Atomoxetine and escitalopram migrate the derangement of the temporomandibular joint morphologic and histologic changes in rats exposed to stress-induced depression

Thanatta Songphaeng; Sarawut Lapmanee; Sakkarin Bhubhanil; Kamonchanok Momdee; Catleya Rojviriya; Kemporn Kitsahawong; Pattama Chailertvanitkul; Jariya U. Welbat; Supawich Morkmued

doi:10.2334/josnusd.23-0077

Abstract

Purpose: The purpose of this in vivo study was to determine the effects of stress-induced depression and antidepressants on depressive-like behavior, microstructure, and histomorphology of the temporomandibular joint (TMJ) using rats.

Methods: Experimentally induced depression in rats was created before being treated with two antidepressants; escitalopram (selective-serotonin-reuptake inhibitors) and atomoxetine (norepinephrine-reuptake inhibitors). Micro-computed tomography (Micro-CT) was performed to measure the change in bone volume and bone porosity of the condyle. Further histological evaluation of the condylar cartilage was performed.

Results: Micro-CT scanning revealed a decrease in bone volume in the depression group. The bone porosity percentage significantly increased in both the escitalopram and atomoxetine groups compared with the control group and the depression group. Histopathological analysis showed increased thickness of cartilage layers in the depression group. In the atomoxetine group, there was a significant increase in the pre-hypertrophic and hypertrophic layer thickness and cell count, but a significant decrease in proteoglycans.

Conclusion: The present study findings indicated the change in TMJ characteristics, especially on the superficial part of the condylar head in the depression group. Concerning the applicability of the different antidepressants, depression with the treatment of atomoxetine has the most disadvantages due to bone porosity and cartilaginous condyle changes.

Introduction

The temporomandibular joint (TMJ) is one of the body’s most complex joints and performs essential functions that are necessary for many daily movements, as well as withstanding strong and constant forces. Temporomandibular disorders (TMDs) are referred to as a general term for a wide range of TMJ pathologies, most often degenerative joint diseases or osteoarthritis (OA) [1]. This disease involves systemic and local mechanical and biochemical degradation of condylar cartilage, subchondral bone, articular disc, and synovium [2]. According to several studies, TMDs are a significant health issue for children and adolescents and their frequency increases with age from childhood to adolescence [3]. In most elderly patients, symptoms of TMDs are mild and self-limiting and usually treated by patient self-management. TMDs in young patients may have a significant impact on airway dimensions, occlusion, and facial growth [3,4]. The etiopathogenesis of osteoarthritis is still complex, and is associated with multiple risk factors. Hence, a deeper understanding of what drives the higher TMD occurrence at a younger age remains a major milestone for the field.

Additionally, the existence of TMD will have a negative impact on the patient’s quality of life, leading to higher medical care needs that are time-consuming and costly [3]. In terms of TMD diagnosis, psychological experts have provided instruments to assess depression and anxiety, sleep disorders, catastrophizing, stress, and resilience in children and adolescents with TMD symptoms [4]. At the peak of the COVID-19 pandemic, Child caregivers had serious negative emotions, including anxiety, depression, frustration, and anger. There are many theories that attempt to explain the causes of depression in children and adolescents, which show the complexity of the pathology [5]. In addition, the occlusal force and masticatory muscles also play an important role in TMJ [6,7]. Nevertheless, previous results showed that emotional problems seem to play a more prominent role than dental factors [8]. Since psychological factors are risk factors for TMD, psychological rat models can produce changes in behavior, as well as degenerative changes in the TMJ condyles and masseter muscles [9]. Consequently, to understand robust TMD pathophysiology, this investigation included the rat as an excellent model to explore the potential pathologic mechanisms. Thus, it is crucial at the level of preclinical study to investigate more comprehensively the TMJ of animal models of depressive phenotypes.

Even the best prevention and management of depression in children and adolescents fundamentally requires the cooperation of parents to help appropriately in accordance with the guidelines, promoting an anti-depressive syndrome by socio-behavioral therapy is the best method, but it is usually not applicable [10]. Treatment of depression mostly requires medication, which may affect many body systems [11]. Furthermore, antidepressants’ effects on oral health directly are still limited especially at the jaw joints. Therefore, the purpose of this study was to investigate the relationship between depressive behavior, three-dimensional microarchitecture, and histomorphology changes of mandibular condyles using a psychological stress-induced inflamed TMJ rat model with and without commonly used antidepressants, norepinephrine-reuptake inhibitors (NRI) and selective-serotonin-reuptake inhibitors (SSRI). These could provide valid evidence for further research related to TMJ-OA that could help in the early diagnosis of suspected depression in children and adolescents, as well as the undesirable effects of receiving such medications.

Materials and Methods

Animals models

The Institutional Animal Care and Use Committee of Thammasat University and Khon Kaen University approved the animal protocols used in this study. All of the experimental methods and procedures were performed according to the guide for the care and use of laboratory animals, national research council, and complied with all relevant ethical regulations for animal testing research (IACUC-KKU-39/63). The timeline of the experiment is illustrated in Fig. 1.

Twenty-eight male, 8-week-old, Wistar rats, weighing from 180 to 200 g were used. Male rats were used to avoid female estrous cycle-induced anxiety-like behavior. The rats were randomly divided into four groups (n = 7 in each group), comprised of a control group and three experimental groups. In the experimental group, depression was induced by restrain stress. Each rat was subjected to being immobilized in a 24 × 6 cm transparent plastic cylinder fixed with plastic tape for 2 h/day, 7 days/week for 2 weeks [12,13]. There was a 1-cm hole at the end of the cylinder for breathing (Fig. 2a). The restraint stress procedure was performed in a quiet room before being treated with an antidepressant. The antidepressant doses followed previous studies [14,15]. One subgroup was administered antidepressant treatment with escitalopram (SSRI, Sun Pharmaceutical Industries Ltd., Mumbai, India) 10 mg/kg orally once per day for a duration of 2 weeks. The second sub-group was administered antidepressant treatment with atomoxetine (NRI, Lilly Del Caribe Inc., Carolina, Puerto Rico) 10 mg/kg orally once per day for a duration of 2 weeks. The third sub-group was administered 5 mL/kg saline (placebo) orally once per day for a duration of 2 weeks, at the same volume as the antidepressant treatment group. In the control group, rats were administered 5 mL/kg saline (placebo) orally once per day for a duration of 2 weeks without depression induction. On day 15, a behavior test was performed to evaluate depressive-like behaviors. At the end of the study, all rats underwent euthanasia and had their skulls removed. Samples containing TMJ tissue were fixed in 10% neutral-buffered formalin. TMJs were selected for micro-computed tomography (Micro-CT) scan before being used for histopathological analyses.

Fig. 1

Timeline diagram shows pharmacological treatment protocols. After one week of acclimatization, rats were divided into one control and three experimental groups, i.e., depression, escitalopram-treated, and atomoxetine-treated groups. Drug administration was given every day for 2 weeks after the restraint procedure. Behavioral tests were performed at the end of the protocol.

Fig. 2

The experimental procedures for inducing depression-like behavior in rats were as follows: (a) The rats were placed inside a transparent plastic cylinder that was secured with plastic tape, with the aim of inducing depression. (b) To assess their behavior, the rats underwent the Porsolt forced swim test. This test involved placing the rats inside a Plexiglas cylinder measuring 45 cm in height and 25 cm in diameter, filled with tap water at a temperature of 25°C.

Forced swim test (FST)

The FST is a hallmark of depressive-like behavior in rodents and is often used to test the efficiency of antidepressant drugs. The test consists of training and testing sessions on two consecutive days. Rats had an initial 15-min swimming session for training purposes with no data collection. After the first 15-min swimming session, rats were removed from the cylinders and dried with towels before being returned to their cages. On the day of the test, a 5-min test was performed, and their behavior was digitally recorded. The Porsolt forced swim test was conducted by placing rats in individual glass cylinders (45 cm tall and 20 cm in diameter) containing 25°C tap water following the standard study [16] (Fig. 2b). In experiments, infrared video cameras were used for post-recording measurements of the number and duration of swimming behavior (active movement of the forepaws with goal-directed horizontal actions, such as crossing between quadrants of the cylinder and turning), climbing (upward goal-directed movements of the forepaws along the side of the cylindrical container), immobility behavior (floating in water with only movement necessary to keep the head above water), and the number of fecal pellets. The increase in immobility duration and fecal pellet number represented high depression-like behaviors in rats [17].

Sucrose consumption test

As described by Charoenphandhu et al. in 2011, this study conducted the following behavioral protocol [18]. All rats were housed individually and trained to become familiar with the drinking of 2% w/v sucrose solution (Ajax Finechem, Taren Point, Australia) after completing acclimatization. Then, throughout the 2-week experimental period, rats were weekly subjected to the same 100-mL sucrose test 1 h/day after food and water deprivation for 6 h. Total consumption was determined by weighing the sucrose bottle before and after each 1-h test session, and the consumed volume was calculated from the density of 1.0098 g/cm³. An increase in sucrose consumption (i.e., increased sensitivity to reward) indicated an emotional stress response, whereas physical stress induced by electrical foot shock or painful exposure decreased the amount of sucrose consumption (i.e., anhedonic response) [18,19].

Serum corticosterone

At the end of the study, trunk blood samples (10 mL) were collected from rats after being exposed to 5% isoflurane anesthesia. Blood was then centrifuged at 3,000 g for 10 min, and serum was removed and stored at −20°C until analysis for corticosterone using a commercially available enzyme immunoassay kit (Immunodiagnostic Systems Ltd., Tyne and Wear, UK). All serum samples were assayed in duplicate, and the mean values were derived.

Micro-CT analysis

The bilateral TMJ condyles were collected and stabilized in the sample holder for micro-CT scan. The samples were scanned using synchrotron radiation X-ray tomographic microscopy (SRXTM) at beamline 1.2W, Siam Photon Laboratory (SPS) to examine the TMJ bony change. The X-ray beam was generated from the 2.2-T multipole wiggler in the 3-GeV Siam Photon Source, which was operated at 150 mA. The tomographic scanning of each sample was performed with the filtered X-ray beam at the mean energy of 13 keV. The X-ray projections were normalized and used in tomographic reconstruction based on filter-back projection by Octopus Reconstruction software (University of Ghent, Ghent, Belgium). The resulting tomograms of the samples were presented and visualized in 3D by using Drishti version 2.6.4 software (Australian National University, Canberra, Australia). The parameters such as bone volume fraction (BV/TV) and percentage of porosity were measured to analyze trabecular microstructure using Octopus Analysis software (University of Ghent). The bone volume is the region of interest (ROI) that covers the upper part of the condylar head to the neck of the condyle. As for porosity measurements, the pore canals at the widest point of the condylar head were segmented down horizontally for 300 sections and analyzed as a fraction of the void volume averaged five times in each specimen, expressed in terms of the percent void volume as the porosity.

Histological analysis

TMJ tissue was fixed in 10% neutral-buffered formalin, followed by decalcification with 10% ethylene diamine tetra acetic acid (EDTA) (Elabscience Biotechnology Inc., Houston, TX, USA) for 14 days. For histological procedures, consecutive mid-sagittal 5-µm-thickness were obtained from paraffin blocks. The samples were stained with hematoxylin and eosin (H&E, Sigma-Aldrich, Saint Louis, MO, USA) and Safranin O (Loba Chemie Pvt. Ltd., Mumbai, India). H&E staining was performed to determine the condyle cartilage thickness and chondrocyte number. The histological analysis was performed by one investigator who was blinded to the experimental conditions. Condyle cartilage thickness was measured using the Image framework system (DS Camera Control Unit DS-L2, Nikon corporation, Tokyo, Japan). Briefly, four bold lines were used to divide the condylar cartilage into anterior (AP), central (CP), and posterior (PP) thirds, and three thin lines were used to further divide the AP, CP, and PP thirds into four smaller portions. The cartilage thicknesses of the fibrous, proliferative, pre-hypertrophic, and hypertrophic layers of the AP, CP, and PP thirds of the TMJs were measured as the average lengths of the three thin lines in each corresponding third. The prominent gap within the section was excluded from the length measurement. The chondrocyte cells were counted at the pre-hypertrophic layer. Safranin O was used to detect proteoglycan changes using the UCLA staining protocol for cartilage instructions. Briefly, for Safranin O staining, paraffin sections were stained in 0.001% fast green (Loba Chemie Pvt. Ltd.) for 5 min after dewaxing and then rinsed in 1% acetic acid solution, followed by staining in 0.1% Safranin O for 5 min, and washing in 95% alcohol. The proteoglycan area was measured at the posterior of the condylar cartilage using area fraction in the ImageJ software version 1.53t (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Nine sections representing a whole condyle of each rat were selected to be analyzed in triplicate.

Statistical analysis

All data are presented as the mean ± standard deviation (S.D.), as indicated in the figure legends and tables. Statistical analyses were performed using the SPSS version 23 software (IBM Corp., Armonk, NY, USA). The procedures for quantifying parameters from the images and micro-CT analysis were all performed twice in a blinded fashion with no knowledge of the groups and the data were double-checked by another investigator. All data acquisition and analysis were completed blindly, and each experiment was performed by different observers independently. The paired t-test was used for the data analysis between the left and right TMJs. The one-way ANOVA and nonparametric Kruskal–Wallis were used for the data analysis between the four groups. Results with P < 0.05 were considered significant.

Results

Antidepressant drugs clinically alleviate stress-induced depression-like behaviors

In the depression group, restraint stress significantly decreased swimming duration (50.20 ± 21.38 s, P < 0.05) and increased immobilization duration, but there was no statistical difference (189.49 ± 40.66 s) without changes in climbing duration in FST, suggesting that this method could induce depression-like behavior (Table 1). The same trend in behavioral tests was found in the escitalopram group, but there was no statistical difference compared with the control group. On the other hand, the atomoxetine group showed significantly longer swimming duration (99.95 ± 40.90 s, P < 0.05) and less immobilization duration (127.13 ± 41.40 s, P < 0.05) than the depression group (Table 1). Moreover, serum corticosterone levels were higher in the depression group (100.17 ± 29.41 ng/mL) than in the control group, while a decrease in serum corticosterone levels was found in depressive rats treated with escitalopram (53.21 ± 15.57 ng/mL), but there was no statistical difference (Fig. 3). At 1 week, the depression group had a significantly increased sucrose intake volume compared with the control group (22.93 ± 2.48 mL, P < 0.05). In contrast, there was a significant reduction in sucrose intake volume in the escitalopram group (19.37 ± 1.08 mL, P < 0.05) and the atomoxetine group (18.25 ± 2.9 mL, P < 0.01) compared with the depression group (Fig. 4>). The same trend was found at baseline and 2 weeks, but there was no statistical difference (Fig. 4). These results suggested restraint stress induced depression-like behaviors and highly emotional responses in male rats. In addition, antidepressants, i.e., escitalopram and atomoxetine could reverse the worst effects of stress induction.

Table 1 Swimming duration, climbing duration and immobilization durations measured using the forced swimming test (FST) in control, depressed, and depressed rats given escitalopram or atomoxetine treatment

	Group	Mean (s)	SD
Swimming duration	control	102.80^a	31.65
	depression	50.20^{a, b}	21.38
	escitalopram	79.90	29.53
	atomoxetine	99.95^b	40.90
Climbing duration	control	47.88	13.31
	depression	54.79	20.47
	escitalopram	53.87	18.47
	atomoxetine	56.32	29.75
Immobilization duration	control	159.98	16.24
	depression	189.49^c	40.66
	escitalopram	161.68	47.48
	atomoxetine	127.13^c	41.40

Lowercase letters are used to compare means. ^aSwimming duration shows statistically significant differences between control and depression groups (P < 0.05). ^bSwimming duration shows statistically significant differences between depression and atomoxetine groups (P < 0.05). ^cImmobilization duration shows statistically significant differences between depression and atomoxetine groups (P < 0.05).

Fig. 3

Serum corticosterone levels in controlled and depressed rats subjected to each treatment.

Fig. 4

Sucrose intake at baseline and at the end of every week during the treatments for 2 weeks. *P < 0.05, **P < 0.01

Stress-induced depression causes deterioration of the surface of the condylar bone, meanwhile antidepressants affect bone porosity

The micro-CT results showed that the condyle has multi-erosion lesions on the bone surface, and the shape of the condyle also changed in the depression group. Nevertheless, the bone surface in the control group was smooth and continuous (Fig. 5). Micro-CT analysis showed that there were significant differences in mean bone volume between the four groups (P < 0.05). The bone volume to tissue volume (BV/TV) ratio decreased significantly from 13.3 ± 1.26% in the control group to 10.29 ± 1.7% in the depression group (P < 0.05, Table 2). The same trend was found in the escitalopram group (12.17 ± 2.12%) and atomoxetine group (11.82 ± 1.48%), but there was no statistical difference compared with the control group (Table 2).

Subchondral bone loss was observed in the depression and antidepressant groups. The bone porosity of subchondral cortex significantly increased in the escitalopram and atomoxetine groups compared to the control group (22.25 ± 1.35%, P < 0.01 and 24.28 ± 2.3%, P < 0.001, Table 3). The severity tended to be more severe at the left side (Table 2 and 3). The bone porosity percentage of the atomoxetine group increased the most when compared with other groups. In contrast, no difference in the bone porosity was observed between the depression group and the control group, though this percentage was slightly higher in the depression group (Table 3).

Fig. 5

Micro-CT scanning represented images of the condyles with sagittal view and top view. (i, ix) The control condyles showed intact subchondral bone with smooth and continuous surface. (ii, x) Subchondral bone loss (arrow) was observed in the condyles of the depression group. (vi, xiv) The condyles in the depression group were smaller than those in the other groups. The condyles in the escitalopram (iii, vii, xi, xv) and atomoxetine groups (iv, viii, xii, xvi) also showed high porosity compared with the other groups.

Table 2 Bone volume to tissue volume ratio (BV/TV) of subchondral bone on the left, right and both sides

	Group	Mean (%)	SD
BV/TV (left)	control	13.59^a	1.31
	depression	10.36^a	2.05
	escitalopram	12.15	1.97
	atomoxetine	10.97	2.01
BV/TV (right)	control	13.01	1.67
	depression	10.22	1.94
	escitalopram	12.19	2.42
	atomoxetine	12.67	1.94
BV/TV	control	13.30^b	1.26
	depression	10.29^b	1.70
	escitalopram	12.17	2.12
	atomoxetine	11.82	1.48

Lowercase letters are used to compare means. ^aLeft bone volume to tissue volume ratio shows statistically significant differences between control and depression groups (P < 0.05). ^bBone volume to tissue volume ratio shows statistically significant differences between control and depression groups (P < 0.05).

Table 3 Subchondral cortical bone porosity percentage (%) on the left, right and both sides

	Group	Mean (%)	SD
Porosity (left)	control	17.27^{a, b}	2.83
	depression	18.91^c	3.44
	escitalopram	22.50^a	2.60
	atomoxetine	24.44^{b, c}	2.82
Porosity (right)	control	18.43^d	2.44
	depression	19.06^e	2.65
	escitalopram	21.00	0.66
	atomoxetine	24.13^{d, e}	3.48
Porosity	control	17.85^{f, g}	2.05
	depression	18.99^{h, i}	2.42
	escitalopram	22.25^{f, h}	1.35
	atomoxetine	24.28^{g, i}	2.30

Lowercase letters are used to compare means. ^aLeft subchondral cortical bone porosity percentage shows statistically significant differences between control and escitalopram groups (P < 0.05). ^bLeft subchondral cortical bone porosity percentage shows statistically significant differences between control and atomoxetine groups (P < 0.01). ^cLeft subchondral cortical bone porosity percentage shows statistically significant differences between depression and atomoxetine groups (P < 0.05). ^dRight subchondral cortical bone porosity percentage shows statistically significant differences between control and atomoxetine groups (P < 0.01). ^eRight subchondral cortical bone porosity percentage shows statistically significant differences between depression and atomoxetine groups (P < 0.01). ^fSubchondral cortical bone porosity percentage shows statistically significant differences between control and escitalopram groups (P < 0.01). ^gSubchondral cortical bone porosity percentage shows statistically significant differences between control and atomoxetine groups (P < 0.001). ^hSubchondral cortical bone porosity percentage shows statistically significant differences between depression and escitalopram groups (P < 0.05). ⁱSubchondral cortical bone porosity percentage shows statistically significant differences between depression and atomoxetine groups (P < 0.001).

Stress-induced depression and atomoxetine deteriorate histologically cartilage changes

The normal condylar cartilage in the control group displayed characteristic zonal cellular arrangements with four distinct regions, i.e., the fibrous, proliferative, pre-hypertrophic and hypertrophic layers (Fig. 6). In contrast, the atomoxetine group showed local pathologic changes in the condylar cartilage (Fig. 6) and degenerative change was found in the proliferative and pre-hypertrophic layers. In the depression group, the thickness of all four layers significantly increased to 52.09 ± 24.82 μm in the fibrous layer, 56.63 ± 19.44 μm in the proliferative layer, 71.42 ± 21.29 μm in the pre-hypertrophic layer, and 86.87 ± 33.05 μm in the hypertrophic layer, respectively (P < 0.001) compared with the control group (Table 4). In the atomoxetine group, a significant increase in the pre-hypertrophic and hypertrophic layers thickness was seen when compared with the control group (84.99 ± 31.66 μm, 85.93 ± 34.97 μm, P < 0.001, Table 4). Of the four groups, the atomoxetine group exhibited the highest values of thickness calculated without the thickness of the horizontal cleft/gap, most expressively in the posterior part of the pre-hypertrophic layers (Table 5). Concurrently, the Safranin O staining was performed to compare the expressive presence of proteoglycans (Fig. 6). The atomoxetine group exhibits a significantly reduced amount of proteoglycan (Fig. 7) and this group also had severe cartilage degeneration compared to the control group (Fig. 6).

Fig. 6

Histopathological changes in TMJ condylar cartilage. A representative sagittal section of condylar cartilage stained with hematoxylin and eosin (H&E) and Safranin O (S.O). Scale bar: 10 μm

Table 4 Comparison of layer thicknesses between control and experimental groups

	Control	Depression	Escitalopram	Atomoxetine	P value
Fibrous layer	32.89 ± 12.47^a	52.09 ± 24.82^{a, b, c}	37.58 ± 19.15^b	40.61 ± 17.28^c	< 0.001
Proliferative layer	26.71 ± 8.43^{d, e, f}	56.63 ± 19.44^{d, g, h}	33.51 ± 13.03^{e, g}	36.82 ± 12.82^{f, h}	< 0.001
Pre-hypertrophic layer	36.32 ± 16.33^{i, j}	71.42 ± 21.29^{i, k}	41.46 ± 17.32^{k, l}	84.99 ± 31.66^{j, l}	< 0.001
Hypertrophic layer	43.98 ± 16.72^{m, n}	86.87 ± 33.05^{m, o}	41.04 ± 16.02^{o, p}	85.93 ± 34.97^{n, p}	< 0.001

Lowercase letters are used to compare means in the same row; means sharing a superscript letter are significantly different. Data are mean ± S.D. (µm). ^eP < 0.05, ^cP < 0.01 and ^{a, b, d, f-p}P < 0.001

Table 5 Comparison of the thickness of each layer of the anterior, middle and posterior condyles between control and experimental groups

	Control	Depression	Escitalopram	Atomoxetine	P value
Fibrous layer
anterior	23.73 ± 6.42	40.30 ± 14.43	23.21 ± 3.94	25.79 ± 8.07	< 0.001
middle	30.27 ± 6.24	43.90 ± 15.76	31.87 ± 8.57	40.19 ± 15.96	0.001
posterior	44.67 ± 12.65	72.07 ± 28.46	55.66 ± 19.30	55.85 ± 11.37	< 0.001
Proliferative layer
anterior	20.99 ± 5.46	54.82 ± 20.39	23.73 ± 5.33	29.77 ± 12.22	< 0.001
middle	29.11 ± 6.82	60.47 ± 24.11	36.09 ± 12.81	44.32 ± 20.43	< 0.001
posterior	30.02 ± 9.54	54.60 ± 11.95	40.71 ± 13.04	44.89 ± 9.28	< 0.001
Pre-hypertrophic layer
anterior	24.22 ± 11.22	73.44 ± 23.42	25.77 ± 9.64	63.44 ± 38.95	< 0.001
middle	46.88 ± 17.77	76.43 ± 17.41	54.66 ± 16.23	86.23 ± 19.99	< 0.001
posterior	37.86 ± 10.44	64.40 ± 21.48	43.95 ± 11.13	105.30 ± 15.99	< 0.001
Hypertrophic layer
anterior	30.78 ± 10.50	84.54 ± 22.14	34.03 ± 13.60	94.89 ± 51.46	< 0.001
middle	56.17 ± 17.03	100.84 ± 44.49	50.35 ± 18.23	83.57 ± 24.89	< 0.001
posterior	45.00 ± 11.11	75.22 ± 23.27	38.74 ± 11.23	79.32 ± 19.01	< 0.001

Data are mean ± S.D. (µm).

Fig. 7

Percentages of proteoglycan areas in condylar cartilage. **P < 0.01, ***P < 0.001

Escitalopram affects bone porosity but has little change on the cartilage of the condylar head

The microstructural changes in the subchondral cortex were defined by micro-CT in the escitalopram group with more porosity (Fig. 5 (iii, xi)). The subchondral bone porosity significantly increased in the escitalopram group compared to the control and the depression groups (22.25 ± 1.35%, P < 0.01, P < 0.05, Table 3). Nevertheless, the escitalopram group (12.17 ± 2.12%) decreased the percentage of BV/TV, but there was no statistical difference compared to the control group (13.3 ± 1.26%, Table 2). For the cartilage changes, only the proliferative layer of the escitalopram group had a significant increase in the layer thickness compared with the control group (33.51 ± 13.03 μm, P = 0.015) (Table 4).

Stress-induced depression and atomoxetine accelerate cartilage turnover

The pre-hypertrophic layer contains chondrocytes at various stages of maturation [20]. Histomorphometric analysis of this layer showed significant differences in the chondrocyte cell number between the four groups (P < 0.001). The chondrocyte cell number significantly increased from 60.07 ± 21.11 cells in the control group to 88.482 ± 23.749 cells in the depression group, and 107.56 ± 27.57 cells in the atomoxetine group, respectively (P < 0.001, Fig. 8). The chondrocyte cell number of the atomoxetine group increased most when compared with other groups. Conversely, the chondrocyte cell number was similar between the control group and the escitalopram group.

Fig. 8

Number of chondrocyte cells in condylar cartilage. **P < 0.01, ***P < 0.001

Discussion

TMJ-OA is an accumulative defect, in which a psychological depressive animal model causes this disease, but it has not been widely investigated. Many clinical studies have demonstrated that psychological stress, such as anxiety and depression, is related to TMJ disorder [21] and also has a negative impact on quality of life [22]. Thus, depressed patients require prescriptions for antidepressant drugs which act within the central nervous system to alter monoamine neurotransmitter function [23]. In the present study, to supply more evidence of the correlation between depression, antidepressants, and TMJ-OA, firstly, experimental models were based on the induction and treatment of depressive phenotype, and testing of the antidepressants to relieve symptoms [17]. The main objective of the experiment is to relieve depression-like symptoms and maintain appropriate brain function, while screening for TMD is optional. As expected, antidepressants alleviated depression-like behaviors, referring to the level of despair, to restrain stress. The despair symptoms were slightly better in the atomoxetine group than in the escitalopram group which showed the effectiveness of the treatment of depression. Clinical despair can be conceptualized as a profound and existential hopelessness and powerlessness over one’s life and the future [24]. The depression group in this study may represent a population who encounter problems with life, family, health, finances, or world events, and still remain in a period of loss of faith in their ability to find happiness or to create a satisfactory future for themselves.

Condylar bone investigation by micro-CT, in the depression group, some of the condylar surfaces are locally altered as the reduction of bone which is also found in the previous psychological stress murine TMJ accordingly [25] or the other model of TMD progression [26]. The lesion was mainly on the surface of the depression group and may have been caused by the results of synovitis [27]. These erosions and irregularities were also found mostly in systemic sclerosis patients suggesting the inflammatory infiltrate at the early stage of TMD [2]. Another remarkable finding was an incremental increase in subchondral bone porosity when treated with both escitalopram and atomoxetine. The results that tend to reduce subchondral bone formation are supported by evidence from a previous study that found reduced bone formation and enhanced bone resorption of escitalopram [28]. According to several studies, SSRI increased the number of osteoclasts while decreasing the number of osteoblasts, resulting in bone loss [23,29,30]. Another study showed that NRI may have deleterious effects on the homeostasis of bone [31]. The findings of the present study suggest SSRI and NRI can play a role in subchondral bone remodeling during the treatment of depression.

Regarding the condylar cartilage investigation, these results showed that the fibrous and proliferative layers of the depression group, which are outer to the condylar surface, were significantly thicker than in the control group. Previous studies in the same group of psychological stressed induced TMD in rats conducted as in this study, showed a TMJ-OA with the decreased thickness of the condylar cartilage beginning at 3 weeks [25,32]. Another study in sleep-deprived rats showed that the surface of the fibrous layer was cracked and exfoliated [33]. Moreover, similar thickening of the entire cartilage layer was found in rats with disorganized occlusions, implying that this may be the result of an adaptive response to depression [34]. The proliferative layer is also the region that has the ability to proliferate and differentiate, and its pathway is mainly regulated by biomechanical force [35]. The mice with bilateral anterior elevation of occlusion and the rats with anterior disc displacement also exhibited the greatest increase in cartilage thickness during the OA process [26,36].

However, this finding is in contrast to some previous studies on rat models with unilateral anterior crossbite and CFA injected-induced inflamed TMJ, which has revealed a thinning of the proliferative layer [27] and many invasive methods that induced TMD have similar results accordingly [35]. It was also found that the severity tended to occur more on the left side, which may be related to the dominant side, but still unclear. This suggests the occlusive force or masticatory muscles might contribute to the pathogenesis of restraint stress-induced depression such as the unilateral posterior cross-bite can induced the integrin expression on the opposite side [6,7]. These results showed distinct histomorphological changes even within a short period of depressive time, which can link to inflammation inside the synovial fluid that may be induced by serum corticosteroid, or the mechanical force related to depressive symptoms. Still, the effects of depression that induced TMD depend on multiple factors which should also be taken into account such as duration, adaptive response of chewing, context of exposure, and individual variability.

Nevertheless, the use of animal models to study stress and psychopathology is challenging because the complexity of these disorders makes it difficult to model them entirely. Hence, some specific features of human psychopathology are difficult and even animal models should be used with great caution [37]. Even though antidepressants are not the first line of treatment for mild depression and should not be used for treating depression in children, they are not the first line of treatment in adolescents, among whom they should be used with extra caution. Yet, the development of drugs for depression-related disorders relies heavily on animal models. Furthermore, in this study, an obvious histological change in cartilage thickness and the percentage of proteoglycans at the pre-hypertrophic and hypertrophic layers was found in the atomoxetine group, but there were no significant changes in the escitalopram group when compared to the control group. Many severe TMJ-OA-induced models also show a similar loss of proteoglycan [27,38,39]. Additionally, SSRI previously induced cartilage degradation and ameliorated OA progression [40]. Therefore, the degeneration of cartilaginous layers can be worse in atomoxetine than in escitalopram, meanwhile, there are still limitations concerning the knowledge of the effect on cartilage remodeling and antidepressants, especially on NRI drugs.

The present findings showed the consequence of TMD pathogenesis induced by depression, which is important for clinicians to be able to recognize stages of osteoarthritis in order to provide contingent prevention. Even though the results were investigated in a blinded manner, there is still a limitation to explain the effect of drugs individually without combining with depression.

However, this study is a descriptive investigation that screens the differences between experimental groups in a blinded manner. The effects of the antidepressants in this study are the combinations when used with the treatment of depressive-like symptoms, so understanding of the mechanism of these drugs individually related to TMD needs to be further investigated. Further research into these changes would be quantitative, particularly on signaling pathways such as identifying the pathway of TMD inflammation with advanced technology such as next-generation sequencing of synovial fluid, immunohistochemistry at different time-points, proteoglycans mechanisms, or muscular activity during depression. Certainly, there is no denying that the complexity of this depressive model is difficult, which limits its investigation to fully reflect the etiology and progression of TMJ-OA.

In summary, these findings indicate that psychological depression can affect temporomandibular disorder (TMD) and is associated with the use of antidepressant medications. Through a comparison between rats exhibiting a depressive-like phenotype and those treated with escitalopram (SSRI) and atomoxetine (NRI), the present study observed distinct characteristics in each group. Specifically, the depressive group showed damage to the bone of the condylar head and changes in condylar cartilage in terms of morphology and histology, suggesting a significant impact on both bone and cartilage in understanding the pathogenesis of TMD. Interestingly, treatment with atomoxetine, which alleviated the depressive-like behavior, exhibited additional disadvantages such as increased bone porosity and substantial changes in condylar cartilage. Conversely, treatment with escitalopram induced bone porosity but had minimal effects on the cartilage layer and the surface of the condylar head. In conclusion, the present investigation highlights the role of depression as a significant causative factor in the development of TMD and suggests that the use of antidepressants may exacerbate the condition.

Acknowledgments

The authors thank Associate Professor Dr. Jarin Paphangkorakit, Assistant Professor Dr. Poramaporn Klanrit, Faculty of Dentistry for their critical suggestions, Dr. Ram Prajit, Faculty of Medicine, Khon Kaen University for histological process, Associate Professor Dr. Jantarima Charoenphandhu, Faculty of Medicine and Laboratory Animal Center, Thammasat University for behavioral testing facility. This research was funded by RGJ Advanced Program, Thailand Science Research and Innovation (TSRI; RAP61K0020) and the Young Researcher Development Project of Khon Kaen University Year 2020.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

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Corresponding author

Funder information

1.Fund name: RGJ Advanced Program, Thailand Science Research and Innovation

2.Fund name: Young Researcher Development Project of Khon Kaen University Year 2020

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