Age-dependent down-regulation of orexin receptors in trigeminal nucleus caudalis correlated with attenuation of orexinergic analgesia in rats

Aging is related to a variety of physiological organ changes, including central and peripheral nervous systems. It has been reported that the orexin signaling has a potential analgesic effect in different models of pain, especially inflammatory pulpal pain. However, the age-induced alteration in dental pain perception and orexin analgesia has not yet been fully elucidated. Here, we tested that how aging may change the effect of orexin-A on nociceptive behaviors in a rat dental pulp pain model. The expression levels of orexin receptors and the nociceptive neuropeptides substance P (SP) and calcitonin-related gene peptide (CGRP) were also assessed in the trigeminal nucleus caudalis (TNC) of young and aged rats. Dental pulp pain was induced by intradental application of capsaicin (100 μ g). The immunofluorescence technique was used to evaluate the expression levels. The results show less efficiency of orexin-A to ameliorate pain perception in aged rats as compared to young rats. In addition, a significant decrease in the number of orexin 1 and 2 receptors was observed in the TNC of aged as compared to young rats. Dental pain-induced SP and CGRP overexpression was also significantly inhibited by orexin-A injection into the TNC of young animals. In contrast, orexin-A could not produce such effects in the aged animals. In conclusion, the older age-related reduction of the antinociceptive effect of orexin may be due to the downregulation of its receptors and inability of orexin signaling to inhibit the expression of nociceptive neuropeptides such as SP and CGRP in aged rats.


Introduction
Adults aged 65 years or older are the most rapidly growing proportion of the population, and currently greater impact is being placed on their quality of life, rather than extending a person's length of life (Lobbezoo et al., 2022).Generally, a decline of the oral health conditions of the elderly, such as dental caries and periodontal disease, is to be expected due to poor oral health in the elderly (López et al., 2017).Morphological, histological and functional changes to the enamel, dentine and pulp occur throughout lifetime (Ketterl, 1983).Certain physiological changes seem to be an integral part of the aging process, and those should be carefully distinguished from pathological alterations to the teeth, especially when they induce pain or have a negative impact on the quality of life of the older individuals (Carvalho and Lussi, 2017).
Dental pain is the most common type of orofacial pain, and its assessment and treatment in aged subjects present special challenges for health care services (Jain et al., 2013).In addition, little is known about aging-related molecular changes in the peripheral and central nervous system that affect inflammatory dental pain transmission and perception.
Orexins A and B, which are also known as hypocretins, are derived from the same precursor protein (pro-orexin) (Wang et al., 2018).Their receptors are G protein-coupled receptors and include orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) (Wang et al., 2018).Orexin-A has almost the same affinity for OX1R as for OX2R, whereas orexin-B has a tenfold higher affinity for OX2R than for OX1R (Sakurai et al., 1998).Histological studies revealed that orexinergic neurons are able to project to many pain-related brain regions, including the thalamus, limbic system, dorsal raphe nucleus, locus coeruleus, periaqueductal gray matter, dorsal hippocampus, reticular formation, and trigeminal caudate nucleus (Broberger et al., 1998).The orexin receptors (OXR) are also widely expressed throughout the central nervous system.(Boss and Roch, 2015).
There is much evidence showing that the neuropeptide orexin-A plays a critical role in inhibiting nociceptive transmission (Kumamoto, 2019).Surprisingly, a loss of orexinergic neurons is has been demonstrated with aging (Kessler et al., 2011).An age-related defect in the orexin signaling has been widely demonstrated in animals, and parallels physiological alterations in orexin dependent body function during aging (Nixon et al., 2015).
It has been reported that substance P (SP) and calcitonin-related gene peptide (CGRP), as nociceptive neuropeptides, have indirect contributions to nociceptor activity during inflammation and modulate orofacial pain (Kidd and Urban, 2001;Liang et al., 2021).According to the peripheral noxious stimuli, trigeminal neurons significantly produce neuropeptides like SP and CGRP (Goto et al., 2017).In addition, it has been documented that the expression of SP and CGRP can be affected by aging (Mohammed and Santer, 2002).It has been demonstrated that orexin receptor activation in the trigeminal pathway can inhibit prejunctional release of CGRP from trigeminal neurons (Holland et al., 2005).
Taken together, pain is poorly understood in elderly.Aging is associated with the changes in pain thresholds and pain inhibitory systems (Lautenbacher et al., 2017).We have recently reported that age-related changes are also observed in dental pain, and pulpal stimulation elicits increased nociceptive behaviors and induces more neuroinflammation in the trigeminal pathway of aged rats as compared to young rats (Torkzadeh-Mahani et al., 2023).However, although the findings of agerelated abnormalities at the behavioral level have been repeatedly reported, functional changes in the central nervous system responsible for such changes are still unknown.
In addition, orexin-A has a significant antinociceptive effect in an orofacial pain model in young animals (Bahaaddini et al., 2016;Kooshki et al., 2016).However, orexin-related dental pain modulation in aged rats has not yet been clarified.Therefore, evaluating the age-related pain intensity and age-associated molecular changes in the orexin signaling can be helpful in dental pain management in elderly.
The first aim of this study was to test how aging may change orexin analgesic signaling in a rat inflammatory pulpal model.Since the nociceptive mediators SP and CGRP have critical roles in both pain perception and aging development, the second aim of this study was to evaluate the possible changes in the expression of such substances in the TNC of young and aged rats with dental pain.

Animals
Young and aged (3 and 24 months, respectively) male Wistar rats were used in this study.The rats were housed in individual cages with a 12-h light/dark cycle (lights on at 7:00a.m) and a constant room temperature of 23 ± 2 • C, with unlimited access to standard rat chow and water before and during the study.The animals were allocated randomly to three experimental groups within each age (young 3 months and aged 24 months) including control, capsaicin-and capsaicin+orexin-treated groups.Each group comprising eight animals (total number = 48, with 8 rats in 6 experimental groups).A proper sample size calculation was performed based on the scientific and ethical imperative.The experimental procedures were approved by the Animal Research Ethics Committee of Shahid Bahonar University of Kerman (IR.VETMED.REC.1401.006).This committee follows the international standards.All efforts were made to minimize discomfort for the animals.

Surgery and experimental design
The rats were anesthetized by an intraperitoneal injection of ketamine in combination with xylazine (100 and 2.5 mg/kg, respectively).The rats were fixed on a stereotaxic instrument and surgical implantation of stainless-steel guide cannulas, bilaterally into the trigeminal nucleus caudalis (TNC), was performed according to the rat brain atlas (Paxinos and Watson, 2006).For the dental procedure, the distal part of the right mandibular incisor was carefully cut off without pulp exposure.
Capsaicin (Sigma-Aldrich, USA) solution (Cap, 100 μg) on a small cotton pellet was placed in the cavity under a light-cured glass-ionomer (Fuji II, GC, Tokyo, Japan) restoration (Raoof et al., 2015).Orexin-A (Tocris, USA) was dissolved in distilled water and microinjected to the TNC using a Hamilton micro-syringe.The injection needle was inserted 1 mm beyond the tip of the guide cannula, and infusions were delivered in a total injection volume of 2 μl (1 μl per side) during 1 min.The animals received intra-TNC microinjections of orexin-A (100 pM) 20 min prior to the injection of Cap.

Assessment of nociceptive behavior
Immediately after the administration of Cap, each rat was transferred to a clear plastic box (30 × 30 × 30 cm) with a mirror being placed underneath at a 45 • angle to allow an unimpeded view of the animals.The animals were observed (blind to the randomization), and the typical patterns of facial nociceptive behaviors were used to measure pain levels.The nociceptive behaviors were scored according to the following behaviors: 0-Calm, normal behavior such as grooming.1-Mild head shaking or continuous placement of the jaw on the floor or the wall of the cage.2-Abnormal continuous shaking of the lower jaw.3-Excessive rubbing of the mouth with foreleg movements that concentrated consistently and mainly on the lower jaw (Chidiac et al., 2002).
In addition, the total time spent in each score (time duration in seconds) was recorded and analyzed.

Immunofluorescence staining
The immunofluorescence technique was used to find orexin receptors, CGRP and SP expressing cells in TNC sections.In this section of the study, the rats were anesthetized by injection of ketamine and xylazine (100 and 10 mg/kg i.p., respectively) and perfused transcardially with 100 ml of 0.9 % saline followed by 500 ml of 4 % paraformaldehyde.The brains were fixed overnight in 4 % paraformaldehyde and then embedded in paraffin, after which 2-μm serial sections (three sections for each rat) were prepared and deparaffinized before immunostaining.The sections were then treated for antigen retrieval by microwave treatment for 30 min in citrate buffer (pH = 6) and washed for 3 min in phosphate-buffered saline solution (PBS).The slices were dipped in hydrogen peroxide for 10 min and then exposed to the primary antibodies diluted at 1:500 (sc-8073 and sc-8074, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) overnight in a humidity chamber.Control experiments were performed by removing the primary antibody.
The slices were then washed in PBS and incubated for 90 min with goat anti-rabbit IgG-CFL 488 secondary antibody diluted at 1:1000 (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) and washed again by PBS.Then, they were exposed to 4′,6-diamidino-2-phenylindole (DAPI) at room temperature, and immediately washed by PBS.Finally, the images were captured using a fluorescence microscope (Olympus) with 40× magnification.After finding the desired region of interest according the standard landmarks (Supplementary figure), the desired S. Torkzadeh-Mahani et al. portion of the specimen (TNC) were analyzed and the ratio of protein expressing cells to the total number of stained nucleuses were counted.Immunoreactivity for orexin receptors, CGRP or SP was illustrated in green and that for DAPI as blue.The merge image was obtained by fusing the green and the blue filters.Merge imaging (yellow) was used to demonstrate the possible overlap between the fluorescence of the orexin 1 and 2 receptors, CGRP, and SP with the DAPI signals.Yellow signals reveal TNC expressing orexin receptors, CGRP, and SP cells.An automatic threshold was set for DAPI staining images since strong and contrasted signals were expected for all these images in order to separate nearby nuclei for accurate counting.The population of stained nucleus in the TNC region (500 mm 2 ) was counted by Image J software (NIH, Bethesda, MD, USA).

Statistical analysis
The analyses were performed using IBM SPSS Statistics 22 software (Chicago, IL, United States).All data are presented as mean ± SEM, and the differences among the mean of pain scores were evaluated by twoway analysis of variance (ANOVA), followed by post-hoc Tukey's test.In addition, data from orexin receptors immunofluorescence labeling were analyzed by t-test, and from CGRP and SP by one-way ANOVA with post-hoc Tukey's test.The significance level was set at p < 0.05 for all analyses performed.

Dental pain behavioral analysis in young and aged rats
The mean of pain scores showed that intradental administration of Cap produced potent pronociceptive effects in both young and aged rats (Figs. 1 and 2).In young rats, the nociceptive behaviors were attenuated by intra TNC orexin-A microinjection, which was significant at 15 to 30 min after injection (Fig. 1).The data in Fig. 2 show that orexin-A had no analgesic effect in Cap-administrated aged animals.
In addition, area under curves (AUC) analysis of the data showed numerically similar pain pattern over the time course of the study (2.35 for young control group, 63.5 for Cap-injected group and 47.3 for orexin-treated animals with a significant, p < 0.01, it means a decrease in the AUC following orexin treatment in young animals).In aged animals, the values were 3.35, 92.5 and 88.22 for control, Cap and Cap+orx groups, respectively.Orexin-A treatment had no significant decreasing effect (5.2 %) on the AUC parameter in aged animals.AUC analysis also indicates that the 100 pM orexin-A produced a 26 percent decrease in total nociceptive scores (AUC) just in young rats.

Molecular analysis
To explain the differences in the dental antinociceptive effect of orexin-A between young and aged rats, the orexin 1 and 2 receptors (OX1R and OX2R) density in the TNC of young (3 months) and aged (24 months) animals was assessed.Immunofluorescence data showed that the percentage of the numbers of OX1R (Fig. 3) and OX2R (Fig. 4) expressing cells were significantly decreased in the TNC of aged rats as compared with young animals (p < 0.001).
The data showed that the OX1R expressing cells dramatically (p < 0.001) decreased with aging (Fig. 3).In addition, less selective receptors for orexin-A (OX2R) had also lower density (p < 0.001) in aged rats than that in young rats (Fig. 4).
In addition, the data showed that the numbers of CGRP positive cells were significantly (p < 0.001) increased following Cap administration in both young and aged rats (Fig. 4).Although intra-TNC orexin-A injection could significantly decrease pain-induced CGRP overexpression in young rats, it failed to inhibit such phenomenon in aged animals.
Immunofluorescence labeling of CGRP in the TNC of animals revealed that the numbers of CGRP-positive cells in aged animals were significantly lower (p < 0.001) than those in the young control rats (Fig. 5).
The results also showed that the numbers of SP labeling cells were significantly increased following Cap administration in both young and aged rats (p < 0.001).Intra-TNC injection of orexin-A before intradental Cap administration significantly (p < 0.001) decreased SP overexpression in the TNC of the young rats.However, orexin-A had no effect on Cap-induced SP overexpression in aged animals.Furthermore, aging elicited a significant (p < 0.001) decrease in the TNC expression level of SP in rats (Fig. 6).

Fig. 1.
The effect of intra trigeminal nucleus caudalis administration of orexin-A (Orx, 100 pM/rat) on nociceptive scores in capsaicin (Cap)-administrated young rats over the time course of the study.The data were analyzed by two-way ANOVA, followed by the Tukey test.The results were expressed as mean ± SEM (n = 8).*p < 0.05 versus Cap+Orx-administrated group at the same time.

Discussion
The present study showed that capsaicin-induced pulpal pain occurred in both young and aged rats and, interestingly, that orexin-A could not alleviate pain in the aged group as opposed to its ability in young animals.Our hypothesis was that maybe such defect in orexin analgesia occurred due to the decrease in the level of orexin receptor density or their signaling in the trigeminal pathway of the aged group as compared to the normal young subjects.In accordance with the hypothesis, the data revealed that the OX1R and OX2R are significantly downregulated in aged rats, and activation of those receptors could not inhibit pain-induced SP/CGRP production.It seems that the orexin system in elderly is inefficient to elicit appropriate signaling to reduce dental pain perception.However, little is known about the molecular mechanisms of age-induced alterations in cell signal transduction networks and age-related pathology of pain.
Orexin antinociceptive effects and mechanisms have been reported in various types of pain, including neuropathic pain, visceral pain, cluster headache, and orofacial pains (Razavi and Hosseinzadeh, 2017).Although age-related deterioration of the orexin system has been demonstrated in some physiological processes like the regulation of energy balance, food intake, vigilance, and several endocrine and neural functions (Takano et al., 2004;Nixon et al., 2015), its impact on aging pain perception has not yet been studied.
From the molecular aspect, receptor downregulation and desensitization may play critical roles in the reduced trigeminal orexin signaling in aged rats.Such probable reason can be determined by immunohistological analysis of the receptors and post receptor effectors.The present data (Fig. 3) showed that the selective (OX1R) and less selective (OX2R) orexin receptors expression is dramatically decreased with aging.It can be concluded that one of the possible reasons, at least in part, for the orexin insensitivity and the disappearance of its analgesic effect in aged rats is orexin receptors down-regulation in the pain pathway, especially in the site of nucleus caudalis.
Previous reports have illustrated the age-related alterations in the orexin system in rats and other species.It has been reported that dysregulation or even loss of orexin signaling can be linked to the agerelated disorders (Nixon et al., 2015).Ran et al. (2018) reported that although plasma orexin concentration is increased and the OX1R density is decreased, the number of orexinergic neurons does not change with aging in rats.They also reported that such age-related decrease in OX1R expression is associated with delayed emergence from isoflurane anesthesia in aged rats (Ran et al., 2018).In addition, the degeneration of orexinergic neurons is associated by sleep-wake disturbance in aged cats (Zhang et al., 2002).
It has also been reported that aged rats exhibit a loss of >40 % of orexin-immunoreactive neurons in the lateral hypothalamus and contiguous perifornical area (Kessler et al., 2011).With the aging in rats, the expression level of prepro-orexin gene and the amount of orexin-A and B are significantly decreased in the lateral hypothalamus (Porkka-Heiskanen et al., 2004).Age-related changes in orexins gene expression have been reported in different mouse brain areas, including the hippocampus, thalamus, pons, and medulla (Terao et al., 2002).
It has been demonstrated that orexin neurons are continuously increased during maturation and then significantly decrease by 24 months old (aged), which indicates the relation of orexin system in the physiological changes in aging rats (Sawai et al., 2010).Consistent with other studies, such age-related oreginergic degradation was also observed in the TNC of rats in our experiment.This means that the ageinduced downregulation of orexins receptors in the trigeminal pathway may be involved in the decline of pharmacological effect of orexin-A to elicit antinociception in aging rats.However, no studies have examined whether orexin-producing neurons decline with aging in human.If the same is true in humans, reduction in orexin receptors may require greater doses of peptide or potent synthetic receptor agonist to overcome reduced orexin sensitivity for pain therapy.Hence, determining the contribution of defected orexin signaling to pain pathophysiology in aging may lead to improved therapeutic avenues.
Other findings in our study illustrate that the expression of CGRP and SP was significantly increased following Cap administration in both young and aged groups.In contrast to young animals, orexin-A failed to inhibit pain-induced CGRP or SP overexpression in aged animals.It is well known that Cap produces inflammatory pain through activating the transient receptor potential cation channel subfamily V member 1 (TRPV1) via the release of pro-inflammatory neuropeptides such as neurokinin A, SP, and CGRP in the trigeminal nerves (Zhou et al., 2016;Kistner et al., 2016).CGRP plays an important role in the transmission and modulation of orofacial inflammatory pain (Cady et al., 2019;Capuano et al., 2009;Eberhardt et al., 2008;Kaiser and Russo, 2013).Moreover, CGRP, often co-localized with SP, is found in nerve endings of Fig. 2. The effect of intra nucleus caudalis administration of orexin-A (Orx, 100 pM/rat) on nociceptive scores in capsaicin (Cap)-administrated aged rats over the time course of the study.The data were analyzed by two-way ANOVA, followed by the Tukey test.The results were expressed as mean ± SEM (n = 8).a subset of sensory nerves (Gibbins et al., 1985;McWan et al., 1998).
It has been documented that orexin-A is able to suppress neurogenic dural vasodilation and inhibit prejunctional release of CGRP from trigeminal neurons by OX1R activation (Holland et al., 2005).
finding of the present study is that the expression levels of CGRP SP in the aged rat TNC is lower than in young rats.Moreover, orexinergic system is not efficient enough to alleviate pain and paininduced CGRP-SP overexpression in aged animals.Surprisingly, ageinduced changes in CGRP and SP content have been demonstrated in a number of rat tissues, and most can be explained by the physiological and pathological processes during aging.For example, in some specific areas of the brain (the substantia nigra and striatum), peripheral nervous system (dorsal root and trigeminal ganglion) and some cardiovascular tissues such as atria and arteries, the CGRP content declines with age (Wimalawansa, 1992).It has been previously reported that the spinal levels of SP in old rats are lower than the levels in younger rats (Bergman et al., 1996).In humans and mice, SP levels are significantly reduced in old subject corneas (Barbariga et al., 2018).The density and staining intensity of SP-immunopositive nerve fibers in the myenteric plexus of old rats are lower than those in young rats (Lazarov et al., 2010).In addition, aging alters the sensitizing effect of SP in the joint of Guinea pigs (McDougall and Schuelert, 2007).It seems that degenerative alterations in SP-and CGRP-containing cells in trigeminal nucleus caudalis during aging may has a role in dental nociceptive changes and orexin analgesic tolerance in aging.However, this issue needs to be further clarified in future studies.
It is widely known that many orexinergic neurons co-express glutamate as a transmitter and the glutamatergic transmission could easily compensate for the attenuation of orexinergic transmission (Eyigor et al., 2012).Therefore, the confirmation of postsynaptic cells (TNC cells) activity is critical.Immediate-early genes such as c-fos are powerful tools for detecting activated neurosecretory neurons in physiological and pathological conditions.It has been reported that tooth movement or pain resulted in an increase in the Fos expression in the trigeminal sensory subnucleus caudalis and other brain nuclei in rats which means a recruitment of neurons related to nociception and to antinociception in dental pain (Magdalena et al., 2004;Reis et al., 2023).However, the response of TNC neurons to dental Cap treatment in the aged rats has not been fully determined and further study needs to be done to evaluate the response (activation) of TNC neurons to the treatment by c-Fos expression.
The strength of our study is the application of a validated experimental protocol to assess aging-related molecular changes in rat brain, which may affect dental pain perception in aging.By using such animal experiments, we can better understand fundamental processes under precise experimental conditions.In addition, by having brain tissue sampling, we are able to find the molecular changes that would be difficult or impossible to find in humans.Although there are some key similarities in basic biology, cells, and organ systems between laboratory animals and human, there are also differences, which makes it difficult to assume the relevance of experimental data for humans.
However, future researches are needed to assess the expression level

Conclusion
It can be concluded one of the possible reasons, at least in part, for the orexin insensitivity and the disappearance of its effect in elder rats is orexin receptor down-regulation in the site of the nucleus caudalis, suggesting a possible defect in orexin signaling for inhibition of nociceptive neuropeptides such as SP and CGRP in aged rats.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 3 .
Fig. 3. Immunofluorescence double-labeling for orexin 1 receptors (OX1R, green signal in upper panel) and DAPI-stained nuclei (blue, middle panel) in the trigeminal nucleus caudalis (TNC) of young (3 months) and aged (24 months) rats.The data show a significant decrease in the number of OX1Rs immunoreactive cells in aged rats.Overlap between the fluorescence of the OX1R and DAPI staining cells (merged) is shown in lower panel.The bars show the comparison of the mean number of OX1R immunoreactive cells in the TNC sections of experimental groups.Data are presented as mean ± SEM. ***p < 0.001 vs control young animals.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5 .
Fig. 5. Immunofluorescence labeling of calcitonin gene related peptide (CGRP) in the trigeminal nucleus caudalis (TNC) of three experimental groups within each age including control, capsaicin (Cap)-or capsaicin plus 100 pM orexin-A (Cap+orx)-treated rats.Green highlights show immunoreactivity for CGRP in the TNC sections (upper panel), blue (4′,6-diamidino-2-phenylindole, DAPI)-stained points remark cell bodies (middle panel), and the lower panel shows the merged staining.The merge image documents the overlap between the fluorescence of the CGRP and DAPI signals.The bars show the mean number of CGRP expressing cells in the TNC sections of control, Cap-, and Cap+orx-treated rats.Data are presented as mean ± SEM. ***p < 0.001 and *p < 0.05 vs control animals in same age-matched group; + p < 0.05 vs Cap-treated young animals; ### p < 0.001 vs young control animals.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Shima Torkzadeh-Mahani: Conceptualization, Data curation, Investigation, Methodology, Investigation, Methodology, Writingoriginal draft, Mehdi Abbasnejad: Data curation, Supervision, Formal analysis, Writingreview and editing.Maryam Raoof: Data curation, Methodology, Formal analysis, Review & editing.Ghizlane Aarab: Writingreview & editing.Saeed Esmaeili-Mahani: Data curation, Formal analysis, Writingreview & editing.Frank Lobbezoo: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writingoriginal draft, Writing-review & editing.

Fig. 6 .
Fig. 6.Immunofluorescence labeling of substance P (SP) in the TNC of control, capsaicin (Cap)-, and capsaicin plus 100 pM orexin-A (Cap+orx)-treated young or aged rats.Green highlights show SP expressing cells in the TNC (upper panel), blue (4′,6-diamidino-2-phenylindole, DAPI)-stained cell bodies (middle panel) and the lower panels are merged staining (overlap between SP and DAPI signals).The bars show the mean ± SEM. ***p < 0.001 and **p < 0.01 vs control animals in same age-matched group; + p < 0.05 vs cap-treated young animals; ### p < 0.001 vs young control animals.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)