Nerve injury triggers time-dependent activation of the locus coeruleus, influencing spontaneous pain-like behavior in rats.

Pain Medicine Anesthesiology, V 141 • NO 1 July 2024 131 ediTOR’S PeRSPecTiVe What We Already Know about This Topic • Increasing evidence suggests the involvement of the noradrenergic locus coeruleus in the dynamic modulation of nociception • The role of neuronal projections from the locus coeruleus to the spinal cord and to the rostral anterior cingular cortex in the modulation of neuropathic pain over time is incompletely understood What This Article Tells us That Is New • In an experimental model of chronic constriction nerve injury in rats, a combination of genetic and histologic approaches revealed a biphasic time-dependent role for locus coeruleus neurons in modulating nociceptive responses • After 2 days of nerve injury, activation of locus coeruleus neurons projecting to spinal cord played a role in attenuating pain-like behavior while activation of locus coeruleus neurons projecting to the rostral anterior cingular cortex amplified nociceptive responses • After 30 days of nerve injury, only the projections from locus coeruleus neurons to the rostral anterior cingular cortex contributed to the modulation of pain-like behavior in this experimental model The locus coeruleus (LC) noradrenergic system is the main source of noradrenaline in the central nervous system, and it is a key brain area involved in pain plasticity.1–3 Numerous studies indicate that the LC is engaged by acute noxious stimuli or inflammation, promoting feedback inhibition of pain.4–6 However, recent studies suggest that the LC does not fulfill a uniform role in chronic pain, but rather, it changes dynamically through the activation of specific LC projections while others are silenced as pain Nerve Injury Triggers Time-dependent Activation of the Locus Coeruleus, Influencing Spontaneous Pain-like Behavior in Rats Irene Suárez-Pereira, Ph.D., Carolina López-Martín, M.Sc., Carmen Camarena-Delgado, Ph.D., Meritxell Llorca-Torralba, Ph.D., Francisco González-Saiz, M.D., Rocío Ruiz, Ph.D., Martiniano Santiago, Ph.D., Esther Berrocoso, Ph.D. Anesthesiology 2024; 141:131–50 aBSTRacT Background: Dynamic changes in neuronal activity and in noradrenergic locus coeruleus (LC) projections have been proposed during the transition from acute to chronic pain. Thus, the authors explored the cellular cFos activity of the LC and its projections in conjunction with spontaneous pain-like behavior in neuropathic rats. Methods: Tyrosine hydroxylase:Cre and wild-type Long–Evans rats, males and females, were subjected to chronic constriction injury (CCI) for 2 (shortterm, CCI-ST) or 30 days (long-term, CCI-LT), evaluating cFos and FluoroGold expression in the LC, and its projections to the spinal cord (SC) and rostral anterior cingulate cortex (rACC). These tests were carried out under basal conditions (unstimulated) and after noxious mechanical stimulation. LC activity was evaluated through chemogenetic and pharmacologic approaches, as were its projections, in association with spontaneous pain-like behaviors. Results: CCI-ST enhanced basal cFos expression in the LC and in its projection to the SC, which increased further after noxious stimulation. Similar basal activation was found in the neurons projecting to the rACC, although this was not modified by stimulation. Strong basal cFos expression was found in CCI-LT, specifically in the projection to the rACC, which was again not modified by stimulation. No cFos expression was found in the CCI-LT LCipsilateral (ipsi)/contralateral (contra)→SC. Chemogenetics showed that CCI-ST is associated with greater spontaneous pain-like behavior when the LCipsi is blocked, or by selectively blocking the LCipsi→SC projection. Activation of the LCipsi or LCipsi/contra→ SC dampened pain-like behavior. Moreover, Designer Receptor Exclusively Activated by Designer Drugs (DREADDs)–mediated inactivation of the CCI-ST LCipsi→rACC or CCI-LT LCipsi/contra→rACC pathway, or intra-rACC antagonism of α-adrenoreceptors, also dampens pain-like behavior. conclusions: In the short term, activation of the LC after CCI attenuates spontaneous pain-like behaviors via projections to the SC while increasing nociception via projections to the rACC. In the long term, only the projections from the LC to the rACC contribute to modulate pain-like behaviors in this model. (Anesthesiology 2024; 141:131–50) Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are available in both the HTML and PDF versions of this article. Links to the digital files are provided in the HTML text of this article on the Journal’s Web site (www.anesthesiology.org). Part of the work presented in this article has been presented as a poster at International Association for the Study of Pain, Toronto, Canada, September 19 through 23, 2022; and at European Pain Federation, Dublin, Ireland, April 27 through 30, 2022. I.S.P. and C.L.M. contributed equally to this article. Submitted for publication July 14, 2023. Accepted for publication March 26, 2024. Published online first on April 10, 2024. Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc., on behalf of the American Society of Anesthesiologists. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Anesthesiology 2024; 141:131–50. DOI: 10.1097/ALN.0000000000005006 The article processing charge was funded through the authors’ institution. 132 Anesthesiology 2024; 141:131–50 Suárez-Pereira et al. Pain Medicine transitions from acute to chronic, contributing to both analgesia and pain.7 Indeed, 2 days after nerve lesion, we find stronger basal activity in the LC ipsilateral to the side of lesion (LCipsi) than in the contralateral LC (LCcontra), as witnessed through the expression of cFos.8 Stronger cFos expression was also evident in the LCipsi neurons projecting to the spinal cord (SC), and similar strong cFos expression was evident after long-term nerve damage (30 days). However, in this latter case, the enhanced cFos expression was bilateral in the LC, and it was also evident in neurons projecting to the rostral anterior cingulate cortex (rACC).8 The expression of cFos is a common marker for recent cellular activity, reflecting cell activation induced by acute events.9 For example, acute stress or nociceptive stimulus increases cFos expression in the LC within 1.5 to 2 h of the insult.10–13 Therefore, the physiologic significance of an increase in cFos 2 days or longer after nerve injury is intriguing. Furthermore, it is unclear whether the critical circuits for the normal processing of acute noxious inputs are disrupted by nerve damage. It has been proposed that spontaneous strong basal cFos expression might possibly reflect the participation of the LC in processing spontaneous pain. Sensitivity to evoked nociceptive stimuli is enhanced in patients experiencing neuropathic pain, as it is to spontaneous pain. However, while paroxysmal or ongoing spontaneous pain is consistently identified as a major clinical complaint (see review14), the underlying mechanisms remain unclear. To further explore this issue, we assessed whether constriction damage to the sciatic nerve alters the activity of LC cells at early and late time points after lesion, both under basal conditions (unstimulated) and after the application of a noxious mechanical stimulus. Furthermore, as the LC is apparently composed of modules that might produce targeted neuromodulation,2,3,15 we assessed the role of the LC as a whole, and both the LCipsi or LCcontra to the lesion. In addition, we assessed the temporal modulation of pain by examining the descending LC pathway to the SC, as noradrenaline released in the SC suppresses pain-like behaviors.6,16 We also studied the rACC, an area connected to the LC and implicated in neuropathic pain.17 These studies were prompted by findings associating bilateral increases in noradrenaline in the prefrontal cortex with long-term neuropathy,18 suggesting overactivation of the noradrenergic system in prolonged pain conditions. Consequently, cFos expression and FluoroGold (FG; Fluorochrome, USA) staining were explored in the LC of Long–Evans rats subjected to chronic constriction injury (CCI) for 2 days (short-term, CCI-ST) or 30 days (long-term, CCI-LT), analyzing how this responded to noxious mechanical stimuli. Chemogenetics were used to manipulate LC activity and that of the specific LC pathways to the SC or rACC, particularly in relation to spontaneous pain-like behaviors.8,18 In addition, pharmacologic studies were performed to evaluate the involvement of rACC adrenergic receptors in spontaneous pain-like behaviors. Materials and Methods Animals Transgenic rats were obtained by crossing hemizygous tyrosine hydroxylase–Cre (TH:Cre) transgenic and wild-type (WT) Long–Evans rats at the University of Cádiz (Cádiz, Spain) from founders provided by the Rat Resource and Research Center (Columbia, Missouri; donated by K. Deisseroth, Ph.D.), or WT Long–Evans rats produced in house or obtained commercially from Charles River (Italy) and from Janvier Laboratories (France). Experiments were carried out on male TH:Cre transgenic or on male and female WT Long–Evans rats (300 to 450 g), all maintained at the University of Cadiz under standard laboratory conditions (22ºC, 12-h light/dark cycle, lights on 8:00 am, food and water ad libitum). All animal handling and procedures were carried out in accordance with the European Commission (Brussels, Belgium) directive (2010/63/ EU) and Spanish law (RD 53/2013) regulating animal Irene Suárez-Pereira, Ph.D.: Biomedical Research Networking Center for Mental Health (CIBERSAM), Institute of Health Carlos III (ISCIII), Madrid, Spain; Neuropsychopharmacology and Psychobiology Research Group, Department of Neuroscience, Faculty of Medicine, University of Cádiz, Cádiz, Spain; Biomedical Research and Innovation Institute of Cádiz (INIBICA), Puerta del Mar University Hospital, Cádiz, Spain. Carolina López-Martín, M.Sc.: Biomedical Research Networking Center for Mental Health (CIBERSAM), Institute of Health Carlos III (ISCIII), Madrid, Spain; Neuropsychopharmacology and Psychobiology Research Group, Department of Neuroscience, Faculty of Medicine, University of Cádiz, Cádiz, Spain; Biomedical Research and Innovation Institute of Cádiz (INIBICA), Puerta del Mar University Hospital, Cádiz, Spain. Carmen Camarena-Delgado, Ph.D.: Biomedical Research and Innovation Institute of Cádiz (INIBICA), Puerta del Mar University Hospital, Cádiz, Spain; IRCCS Humanitas Research Hospital, Milan, Italy; Institute of Neuroscience (IN-CNR), National Research Council of Italy, Milan, Italy. Meritxell Llorca-Torralba, Ph.D.: Biomedical Research Networking Center for Mental Health (CIBERSAM), Institute of Health Carlos III (ISCIII), Madrid, Spain; Biomedical Research and Innovation Institute of Cádiz (INIBICA), Puerta del Mar University Hospital, Cádiz, Spain; Neuropsychopharmacology and Psychobiology Research Group, Department of Cell Biology and Histology, University of Cádiz, Cádiz, Spain. Francisco González-Saiz, M.D.: Biomedical Research Networking Center for Mental Health (CIBERSAM), Institute of Health Carlos III (ISCIII), Madrid, Spain; Department of Neuroscience, Faculty of Medicine, University of Cádiz, Cádiz, Spain; Community Mental Health Unit of Villamartin, University Hospital of Jerez de la Frontera, Cádiz, Spain. Rocío Ruiz, Ph.D.: Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Institute of Biomedicine of Sevilla (IBiS) - University Hospital Virgen del Rocío/CSIC/University of Sevilla, Sevilla, Spain. Martiniano Santiago, Ph.D.: Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Institute of Biomedicine of Sevilla (IBiS) - University Hospital Virgen del Rocío/CSIC/University of Sevilla, Sevilla, Spain. Esther Berrocoso, Ph.D.: Biomedical Research Networking Center for Mental Health (CIBERSAM), Institute of Health Carlos III (ISCIII), Madrid, Spain; Neuropsychopharmacology and Psychobiology Research Group, Department of Neuroscience, Faculty of Medicine, University of Cádiz, Cádiz, Spain; Biomedical Research and Innovation Institute of Cádiz (INIBICA), Puerta del Mar University Hospital, Cádiz, Spain. Anesthesiology 2024; 141:131–50 133Suárez-Pereira et al. Pain Changes Locus Coeruleus Activity research. Furthermore, all experimental protocols were approved by the Committee for Animal Experimentation at the University of Cadiz. The study was conducted and the data reported in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.19 Neuropathic Pain Model CCI of the sciatic nerve was used as a model of neuropathic pain.20,21 Rats were anesthetized with isoflurane (induction with 3 to 4% and maintenance with 1.5 to 2.5%), and the left sciatic nerve was then exposed at the mid-thigh level, proximal to the sciatic trifurcation. Four chrome gut (4–0) ligatures were tied loosely around the nerve, separated by 1.0 to 1.5 mm so as not to compromise the vascular supply. The overlying layers of muscle were then closed with 4–0 nonabsorbable silk thread, and the skin was sutured with 2–0 silk thread. Sham operations were performed in the same manner but without nerve ligation. The experimental procedures were carried out 2 (ST) and 30 (LT) days after CCI. Experimental Design and Groups All animals used in this study were initially randomized into groups that were assigned different treatments. In the experiments involving female rats, they were initially assigned randomly to the different groups, and subsequently, their hormonal status was assessed by examining vaginal smears while they were under anesthesia before applying noxious hind paw stimulation.22 All stages of the estrous cycle were represented in each group, as determined by vaginal cytology. Before the behavioral experiments, all the animals underwent initial training and were habituated to both the experimental room and handling. All the experiments and the analysis of the data were performed blind to the group and treatment assignments. Furthermore, power analyses were performed with G*Power23 (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) to determine the minimum sample size required for the experiments in order to detect a significant effect using two-tailed t tests at an α level = 0.05, power = 0.80, and effect size based on similar published studies of cFos expression in the LC8 and paw flinches in spontaneous pain tests.24 Detailed sample size tables for each figure (figs. 1 through 7) are shown in Supplemental Digital Content 2 (Supplemental Tables 1 through 7, https://links.lww.com/ ALN/D532). Some experimental units or data points were excluded from the experiment or statistical analysis if histologic verification indicated retrograde tracer injection or the site of catheter/cannula placement was inaccurate, or if there was no Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) expression in the LC. Furthermore, a Grubbs’s statistical test was employed to detect outliers within the dataset. These exclusion criteria were predefined before conducting the analysis. Schemes representing the experimental procedure are shown in each figure. In general, CCI was induced in male and female adult Long–Evans rats at two different time points after nerve injury: 2 days (CCI-ST) and 30 days (CCI-LT). As control groups, rats were subjected to a sham surgical procedure (sham group) or left unoperated (naive group). To explore changes in LC activity and its projections over time, and with respect to noxious stimulation, CCI and control animals (sham or naive) were initially randomized into the following experimental groups: Stim(–), unstimulated; or Stim(+), stimulated. Alternatively, for the chemogenetic modulation of global noradrenergic LC neuron activity or of specific noradrenergic LC projections, adeno-associated virus (AAV)–Gi–mCherry (DREADDs for chemogenetic inhibition), AAV-Gs-mCherry (DREADDs for chemogenetic activation), or AAV-mCherry (control virus) was injected into the LCipsi and/or LCcontra of TH:Cre rats. Clozapine-N-oxide (CNO, designer ligand) was administered via intraperitoneal injection, or locally (intrathecal or intra-rACC) via microinjection into the target site 20 min before performing the behavioral tests. In all these experiments, a group administered the vehicle alone was included as a control (saline [Sal] group). Finally, for the pharmacologic blockade of adrenoreceptors in the rACC, a cannula was implanted bilaterally into the rACC of all experimental rats, and the drugs were administered 30 or 20 min before performing the behavioral tests (see section “Drugs”). Similarly, a group administered with the vehicle alone was included as a control (saline group) in these experiments. Noxious Hind Paw Stimulation The hind paw of rats was stimulated by applying pinched pressure to the ipsilateral (left) hind paw (600 g, 5 min) using a Analgesy-Meter (Ugo Basile, Italy), defined as a mechanical noxious stimulation, to enhance cFos expression. This stimulus was administered under mild anesthesia with sodium pentobarbital (50 mg/kg, intraperitoneally).26 Two hours after noxious stimulation, all the rats were anesthetized deeply with an overdose of sodium pentobarbital, and they received a transcardial perfusion with saline, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), after which their SC and brain were removed.27 DREADDs Approaches DREADDs approaches were carried out as indicated previously, whereby strong colocalization between mCherry and noradrenergic neurons was found in the LC of TH:Cre rats. For methodologic validation of the DREADDs approach, see Llorca-Torralba et al.,8,28 as well as Supplemental Figures 1 and 2 (Supplemental Digital Content 1, https://links. lww.com/ALN/D531). Stereotaxic Surgery. The following vectors were used in these studies: DREADDs (Virus Vector Core, Gene Therapy Center Vector Core at the University of North Carolina, USA); AAV2/hSyn-DIO-hM4D(Gi)-mCherry 134 Anesthesiology 2024; 141:131–50 Suárez-Pereira et al. Pain Medicine Fig. 1. Study of locus coeruleus (lC) activation over time after neuropathy in unstimulated and stimulated male rats. (A) Scheme representing the experimental procedure. The paw ipsilateral to the injured one was stimulated in anesthetized rats by paw compression using an Analgesy-Meter (ugo Basile, Italy; 600 g, 5 min). The terms ipsilateral and contralateral are relative to the chronic constriction injury (CCI). (B) Representative immunohistochemistry of cFos labeled neurons in the ipsilateral central lC (–9.72 to –9.96 from bregma25) in conditions of no stimulation (Stim[–], black borders) or stimulation (Stim[+], red borders) in naive, sham–short-term (ST), sham–long-term (lT), CCI-ST, and CCI-lT wild-type rats (scale bar, 100 μm: cFos, red; dopamine beta-hydroxylase [DBH], green). The white arrows illustrate examples of DBH-positive cells with cFos-positive nuclei in lC. (C) Experimental timeline of neuropathy and ST (2 days) quantification of the cFos-positive neurons in the central ipsilateral (I) and contralateral (C) lC of naive, sham-ST, and CCI-ST rats without stimulation (Stim[–]) or after applying a noxious stimulus (Stim[+]). The mean + SD neurons per slice are shown (each point represents an individual rat; n = 4–6 animals/group): **P < 0.01, ***P < 0.001 vs. Stim(–); +P < 0.05, ++P < 0.01, +++P < 0.001 vs. lCipsi; #P < 0.05, ###P < 0.001 vs. naive or sham-ST (three-way ANOVA with Tukey post hoc test between naive and sham-ST groups, and between sham-ST and CCI-ST groups). (D) Experimental timeline of neuropathy and lT (30 days) quantification of the cFos-positive neurons in the central ipsilateral (I) and contralateral (C) lC of sham-lT and CCI-lT rats without stimulation (Stim[–]) or with noxious stimulation (Stim[+]). The mean + SD neurons per slice are shown (each point represents an individual rat; n = 6 animals/group): ***P < 0.001 vs. Stim(–); ##P < 0.01, ###P < 0.001 vs. sham-lT (three-way ANOVA with Tukey post hoc test). Anesthesiology 2024; 141:131–50 135Suárez-Pereira et al. Pain Changes Locus Coeruleus Activity Fig. 2. Study of the activation of the locus coeruleus (lC)→spinal cord (SC) pathway over time after neuropathy in unstimulated and stimulated male rats. (A) Scheme of the retrograde Fluoro-Gold (FG; Fluorochrome, uSA) tracer strategy and representative images (scale bar, 500 μm) of FG administration site and lC neurons that project to the ipsilateral spinal cord (SC) in wild-type rats. Distribution of FG labeling in ipsilateral (ipsi) and contralateral (contra) lC neurons (mean + SD of the number of neurons per slice; each point represents an individual rat; n = 4 animals: Student’s t test). (B) Representative immunofluorescence of the central lC (–9.72 to –9.96 from bregma25) showing the DBH, cFos, and FG expression, as well as a merged image. The white arrows illustrate examples of cFos-positive cells, and the inset shows an example of cFos/FG-labeled DBH-positive neurons (scale bar, 100 μm: DBH, green; cFos, red; FG, yellow). (C) Experimental timeline of neuropathy and short-term (ST, 2 days) quantification of the cFos/FG labeled neurons in the central ipsilateral (I) and contralateral (C) lC of sham-ST and CCI-ST rats without stimulation (Stim[–]) or with noxious stimulation (Stim[+]). The mean + SD neurons per slice are shown (each point represents an individual rat; n = 6 animals/group): *P < 0.05, **P < 0.01 vs. Stim(–); +P < 0.05, ++P < 0.01 vs. lCipsi; #P < 0.05 vs. sham-ST (three-way ANOVA with Tukey post hoc test). (D) Experimental timeline of neuropathy and long-term (lT, 30 days) quantification of the cFos/FG-labeled neurons in the central ipsilateral (I) and contralateral (C) lC of sham-lT and CCI-lT rats without stimulation (Stim[–]) or with noxious stimulation (Stim[+]). The mean + SD neurons per slice are shown (each point represents an individual rat; n = 6 animals/group): non-significant differences (three-way ANOVA with Tukey post hoc test). CCI, chronic constriction injury; DBH, dopamine beta-hydroxylase; 4V, fourth ventricle. 136 Anesthesiology 2024; 141:131–50 Suárez-Pereira et al. Pain Medicine Fig. 3. Study of the activation of the locus coeruleus (lC)→rostral anterior cingulate cortex (rACC) pathway over time after neuropathy in unstimulated and stimulated male rats. (A) Cartoon of the retrograde Fluoro-Gold (FG; Fluorochrome, uSA) tracer strategy targeting lC neurons that project to the bilateral rACC and representative images (scale bar, 500 μm) of FG administration site and lC neurons that project bilaterally to the rACC in wild-type rats. The distribution of FG label in the ipsilateral (ipsi) and contralateral (contra) lC neurons was evaluated (mean + SD of the number of neurons per slice; each point represents an individual rat; n = 4 animals: Student’s t test). (B) Representative immunofluorescence of the central lC (–9.72 to –9.96 from bregma25) showing the DBH, cFos, and FG expression, as well as a merged image. The white arrows illustrate examples of cFos-positive cells, and the inset shows an example of cFos/FG-labeled DBH-positive neurons (scale bar, 100 μm: DBH, green; cFos, red; FG, yellow). (C) Experimental timeline of neuropathy and short-term (ST, 2 days) quantification of the cFos/FG-labeled neurons of the central ipsilateral (I) and contralateral (C) lC of sham-ST and CCI-ST rats without stimulation (Stim[–]) or after applying a noxious stimulus (Stim[+]). The mean + SD neurons per slice are shown (each point represents an individual rat; n = 5 animals/ group): **P < 0.01, ***P < 0.001 vs. (Stim[–]); ++P < 0.01, +++P < 0.001 vs. lCipsi; ##P < 0.01, ###P < 0.001 vs. sham-ST (three-way ANOVA with Tukey post hoc test). (D) Experimental timeline of neuropathy and long-term (lT, 30 days) quantification of the cFos/FG-labeled neurons of the central ipsilateral (I) and contralateral (C) lC of sham-lT and CCI-lT rats without stimulation (Stim[–]) or with noxious stimulation (Stim[+]). The mean + SD neurons per slice are shown (each point represents an individual rat; n = 5 or 6 animals/group): **P < 0.01, ***P < 0.001 vs. (Stim[–]); +++P < 0.001 vs. lCipsi; ##P < 0.01, ###P < 0.001 vs. sham-lT (three-way ANOVA with Tukey post hoc test). CCI, chronic constriction injury; DBH, dopamine beta-hydroxylase; 4V, fourth ventricle. Anesthesiology 2024; 141:131–50 137Suárez-Pereira et al. Pain Changes Locus Coeruleus Activity (hM4D[Gi]-DREADD) inhibitor virus, titer 3 × 1012 vg/ ml, 1.4 µl/ LC; AAV2/hSyn-DIO-rM3D(Gs)-mCherry (rM3D[Gs]-DREADD) activator virus, titer 3 × 1012 vg/ ml, 1.4 µl/LC; and AAV2/hSyn-DIO-mCherry (controlDREADD), titer 5.6 × 1012 vg/ml, 1.4 µl/LC. This control vector contained a mCherry reporter protein without the DREADD reporter. DREADDs vectors were injected into the LC of TH:Cre rats anesthetized (intraperitoneally) with ketamine (100 mg/kg, Richter Pharma, Spain) and xylazine (20 mg/ Fig. 4. Effect of chemogenetic inhibition and activation of the ipsilateral and contralateral locus coeruleus (lC) on spontaneous pain-like behavior at different times after nerve injury. (A) Scheme of the noradrenergic lC inhibition or activation using the DREADD strategy in TH:Cre male rats and representative immunofluorescence image (scale bar, 10 μm) of mCherry expression in the noradrenergic lC neurons (DBH, green; mCherry, red). (B) Experimental timeline of neuropathy and quantification of the short-term (ST, 2 days) and long-term (lT, 30 days) ipsilateral paw flinches of sham and CCI rats in the spontaneous pain test after ipsilateral lC (lCipsi) or contralateral lC (lCcontra) noradrenergic inhibition by CNO (1 mg/kg intraperitoneal, mean + SD, each point represents an individual rat; n = 4–6 animals/group: *P < 0.05, ***P < 0.001, vs. sham-Sal; ++P < 0.01 vs. CCI-Sal, three-way repeated measures ANOVA with Tukey post hoc test). (C) Experimental timeline of neuropathy and long-term (lT, 30 days) quantification of the ipsilateral paw flinches of sham and CCI rats in the spontaneous pain test after ipsilateral lC (lCipsi) or contralateral lC (lCcontra) noradrenergic activation by CNO (1 mg/kg intraperitoneal, mean + SD, each point represents an individual rat; n = 8 or 9 animals/group: ***P < 0.001 vs. sham-Sal; +++P < 0.001 vs. CCI-Sal, Kruskal–Wallis test followed by Mann– Whitney u test). AAV, adeno-associated virus; CCI, chronic constriction injury; CNO, clozapine N-oxide; DBH, dopamine beta-hydroxylase; DREADD, Designer Receptor Exclusively Activated by Designer Drug; Sal, saline; SP, spontaneous pain-like test; TH, tyrosine hydroxylase. 138 Anesthesiology 2024; 141:131–50 Suárez-Pereira et al. Pain Medicine Fig. 5. Effect of chemogenetic inhibition and activation of ipsilateral and contralateral locus coeruleus (lC)→spinal cord (SC) pathway on spontaneous pain-like behavior over time after nerve injury. (A) Experimental timeline of lCipsi-SC or lCcontra-SC pathway inhibition at shortterm (ST, 2 days) and long-term (lT, 30 days) after neuropathy using the DREADD strategy in male TH:Cre rats. Quantification of the ipsilateral paw flinches of sham and CCI rats in the spontaneous pain-like test after ipsilateral lC (lCipsi) or contralateral lC (lCcontra) noradrenergic inhibition by CNO (3 μM intrathecal, mean + SD, each point represents an individual rat; n = 4 animals/group: *P < 0.05, ***P < 0.001, vs. sham-Sal; +P < 0.05 vs. CCI-Sal, three-way repeated measures ANOVA with Tukey post hoc test). (B) Experimental timeline of lCipsi→SC or lCcontra→SC pathway activation in the short-term (ST, 2 days) and long-term (lT, 30 days) after neuropathy using the DREADD strategy in TH:Cre rats. Quantification of the ipsilateral paw flinches of sham and CCI rats in the spontaneous pain test after ipsilateral lC (lCipsi) or contralateral lC (lCcontra) noradrenergic activation by CNO (3 μM intrathecal, mean + SD, each point represents an individual rat; n = 3–7 animals/ group: **P < 0.01, ***P < 0.001 vs. sham-Sal; ++P < 0.01, +++P < 0.001 vs. CCI-Sal, three-way repeated measures ANOVA with (Continued ) Anesthesiology 2024; 141:131–50 139Suárez-Pereira et al. Pain Changes Locus Coeruleus Activity kg, Calier Laboratory, S.A., Spain), which were placed into a stereotaxic frame with the skull level along both the anteroposterior (AP) and medio-lateral (ML) axis for precise targeting. Cranial windows were opened above the LC coordinates (AP, –3.2 mm; ML, ±1.3 mm; dorso-ventral [DV], 6.2 mm), with the head oriented at a 15-degree angle to the horizontal plane. After injection, the wound was cleaned with 0.9% saline and disinfected with iodine polyvidone, and the skin was sutured with 4-0 nonabsorbable silk thread. To enhance vector expression, the behavioral studies were carried out 3 weeks after the injections. Viral Expression. Animals were perfused with PFA (4%) at the end of the experiments, and their brains were collected and processed28,29 to verify the viral injection site and the expression of mCherry in the LC by immunofluorescence. All the LC sections (30 µm) were incubated for 48 h at 4ºC with an antibody against red fluorescent protein (1:500 [5F8], Chromotek, Germany) and mouse antidopamine beta hydroxylase (DBH, 1:1,000, Merck Chemicals & Life Science S.A., Spain). Subsequently, the antibodies were detected with a biotinylated donkey antirat antibody (1:200, Jackson ImmunoResearch Europe, United Kingdom), which was visualized with Alexa Fluor 568 streptavidin or a donkey antimouse Alexa Fluor 488 (1:1,000, Invitrogen, USA). The sections were then washed and coverslipped in fluoro-gel aqueous mounting medium, and the images were acquired on a Zeiss LSM 900 Confocal microscope with Airyscan 2 (Carl Zeiss Microscopy GmbH, Germany). The selective expression of DREADD was also assessed in the A5 noradrenergic nucleus and ventral tegmental area, the latter probed with a rabbit anti-TH primary antibody (1:1,000) that was visualized with a donkey antirabbit Alexa Fluor 488 (Invitrogen, USA). Tracer Approach FG was used as a retrograde tracer (4%/0.4 µl) for the rACC or SC. For FG injections at the lumbar level of the SC,8 rats were anesthetized with sodium pentobarbital (50 mg/ kg, intraperitonally, Vetoquinol S.A., Spain), and local analgesia was administered subcutaneously with carprophen (10 mg/kg, Zoetis, Spain) and bupivacaine (2 mg/kg, B. Braun Medical S.A., Spain). Additional doses of pentobarbital were administered as necessary. Rats were placed in a stereotaxic frame, and a laminectomy was performed at the level of the T12–L2 vertebrae to expose the L4–L6 segments of the SC. The dura was then removed, and FG was administered at four sites along the ipsilateral L4–L6 dorsal horn of the SC (SCipsi). After FG injection, the skin was closed with staples, and the wound was cleaned with 0.9% saline and disinfected with iodine polyvidone. In another set of experiments, the FG was microinjected unilateral or bilaterally into the rACC according to the same stereotaxic surgery performed in the DREADD experiments (see section “DREADDs Approaches”).8 The target coordinates for rACC were AP + 3.0 mm, ML ± 1.0 mm, and DV 1.2 mm, with the head oriented at a 0-degree angle to the horizontal plane. To enhance tracer expression, the neuroanatomical studies were carried out 4 days after the injections, and the location of the FG injection site was verified to be within the SC and rACC in all the animals studied. Sequential SC and rACC (40 µm) coronal sections were visualized directly on an Olympus BX60 fluorescence microscope equipped with an Olympus DP74 camera (Spain). After verification, the animals in which the FG injection was not appropriately located in the target area were excluded from the analysis. rACC Cannula Implantation For intra-rACC drug administration, cannulae were implanted by stereotaxic surgery.8 The rats were anesthetized with an intraperitoneal injection of ketamine (100 mg/ kg) and xylazine (20 mg/kg), and then placed in a stereotaxic frame. The cannulae were implanted bilaterally into the rACC (AP + 3.0 mm, ML ± 1.0 mm, and DV 1.2 mm) with the head oriented at a 0º angle to the horizontal plane. The cannulae were fixed in place with dental cement and four anchor screws, and maintained closed by inserting a stainless steel wire until the test session. After cannula implantation, the rats were allowed to recover for 1 week before the behavioral tests began. To verify cannula placement, 0.5 μl Pontamine Sky Blue (Sigma–Aldrich, Spain) was injected into the intra-rACC just before the animals were sacrificed. Sequential coronal sections (40 µm) of the rACC were ultimately obtained on a Sliding Microtome (Microm HM 450, Fisher Scientific SL, Spain) coupled to BFS-MP freezing stage (Physitemp Instruments, USA). After verification, the animals in which the cannula was not appropriately located in the target area were excluded from the analysis (Supplemental Figures 3, 4, and 5, Supplemental Digital Content 1, https://links.lww.com/ALN/D531). Intrathecal Catheter Installation For intrathecal drug injection, a PE-10 catheter (0.61 mm diameter) was implanted into the lumbar SC under isoflurane anesthesia.30 An incision was made in the skin, and a cannula (20-gauge, 0.9 × 40 mm) was introduced Fig. 5. (Continued) Tukey post hoc test). (C) Experimental timeline of neuropathy and long-term (lT, 30 days) quantification of the ipsilateral paw flinches of sham and CCI rats in the spontaneous pain test after clonidine (20 μg intrathecal) administration (mean + SD, each point represents an individual rat; n = 5 or 6 animals/group: **P < 0.01 vs. sham-Sal; ++P < 0.01 vs. CCI-Sal, Kruskal–Wallis test followed by Mann–Whitney u test). AAV, adeno-associated virus; CCI, chronic constriction injury; Cloni, clonidine; CNO, clozapine N-oxide; DREADD, Designer Receptor Exclusively Activated by Designer Drug; Sal, saline; SP, spontaneous pain; TH, tyrosine hydroxylase. 146 Anesthesiology 2024; 141:131–50 Suárez-Pereira et al. Pain Medicine SC, which enhances noradrenaline release and in turn contributes to the blockade of ascending nociceptive inputs.6,8,16 Another relevant pathway is that of the LC→rACC, as lesion47,48 or injection of a brain-derived neurotrophic factor (BDNF)–tropomyosin receptor kinase B (TrkB) antagonist into the rACC completely blocks the conditioned place preference induced by the α-2 adrenoreceptor agonist Cloni.48,49 Hence, the ACC appears to be necessary to regulate spontaneous pain. In CCI-ST animals, there is also a strong and lateralized activity in this region, such that chemogenetic blockade of the LCipsi→rACC dampens spontaneous pain, yet no effect is found when the LCcontra is manipulated. Overall, this suggests that lateralized cFos expression is related to spontaneous pain-like behavior, albeit in opposing directions depending on the projection. In long-term neuropathy, lateralized cFos expression is lost, and while strong expression is found in the LC globally,8,50 this is not the case for cells projecting to the SC. DREADDs-mediated blockade of either the LCipsi or the LCcontra globally or of the specific projection to the SC does not modify spontaneous pain-like behaviors. This response is consistent with the belief that the LC does not induce endogenous analgesia through the descending projection to the SC when neuropathy becomes long-term.8,51 There is also a robust bilateral increase in cFos at the ACC level in CCI-LT, and there is a reduction in spontaneous painlike behaviors when the LCipsi/contra is blocked. Hence, the LC→rACC projection appears to fulfill a pronociceptive role in both shortand long-term pain. This aligns with previous findings showing that chemogenetic activation of the LC→Prefrontal cortex projection exacerbates spontaneous pain, produces aversion, and increases anxiety-like behavior in neuropathic pain animals.16 On the other hand, when the LC is chemogenetically activated globally, or simply its projection to the SC, there is significant relief of pain-related behaviors.8 These chemogenetic studies are consistent with the pharmacologic effects of the acute intrathecal administration of the selective noradrenergic reuptake inhibitor reboxetine, which relieves evoked pain and induces conditioned place preference. However, acute systemic reboxetine administration also relieves evoked pain, although it is aversive in the conditioned place preference paradigm.52 Hence, the beneficial effect of noradrenaline at the SC is probably counteracted by the activation of other supraspinal noradrenergic projections. Our data also suggest that the hyperactivity of the LC→rACC projection that induces spontaneous pain-like behavior is mediated by activation of α-1 adrenoreceptors over the short term, and by α-1 and α-2 adrenoreceptors in the long term. Previous findings showed microinjection of the α-2 adrenoreceptors agonist Cloni into the ACC alleviates spontaneous pain-like behavior at day 7 but not at 14 days after nerve injury in common peroneal nerve– ligated mice.48 These data, in a different animal model, suggest an analgesic effect of ACC α-2 adrenoreceptor activation, which contrasts with our findings, although it is consistent with the dynamic regulation of cingulate αadrenoreceptors over time after nerve injury. We also explored cFos levels after mechanical noxious stimulation of a hind paw. In naive animals, cFos was only weakly expressed, but it increased bilaterally and uniformly in the LC when noxious stimulation was applied to one hind paw, consistent with previous data.11,13 Sham animals behave like naive animals in the long term, which may indicate satisfactory resolution of the damage produced by the sham intervention. Findings from naive and sham-LT animals suggest that bilateral activation of the LC during acute unilateral hind paw pain could partly reflect the modulation of nociceptive processing in the SC dorsal horn. However, this is unlikely because this pathway is not endogenously activated when FG/cFos colabeling of neurons is explored in sham-LT animals. Furthermore, chemogenetic inhibition of LC-noradrenergic neurons or intra-LC lidocaine administration did not modify sensorial responses in sham-LT animals.2,8,24,53 Another possibility is that this bilateral LC activation is related to other nonsensorial aspects of pain, such as the inherent stress associated with the pain experience itself.7,54 In this sense, restraint stress increases anxiety behavior and cFos immunoreactivity in LC neurons, and the chemogenetic inhibition of LC-noradrenergic neurons prevents stress-induced anxiety.40 Therefore, LC activity appears to have a minor influence on evoked hypersensitivity in these conditions, which could be related to a stressrelated response. In sham-ST animals, the lateralized basal cFos expression is further enhanced by noxious stimulation of the hind paw. Interestingly, similar data were obtained from the LCipsi of CCI-ST rats, although cFos expression was significantly stronger, suggesting that the LC responses at this point of neuropathy resemble other forms of short-term injury, yet they are more intense. Furthermore, cFos expression in the CCI-ST LCcontra did not differ significantly from that in the LCcontra of sham animals, and it did not change upon noxious stimulation, perhaps suggesting that the LCcontra had already reached its maximum possible response. These data are consistent with the significant increase in hypersensitivity to a cold stimulus in CCI-ST animals after chemogenetic inhibition of the LCipsi.8 Furthermore, we previously found that pharmacologic blockade with lidocaine significantly increased the nociceptive threshold of Sprague–Dawley rats.24 As no effect was found when blocking the LCcontra,8,24 endogenous LC-driven analgesia in the short term under evoked conditions appears to be mediated through the LCipsi. A very different profile is found in the long term after neuropathy, whereby the baseline increase in cFos expression is not altered by noxious stimuli, either globally in the LC or in the specific projections to the SC or rACC. This is consistent with earlier data showing that 11 days of nerve injury attenuated restraint-induced global LC cFos expression,55 suggesting that long-term nerve injury might Anesthesiology 2024; 141:131–50 147Suárez-Pereira et al. Pain Changes Locus Coeruleus Activity impair the ordinary cellular response of the LC to an acute stimulus. This is again in line with reduced LC-mediated descending inhibitory controls or diminished analgesic efficacy of drugs targeting LC pathways described previously.56–60 Accordingly, chemogenetic blockade of the LCipsi or LCcontra at CCI-LT does not modify the response to acetone,8 suggesting that this increase in cFos is not related to the sensory dimension of pain. However, blockade of the LCcontra with lidocaine elevates the nociceptive threshold in CCI-LT Sprague–Dawley rats.24 Furthermore, enhanced LC electrophysiologic responses are produced by mechanical stimulation of the nerve-injured paw at CCI-LT, predominantly mediated by the LCcontra.61 These findings align with the responses of Sprague–Dawley rats to bilateral microinjections of lidocaine into the LC, which completely reverse the behavioral signs of neuropathy 2 weeks after nerve injury.62 Overall, the LC seems to fulfill distinct roles depending on the rat strain, with a lateralized pronociceptive activity of the LC associated with long-term neuropathy in Sprague–Dawley but not Long–Evans rats. These discrepancies may reflect the reported strain differences in the noradrenergic pathways of these animals.63–66 The new data presented here suggest that bilateral LC or LC→SC projections fulfill an analgesic role in spontaneous and evoked conditions, mainly due to the activation of the LCipsi, although activation of the LCipsi→rACC pathway partially counteracts endogenous analgesia in response to spontaneous pain. In the long term, cFos is expressed strongly on a global level in response to pain, and in the rACC, yet it is no longer lateralized and there is no activation in the SC. Importantly, cFos expression is not modified by nociceptive stimulation after long-term pain. At this time point, the analgesic contribution of the LC is lost, and the activity of the LCipsi→rACC pathway in promoting spontaneous pain predominates. Anticipating forthcoming electrophysiologic studies to complement these experiments, specific LC projections to the SC or rACC appear to have different behavioral effects depending on the time that has passed since the injury. Moreover, other projection areas like the dorsal reticular nucleus24 or insular cortex67 could also potentially contribute to the spontaneous pain mediated by the LC and warrant further exploration. Future studies utilizing the targeted recombination of active populations (TRAP) approach68–70 could provide insights as to whether distinct subpopulations within the LC are recruited for spontaneous pain, and help elucidate their physiologic and molecular characteristics. Finally, although our study did not reveal differences between male and female rats, it is crucial to include both sexes in such research to comprehensively understand any sexual dimorphism or common behaviors related to these phenomena. Acknowledgments The authors are very grateful to Jose Antonio Garcia Partida, M.Sc., University of Cádiz (Cádiz, Spain), and Elena Marín Álvarez, Higher Technician, University of Cádiz (Cádiz, Spain) for their excellent technical assistance. They also acknowledge the assistance of the Central Services of Scientific and Technological Research, Health Sciences and Animal Research at the University of Cádiz. Research Support This study was supported by grants PID2022-142785OB-I00 and PDC2022-133987-100 funded by MCIN/AEI/ 10.13039/501100011033 and, as appropriate, by “ERDF A way of making Europe,” by the European Union and European Union NextGenerationEU/PRTR respectively, by Andalusian Regional Ministry of Health (Spain) (PI0134-2018; No. P20-00958), by the Regional Operational Program for Andalucía FEDER, by the Biomedical Research and Innovation Institute of Cádiz (INIBICA, Spain) (INC09), by the Andalusian Regional Ministry of Economy, Innovation, Science and Employment (Spain) (CTS-510), and by the Biomedical Research Networking Center for Mental Health (CIBERSAM, Spain) (CB07/09/0033). The figures were generated with BioRender.com (BioRender, Canada). Competing Interests The authors declare no competing interests. Correspondence Address correspondence to Dr. Berrocoso: Neuropsychopharmacology and Psychobiology Research Group, Department of Neuroscience, Faculty of Medicine, University of Cádiz, 11003 Cádiz, Spain. esther.berr[email protected] Supplemental Digital Content Supplemental Digital Content 1: Supplemental Figures, https://links.lww.com/ALN/D531 Supplemental Digital Content 2: Sample Size Tables 1 through 7, https://links.lww.com/ALN/D532 Supplemental Digital Content 3: Statistical Tables 8 through 14, https://links.lww.com/ALN/D533 Supplemental Digital Content 4: Table 15: HPLC Results, https://links.lww.com/ALN/D534 References 1. Llorca-Torralba M, Borges G, Neto F, Mico JA, Berrocoso E: Noradrenergic locus coeruleus pathways in pain modulation. Neuroscience 2016; 338:93–113 2. Schwarz LA, Luo L: Organization of the locus coeruleus-norepinephrine system. Curr Biol 2015; 25:R1051–6 148 Anesthesiology 2024; 141:131–50 Suárez-Pereira et al. Pain Medicine 3. 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