Suppression of the Excitability of Nociceptive Secondary Sensory Neurons Following Systemic Administration of Astaxanthin in Rats

Abstract

Although astaxanthin (AST) has demonstrated a modulatory effect on voltage-gated Ca²⁺ (Cav) channels and excitatory glutamate neuronal transmission in vitro, particularly on the excitability of nociceptive sensory neurons, its action in vivo remains to be determined. This research sought to determine if an acute intravenous administration of AST in rats reduces the excitability of wide-dynamic range (WDR) spinal trigeminal nucleus caudalis (SpVc) neurons in response to nociceptive and non-nociceptive mechanical stimulation in vivo. In anesthetized rats, extracellular single-unit recordings were carried out on SpVc neurons following mechanical stimulation of the orofacial area. The average firing rate of SpVc WDR neurons in response to both gentle and painful mechanical stimuli significantly and dose-dependently decreased after the application of AST (1–5 mM, i.v.), and maximum suppression of discharge frequency for both non-noxious and nociceptive mechanical stimuli occurred within 10 min. These suppressive effects persisted for about 20 min. These results suggest that acute intravenous AST administration suppresses the SpVc nociceptive transmission, possibly by inhibiting Cav channels and excitatory glutamate neuronal transmission, implicating AST as a potential therapeutic agent for the treatment of trigeminal nociceptive pain without side effects.

1. Introduction

The trigeminal ganglion (TG) transmits sensory information from the orofacial region that is innervated by small-diameter Aδ fibers and unmyelinated C fibers, to second-order neurons in the spinal trigeminal nucleus (SpV) [1]. The SpV caudalis (SpVc) serves as a crucial intermediary for the transmission of pain signals [1,2,3]. Two distinct types of nociceptive neurons are present in the superficial dorsal horn of the spinal cord: nociceptive-specific (NS) neurons and wide-dynamic-range (WDR) neurons [1,2,3]. The trigeminal primary afferents, both nociceptive (Aδ-/C-) and non-nociceptive (Aβ-), that end in the deep layers (IV–V) of WDR neurons, show responses to noxious and non-noxious stimulation [1,2]. The application of graded nociceptive stimuli to the most sensitive area within the receptive field leads to a rise in firing frequency that is proportional to the stimulation, suggesting that WDR neurons encode stimulus intensity [1,2]. The WDR neurons located in the SpVc region are crucial for the development of hyperalgesia, allodynia, and referred pain linked to orofacial pain. This is because these neurons frequently receive input from the tooth pulp, jaw muscles, and facial skin and masseter muscles, and exhibit notable alterations in excitability following tissue injury or inflammation [1,4,5].

Astaxanthin (AST) is a naturally occurring carotenoid widely distributed in a variety of living organisms, such as plants, microalgae, crustacean shells (crabs, shrimps) and salmon [6]. AST is a more powerful antioxidant than other carotenoids, including lutein and zeaxanthin [7,8,9] and has many biological activities, including anti-inflammatory, anti-tumor, anti-diabetic and immunomodulatory effects [10,11,12,13]. A previous in vitro study reported that AST reduced the release of glutamate from rat cortical synaptosomes in a dose-dependent manner by inhibiting presynaptic voltage-gated Ca²⁺ channels (Cav) and the mitogen activated protein kinase (MAPK) signaling cascade [14]. A recent study indicated that AST administration ameliorated neuropathic pain by antagonizing the effect of n-methyl-D-aspartate (NMDA) glutamate receptors [15]. Furthermore, studies have demonstrated that AST is capable of crossing the blood–brain barrier [16]. Based on these findings, we hypothesized that intravenous AST administration would attenuate the noxious stimulation-induced excitability of SpVc WDR neuronal activity through central mechanisms, as is the case for analgesic drugs. Nonetheless, the immediate impact of AST on trigeminal neuronal activity when subjected to both painful and non-painful mechanical stimuli in vivo has not yet been clarified. Thus, the aim of the present study was to investigate whether acute intravenous administration of AST in rats could attenuate the excitability of nociceptive SpVc WDR neuronal activity in vivo in response to mechanical stimulation. In the present study, we found that the mean firing frequency of SpVc WDR neurons in response to mechanical stimuli was inhibited by AST in a dose-dependent and reversible manner, implicating AST as a potential therapeutic agent for the treatment of trigeminal nociceptive pain without side effects.

2. Materials and Methods

The Animal Use and Care Committee of Azabu University granted approval for the experiments (No. 200529-3) and these were performed in accordance with the ethical guidelines of the International Association for the Study of Pain [17]. All possible measures were taken to reduce the number of animals used and to alleviate their suffering.

2.1. Recording Single Units of WDR Neuron Activity Extracellularly in the SpVc

Adult male Wistar rats, with weights ranging from 215 to 255 g, were kept under constant lighting conditions (lights on: 07:00–19:00). The room temperature was maintained at 23 ± 1 °C. Food and water were provided ad libitum. We carried out electrophysiological assessments on 11 rats. Every rat was sedated with 3–5% isoflurane and sustained with further administration of a combined anesthetic (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam and 5.0 mg/kg of butorphanol) at appropriate doses (as judged from the presence or absence of a flexion reflex) as required, through a cannula into the jugular vein. Throughout the recording, the anesthesia level was validated by the absence of any response when the paw was pinched. The rectal temperature was maintained at 37.0 ± 0.5 °C with a homeothermic blanket (Temperature Controller, 40-90-8D; FHC, Bowdoin, TX, USA) during recording. Throughout the experiments, a local anesthetic, 2% lidocaine (xylocaine), was continuously applied to all the wound borders. The animals were then placed in a stereotaxic apparatus (SR-50; Narishige, Tokyo, Japan), and the neck muscles were divided along the midline. By making an incision in the atlanto-occipital ligament and the dura mater below, the medullary brain stem was exposed. Extracellular recordings of single-unit activity in the SpVc area were obtained using a tungsten microelectrode (3–5 MΩ impedance) placed into the ipsilateral side and advanced or retracted in 10 μm steps using a micromanipulator (SM-11 and MO-10; Narishige), according to the stereotaxic coordinates of the rat brain atlas of Paxinos and Watson [18]. Neuronal activity was amplified (DAM80; World Precision Instruments, Sarasota, FL, USA), filtered (0.3–10 KHz), monitored with an oscilloscope (SS-7672; Iwatsu, Tokyo, Japan), and recorded for off-line analysis by PowerLab and Chart 5 software (ADI Instruments, Oxford, UK) as described previously [19,20].

2.2. Procedures for Carrying Out Electrophysiological Experiments

The response of extracellular single-unit SpVc WDR activity to mechanical stimulation of the whisker pad, was examined using a series of procedures. To avoid sensitization of peripheral mechanoreceptors, a paint brush was quickly used as a search stimulus to identify the approximate area of the receptive field on the left side of the whisker pad. Subsequently, single units responding to a series of von Frey hairs were investigated on the left side of the whisker pad (Semmes–Weinstein Monofilaments, North Coast Medical, Morgan Hill, CA, USA) with non-noxious (2, 6, and 10 g) and noxious (15, 26, and 60 g) mechanical stimulation for 5 s at intervals of 5 s [19,20]. In this study, we identified the criteria for WDR neurons as follows: graded non-noxious and noxious mechanical stimulation applied to the receptive field produces an increased firing frequency in proportion to stimulus intensity. Following the identification of nociceptive SpVc WDR neurons that respond to the whisker pad, we measured the threshold for mechanical stimulation and the size of the receptive field. The mechanical receptive field of neurons was identified by applying von Frey hairs to the facial skin, and then traced onto a life-sized illustration of the face [19,20].

To quantify the WDR neuronal discharges triggered by mechanical stimulation, the base line activity was subtracted from the activity induced by the stimulus. Spontaneous discharge frequencies were determined for 2–5 min.

Previous research has shown that WDR neurons in the SpVc area are crucial in the mechanism responsible for hyperalgesia and referred pain associated with orofacial pain [1,4,5]. The main objective of the current research was to analyze the impact of AST on nociceptive SpVc WDR neuronal functions, without considering NS neurons. Post-stimulus histograms (bin = 100 ms) were generated for each stimulus. The impact of administering AST intravenously (0.2 mL of 1 mM and 5 mM) using a Hamilton microsyringe (Sigma-Aldrich, Milano, Italy), was assessed and evaluated 5, 10, 15, 20, 25, and 30 min after administration because the peak effect and recovery were thought to occur within this timeframe. AST was dissolved in dimethyl sulfoxide to obtain a 10 mM stock solution. The stock solution was stored at −20 °C until use. The stock solution was immediately diluted to the required concentration using saline before use. The average spontaneous and mechanically evoked discharge rates, as well as the mechanical threshold, were measured before and after intravenous injection of AST and were analyzed in the present study. The positions of the individual unit recordings within the SpVc region were determined using the micromanipulator, measuring the distance from the obex along the medial-lateral axis and surface of the medullary dorsal horn, with reference to the rat brain atlas, as described in our previous studies [18,19,20].

2.3. Data Analysis

Values are expressed as the mean ± standard error of the mean. We performed statistical analysis utilizing one-way repeated-measures ANOVA, with subsequent post hoc testing using the Tukey–Kramer or Dunnett’s methods and the Student’s t-test for electrophysiological data. A significant difference was identified when the two-sided p-value was less than 0.05.

3. Results

3.1. Characteristics of SpVc WDR Neurons That Provide Innervation to the Facial Skin

This investigation involved recording extracellular single-neuron activity in 11 neurons within the SpVc. The effects of intravenous injections of AST were tested in eleven SpVc neurons. An illustrative example of the receptive field of SpVc neurons, that react to both non-noxious and noxious mechanical stimulation in the whisker pad, is displayed in Figure 1A. Figure 1B indicates that recording sites were mainly distributed in maxillary branches and layers III–IV in SpVc (0.5 to 1.5 mm distance from obex). An example of a standard histological identification unit is presented in the inset of Figure 1B. Typical examples of SpVc WDR neuronal unit responses are shown in Figure 1C. Incremental mechanical stimulation was administered to the most sensitive region of the receptive field, leading to an increase in firing frequency of the neurons. Spontaneous discharges were observed in 2 of the 11 SpVc neurons examined. The mean mechanical stimulation-induced spike threshold was 1.7 ± 0.5 g. Each neuron that was recorded fell within the WDR category of neurons [5,6,19,20].

3.2. Influence of AST Administered Intravenously on the Excitability of SpVc WDR Neurons in Reaction to Noxious and Non-Noxious Stimuli

Following an intravenous injection of 5 mM AST, ten minutes later into the center of the receptive field, non-noxious (2–10 g) mechanical stimulation-evoked SpVc WDR neuronal activity was inhibited, with activity returning to control levels within approximately 20 min (Figure 2). The receptive field’s size remained largely the same prior to and following AST administration. Post AST administration, no significant changes in the mechanical threshold were detected. The summary of how AST affects SpVc WDR neuronal activity in response to non-noxious mechanical stimulation is presented in Figure 3. Following non-noxious mechanical stimulation, the mean firing rates evoked in SpVc WDR neurons tended to decrease after AST injection (10 min) compared to the baseline, and they returned to control levels within 20 min. Intravenous injection of AST did not result in any significant changes in spontaneous firing. Inhibition of SpVc WDR neuronal activity induced by nocuous mechanical (15–60 g) stimulation was observed 10 min post AST administration, but neuronal activity returned to control levels within approximately 20 min (Figure 2). As shown in Figure 3, the mean firing rates of SpVc WDR neurons evoked by noxious mechanical stimulation decreased significantly after the injection of AST compared with controls (Figure 3, p < 0.05; n = 7). There were no significant changes in the size of the receptive field before and after AST administration. Local injection of vehicle (DMSO) had no significant effect on spontaneous or non-noxious, noxious mechanical stimulation-evoked SpVc WDR neuronal activity (n = 3).

The firing of SpVc WDR neurons, induced by non-noxious mechanical stimulation, was considerably suppressed by AST in a dose-dependent fashion (1–5 mM, Figure 4; 1 mM vs. 5 mM, p < 0.05). AST showed a marked, dose-dependent (1–5 mM) reduction in SpVc WDR neuron firing in response to noxious mechanical stimulation (Figure 4; 1 mM vs. 5 mM, respectively, p < 0.05).

3.3. SpVc WDR Neuronal Activity in Response to Noxious vs. Non-Noxious Stimuli after AST

We evaluated how 10 mM intravenous administration of AST affects responses to both non-noxious and noxious stimuli. As shown in Figure 5, there was no noticeable difference in the average inhibition discharge frequency caused by non-noxious and noxious stimuli.

4. Discussion

4.1. The Excitability of SpVc WDR Neurons Is Diminished by Acute Intravenous Administration of AST

This study explored whether administering acute intravenous AST to rats reduces the excitability of SpVc WDR neurons in response to mechanical stimuli. The significant results of the present investigation are the following: (i) AST dose-dependently reduced the firing rate of SpVc WDR neurons (1–5 mM, i.v.) in response to both non-noxious and noxious mechanical stimuli; (ii) This inhibition of discharge was reversible in around 20 min.; (iii) The vehicle administered intravenously showed no significant impact on both the spontaneous and the evoked activity of SpVc WDR neurons. It is known that AST can cross the blood–brain barrier [16], and an in vitro study reported that 50 μM AST significantly inhibited glutamate release from rat cortical nerve terminal preparations [14]. Since it can be assumed that following intravenous administration, 5 mM AST would be diluted in extracellular fluid to produce a concentration of approximately 0.05 mM (50 μM), in this study, we tested the systemic administration of 5 mM AST on the SpVc WDR neuronal activity and found that this concentration had a significant inhibitory effect on the nociceptive transmission of SpVc WDR neuronal firing. Taken together, these observations imply that under in vivo conditions, a single intravenous dose of AST reduces trigeminal nociceptive signaling in the SpVc.

4.2. The Excitability of SpVc WDR Neurons Is Suppressed by AST via Mechanisms Involving Both the Peripheral and Central Systems

Studies conducted previously, indicate that Cav channels open in reaction to membrane depolarization, facilitating the influx of Ca²⁺ ions. This influx has an important consequence: Ca²⁺ acts as a second messenger, leading to the activation of many cellular processes, such as contraction, secretion and gene transcription [21]. Voltage-gated Ca²⁺ channels can be categorized into two groups: low-voltage activated (or T-type) calcium channels and high-voltage activated channels (L, P/Q, N, and R types) [22]. The primary afferent signaling pathway is significantly mediated by both N-type and T-type Cav channels; N-type channels activated by high voltage are primarily situated in the presynaptic regions of laminae I and II within the dorsal horn [22,23,24]. Action potentials traveling through the dorsal root ganglion neurons, including C and Aδ afferents, cause the opening of pre-synaptic N-type calcium channels, which in turn initiate the release of nociceptive transmitters, such as glutamate, substance P, and calcitonin-gene-related peptide onto spinal interneurons and projection neurons [22]. According to a former in vitro analysis, AST was found to inhibit glutamate release from rat cortex nerve terminals in a dose-dependent way, primarily through the suppression of the presynaptic Cav and MAPK signaling cascades [14]. They also showed that the impact of AST on triggered glutamate release was eliminated by the use of N-, P-, and Q-type Ca²⁺ channel blockers [14]. This investigation found that the mean firing frequency of SpVc WDR neurons in reaction to both non-noxious and noxious mechanical stimuli increased significantly and in a dose-dependent manner (1–5 mM, i.v.) and maximum inhibition of the discharge frequency of both non-noxious and noxious mechanical stimuli was seen within 5–10 min. Considering the sum of the results, we speculate that intravenous administration of AST suppresses trigeminal nociceptive neuronal excitability via N-type Ca²⁺ channels, probably at the presynaptic terminal of trigeminal ganglion (primary) neurons.

Since it has been proven that the suppression of NMDA and non-NMDA receptor-mediated glutamatergic activity leads to a considerable decrease in excitatory functions, glutamatergic signaling pathways in the SpVc are clinically important for addressing primary headache syndromes, including migraines and cluster headaches [25]. Cahn and Maassen Van Den Brink [26] indicated that glutamate receptor antagonists played an important role in the management of migraine. Both NMDA and non-NMDA receptor antagonists affect the majority of spinal dorsal horn neurons in untreated rats, indicating the presence of both excitatory amino acid receptor types on the spinal neurons of naïve rats [27]. In the trigeminal system, the administration of NMDA and non-NMDA receptor blockers inhibits the excitatory reactions in upper cervical (C1-C2) neurons to electrical stimulation of the sagittal sinus [26]. Since administering selective NMDA receptor antagonists intrathecally reduces nociceptive behavior (such as in tail-flick and hot plate tests) [28,29], it is more probable that NMDA receptors play a role in nociceptive spinal transmission [27]. According to a recent study, administering AST helps alleviate neuropathic pain through the inhibition of NMDA glutamate receptor activity, particularly NMDA receptor subtype 2B (NR2B) protein, which is involved in nociception [15]. The NR2B subunit is the most important tyrosine-phosphorylated protein, and the phosphorylation of NR2B receptor subunits has been proposed to lead to increased Ca²⁺ entry through the receptor in both central sensitization and NMDA-dependent synaptic plasticity [30,31]. Thus, it is possible that AST suppresses glutaminergic excitatory synaptic transmission of SpVc neurons by inhibiting post-synaptic glutamate receptors. Previously, using a multi-barrel electrode, we demonstrated iontophoretic application of L-glutamate induced the mean firing frequency of a SpVc WDR neuron responding to noxious mechanical stimulation and was inhibited by intravenous administration of resveratrol [19]. Therefore, further confirmational studies are needed on whether AST modulates iontophoretic application of glutamate-induced SpVc neuronal discharge using a multi-barrel electrode.

Alternatively, research has shown that T-type Ca²⁺ channels are primarily found in small- (15–30 µm) and medium-sized (31–40 µm) sensory neurons [32,33]. These neurons are generally categorized as unmyelinated C-neurons and myelinated Aδ-neurons [34]. Jevtovic-Todorovic and Todorovic [35] found that the amplitude of T-type Ca²⁺ currents was increased, causing a reduction in the excitability threshold and consequently an increase in the probability of burst-firing of neurons. Although there is no evidence that AST inhibits T-type Ca²⁺ channels, it is likely that AST reduces the amplitude of T-type Ca²⁺ currents, which in turn leads to a decrease in the firing rate of SpVc WDR neurons in response to noxious mechanical stimuli. However, further studies are needed to elucidate this possibility.

4.3. Functional Impact of AST’s Inhibition on the Excitability of SpVc Neurons Caused by Nociceptive Stimulation

We have previously demonstrated that polyphenol compounds, carotenoids, and polyunsaturated fatty acids can attenuate the excitability of nociceptive neuronal activity under in vivo nociceptive and inflammatory pain conditions, via voltage-gated ionic channels and transient receptor potential and ligand-gated channels [19,20,36,37]. Concerning carotenoids and marine fish-derived dietary constituents, we have previously shown that systemic administration of docosahexaenoic acid (marine fish oil), carotenoids and lutein (from leafy green vegetables) in a rat inflammatory pain model inhibited inflammatory mechanical hyperalgesia and associated nociceptive neuronal hyperexcitability induced by complete Freund’s adjuvant, by inhibiting the cyclooxygenase-2 enzyme from converting arachidonic acid to prostaglandin E₂ [36,37].

Although in the present study we could not examine whether AST administration attenuated the inflammatory pain, we found that acute intravenous AST administration suppressed the SpVc nociceptive transmission, possibly by inhibiting Cav channels and excitatory glutamate neuronal transmission, implicating AST as a potential therapeutic agent for the treatment of trigeminal nociceptive pain without side effects. AST suppresses the production of cytokines such as prostaglandin E2 and tumor necrosis factor-α, which are produced in tissues during inflammation and enhance the inflammatory response, in a concentration-dependent manner [38]. Recent findings also produced evidence that AST administration alleviated neuropathic pain through inhibition of MAPKs and nuclear factor kappa B signal cascades in animal models [39]. Taken together, these findings suggest that AST alleviates pathological pain, including inflammatory pain, by suppressing the production of inflammatory signal mediators and inflammatory cytokines expressed in neurons and glial cells. However, further studies are needed to elucidate the mechanism underlying the effects of AST on trigeminal inflammatory pain.

5. Conclusions

The present study provides the first evidence that, in the absence of inflammatory or neuropathic pain, acute intravenous administration of AST produces a shot-term inhibition of trigeminal sensory transmission, including nociception, possibly by inhibiting Cav channels and excitatory glutamate neuronal transmission (Figure 6). Although further studies are needed to elucidate this mechanism, AST may be considered a potential therapeutic agent for the treatment of trigeminal nociceptive pain without side effects.