pain in general. Thus, results derived from this functional (2- observed in the medullary trigeminal nuclei, the increased neu- DG) activity mapping study may serve as guidelines for future ral activity within the ventral posteromedial thalamic nucleus electrophysiological and anatomic studies to determine the (and possibly other brain areas) is likely to be related to the physiological nature of neurons activated in neuropathic pain divergence of excitatory activity within the brain itself (Willis, as well as to delineate the neural circuitry involving supraspinal 1985; Price, 1988). The extension of neural activity to brain nociceptive processing under chronic pain conditions. areas beyond those related to the anatomic territory of the in- jured sciatic nerve may account for pain radiation and for the occurrence of sensory abnormalities well beyond the territory References of original injury in neuropathic pain patients (Bonica, 1979; Ackermann RF, Finch DM, Babb TL, Engel J Jr (1984) Increased Price et al., 1989). Thus, it will be ofinterest to examine whether glucose metabolism during long duration recurrent inhibition of hip- similar phenomena can be seen, for instance, in the facial area pocampal pyramidal cells. 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