Activation of the Locus Coeruleus Mediated by Designer Receptor Exclusively Activated by Designer Drug Restores Descending Nociceptive Inhibition after Traumatic Brain Injury in Rats

Abstract

Disruption of endogenous pain control mechanisms including descending pain inhibition has been linked to several forms of pain including chronic pain after traumatic brain injury (TBI). The locus coeruleus (LC) is the principal noradrenergic (NA) nucleus participating in descending pain inhibition. We therefore hypothesized that selectively stimulating LC neurons would reduce nociception after TBI. All experiments used a well-characterized rat lateral fluid percussion model of TBI. NA neurons were stimulated by administering clozapine N-oxide (CNO) to rats selectively expressing a designer receptor exclusively activated by designer drug (DREADD) viral construct in their LC's. Mechanical nociceptive thresholds were measured using von Frey fibers. The efficacy of diffuse noxious inhibitory control (DNIC), a critical endogenous pain control mechanism, was assessed using the hindpaw administration of capsaicin. Immunohistochemical analyses demonstrated the selective expression of the DREADD construct in LC neurons after stereotactic injection. During the 1st week after TBI, when rats demonstrated hindlimb (HL) nociceptive sensitization, CNO administration provided transient anti-allodynia in DREADD-expressing rats but not in rats injected with control virus. Seven weeks after TBI we observed a complete loss of DNIC in response to capsaicin. However, CNO administration largely restored DNIC in TBI DREADD-expressing rats but not those injected with control virus. Unexpectedly, the effects of LC activation in the DREADD-expressing rats were blocked by the α-1 adrenergic receptor antagonist prazosin, but not the α-2 adrenergic receptor antagonist atipamezole. These results suggest that directly stimulating the LC after TBI can reduce both early and late manifestations of dysfunctional endogenous pain regulation. Clinical approaches to activating descending pain circuits may reduce suffering in those with pain after TBI.

Introduction

Traumatic brain injury (TBI) occurs when an external force, such as a violent blow, pressure wave, deceleration, or jolt to the head causes injury to the brain. Such injuries often result in functional disability and additional adverse outcomes. These outcomes include impairments in cognition, sensory processing, motor control, and, importantly, chronic pain.^1

–5 Therefore, understanding the mechanisms through which TBI patients develop chronic pain and identifying potential treatments would be of tremendous benefit to the field. Endogenous pain modulation involves a balance of descending inhibitory and facilitatory pathways from the brain to the spinal cord that can decrease or increase pain respectively.⁶ The periaqueductal gray (PAG) is a major regulator of these descending pathways, which indirectly communicates with the spinal cord largely via the rostral ventromedial medulla (RVM).^7
–9 Fibers from the RVM that innervate the spinal dorsal horn are predominantly serotonergic, the effect of which can be either inhibitory (5-HT_1A, 5-HT_1B, 5-HT_1D, and 5-HT₇₎ or facilitatory (5-HT₃ and also 5-HT_2A), depending on the receptor subtype activated.^6,10
–12 Descending inhibitory modulation is additionally provided by noradrenergic innervation of the spinal dorsal horn from the locus coeruleus (LC), as well as the A5 and A7 noradrenergic subnuclei via spinal α2-adrenoreceptors (α2-AR).¹³ Experimental evidence from our team and others suggests that TBI causes damage to endogenous pain control centers in the brainstem, tipping the balance of descending pain modulation in favor of facilitation.^3,14

–18 This would enable nociceptive signaling and support the development of central sensitization, key features of patients with chronic pain.^19
–21

Our studies using experimental models of TBI in rats and mice have shown that changes in nociceptive signaling after TBI manifest in two distinct phases. The initial serotonin-dependent phase occurs within 24 h of TBI and involves mechanical allodynia of the HL that resolves within 28 days post-injury (DPI).²² During the initial phase there is a significant increase in neuroinflammation within the PAG, LC, RVM, and superficial dorsal horn, which are all key areas involved in the descending modulation of pain.¹⁴ This is followed by a more slowly developing second phase characterized by the failure of a critical endogenous pain control mechanism known as “diffuse noxious inhibitory control” (DNIC), which can persist to 180 DPI.¹⁴ We assess the presence of a DNIC response using an established capsaicin-based protocol^23
–25 (see Methods section for details). Briefly, prostaglandin (PG)E-2 is injected into the plantar surface of the left hindpaw (contralateral [CL] to the injury) to produce brief hypersensitivity. Sixty minutes post-injection, capsaicin is injected into the dorsum of the ipsilateral forepaw (FP). In uninjured rats, capsaicin induced a significant decrease in mechanical hyperalgesia of the left hindpaw within 5 min, which progressively increased over a period of 3 h.^14,16 In contrast, TBI rats showed no significant changes in the mean withdrawal threshold of the left hindpaw after capsaicin injection.^14,16 Thus, an anti-noceptive effect, a manifestation of DNIC, was observed in response to remote capsaicin injection, whic was absent after TBI.

Noradrenergic signaling has been shown by a number of investigators to play a key role in the DNIC response.^26

–29 In a rat model of TBI, however, it was observed that pharmacologically blocking noradrenaline (NA) reuptake was insufficient to restore DNIC in TBI rats,¹⁶ whereas enhancement of descending serotonergic signaling successfully restored DNIC.¹⁶ Because of the importance of functional descending noradrenergic signaling to normal pain regulation, we wanted to further examine the function of the LC and descending noradrenergic signaling to determine whether this pathway could potentially be augmented to reduce pain after TBI. To address this goal, we directly and selectively stimulated LC neurons using a recently described designer receptor exclusively activated by designer drug (DREADD) construct.³⁰

Methods

Animals

All studies were approved by the Veterans Affairs Palo Alto Health Care System Institutional Animal Care and Use Committee (Palo Alto, CA, USA) and followed the animal guidelines of the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory animals (NIH Publications No. 8023, revised 1978). Male Sprague Dawley rats (250–300 g; Envigo, Indianapolis, IN, USA), were housed under standard conditions with a 12 h light–dark cycle (6:30 am to 6:30 pm) and were given food and water ad libitum. The animals were housed in pairs in 30 × 30 × 19-cm isolator cages with solid floors covered with a 4 cm layer of wood chip bedding. Experimenters were blinded to the identity of treatments and experimental conditions. All studies were designed to minimize the number of rats required. All in vivo experiments were performed between 7 am and 3 pm in the Veterinary Medical Unit.

Drugs

Drugs used were: PGE-2 (1 μg/50 μL], i.pl (14010, Cayman Chemicals, MI, USA) a principal mediator of inflammation and pain hypersensitivity; capsaicin (CAP) (10 μg/10 μL), i.pl. (M2028, Millipore-Sigma, MO, USA) that causes hyperalgesia to mechanical stimuli at and around the injection site; clozapine N-oxide (CNO) (3 mg/kg), i.p., (16882, Cayman Chemicals) a ligand used to selectively activate DREADDs and is otherwise pharmacologically and behaviorally inert in animals at this dose; reboxetine mesylate (RBX) (10 μg/10 μL), i.t., (R1126, Ontario Chemicals, Canada) a noradrenergic reuptake inhibitor (NRI); atipamezole hydrochloride (ATZ) (1 mg/kg), s.c., (9001181, Cayman Chemicals) an α-2-adrenoceptor (AR) antagonist; and prazosin (PRZ) (1 mg/kg), i.p., (15023, Cayman Chemicals) an α-1-AR antagonist.

Viral vectors

Viral vectors, kindly provided by Dr. Christopher Peters, had been custom subcloned and packaged by the University of Pennsylvania Viral Vector Core. In an AAV10 vector, the synthetic PRSx8 promoter³¹ was used to restrict expression of the hM3Dq DREADD gene with a haemagglutinin (HA) tag³² to noradrenergic neurons in the LC area. Vazey and Aston-Jones³⁰ demonstrated 97% co-localization of tyrosine hydroxylase (TH), expression of which denotes noradrenergic neurons and the HA tag that identified hM3Dq expression in the LC. Quantification of the proportion of LC neurons that were hM3Dq positive was not reported. However, CNO-induced activation of the DREADD-expressing neurons of the LC in these studies was successful in altering the anesthetic state of rats while under isoflurane anesthesia.³⁰ The control AAV10 vector contained the PRSx8 promoter with only an mCherry reporter protein without the DREADD gene, hM3Dq.

DREADD microinjection surgeries

All surgeries were performed under isoflurane anesthesia (5% for induction and 2% for maintenance). Rats were secured in a stereotactic frame, and the coordinates for the left and right LC (From lambda: +1.5 mm A-P, + 1.5mm M-L, 7.0 mm D-V) were entered and marked on the skull with a surgical pen.³³ A 2-mm craniotomy was made into both marked locations using a mini-drill and a 1.4 mm drill burr (Fine Science Tools, CA, USA), and 1.4μl of the excitatory DREADD (AAV10 PRSx8hM3D) or the control virus (AAV10-PRSx8-mCherry) was injected at a rate of 0.1 μL/min using a 5-cm 30G beveled needle (7803-07, Hamilton, NV, USA) connected to a Hamilton 5 μL syringe (7634-01, Hamilton). The needle was left in place for 10 min after infusion and then slowly removed. The overlying scalp wound was closed using staples, and rats were allowed 4 weeks for recovery and viral expression.

TBI surgery

The lateral fluid percussion (LFP) rat model of TBI was used as described previously.^14,16,22 Briefly, a midline incision was made in the scalp, and a 5-mm craniotomy was made on the right side of the skull using a mini-drill and a 5-mm trephine burr (Fine Science Tools). The craniotomy was placed midway between the bregma and lambda sutures and 2 mm right of the midline suture. Using cyanoacrylate glue and dental acrylic, a female luer attachment was affixed to the craniotomy opening. Following recovery of pinch reflexes, the luer attachment was connected to the LFP apparatus (Amscien Instruments, VA, USA), and a pressure wave of 1.3 (±0.1 atm) to produce mild level injuries or no pressure wave (uninjured) was applied to rat dura based on previous reports. Thereafter, the luer attachment and dental acrylic were removed, the bone flap replaced, and the overlying wound closed using staples. Rats were allowed to recover in their home cages.

Intrathecal injection

Rats were anesthetized with isoflurane throughout the procedure and the paw pinch reflex was used to ensure the state of anesthesia. A 3 cm² window of fur, near the base of the tail, was shaved and washed with 70% ethanol to maximize visualization during needle insertion. An empty 50 mL falcon tube was placed underneath the rat to raise up the lumbar vertebral column, and the spinous process of L5 was located. The spinous process of L5 was pulled in a cranial direction and the vertebral body of L6 was pulled caudally toward the tail. This maximized the space within the groove between L5 and L6 vertebrae, into which a 25G needle was carefully inserted. Successful entry of the needle into the intradural space was confirmed by the observation of a tail flick. The needle was immediately secured into position with one hand, and the desired volume of substance was slowly injected with the other hand. Once injection was performed, the rat was placed on a warming pad prior to returning to their home cage.

Behavioral testing

Mechanical withdrawal thresholds were measured using a modification of the up-down method and von Frey filaments as described previously.³⁴ Animals were placed in clear Plexiglas boxes (17 cm length x 11 cm width x 13 cm height) on an elevated horizontal wire mesh stand (IITC Life Science Inc, CA, USA). After 60 min of acclimation, fibers of increasing stiffness with initial bending force of 4.31 N (ranging from 4.31 N up to 5.18 N) were applied to the plantar surface of the hindpaw, and left in place for 5 sec with enough force to slightly bend the fiber. Withdrawal of the hindpaw from the fiber was scored as a response. When no response was obtained, the next firmer fiber in the series was used in the same manner. If a response was observed, the next less stiff fiber was applied. Testing continued until four fibers had been applied after the first one causing a withdrawal response, allowing the estimation of the mechanical withdrawal threshold using a curve fitting algorithm.³⁵

DNIC paradigm

Assessment of post-traumatic mechanical hypersensitivity was performed using a variant of a widely used noxious stimulation–induced anti-nociceptive protocol.^23
–25 Briefly, it was confirmed that the rats had recovered to baseline mechanical thresholds after TBI (28 days post-injury). Next, intraplantar injection of PGE-2 (test stimulus) into the hindpaw (HL) contralateral to the TBI was used to produce brief hypersensitivity.³⁶ After 1 h, the PGE-2 injection HL withdrawal thresholds were measured using the von Frey filaments as described. Subsequent to mechanical threshold evaluations, rats were given a capsaicin injection (conditioning stimulus) into the dorsal surface of the forepaw (FP) ipsilateral to the TBI to induce DNIC. All intraplantar injections were administered using light isoflurane anesthesia. At 15, 30, 60, 120, 180, and 240 min after capsaicin injection, withdrawal thresholds were measured using the von Frey filaments as described.

Drug treatments during DNIC assessment

For studies involving CNO, ATZ and PRZ, an injection of the drugs was given after the withdrawal threshold measurements from the test stimulus (PGE-2) were recorded. In studies involving two drugs, CNO was always given first.

Immunohistochemistry

This protocol has been described in detail previously.^14,22 Briefly, rats were perfused with 4% paraformaldehyde, and brains were then removed and cryoprotected in 20% sucrose in phosphate buffered saline (PBS) for 2 days at 4°C. The brainstems were cut into 12-μm transverse sections using a cryostat. Immunostaining was performed using an antibody against (1) Tyrosine Hydroxylase (TH) (1:500, AB152, Millipore-Sigma), which is the rate-limiting enzyme of catecholamine synthesis of which noradrenaline is a product; (2) HA-tag (1:500, C29F4, Cell Signalling Technology, MA, USA), which is a small 9 amino acid epitope of the haemagglutinin of the influenza virus; (3) c-Fos (1:4000, ABE-457, Millipore-Sigma) for neuronal activation; and (4) Dopamine-beta-Hydroxylase (DβH; 1:500, MAB308, Millipore-Sigma), the enzyme that catalyzes the conversion of dopamine to norepinephrine, which is routinely used to label NA neurons of the LC. Blocking of all the sections took place at room temperature for 2 h in PBS containing 10% normal goat serum (Vector Laboratories, CA, USA), followed by exposure to the primary antibodies overnight at 4°C. Sections stained with c-Fos or TH were then treated with 0.3% hydrogen peroxide to quench endogenous peroxidases. After washing, TH and c-Fos were visualized, using biotin-conjugated second antibodies (Vector Laboratories). For TH, this was followed by incubation with the Vectastain Elite ABC reagent (Vector Laboratories) and developed using the DAB peroxidase substrate kit (Vector Laboratories). For c-Fos, this was followed by incubation with the Vectastain ABC AP reagent (Vector Laboratories) and developed using the ImmPACT Vector Red substrate kit (Vector Laboratories). HA-tag and DβH were visualized Alexa Fluor-conjugated second antibodies (ThermoFisher, MA, USA). Controls prepared with the primary antibody omitted showed minimal background fluorescence, DAB, and vector red staining under the conditions employed. Staining was performed concurrently for each group of sections compared with one another, and photographed under identical conditions.

Image analysis

In all data assessments, both the photography and image analysis were performed by observers who were blinded to the experimental conditions. c-Fos, HA-tag, DβH, and TH expression was evaluated bilaterally in the LC (A-P -9.0 to -10.5 mm) on three coronal sections per brain spaced 400 μm apart. Sections from uninjured, untreated rats were used to establish a threshold level that excluded all non-specific staining. This threshold level was then applied to all experimental groups.

Data analysis

An independent statistician (A.R.F.) tested statistical assumptions, and performed missing values analysis and descriptive statistical tests, followed by inferential tests. Data met normality, homogeneity of variance, and independence assumptions sufficiently for application of general linear models (GLM). Group comparisons were therefore performed according to a priori balanced experimental designs as two-way, three-way, or four-way mixed analyses of variance (ANOVAs), modeling between- and within-subject factors as appropriate using GLM. Significant effects were followed by interaction plots and Bonferroni's post-hocs.

Statistical analysis

All statistical analyses were performed using IBM SPSS software (v.27, IBM Corp.) with base, regression, missing values, and advanced statistics modules. Statistical code is freely available upon request. Graphs were created using Prism 9.1.0 (GraphPad Software). Data are presented as mean values ± standard error of the mean (SEM). All experimental sample sizes (n) were selected by a priori power calculations based on historical data from our laboratory. The results report precise F values, degrees of freedom, p values, effect sizes, and observed power for all significant effects as well as the ns completed for each group.

Data availability

All the raw data are available through the VA- and NIH-supported open data commons for TBI (odc-tbi.org), in support of funder mandates for findable, accessible, interoperable, and reusable (FAIR) data stewardship policies. Data may be accessed at https://dx-doi-org.web.bisu.edu.cn/10.34945/F5759W upon article publication and reused with attribution via the creative commons 4.0 BY license.

Results

TBI significantly reduces the number of c-Fos positive cells in the LC during DNIC assessment

At 49 DPI, we compared the number of c-Fos positive cells in the LC of TBI and uninjured rats during DNIC testing. Both TBI and uninjured rats were given either the test stimulus, PGE-2, or vehicle (VEH) into the left HL contralateral to the side of injury in TBI rats (Fig. 1A). One hour after HL injection, all rats were then given the conditioning stimulus, capsaicin, into the right FP. All rats were then perfused 30 min post-capsaicin injection. Immunostaining with Tryosine hydroxylase, to outline the borders of the LC (Fig. 1 B.a-d) and c-Fos (Fig. 1 B.e-h) revealed a significantly greater increase in the number of c-Fos positive cells in the uninjured, PGE-2-treated rats than in TBI, PGE-2-treated rats after capsaicin (Fig. 1C, p < 0.01). There was no significant difference in the number of c-Fos-positive cells in VEH-injected, TBI, and uninjured rats. Two-way ANOVA revealed significant main effects of drug (F [1,20] = 25.67, p = 5.9 x10^−5, partial ƞ² = 0.56, observed power [OP] = 0.998), and injury (F [1,20] = 5.59, p = 0.028, partial ƞ² = 0.22, OP = 0.61). Therefore, the combination of the PGE-2 and capsaicin injections, required to stimulate a DNIC response, significantly increased neuronal activation in the LC of uninjured rats. However, neuronal activation in the LC of TBI rats was significantly reduced compared with that in uninjured rats. Therefore, DNIC failure in TBI rats could be the result of reduced activation of the LC neurons. The injection of capsaicin alone did not increase neuronal activation in the LC in either TBI or uninjured rats.

FIG. 1.

The number of c-Fos-positive cells (a marker for neuronal activation) in the locus coeruleus (LC) was significantly decreased following diffuse noxious inhibitory control (DNIC) testing after traumatic brain injury (TBI) compared witht uninjured rats at 49 days post-injury (DPI). (A) Protocol; TBI or uninjured rats were given either the test stimulus (prostaglandin [PG]E-2) or a vehicle into the left hindpaw. All rats were then given the conditioning stimulus, capsaicin, and euthanized 30 min later. (B) Quantification of the number of c-Fos-positive cells in the LC revealed significantly fewer cells in PGE-2-injected TBI rats than in uninjured, PGE-2-injected rats. Data are presented as mean ± standard error of the mean (SEM) (n = 6, all groups). *Uninjured + PGE-2 versus TBI + PGE-2 (p < 0.01) by Bonferroni's post-hocs. (C) Photomicrographs of the LC with tyrosine hydroxylase (TH) staining (C.a-d) were used to establish the boundaries of the LC that were then used to count the c-Fos- positive cells on an adjacent section (C.e-h). Scale bar: 50μm. Color image is available online.

DREADD-mediated activation of the LC can trigger endogenous pain modulation in uninjured rats

Next, we wanted to determine the role that the activation of the neurons in the LC played in eliciting a DNIC response in uninjured rats. To test this, we substituted the conditioning stimulus (capsaicin) with CNO/DREADD-mediated activation of the LC following PGE-2 injection into the hindpaw. For this study we bilaterally injected rats with the DREADD (AAV10 PRSx8hM3DqHA-tag) or the control virus (AAV10-PRSx8-mCherry) into the LC, 28 days prior to behavioral testing. Injection of this DREADD construct into the LC has been previously reported to result in 97% co-localization of TH, expression of which denotes noradrenergic neurons and the HA tag that identifies hM3Dq expression.³⁰ Expression of the DREADD-virus (HA-tag) in our studies was also limited to the DβH-positive neurons of the LC (Fig. 2 B.a-c). DREADD expression in these neurons was restricted to the plasma membrane of the NA cell bodies and axons in all animals (Fig. 2 B.d). DβH-positive neurons of the A5 and A7 noradrenergic cell groups did not co-express HA-tag. On the day of testing, rats were given an intraplantar injection of PGE-2 into the left hindpaw to produce brief hypersensitivity.³⁶ Subsequent to mechanical threshold measurements, rats were given a systemic injection of CNO (i.p. 3 mg/kg), which is used to activate the DREADD virus or the VEH saline (Fig. 2 A). Data in Figure 2 B reveal that the CNO/DREADD-mediated activation of the LC did result in an anti-allodynic response. In contrast, no such response was present in VEH/DREADD-injected rats and or in either CNO/control-injected or VEH/control-injected rats. Three-way repeated measures ANOVA revealed a main effect of drug (F [1,12] = 67.07, p = 3.0 x10⁻⁶, partial ƞ² = 0.85, OP = 1.0), virus (F[1,12] = 73.44, p = 2.0 x 10⁻⁶, partial ƞ² = 0.86, OP = 1.0), and time after CNO (F [6,72] = 9.85, p = 6.5 x 10⁻⁸, partial ƞ² = 0.45, OP = 1.0). There was also a significant interaction between drug by virus (F[1,12] = 62.89, p = 4.0 x10⁻⁶, ƞ² = 0.84, OP = 1.0), time after CNO by drug (F [6,72] = 9.41, p = 1.3 x 10⁻⁷, partial ƞ² = 0.44, OP = 1.0), time after CNO by virus (F [6,72] = 9.04, p = 2.31 x10⁻⁷, partial ƞ² = 0.43, OP = 1.0) and time after CNO by virus by drug (F [6,72] = 9.86, p = 6.4 x 10⁻⁸, partial ƞ² = 0.45, OP = 1.0). No other main effects or interactions were significant. Post-hoc analysis indicated a statistically significant increase in paw withdrawal threshold in DREADD-expressing, TBI rats in the presence of CNO at 10, 30, 60, 90, 120, 150, and 180 min compared with VEH-treated DREADD-expressing TBI rats (p < 0.0001). In summary, activation of LC neurons after the test stimulus (PGE-2) was important for the manifestation of a DNIC response in uninjured rats.

FIG. 2.

Designer receptor exclusively activated by designer drug (DREADD)-mediated activation of the locus coeruleus (LC) can trigger endogenous pain modulation in the absence of a conditioning stimulus in naïve rats. (A) Protocol: rats were injected with either the DREADD virus (AAV10 PRSx8hM3D) or the control virus (AAV10-PRSx8-mCherry) bilaterally into the LC. On the day of testing, 28 days post-injection, all rats were given an intra-plantar injection into the left hindpaw with the test stimulus, prostaglandin (PG)E-2 (Open arrow, i.pl. [1 μg/50 μL). One hour after PGE-2 injection, rats were then split into vehicle-treated (saline, i.p.) or clozapine-N-oxide (CNO)-treated (3 mg/kg i.p) groups. (B) Behavioral assessment revealed that CNO-mediated activation of the DREADD virus in the LC triggered an endogenous pain response that was not present in non-CNO treated, DREADD-injected rats. Further, no change in the PWT was observed in rats injected with the control virus with or without CNO treatment. Data are presented as mean ± standard error of the mean (SEM) (n = 4, all groups). *DREADD/CNO versus DREADD/vehicle (VEH) (p < 0.001) by Bonferroni's post-hocs. CNO is used as a ligand to activate DREADD receptors. Low magnification of haemagglutinin (HA)-tag (C.a), DβH (C.b), and HA/DβH (C.c) immunostaining of the LC. (C.d) High magnification of HA/DβH co-immunostaining of the LC. Co-positive cells labeled with white arrowhead. Color image is available online.

Intra-LC injection of DREADD viral vector did not alter nociceptive changes after TBI

In order to assess the effect of the DREADD in TBI rats we had to ensure that both the surgery and/or the presence of viral vectors in the LC did not affect the early nociceptive sensitization characteristic of this TBI model.¹⁶ Data in Figure 3 revealed that neither the surgery nor the intra-LC injection of the virus (DREADD or control) impacted the nociceptive outcomes of the initial pain phase after TBI. Three-way mixed ANOVA revealed a main effect of group (F [3,20] = 158.71, p = 4.12 x 10⁻¹⁴, partial ƞ² = 0.96, OP = 1.0), and time post-TBI (F [6,120] = 79.97, p = 1.43 x 10⁻³⁹, partial ƞ² = 0.80, OP = 1.0). There was also a significant interaction between time post-TBI by group (F [18,120] = 7.67, p = 6.14 x 10⁻¹³, partial ƞ² = 0.52, OP = 1.0). No other main effects or interactions were significant. In summary, the initial TBI pain phase was unaffected by the addition of the DREADD step.

FIG. 3.

Traumatic brain injury (TBI)-induced nociceptive sensitization of the contralateral hindpaw was unaffected by the injections of the designer receptor exclusively activated by designer drug (DREADD) or control virus into the locus coeruleus (LC) up to 49 days post-injury. Data are presented as mean ± standard error of the mean (SEM) (n = 8, all groups). Color image is available online.

DREADD-mediated activation of the LC reduces mechanical sensitization of the hindpaw during the initial pain phase after TBI

We have previously shown that the first pain stage after TBI is caused by enhanced serotonergic, pronociceptive descending facilitation. In this experiment, we aimed to tip the balance of endogenous pain modulation in favor of descending inhibition by enhancing the level of NA within the dorsal horn of the spinal cord. This was achieved via the intrathecal administration of the NA-selective reuptake inhibitor, RBX. At 7 DPI, the presence of mechanical hypersensitivity of the hindlimb on the contralateral side (CT) was confirmed in all the injured rats prior to treatment (Fig. 4). Following intrathecal injection, RBX significantly reduced the degree of mechanical sensitization in the hindlimb on contralateral side when compared with TBI/VEH rats (Fig. 4). This effect lasted ∼60 min before the TBI/RBX rats began to show signs of their allodynia returning. Hindpaw sensitivity in uninjured rats did not vary from baseline for the duration of the experiment (Fig. 4). Three-way mixed ANOVA revealed a main effect of drug (F [1,20] = 16.39, p = 6.2 x10⁻⁴, partial ƞ² = 0.45, OP = 0.97), injury (F [1,20] = 256.24, p = 7.2 x10⁻¹³, partial ƞ² = 0.93, OP = 1.0), and time after RBX (F [5,100] = 8.13, p = 2.0 x10⁻⁶ partial ƞ² = 0.29, OP = 1.0). There was also a significant interaction between time after RBX by drug (F [5,100] = 8.05, p = 2.0 x10⁻⁶ partial ƞ² = 0.29, OP = 1.0), time after RBX by injury (F [5,100] = 5.46, p = 1.7 x10⁻⁴, partial ƞ² = 0.96, OP = 1.0), and time after RBX by injury by drug (F [5,100] = 5.48, p = 1.7 x10⁻⁴, partial ƞ² = 0.96, OP = 1.0). No other main effects or interactions were significant. Post-hoc analysis indicated a statistically significant increase in paw withdrawal threshold (PWT) in TBI/RBX rats at 30 and 60 min post-RBX injection compared with TBI/VEH rats (p < 0.001). In summary, the initial TBI pain phase at 7 DPI was temporarily ameliorated by intrathecal RBX.

FIG. 4.

The effect of the norepinephrine reuptake inhibitor, reboxetine (RBX) on TBI-induced nociceptive sensitization 7 days after traumatic brain injury (TBI), at the peak of hindpaw hypersensitivity, mechanical hyperalgesia of the contralateral hindpaw was confirmed in all TBI rats prior to treatment. Both uninjured and TBI rats were randomly and equally split into vehicle-treated (VEH, i.t.) or RBX-treated (10 μg/10 μL, i.t.) groups. Assessment of nociceptive sensitivity of the contralateral hindpaw revealed that RBX transiently restored the paw withdrawal threshold to baseline at 60 min post-injection. Data are presented as mean ± standard error of the mean (SEM) (n = 6, all groups). *TBI/RBX versus TBI/VEH (p < 0.01) by Bonferroni's post-hocs. Color image is available online.

With this in mind, we next wanted to see if CNO/DREADD-mediated activation of the LC could affect the manifestation of the initial pain response to TBI. Figure 5 reveals that CNO/DREADD-mediated activation of the LC significantly decreased the degree of mechanical sensitization in the hindlimb at both 7 DPI (Fig. 5A) and 21 DPI (Fig. 5B). This effect lasted ∼60 min before the CNO/DREADD-treated TBI rats began to show signs of their allodynia returning. Hindpaw sensitivity in VEH/DREADD-TBI rats or in both control-TBI rat groups did not vary from baseline for the duration of the experiment (Fig. 5A and B). Four-way mixed ANOVA revealed a main effect of virus (F [1,28] = 8.80, p = 6.0 x10⁻³, partial ƞ² = 0.24, OP = 0.82), injury (F [1,28] = 917.06, p = 6.09 x10⁻²³ partial ƞ² = 0.97, OP = 1.0), and time after CNO (F [5,140] = 7.89, p = 1.0 x10⁻⁶ partial ƞ² = 0.22, OP = 1.0). There was also a significant interaction between virus by injury (F [1,28] = 9.98, p = 3.7 x10⁻³, partial ƞ² = 0.26, OP = 0.86), time after CNO by virus (F [5,140] = 7.87, p = 1.0 x10⁻⁶, partial ƞ² = 0.22, OP = 1.0), time after CNO by injury (F [5,140] = 6.01, p = 4.6 x10⁻⁵, partial ƞ² = 0.18, OP = 0.99), time after CNO by injury by virus (F [5,140] = 10.92, p = 6.9x10⁻⁹, partial ƞ² = 0.28, OP = 1.0), day of testing by time after CNO by virus (F [5,140] = 3.08, p = 0.01, partial ƞ² = 0.1, OP = 0.86), day of testing by time after CNO by injury (F [5,140] = 2.7, p = 0.023, partial ƞ² = 0.09, OP = 0.81), and day of testing by time after CNO by virus by injury (F [5,140] = 2.81, p = 0.019, partial ƞ² = 0.09, OP = 0.82). No other main effects or interactions were significant. Post-hoc analysis indicated a statistically significant increase in paw withdrawal threshold (PWT) in DREADD/TBI/CNO rats at 30 and 60 min after CNO injection compared with DREADD/TBI/VEH rats (p < 0.001). In summary, the initial TBI pain phase was temporarily ameliorated by the CNO/DREADD-mediated activation of the LC at both 7 and 21 DPI.

FIG. 5.

The effect of designer receptor exclusively activated by designer drug (DREADD)/clozapine N-oxide (CNO)-mediated activation of the locus coeruleus (LC) on traumatic brain injury (TBI)-induced nociceptive sensitization at (A) 7 and (B) 21 days post-injury (DPI). Assessment of nociceptive sensitivity of the contralateral hindpaw revealed that DREADD-mediated activation of the LC does increase paw withdrawal threshold (peaking 30 min post-CNO) on both 7 and 21 DPI. Data are presented as mean ± standard error of the mean (SEM) (n = 8, all groups).*DREADD/TBI/CNO versus DREADD/TBI/vehicle (VEH) (p < 0.05). Color image is available online.

DREADD-mediated activation of the LC can restore DNIC after TBI.

At 49 DPI, we tested if DREADD-mediated activation of the LC would restore the DNIC response in TBI rats. The data in Figure 6A demonstrate that the DNIC response was restored in DREADD-expressing, TBI rats that were treated with CNO. This effect remained significant for up to 30 min after injection of the conditioning stimulus capsaicin. In the absence of CNO, the DNIC response was not restored in DREADD-expressing TBI rats (Fig. 6A). Further, CNO treatment did not restore the DNIC response in control virus-expressing TBI rats (Fig. 6B). An intact DNIC response was observed in all uninjured rats (Fig. 6A and B). Three-way mixed ANOVA revealed a main effect of virus (F [1,88] = 7.47, p = 6.0 x10⁻³, partial ƞ² = 0.08, OP = 0.77), and injury (F [1,88] = 381.64, p = 9.4 x10⁻³⁴, partial ƞ² = 0.81, OP = 1.0), and time post-capsaicin (F [6,528] = 125.09, p = 4.87 x10⁻⁹⁸, partial ƞ² = 0.59, OP = 1.0). There was also a significant interaction between virus by injury (F [1,88] = 8.60, p = 4.2 x 10⁻³, partial ƞ² = 0.09, OP = 0.83), virus by drug (F [1,88] = 7.36, p = 8.0 x10⁻³, partial ƞ² = 0.09, OP = 0.83), and injury by drug (F [1,88] = 14.15, p = 3.0 x10⁻⁴, partial ƞ² = 0.14, OP = 0.96), time after capsaicin by virus (F [6,528] = 4.31, p = 3.0 x10⁻⁴, partial ƞ² = 0.05, OP = 0.98), time after capsaicin by injury (F [6,528] = 53.32, p = 2.5 x10⁻⁵¹, partial ƞ² = 0.38, OP = 1.0), time after capsaicin by drug (F [6,528] = 9.62, p = 4.64 x10⁻¹⁰, partial ƞ² = 0.1, OP = 1.0), time after capsaicin by virus by injury (F [6,528] = 8.57, p = 6.7 x10⁻⁹, partial ƞ² = 0.09, OP = 1.0), time after capsaicin by virus by drug (F [6,528] = 7.32, p = 1.57 x10⁻⁷, partial ƞ² = 0.08, OP = 1.0), time after capsaicin by injury by drug (F [6,528] = 4.29, p = 3.1 x10⁻⁴, partial ƞ² = 0.05, OP = 0.98). No other main effects or interactions were significant. Post-hoc analysis indicated a statistically significant increase in PWT in CNO/DREADD/TBI rats at 15 and 30 min post-capsaicin injection compared with VEH/DREADD/TBI rats (p < 0.001). In summary, CNO/DREADD-mediated activation of the LC could restore the DNIC response at 49 DPI.

FIG. 6.

The effect of the designer receptor exclusively activated by designer drug (DREADD)/ clozapine N-oxide (CNO)-mediated activation of the locus coeruleus (LC) on the traumatic brain injury (TBI)-induced failure of the diffuse noxious inhibitory control (DNIC) response at 49 days post-injury (DPI) (A). DREADD/CNO-mediated activation of the LC reinstates DNIC response in TBI rats (B). In absence of CNO-treatment the DNIC response was not restored in DREADD-injected TBI rats (B). Further, DNIC was not restored in the control injected TBI rats with or without CNO treatment (C). Open arrow, prostaglandin (PG)E-2; i.pl. (1 μg/50 μL). Closed arrow, capsaicin; i.pl. (10 μg/10 μL). Data are presented as mean ± standard error of the mean (SEM) (n = 12). *DREADD/TBI/CNO versus DREADD/TBI/vehicle (VEH) (p < 0.001) by Bonferroni's post-hocs. Color image is available online.

Systemic administration of the AR antagonist ATZ does not block the CNO/DREADD-mediated restoration of DNIC after TBI

Normally functioning descending noradrenergic pain inhibition is thought to be caused by the activation of the LC, which results in the release of NA in the dorsal horn of the spinal cord which then acts through the α2-AR to reduce nociceptive signal transmission. To establish the mechanism by which CNO/DREADD-mediated activation of the LC restored the DNIC response after TBI, we used the α2-AR antagonist ATZ. Figure 7A shows that ATZ was administered 1 min after CNO treatment, as both drugs had a similar time of onset (15 min). The dose of ATZ used for this study was the same as that previously shown to block the DNIC response in naïve rats.¹⁶ Unexpectedly, data from Figure 7B reveals that ATZ failed to block the restoration of the DNIC response in CNO/DREADD-injected TBI rats. Three-way mixed ANOVA revealed a main effect of time after capsaicin (F [6,204] = 73.55, p = 2.45 x10⁻⁴⁸, partial ƞ² = 0.68, OP = 1.0). No other main effects or interactions were significant. Data are presented as mean ± SEM (n = 8 [for uninjured groups] to n = 11 [for TBI groups]). In summary, ATZ failed to block restoration of the DNIC response in CNO/DREADD-treated TBI rats.

FIG. 7.

Reinstatement of the diffuse noxious inhibitory control (DNIC) response in clozapine N-oxide (CNO)-treated, designer receptor exclusively activated by designer drug (DREADD)-injected, traumatic brain injured (TBI) rats (as depicted in A) was unaffected by systemic atipamezole (ATZ) compared with vehicle (VEH)-treated CNO/DREADD-injected TBI rats (B). Open arrow, prostaglandin (PG)E-2; i.pl. (1 μg/50 μL). Closed arrow, capsaicin; i.pl. 10 μg/10 μL). Data are presented as mean ± standard error of the mean (SEM) (n = 8–12). Color image is available online.

Administration of the α1-AR antagonist PRZ does block the CNO/DREADD-mediated restoration of DNIC after TBI

To ascertain how CNO/DREADD-mediated activation of the LC restored the DNIC response after TBI, we next used the α1-AR antagonist PRZ (Fig. 8A), as spinal α1-ARs are the other major receptor target of NA released by fibers from the LC in the central nervous system (CNS). Data from Figure 8B demonstrates that PRZ did block the restoration of the DNIC response in CNO/DREADD-injected TBI rats compared with VEH treated CNO/DREADD-injected TBI rats. PRZ had no effect on the DNIC response in the CNO/DREADD-injected uninjured rats. Three-way mixed ANOVA revealed a main effect of injury (F [1,28] = 31.04, p = 6.0 x10⁻⁶, partial ƞ² = 0.53, OP = 1.0), drug (F [1,28] = 9.58, p = 4.4 x10⁻³, partial ƞ² = 0.26, OP = 0.85), time after capsaicin (F [6,168] = 69.33, p = 6.63 x10⁻⁴³, partial ƞ² = 0.71, OP = 1.0). There was also a significant interaction between drug by injury (F [1,28] = 26.46, p = 1.9 x10⁻⁵, partial ƞ² = 0.49, OP = 1.0), time after capsaicin by injury (F [6,168] = 6.61, p = 3.0 x10⁻⁶, partial ƞ² = 0.19, OP = 1.0), time after capsaicin by drug (F [6,168] = 2.86, p = 0.01, partial ƞ² = 0.09, OP = 0.88), time after capsaicin by injury by drug (F [6,168] = 6.77, p = 2.0 x10⁻⁶, partial ƞ² = 0.20, OP = 1.0). Data are presented as mean ± SEM (n = 8). Post-hoc analysis indicated a statistically significant decrease in PWT in CNO/DREADD/TBI rats treated with PRZ post-capsaicin injection compared with VEH-treated, CNO/DREADD/TBI rats (p < 0.001). In summary, PRZ successfully blocked the restoration of the DNIC response in CNO/DREADD-treated TBI rats.

FIG. 8.

Reinstatement of the diffuse noxious inhibitory control (DNIC) response in clozapine N-oxide (CNO)-treated, designer receptor exclusively activated by designer drug (DREADD)-injected, traumatic brain injured (TBI) rats (as depicted in A) was blocked by systemic prazosin compared with vehicle (VEH)-treated CNO/DREADDinjected TBI rats (B). Open arrow, prostaglandin (PG)E-2; i.pl. (1 μg/50 μL). Closed arrow, capsaicin; i.pl.(10 μg/10 μL). No other main effects or interactions were significant. Data are presented as mean ± standard error of the mean (SEM) (n = 8). Color image is available online.

Discussion

Chronic pain is a common consequence of TBI that can increase a patient's suffering and pose a significant challenge to rehabilitative efforts.^5,37,38 Although our understanding of the TBI–chronic pain relationship is poor, our research and that of others suggest that an imbalance in the descending pain modulatory pathways is likely to be involved.^{3,14,16
–18,22} Our previous work using experimental models of TBI suggests that changes in nociceptive processing occurs in two phases. The initial phase involves nociceptive sensitization contralateral to the injury beginning 1–3 days after TBI and lasting 3–4 weeks, resembling the period of acute post-TBI pain in patients. The second phase involves the loss of effective endogenous pain modulation, specifically DNIC, which occurs in the first month after TBI. This phase may provide a model of more persistent and problematic post-TBI pain. We demonstrated previously that DNIC can be restored partially through pharmacological enhancement of descending serotonergic pain inhibition.¹⁶ In contrast, pharmacological NA reuptake inhibitors failed to reinstate the DNIC response in TBI rats, in contrast to effects observed on DNIC failure following peripheral nerve injury.^26,28,39,40 Although our earlier work showed glial activation and possible damage to the brain's principal noradrenergic signaling nucleus, the LC, in the rat LFP model of TBI,¹⁴ we were hopeful that direct stimulation of the surviving tissue could still provide functional descending pain inhibition. Here, we show that the neurons within the LC after a TBI are still functional and capable of activation (Fig. 1). However, the extent of neuronal activation in the LC is significantly reduced post-injury compared with in uninjured rats. In this set of studies, therefore, we revisited the role of descending noradrenergic pain inhibition in TBI, using a more focused approach of DREADD-mediated activation of the LC. Here we demonstrated that transduction of DREADD containing virus, AAV10 PRSx8hM3DHA-tag, into the neurons of the LC could in fact restore the DNIC response in rats up to 49 days post-TBI.

DREADDS are often genetically modified muscarinic acetylcholine receptors that are conjugated to an adeno-associated virus (AAV) and include specific promoters to limit their expression to particular cell populations.^41

–44 Depending on the type of DREADD receptor used, they can either excite (hM3Dq) or inhibit (hM4Di) the activity of the transduced neuron.^32,45 The AAV used in the current study contains the synthetic dopamine-beta-hydroxylase (DβH) promoter PRSx8³¹ to selectively express the excitatory hM3Dq DREADD receptor³² in LC-NA neurons. Activation of the DREADD receptor is accomplished through systemic delivery of the otherwise inert exogenous ligand CNO.^32,45 These tools have seldom, to this point, been used to define or treat the consequences of TBI.⁴⁶

Our results demonstrate that anti-allodynia can be obtained by activating a DREADD construct, AAV10 PRSx8hM3DqHA-tag, selectively expressed in the LC, thus recapitulating endogenous pain modulation that is normally mediated through this center. This observation is consistent with established LC functions and our previous findings of the importance of intact LC-NA signaling mediating endogenous pain modulation (DNIC) in naive rats.^16,23,30,47 We also confirmed that the pattern of nociceptive changes observed in the 7 weeks following TBI was unchanged in the DREADD-transfected animals, suggesting that this tool was unlikely to alter the normally observed post-TBI pain physiology (Fig. 2).

Having established the viability of the novel DREADD tool, we assessed the ability of DREADD-mediated LC activation to block nociceptive sensitization during the initial post-TBI pain phase. Our results demonstrated that the lumbar administration of a NA reuptake inhibitor, RBX, significantly reduced the degree of mechanical sensitization in the hindlimbs of rats 7 days after TBI when compared with VEH-injected animals. This showed that NA in the lumbar spinal cord was available for release and could provide anti-allodynia (Fig. 3). Indeed, we found that CNO/DREADD-mediated activation of the LC in TBI rats did provide a transient (60 min) and significant increase in paw withdrawal threshold at 7 days post-injury that was more pronounced at 21 days post-injury (Fig. 4). When these results are taken together, we interpret them to indicate that in the acute phase of pain sensitization after TBI it may be possible to achieve anti-allodynia through the activation of a noradrenergic endogenous pain control pathway.

The expression of DREADD constructs is sufficiently stable for the study of long-term neurophysiological changes after TBI.³⁰ Even 49 days after injury, CNO administration caused anti-allodynia after hindpaws were sensitized with PGE-2 in LC DREADD-expressing TBI rats. These effects are likely the result of the activated DREADD, as this effect was not observed in DREADD-expressing TBI rats given the VEH. All DREADD-expressing, uninjured rats had a normal DNIC response. Moreover, the DNIC response was not restored in control virus-expressing TBI rats regardless of the presence of CNO. These are key data for our experiments and others considering the use of similar techniques, as there have been some reports that CNO alone can influence an animal's behavior.^48
–50 However, with our balanced experimental design, we were largely able to exclude the possibilities that CNO alone or DREADD expression itself interfered with the behavioral paradigm.

The LC is the major source of noradrenergic fibers projecting to the dorsal horn, and the NA released from these fibers is believed to interact with the α-2 ARs to inhibit nociceptive signal transmission and pain under normal physiological circumstances.^51,52 Although it is tempting to presume that our DREADD-mediated effects involved spinal α-2 ARs, we attempted to confirm that the observed effects were indeed mediated by this mechanism. Surprisingly, the selective α-2 AR antagonist ATZ used at the same dose shown previously by us to block the NA-mediated DNIC response in naïve rats¹⁶ had no effect on reinstatement of the DNIC response in DREADD-expressing TBI rats. As we had shown that expression of the viral construct appeared to be limited to the NA producing LC, and in fact the construct contained the dopamine-beta-hydroxylase promoter, hopefully limiting expression to NA producing cells, we hypothesized that a different adrenergic receptor might be mediating the effects in the TBI rats. In fact, when DREADD-expressing TBI rats were treated with the α-1 AR selective antagonist PRZ, CNO administration was unable to restore the DNIC response. Although analgesic effects through α-2 ARs were expected, it should be recognized that α-1 ARs have also been shown to have an antinociceptive effect through their expression on inhibitory (GABA) interneurons in lamina II/substantia gelatinosa^53
–55 In this situation, NA binds to the α-1 ARs which excites the inhibitory interneurons that then hyperpolarize the neurons of the substantia gelatinosa through the release of GABA.^53
–55 It is possible therefore that restoration of the DNIC response in the DREADD-expressing, TBI rats was caused by an α-1 AR-mediated mechanism. Determining how this system gains functional significance in the rats after TBI will require additional study.

Results such as these demonstrating fundamental changes in neural circuitry after brain injury using DREADD constructs may have implications beyond the study of pathophysiological mechanisms. Specifically, although DREADDS themselves have not to this point proven useful as therapeutic tools, results like ours suggest that augmentation of circuits rendered dysfunctional by TBI could be restored using other modalities for therapeutic effect. Although the placement of electrodes for direct LC stimulation is not common, deep brain stimulation of the brainstem has been described in several studies and case series. Some success in treating pain, movement disorders, sleep, and other maladies has been described.⁵⁶ Less invasive techniques, such as vagal nerve stimulation, may be used to indirectly stimulate the LC.⁵⁷ Transcranial magnetic stimulation (TMS) may offer a non-invasive approach to stimulation of the LC as well.⁵⁸

There are limitations to this study. The model of DNIC employed in these studies measures responses to mechanical stimulation of sensitized tissues with normally non-noxious nylon fibers; that is, anti-allodynia. We hope that this is valuable in understanding the effects of TBI on endogenous pain control related to sensitized tissues, common in chronic pain states. It is important to acknowledge, however, that we did not measure analgesia in tissue that was not sensitized with PGE2. Such measurements might probe at least partially distinct endogenous pain control mechanisms. In addition, there are concerns that CNO and its back metabolism products (clozapine and N-desmethylclozapine) could have off-target effects on behavior.^48
–50 However, we were cognizant of including all the appropriate control groups (i.e., CNO/control-injected rats) and we observed no behavioral effects in rats treated with CNO in the absence of a DREADD. It is also possible that expression of the DREADD virus may have spread to other DβH-positive neuronal cell populations outside of the LC. Spinal noradrenergic terminals also originate from A5 and A7 noradrenergic groups and also contribute to antinociception.^59,60 However, HA/DβH positive cells and their axons were exclusively found within the LC and possibly its subcoerulear region. We also showed a significant reduction in the number of Fos- positive cells in the LC of TBI rats compared with in uninjured rats after the DNIC protocol. However, it is unclear whether this is caused by LC neuronal loss, dysfunction of surviving neurons, or, possibly, damage to afferent fiber tracts. We have previously shown a significant increase in neuroinflammation in the LC at 7 and 28 days after TBI in this model, consistent with all three possibilities for reduced c-fos expression.¹⁴ Taken as a whole, the results strongly suggest that neuromodulation of descending noradrenergic projections emanating from the LC reflect a viable target for treating post-TBI pain.

Funding Information

This work was supported by grants from the United States Department of Defense (MR130295) and the Department of Veterans Affairs (RX001776 and 2 I01 RX001776-05). C.M.P. was supported by grant P01GM113852 from the NIH/National Institute of General Medical Sciences (NIGMs). E.M.V. was supported by the NIH/National Institute of Mental Health (NIMH) (R00MH104716) and the NIH/National Institute of Alcohol Abuse and Alcoholism (NIAAA) (U01AA025481). A.R.F. was supported by a grant from the Department of Veterans Affairs (RX002787) and NIH/National Institute of Neurological Disorders and Stroke (NINDS) (UH3NS106899; U24NS122732).

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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