Doxazosin Alleviates Chronic Orofacial Pain

TL;DR

Doxazosin, a drug that blocks certain brain receptors, reduces chronic facial pain in rats, but its effects on anxiety and other factors differ between males and females.

Contribution

This study shows that doxazosin reverses chronic orofacial pain and glial activation in rats, with sex-specific effects on anxiety and immune markers.

Findings

Doxazosin reversed facial hypersensitivity in both male and female rats.

Doxazosin reduced astrocytic activation in the somatosensory cortex and hippocampus.

Doxazosin improved anxiety in males but recognition memory in females.

Abstract

Central to the linkage of pain circuitry with the limbic system is its initial NAα2-mediated antinociceptive effect in acute pain models, followed by contradictory pronociceptive activation by the locus coeruleus seen in chronic pain models. Rats with a stable, long-term (>10 weeks) inflammatory compression of the trigeminal infraorbital nerve (FRICT-ION) preclinical model were given daily doxazosin, a slow-release NAα1 receptor antagonist, in weeks 8–10. Facial hypersensitivity was reversed back to baseline in male and female rats, but anxiety was only reduced in male animals. Doxazosin-decreased astrocytic activation was indicated by a decrease in both intracranial cathepsin B imaging in vivo and GFAP immunostaining in the somatosensory cortex and hippocampus. Doxazosin reduction in NAα1 receptor activation diminished glial-neuronal interactions, resulting in downstream reduction in pain-related behaviors. Other significant differences by sex included improved elevated zero maze anxiety measures only in males, and improved novel recognition scores only in females. Elevated thymus chemokine CXCL7 levels were reduced by doxazosin but only in male rats. These sexually dimorphic contradictions further complicate the understanding of the noradrenergic system’s involvement in nociception. The findings indicate that by reducing NAα1 receptor drive with doxazosin, the role of the locus coeruleus can be shifted back to NAα2-receptor-mediated pain inhibition.

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Full text

1. Introduction

Trigeminal neuropathy, pain caused after insult to the peripheral nerve, has a higher incidence in women (65%) than in men [1,2]. Approximately 40% of patients with trigeminal neuropathy suffered trauma to the nerve, most commonly due to dental extractions, facial trauma, or vehicular accidents [3,4,5,6,7]. In another 22–38% of patients, trigeminal neuropathy is caused by neoplasms and tumors [2,8,9]. However, trigeminal neuropathy can also be caused by autoimmune diseases, such as multiple sclerosis, or infectious diseases like herpes zoster [2,10]. Patients describe trigeminal neuropathic pain as constant and dull, unlike trigeminal neuralgia which presents as paroxysmal and shock-like [11]. As pain becomes chronic, multifactorial processes are activated. This results in epigenetic, molecular, neuroplastic, and even brain circuitry changes that result in the aberrant recruitment of the limbic system [12,13]. Altered brain connectivity can produce secondary symptoms such as anxiety and depression [14,15]. Neuropathic pain has a poor medical response rate with only 11% of patients having a pain reduction of greater than 50% [16,17,18]. The usual therapy goal is a pain reduction of 30% [11,19]. Adequate pain control is not achieved, and the issue of pain recurrence or relapse despite initial therapeutic success remains. The first line of pharmacological treatments includes tricyclic anti-depressants, serotonin-norepinephrine reuptake inhibitors, and gabapentinoids, which all have unpleasant side effects and are not totally satisfactory [20]. Approximately 15% of patients do not recover within a year; pain remains ongoing [21]. Thus, a better understanding is needed to develop more effective non-opioid treatment approaches [22].

Central to the linkage of the pain circuitry with the limbic system is the activation of the locus coeruleus [23,24,25,26]. This small brain nucleus innervates almost every region of the central nervous system, including the spinal cord, and is the main producer of noradrenaline (also called norepinephrine) [27,28,29]. In healthy individuals, the locus coeruleus (LC) is a descending pain inhibitory system [30,31]. Activation of spinal noradrenergic α2 (NAα2) receptors reduces hypersensitivity in patients and animal models of neuropathic pain [32,33]. However, animal models of neuropathic pain have demonstrated that neuronal inhibition of LC neurons with lidocaine or their ablation with the neurotoxin conjugated antibody anti-dopamine-beta-hydroxylase-saporin (anti-DBH-saporin) is able to eliminate chronic somatic and trigeminal neuropathic pain [34,35,36]. A time course study of the efficacy of the endogenous noradrenergic (NA) system to inhibit pain following tibial nerve injury in rats revealed that by post-injury day 17, yohimbine, a selective NAα2 receptor antagonist, was no longer able to reverse endogenous NA pain inhibition [37]. While descending NA pain inhibition was effective during the acute phase, its efficacy decreased with time. Similarly, medial prefrontal cortex microinjection of benoxathian, a selective NAα1 receptor antagonist, but not idazoxan, a NAα2 receptor antagonist, administered in week 6 post infraorbital nerve chronic constriction injury model induction reduced mechanical hypersensitivity [36]. It has been proposed that in chronic neuropathic pain, the role of the LC shifts from pain inhibition via NAα2 receptors to pain facilitation through activation of NAα1 receptors [26].

The NAα1 receptors are Gq-coupled G-protein coupled receptor proteins made up of 3 subtypes (NAα1A, NAα1B, and NAα1D) which are all expressed within the central nervous system [38,39]. In the present study, we investigated the efficacy of doxazosin, a slow-release NAα1 receptor antagonist, for its ability to alleviate chronic mechanical hypersensitivity and its sequelae in a chronic trigeminal nerve neuropathic pain model. The preclinical foramen rotundum inflammatory constriction trigeminal infraorbital nerve (FRICT-ION) rat model closely models patients’ trigeminal neuropathy [40,41]. This minimally invasive pain model produces mechanical hypersensitivity persisting through at least 14 weeks, allowing the investigation of anxiety- and depression-like behaviors that develop after six weeks [42,43]. The present study investigated both male and female rats, since over 60% of patients with trigeminal neuropathy are women [1,44].

A dose–response study with doxazosin was initiated (0, 10, 30 mg/kg) to determine its effectiveness to alleviate chronic mechanical hypersensitivity and sequelae in naïve, sham, and FRICT-ION animals. A two-week time course of daily treatment with doxazosin was initiated in week 8 post chronic trigeminal pain model induction, the equivalent of over 5 human years of ongoing pain [45]. The effect on cardiac function was assessed. Furthermore, reduction in glial activation was determined in vivo and histologically. Circulating stress hormone cortisol and inflammatory cytokines were quantified. The expression of NAα1 receptor subtypes in the primary sensory cortex and hippocampus was determined immunohistochemically in doxazosin or vehicle-treated animals, with and without the chronic FRICT-ION model.

2. Results

2.1. Doxazosin Reduces Chronic Mechanical Hypersensitivity Was Induced by the FRICT-ION Model in Both Male and Female Rats

The FRICT-ION-model-induced mechanical hypersensitivity was measured in the whisker pad, the innervation territory of the infraorbital nerve, in both male and female animals. One week post FRICT-ION surgery, the mechanical withdrawal threshold was significantly reduced in both male (naïve 18.4 ± 0.2 g; FRICT-ION 2.5 ± 0.8 g; p < 0.001 two-way ANOVA with Tukey post hoc test) and female rats (naïve 18.0 ± 0.2 g; FRICT-ION 1.9 ± 0.4 g; p < 0.001 two-way ANOVA with Tukey post hoc test) compared to naïve control animals (Figure 1). Hypersensitivity was maximal in week 2 and remained so until experiments’ end in vehicle-treated FRICT-ION animals (week 2 male: naïve 18.4 ± 0.2 g; FRICT-ION 0.6 ± 0.2 g; female: naïve 18.6 ± 0.2 g; FRICT-ION 0.4 ± 0.1 g; p < 0.001 two-way ANOVA with Tukey post hoc test). Mechanical sensitivity of male and female control and sham surgery groups remained constant at those levels throughout the experiment.

In week 8, two-week daily oral treatment with doxazosin was started. In males (Figure 1A), treatment dose-dependently reduced mechanical hypersensitivity. Mechanical withdrawal thresholds of male FRICT-ION rats treated with 30 mg/kg doxazosin were not significantly different from those measured in control groups (week 10 male: naïve + vehicle 18.4 ± 0.2 g; FRICT-ION + 30 mg/kg 16.7 ± 0.9 g, p > 0.05). FRICT-ION animals treated with 10 mg/kg daily doxazosin had significantly decreased mechanical hypersensitivity compared to the vehicle-treated FRICT-ION group (FRICT-ION + 10 mg/kg 8.1 ± 1.3 g; FRICT + ION + vehicle 0.4 ± 0.2 g; p < 0.001). However, mechanical withdrawal thresholds remained significantly lower than those measured in control groups. Doxazosin did not alter mechanical sensitivity in the control groups. Only the highest dose of doxazosin was tested in females to reduce required animal numbers (Figure 1B). Female FRICT-ION rats treated with 30 mg/kg doxazosin had significantly increased mechanical withdrawal thresholds compared to the vehicle-treated FRICT-ION group (FRICT-ION + 30 mg/kg 16.1 ± 0.8 g; FRICT-ION + vehicle 0.3 ± 0.1 g; p < 0.001). Similar to males, doxazosin did not alter mechanical sensitivity in controls.

2.2. Neither Chronic Pain nor Treatment with Doxazosin Alters Cardiac Function

Cardiac ultrasound measurements were made at experiment’s end to determine drug safety (Table 1). No experimental group differences were detected when comparing either male or female animals. However, comparison of male and female animals showed that the stroke volume in males was significantly higher than in females (male 218.3 ± 4.0 µL, female 163.0 ± 2.6 µL; p > 0.001, unpaired t-test). Similarly, male rats weighed significantly more than female animals (male 403.6 ± 4.1 g; female 229.3 ± 1.9 g; p > 0.001, unpaired t-test).

2.3. Animals with FRICT-ION Displayed Anxiety-like Behavior in the Light–Dark Place Preference Test

The light–dark-place preference test was used to detect anxiety-like behavior. Vehicle-treated male rats with FRICT-ION spent significantly less time in the brightly illuminated light chamber compared to naïve male rats (FRICT-ION + vehicle 102 ± 32 s, naïve + vehicle 206 ± 14 s; p < 0.05, two-way ANOVA with Fisher’s LSD post hoc test; Figure 2A). Avoidance of the light chamber was dose-dependently decreased by treatment with doxazosin (FRICT-ION + 10 mg/kg 187 ± 37 s; FRICT-ION + 30 mg/kg 250 ± 11 s). Similarly, vehicle-treated female FRICT-ION rats spent significantly less time in the light compared to vehicle-treated naïve female rats (FRICT-ION + vehicle 111 ± 14 s, naïve + vehicle 169 ± 14 s; p < 0.05, two-way ANOVA with Fisher’s LSD post hoc test; Figure 2B). However, in females, this anxiety-like behavior was not improved by treatment with doxazosin (FRICT-ION + 30 mg/kg 159 ± 20 s).

Similar behavioral changes were seen for time spent rearing, an exploratory behavior quantified in the light chamber. Vehicle-treated male FRICT-ION rats spent significantly less time rearing compared to naïve male rats (FRICT-ION + vehicle 25.6 ± 11.3 s, naïve + vehicle 68.8 ± 10.0 s; p < 0.05, two-way ANOVA with Fisher’s LSD post hoc test (Figure 2C)). Treatment with doxazosin dose-dependently increased this exploratory behavior in male rats with FRICT-ION (FRICT-ION + 10 mg/kg, 62.2 ± 12.7 s; FRICT-ION + 30 mg/kg 79.3 ± 6.8 s; p < 0.05, two-way ANOVA with Fisher’s LSD post hoc test). Vehicle-treated female FRICT-ION rats also spent significantly less time rearing than vehicle-treated naïve female rats, yet this was not significant (FRICT-ION + vehicle 30.9 ± 5.2 s, naïve + vehicle 51.9 ± 5.9 s). Treatment with doxazosin had no effect (Figure 2D). Group comparison of the number of rearing events trended to be similar to the other test paradigms. Male and female vehicle-treated animals with FRICT-ION reared significantly less often compared to vehicle-treated naïve animals (male: FRICT-ION + vehicle 11.2 ± 3.7, naïve + vehicle 20.5 ± 2.8; female: FRICT-ION + vehicle 12.0 ± 1.6, naïve + vehicle 18.0 ± 1.5). Treatment with doxazosin did not significantly increase this exploratory behavior (Figure 2E,F).

2.4. Only Male Animals with FRICT-ION Displayed Anxiety-like Behavior in the Elevated Zero Maze

Anxiety-like behavior was also quantified using the elevated zero test. Similar to the light–dark-place preference test, vehicle-treated male rats with FRICT-ION spent significantly less time in the exposed open quadrants compared to vehicle-treated naïve male rats (FRICT-ION + vehicle 75.6 ± 11.0 s, naïve + vehicle 106.6 ± 15.5 s; p < 0.05, two-way ANOVA with Fisher’s LSD post hoc test; Figure 3A). Doxazosin treatment (10 mg/kg) of male rats with FRICT-ION significantly increased open quadrant occupancy while the higher dose had no significant effect (FRICT-ION + 10 mg/kg 108.9 ± 10.9 s; FRICT-ION + 30 mg/kg 92.5 ± 16.7 s). Vehicle-treated female rats with FRICT-ION tended to spend less time in the open quadrants (FRICT-ION + vehicle 82.6 ± 12.6 s, naïve + vehicle 110.9 ± 9.8 s), yet this was not significant (p = 0.0805; Figure 3B). Treatment with doxazosin had no effect. No significant group differences were detected when comparing the number of open quadrant entries (Figure 3C,D).

2.5. Long-Term Memory Tested with the NOR Was Not Altered by FRICT-ION Pain Model

Novel object recognition (NOR), a test during which animals are exposed to a familiar and a novel object in a testing arena, was used to examine long-term memory retention [46,47]. In male animals, the recognition index (RI), the ratio of time spent exploring the novel object and total exploratory time, was not significantly different when comparing groups. Treatment with the highest dose of doxazosin resulted in slight RI increases, but this was not significant (Figure 4A). Vehicle-treated naïve and FRICT-ION female rats had significantly lower RI when compared to their male counterparts (Figure 4B; p < 0.05, one-way ANOVA with Fisher’s LSD post hoc test). Similar to male rats, the trigeminal pain model did not alter the RI in females. However, naïve female rats treated with doxazosin had a significantly higher RI than vehicle-treated ones (p < 0.05, one-way ANOVA with Fisher’s LSD post hoc test). Doxazosin treatment of female FRICT-ION animals increased the RI when compared to vehicle-treated female FRICT-ION rats, but this was not significant. Total exploratory time was compared to elucidate if the FRICT-ION model or drug altered animal engagement with the objects. In males, neither surgical group nor drug treatment altered the total exploratory time (Figure 4C). Vehicle-treated naïve male animals spent significantly more time exploring the objects than vehicle-treated naïve female rats (Figure 4D, p < 0.05, one-way ANOVA with Fisher’s LSD post hoc test). In female rats, treatment with doxazosin increased exploratory time significantly in FRICT-ION animals.

2.6. In Vivo Measurement of Increased Activated Brain Glia in Male Rats with FRICT-ION Was Reduced by Doxazosin Treatment

Cathepsin B is typically localized in endo-/lysosomal cell organelles in glial cells at rest. Activated glial cells in the brain express cathepsin B extracellularly, allowing for transcranial in vivo visualization after injection of a protease-specific, activatable near-infrared-fluorescence (NIRF) imaging agent. Vehicle-treated male rats with FRICT-ION had significantly increased NIRF compared to naïve and sham groups (FRICT-ION + vehicle 3.8 × 10^8^ ± 1.3 × 10^8^, naïve + vehicle 2.8 × 10^7^ ± 1.1 × 10^6^; p < 0.05, two-way ANOVA with Fisher’s LSD post hoc test; Figure 5A). Treatment with doxazosin dose-dependently decreased the NIRF of male animals to naïve levels (FRICT-ION + 10 mg/kg 1.2 × 10^8^ ± 5.4 × 10^6^; FRICT-ION + 30 mg/kg 3.1 × 10^7^ ± 1.5 × 10^6^; p < 0.05, two-way ANOVA with Fisher’s LSD post hoc test). Figure 5B shows sample images overlaying photographs of male FRICT-ION rats treated with different concentrations of doxazosin. Female rats were not tested due to irreparable failure of the IVIS Illumina Optical Imaging System.

2.7. Increased Astrocyte Biomarker GFAP in Primary Somatosensory Cortex and Hippocampus in Animals with FRICT-ION Was Decreased by Prolonged Treatment with Doxazosin

The primary somatosensory cortex was assessed divided into three regions, outer (layers II and III), middle (layer IV) and inner (layers V and VI), for quantitative immunohistochemical analyses. The outer regions had the highest intensity of GFAP staining, and a significant increase in GFAP staining in vehicle-treated male and female rats with FRICT-ION was noted when compared to naïve control animals (Figure 6). Treatment with doxazosin significantly decreased GFAP staining intensity, but only in females. Middle and inner regions of the primary somatosensory cortex had no statistical differences. Similarly, GFAP staining was significantly increased in the hippocampus depending on region (Figure 7D). In the dentate gyrus, vehicle-treated male and female rats with FRICT-ION had significantly increased GFAP expression, which was decreased by treatment with doxazosin (Figure 7A). Staining for GFAP was also significantly increased in the CA1 region, but doxazosin treatment decreased this only in females (Figure 7B). In the CA3 region, GFAP staining was only increased in females, and treatment with doxazosin did not decrease this (Figure 7C).

2.8. Expression of NAα1B Receptor in the Primary Somatosensory Cortex and Hippocampus Is Sex-Dependent

The expression of NAα1B was significantly greater in males than in females in the primary somatosensory cortex (Figure 8A,C) and the hippocampus (Figure 8E,G). No experimental group differences were detected within each gender. In contrast, the expression of NAα1A and NAα1D in the primary somatosensory cortex (Figure 8A–D) and the hippocampus (Figure 8E–H) showed neither experimental group nor gender differences.

2.9. Investigated Circulatory Signaling Molecules Were Minimally Altered by the FRICT-ION Chronic Pain Model

Blood samples collected perimortem were tested for cortisol concentrations in the serum. No significant differences were detected between experimental groups of each gender (Figure 9A). However, as previously reported, cortisol concentrations measured in female samples were significantly higher than in samples from males (male naïve + vehicle 33.8 ± 1.2 ng/mL, female naïve + vehicle 76.9 ± 8.3 ng/mL; Figure 9A,B).

Cytokine levels in the serum were analyzed to determine any potential group differences. Serum samples from male rats were probed using a cytokine test kit simultaneously sampling 29 different chemokines/cytokines. The proinflammatory mediators L-selectin and CXCL7, also known as thymus chemokine, were found to be significantly increased in vehicle-treated male rats with FRICT-ION compared to vehicle-treated naïve male rats. Treatment with doxazosin did not decrease elevated L-selectin levels in male rats with FRICT-ION, but did significantly decrease CXCL7 levels (Table 2).

Females were tested with a larger cytokine kit sampling 79 different signaling molecules to see if this would allow the detection of more differences between experimental groups to identify inflammatory mediators altered in the chronic pain model. In the serum from vehicle-treated female rats with FRICT-ION, only adiponectin (Acrp30), insulin-like growth factor binding protein 3 (IGFBP-3), and lipopolysaccharide-induced CXC chemokine (LIX) were significantly increased when compared to vehicle-treated naïve female samples (Table 3).

3. Discussion

The chronic constriction model of infraorbital nerve pain has been used to study trigeminal neuropathic pain for over 30 years [48]. Despite this, less than 10% of the studies have included female animals even though over 60% of patients with trigeminal neuropathic pain are female [1]. Patients with chronic pain have a higher-than-average suicide rate, with approximately 32% of patients reporting suicidal ideation and 5–14% attempting suicide [49,50]. It is imperative that new, non-opioid treatments for chronic pain are discovered and approved for use. In the present study, an emphasis was placed on comparing male and female results. Similar to previous studies with mice [51], we found no difference when comparing the mechanical hypersensitivity of male and female animals with FRICT-ION. In both genders, the model persists for at least 10 weeks and the intraoral model induction allows group blinding [40,41]. Treatment was started in week 8 after model induction, an equivalent of 5–10 years in humans [45], providing cumulatively increasing efficacy of doxazosin to decrease mechanical hypersensitivity in both genders. This is similar to clinical patients suffering from post-traumatic stress disorder (PTSD) where reports describe significant improvements for some patients in as few as 5 days in 8-week daily treatment studies [52,53]. Little is known about potential gender differences when using prazosin. It can only be postulated that there are little to no differences for reducing symptoms of PTSD [46] or touch sensitivity in patients with complex regional pain syndrome [47], as these studies combined results from primarily males and a few female participants. However, this is in contrast to other animal studies that investigated pain mechanisms at acute time points when anxiety-like behaviors are not yet developed [54,55].

In the present study, doxazosin was dosed based on body weight, which was measured every 2–3 days, since adult male rats can weigh almost twice as much as females (Table 1). Prolonged use of doxazosin did not affect cardiac function. Increased stroke volume in males compared to females reflects their increased body weight (Table 1). A dose–response curve for doxazosin was established in male rats and only the highest, most efficient drug concentration was used in females in order to reduce the number of animals duplicated in this study. Systemic treatment with doxazosin (30 mg/kg bodyweight) increased the mechanical withdrawal threshold, thus decreasing mechanical hypersensitivity with similar efficacy in both male and female rats (Figure 1). A previous study ineffectively used prazosin, a related NAα1 receptor inhibitor, peripherally at a lower dose at 3 weeks post spinal nerve ligation [56], suggesting that the concentration was too low. Also likely is that the site of action for NAα1 that needs to be inhibited is not peripheral, but is in the central nervous system.

The influence of the estrous cycle on anxiety test results in rats is not clear. There is a report that naïve female rats in diestrus showed anxiety-like behavior in the elevated T-maze [57], while newer studies reported that the estrous cycle did not influence anxiety-like behavior in the elevated zero maze in Sprague Dawley rats [58,59]. In the present study, the estrous cycle of females was not determined. Tests to quantify behavioral sequelae to chronic hypersensitivity started in week 8 of our study, well after the reported onset of anxiety-like behaviors in week 6 [42,43]. Group comparisons showed differences in some of the cognition-dependent behavioral test results between male and female experimental groups. In both anxiety tests employed, light–dark-place preference and elevated zero maze tests (Figure 2 and Figure 3), no sex differences were detected in vehicle-treated naïve animals nor vehicle-treated animals with FRICT-ION. This is in contrast to other studies that, depending on the anxiety test, have reported naïve female rats to be less anxious/fearful than male animals [58,60]. Vehicle-treated male and female rats with FRICT-ION spent significantly less time in the “dangerous” highly illuminated or exposed areas compared to vehicle-treated naïve animals of the same gender. However, while doxazosin alleviated this anxiety behavior in male rats, it was not effective in female animals (Figure 2 and Figure 3). This could be due to NAα1 receptor expression sex differences identified here in the primary sensory cortex and hippocampus (Figure 8). The NAα1B receptor subtype was expressed significantly less in females compared to males in both investigated brain regions, and NAα1D receptor had significantly lower expression in the hippocampus of female rats. The images suggest that expression is localized on neurons, but could also be on astrocytes. This finding is similar to a previous study that investigated the expression of alpha-adrenergic receptors using radioligand binding [61]. Surprisingly, the expression patterns of the NAα1 receptor subtypes were not altered by the FRICT-ION injury nor by treatment with doxazosin. This could explain why many patients with PTSD, who stop their treatment due to being asymptomatic, quickly develop symptoms of PTSD again [62]. This also suggests that at least when treating anxiety in clinical patients, NAα1 receptor antagonists are not able to completely reverse the underlying issue. However, a recent case report on three patients who discontinued their PTSD treatment without going into remission contradicts this [63]. One possible mechanism for NAα1 receptor antagonists to reduce dysfunctional brain activity in both neuropathic pain and PTSD is through astrocytes. In neuropathic pain, an increase in activated astrocytes has been reported in multiple brain regions, including the primary somatosensory cortex and hippocampus [64,65]. Immunohistochemical studies for GFAP presented here demonstrated that treatment with doxazosin was able to reduce increased staining for GFAP in both male and female animals (Figure 6 and Figure 7). It is possible that increased GFAP immunoreactivity in vehicle-treated animals with FRICT-ION was caused by astrocytic hypertrophy as opposed to cell proliferation, as in a previous study of neuropathic pain [66]. Future studies will have to discern this. Astrocytes have been reported to express NAα1 receptors. Their stimulation with adrenergic compounds has been shown to be neuromodulatory and to form a bidirectional feedback loop between neurons and astrocytes in the brain [67,68]. Their inhibition through doxazosin may have reduced anxiety-like behavior by interrupting adrenergic signaling at heterosynaptic interactions in the dentate gyrus [69] or another limbic region.

Not surprisingly, we did not see major changes in circulating inflammatory mediators or cortisol. In response to a traumatic brain injury, cytokines are released during the early, acute phase and subside within a week of an insult so that tissue repair and remodeling can occur [70]. Males were analyzed first using the smaller cytokine profiler kit, simultaneously assaying 29 cytokines and chemokines trying to identify changes specific to chronic trigeminal neuropathic pain (Table 2). However, only L-selectin/CD62L, a cell adhesion molecule, expressed on leukocytes [71], and CXCL7, a neutrophil chemoattractant, were found to be significantly increased in the male vehicle-treated FRICT-ION group. Both cytokines have been shown in an acute lower back pain model to be increased after soft tissue manipulation for pain control [72]. Female samples were analyzed using the larger cytokine kit, simultaneously assaying 79 cytokines and chemokines to identify changes in animals with neuropathic pain, and thus potential pharmacologic targets for the treatment of chronic pain (Table 3). While L-selectin/CD62L was not included, sampling of CXCL7 showed no experimental group differences in female rats. Prolactin, a mediator shown to be involved in postoperative pain in females [73], was not elevated at chronic timepoints in the FRICT-ION model. This suggests a role for prolactin in the transition from acute to chronic pain but not in the maintenance of chronic pain.

In the females, two mediators stood out, adiponectin/Acrp30 and IGFBP-3. Both showed a slight increase in the vehicle-treated FRICT-ION animals compared to vehicle-treated naïves, and were doubled in both doxazosin treated groups, identifying a potential anti-inflammatory mechanism activated by doxazosin that may be female-specific [74,75]. Increased levels of proinflammatory LIX in females with vehicle-treated FRICT-ION compared to vehicle-treated naïve females may offer a novel treatment target for chronic pain [76]. However, future studies will have to investigate this.

Similarly, no experimental group differences were noted for the stress hormone cortisol (Figure 9). Cortisol increases in acute pain, but in chronic pain, circulating cortisol levels normalize [77]. Female rats had significantly higher serum cortisol concentrations than males, reflecting its regulation by sex hormones [78].

4. Materials and Methods

4.1. Animals

All animal experiments were approved by the New Mexico Veteran’s Administration Health Care System Animal Component of Research Protocol (ACORP #20-A322) and performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). Adult Sprague Dawley rats (5–6 weeks, 125–149 g, Envigo [now Inotiv], Indianapolis, IN, USA) were housed on a 12/12 h reverse light cycle and fed a soy-protein-free diet (Teklad #2920x, Inotiv) ad libitum. Baseline behavioral tests started after 2-week acclimation to the reverse light cycle. All experimental procedures occurred during the animal’s active dark cycle under red light.

4.2. FRICT-ION Pain Model Induction

As previously described [40,41], animals were anesthetized with inhalant isoflurane (induction: 5% isoflurane in 0.8 L/min oxygen, maintenance: 3.5–4.0% isoflurane in 0.8 L/min oxygen), immobilized in a supine position, and the mouth fixed open with a cotton thread across the frontal incisors. A small, 2 mm incision was made in the buccal margin crease anterior to the sphenomandibular ligament. The infraorbital nerve (ION) was exposed (sham surgery), and in the FRICT-ION group animals, a piece of 4-0 chromic gut suture (4 mm) was inserted into the foramen rotundum along the ION. The suture, coated in chromium salt, likely causes both mechanical and chemical irritation of the ION, thus producing chronic neuropathic pain.

4.3. In Vivo Drug Treatment

Starting in week 8 post model induction, male animals were treated with daily doxazosin (0, 10, and 30 mg/kg bodyweight) per os (p.o.). The drug was dissolved in water and mixed with 1 g of regular chow (Teklad #2920x) ground and formed into a readily eaten “chow cookie” for non-invasive, low-stress oral dosing [79]. Behavioral tests were performed 1 h post drug dosing, on different days to avoid stress. Studies were first conducted with male rats and a dose–response curve established. Only the highest concentration of doxazosin was tested in females to reduce the number of required animals.

4.4. Behavioral Assays

4.4.1. Reflexive Mechanical Sensitivity

Mechanical sensitivity was measured on the whisker pad, the innervation territory of the ION, prior to surgery at baseline and weekly thereafter using the up-down von Frey method as previously described [36,80]. Briefly, animals were restrained by being gently wrapped in a towel. A series of 8 von Frey filaments (0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0, and 15.0 g) (Stoelting, Wood Dale, IL, USA) was used to assess the mechanical sensitivity [81]. Each filament was applied five times perpendicular to the whiskerpad without touching the vibrissae. If the animal withdrew its head at least 3 out of 5 times, the next weaker filament was used. If the response was negative, the next higher filament was applied. A curve-fitting algorithm was then used to determine the mechanical withdrawal threshold, the minimal amount of force required to elicit a response 50% of the time [82].

4.4.2. Cognitive Dependent Anxiety Tests

Animals were acclimated to the behavioral testing rooms in their home cages for 1 h. All animals were tested individually and their behaviors video recorded for off-line analyses by an investigator blinded to animal identity using EthoVision XT 17 software (Noldus, Leesburg, VA, USA).

4.4.3. Light–Dark-Place Preference Test for Anxiety-like Behaviors

The test box (total size 27 × 27 × 27 cm) consisted of two chambers of equal size, a dark one and a brightly illuminated one (700 lumen lamp). The chambers were connected by a small opening (6.5 × 6.5 cm) so that the animal tested could freely move between them. All rats started in the bright chamber. They were recorded for 10 min, after which they were returned to their home cage. Post hoc video analysis quantified the number of entries into the light chamber defined as all 4 paws placed within it, as well as occupancy duration, number of rearing events and time spent rearing [83]. Avoidance of the light chamber is a measure of increased anxiety-like behavior.

4.4.4. Anxiety-like Behaviors in the Elevated Zero Maze Test

The elevated zero maze consisted of a circular runway (10 cm width; 100 cm total diameter) elevated 60 cm above the ground. It was divided into equal-sized quadrants, two closed ones with high, protective walls (30 cm high) at the perimeter, and two open ones, lacking barriers (Maze Engineers, Skokie, IL, USA). The assay was started by placing each animal on the runway of an open quadrant. The animal was allowed to move freely for 5 min while it was recorded and then returned to its home cage. One male rat was eliminated from the results as he repeatedly fell off the track in an open quadrant, losing his balance while trying to turn around. Time spent in open and closed quadrants was quantified using EthoVision XT software for group comparisons. Avoidance of the open quadrants is a measure of increased anxiety-like behavior [84].

4.4.5. Novel Object Recognition Test

The novel object recognition test (NOR) was conducted in an open field arena (40 × 40 cm) using two objects that differed in size, shape, and color [85,86]. Animals were acclimated to the testing arena. On the following day during the training phase, animals were placed in the arena with two identical objects placed into opposite corners and allowed to explore for 15 min each. Four hours later during the test phase, animals were returned to the testing arena with one novel and one familiar object placed in the same locations where the two identical objects had been. Animals were allowed to explore for 5 min each and then returned to their home cage. All behavior was video recorded for post hoc analysis. Time spent with novel object and total exploratory time were measured using EthoVision XT 17 software for group comparisons. The recognition index (RI) was calculated by dividing the time spent investigating the novel object by the total exploratory time. An RI of >50 means that the animal spent more time with the novel object than the familiar one, indicating typical curiosity.

4.5. Cardiac Ultrasounds

All rats were imaged at experiment’s end using the Vevo 3100 high-resolution small animal ultrasound system (FUJIFILM VisualSonics, Bothell, WA, USA). Animals were anesthetized with inhalant isoflurane (induction: 5% isoflurane in 0.8 L/min oxygen, maintenance: 3.5–4.0% isoflurane in 0.8 L/min oxygen), fixed in a supine position on the heated (41 °C) recording platform, and hair from the thorax was removed with depilatory cream. Recordings were conducted using a 21 MHz center frequency linear array ultrasound transducer (15–30 MHz bandwidth; MX250). The probe was positioned lateral to the sternum. The short axis (SAX) view images were taken so that both papillary muscles were visible. Parasternal long axis (PSLAX) view images were taken when the full endocardial length was clearly visible. M- and B-mode sequences of around 300 frames were taken for offline analyses by a scientist blinded to animal identity.

4.6. In Vivo Extracellular Cathepsin B on Activated CNS Glia Imaging

At experiment’s end just prior to euthanasia, extracellular cathepsin B, an in vivo biomarker of activated glial cells, was labeled with the activatable near-infrared fluorescent compound CatB750 FAST (PerkinElmer, Waltham, MA, USA) and imaged in the living animal [87]. Animals were anesthetized with isoflurane (induction: 5% isoflurane in 0.8 L O_2_; maintenance: 3.5–4.5% isoflurane in 0.8 L O_2_), their head and neck were shaved, and they were disinfected with povidone-iodine, followed by 70% ethanol. Animals were placed in a prone position, the neck hyperextended, and the cisterna cerebellomedullaris, located between the atlas and the skull’s occipital bone, was identified with manual palpation. The imaging probe, 50 μL CatB750 FAST (100 pmol/0.2 kg), was diluted in sterile phosphate-buffered saline (PBS) in accordance with manufacturer’s instructions, and slowly injected into the cerebral spinal fluid through the cisterna cerebellomedullaris with a needle (26-gauge) held in place for an additional 30 s to prevent leakage [88]. The animal was allowed to recover completely in its home cage before being returned to the housing room.

After 24 h, animals were scanned using the IVIS Illumina Optical Imaging System (PerkinElmer) while under inhalant anesthesia (induction: 5% isoflurane in 0.8 L O_2_; maintenance: 3.5–4.5% isoflurane in 0.8 L O_2_). Images were acquired and analyzed with the Living Image 4.3.1 software (Perkin Elmer). The fluorescent lamp was set to high, excitation filter 745 nm, emission filter indocyanine green (a dye-emitting fluorescence at 750–950 nm), with an exposure time of 20 s. Each image consisted of the detected near-infrared fluorescent (NIRF) signal overlaying a photograph of the rat’s head. A signal region of interest (ROI) was drawn over the cerebrum to measure the NIRF efficiency signal, and background was measured with an ROI placed on an ear and subtracted from the signal measurement. Imaging experiments were only conducted in male rats because the IVIS Illumina Optical Imaging System was broken beyond repair before female rats could be studied.

4.7. Postmortem Tissue Collection and Analyses

Animals were deeply euthanized with inhalant isoflurane (5% isoflurane in 0.8 L/min O_2_) and blood collected transcardially from all animals using a 5 mL syringe with a 25-gauge needle. Each experimental group was subdivided into two groups for different postmortem analyses. Animals used for fresh frozen tissue assays were transcardially perfused with cold phosphate buffer, and the CNS regions of interest and trigeminal ganglia were dissected, snap frozen, and stored at −80 °C until use. Rats used for immunohistochemistry were transcardially perfused with phosphate buffer followed by cold 4% paraformaldehyde. Trigeminal ganglia (TG) and CNS were dissected, cryoprotected with 30% sucrose overnight, tissue blocked, embedded in tissue-plus O.C.T. compound (Fisher Scientific, Waltham, MA, USA), and stored at −80 °C until use.

4.7.1. Brain Section Immunohistochemistry

Coronal brain slices (40 μm thickness) at approximately bregma −3.14 mm were sectioned with the cryostat and collected in PBS. Free-floating sections were washed, blocked, and reacted with either rabbit anti-NAα1A (1:1000; Cat. # AB137123, Abcam, Waltham, MA, USA), goat anti-NAα1D (1:1000; Cat. # AB166925, Abcam), and guinea pig anti-NeuN (1:2000; Cat. # ABN90; EMD Millipore Sigma, St. Louis, MO, USA) or rabbit anti-NAα1B (1:1000; Cat. # AB169523, Abcam) and chicken anti-GFAP (1:2500; Cat. # 4674, Abcam). Primary antibodies were visualized using a combination of either goat anti-rabbit conjugated to Alexa Fluor 568 (1:1000; Cat. # A-11011, Thermo Fisher Scientific, Waltham, MA, USA), donkey anti-goat conjugated to Alexa Fluor 488 (1:1000; Cat. # A-11055, Thermo Fisher Scientific), and goat anti-guinea pig conjugated to Alexa Fluor 647 (Cat. # A-21450, Thermo Fisher Scientific) or goat anti-rabbit conjugated to Alexa Fluor 568 (1:1000) and goat anti-chicken conjugated to Alexa Fluor 488 (1:1000; Cat. # A-11039, Thermo Fisher Scientific). Tissue was coverslipped using Fluoromount-G mounting medium with DAPI (Cat. # 00-4959-52, Thermo Fisher Scientific). Slides were imaged using the Olympus Fluoview FV1200 laser scanning confocal microscope and software (Olympus Scientific, Waltham, MA, USA). Stitched images of optical stacks (z = 2 μm) were acquired with a 10x objective to include the entire brain region of interest. Images were z-projected and analyzed with NIH ImageJ [89]. All images were taken using identical laser settings. The primary somatosensory cortex was divided into 3 regions, outer (layers II and III), middle (layer IV) and inner (Layers V and VI) for quantitative immunohistochemical analyses and comparisons. The hippocampus was divided into dentate gyrus (DG), CA1, and CA3. ROIs were placed to exclude edge artifacts.

4.7.2. Serum Cytokine Detection

Serum cytokine levels in male rats were quantified using the Proteome Profiler Rat Cytokine Array Kit, Panel A (Cat. # ARY008; R&D Systems, Minneapolis, MN, USA) that simultaneously detects 29 different cytokines following the manufacturer’s protocol. Serum samples from female rats were analyzed using the Proteome Profiler Rat XL Cytokine Array (Cat. # ARY030; R&D Systems), which simultaneously detects 79 different cytokines. Briefly, 40 µg serum protein from each individual rat was sampled. A single kit was used to simultaneously sample 4 different experimental groups (naïve + vehicle, naïve + 30 mg/kg doxazosin, FRICT-ION + vehicle, and FRICT-ION + 30 mg/kg doxazosin). Blots were visualized using IRDye secondary antibodies, imaged with the Odyssey Fc Imager, and signal intensity measured using Image Studio 5.2 Software (LI-COR).

4.7.3. Serum Cortisol Measurements

Circulating cortisol levels were measured to determine if animals with chronic pain from the FRICT-ION model showed signs of stress [90]. The Cortisol Parameter Assay Kit (Cat. # KGE008B, R&D Systems, Minneapolis, MN, USA) was used in accordance with the manufacturer. In brief, serum samples (10 µL/rat in duplicate) and samples for a standard curve were subjected to ELISA. Sample absorbance was measured at 450 nm with the Synergy HTX multimode plate reader (BioTek/Agilent, Santa Clara, CA, USA). A standard curve was generated with a four-parameter logistic (4-PL) curve-fit and the cortisol sample concentration calculated using GraphPad Prism v10.6.1 software (Dotmatics, Boston, MA, USA).

4.8. Statistical Analysis

All data are presented as mean ± standard error of the mean (SEM). Depending on experimental design, data were analyzed with either Student’s t-test, one-way and two-way ANOVA with Tukey, or LSD post hoc test using GraphPad Prism v10.6.1 software (Dotmatics, Boston, MA, USA). No data points were excluded. There were no statistical outliers. A p value of p ≤ 0.05 was considered significant.

5. Conclusions

The present study demonstrates for the first time that the NAα1 receptor antagonist doxazosin can reduce chronic trigeminal neuropathic pain and resulting sequelae in a chronic rodent model with major sex differences. Dysfunctionally strengthened heterosynaptic interactions between astrocytes and neurons in noradrenergic LC target regions in the somatosensory cortex and hippocampus are inhibited by doxazosin, resulting in improved noradrenergic balance and diminished pain-related behavioral measures. Also noted was doxazosin reduction in the elevated adiponectin and IGFBP-3 levels observed in the untreated rats with chronic trigeminal neuropathic pain. The main goal of the study was accomplished with demonstration of LC-noradrenergic signaling, α1 receptor activity, and glia-neuron interactions. Results taken at 10 weeks in this chronic model differ from acute model results reported in the literature. Much additional evidence is required to provide a more direct causal relationship.