Dependence of cytokine levels on the sex of experimental animals: a pilot study on the effect of oestrogen in the temporomandibular joint synovial tissues

Abstract

The aim of this study was to evaluate the effects of 17β-oestradiol (E2) on cartilage thickness and cytokine levels in the temporomandibular joint (TMJ). Thirty rats (15 female, 15 male) were orchidectomized (ORX), ovariectomized (OVX), or sham-operated. After 21 days, animals were assigned to six groups: (1) sham-ORX; (2) ORX; (3) ORX+E2; (4) sham-OVX; (5) OVX; and (6) OVX+E2. Treatments were administered daily for 21 days. The thickness of cartilage layers (fibrous, proliferative, maturation, and hypertrophic) and cytokine levels (interleukins IL-1α, IL-1β, IL-6, and tumour necrosis factor alpha (TNF-α)) were measured by histomorphometry and ELISA, respectively. Kruskal–Wallis/Dunn’s tests were used (alpha = 5%). Sham-ORX showed thicker layers than ORX+E2, but not thicker than ORX. All layers, except the hypertrophic layer, were thicker in sham-OVX than OVX or OVX+E2. Although IL-1β levels were higher in castrated animals, E2 did not affect the level of this cytokine. IL-1α levels were higher in both ORX ( P = 0.0010) and ORX+E2 ( P = 0.0053) than in sham-ORX. However, E2 decreased IL-1α levels in OVX ( P = 0.0129). When compared to sham-ORX/OVX, IL-6 levels were not affected by E2 in males but were reduced in OVX ( P = 0.0079) and increased in OVX+E2 ( P = 0.0434). Levels of TNF-α were reduced by E2 in both ORX+E2 and OVX+E2. E2 treatment caused gender- and layer-dependent changes in the cartilage. Castration increased all cytokine levels, except for IL-6, without respect to gender.

The degeneration of joint cartilage in temporomandibular joint diseases (TMD) is more frequent in females than in males, suggesting a possible contribution of gonadal hormones to the pathophysiology of this condition. Inflammation also plays a role in TMD, especially in cases associated with arthritis, acute trauma, and cartilage derangement.

Levels of interleukins, such as IL-1β, IL-8, and IL-6, and of tumour necrosis factor alpha (TNF-α) are increased in the synovial fluid of patients with TMD. Some interleukins, such as IL-6 and IL-11, have also been associated with normal temporomandibular joint (TMJ) adaptive processes.

Oestrogen is a gonadal steroid hormone known to regulate diverse physiological processes in target tissues of both genders. At physiological concentrations, oestrogen may play a role in TMJ remodelling. Gonadal hormones have a significant impact on the development of autoimmune diseases in humans and in rodents.

The anti-inflammatory effects of both testosterone and oestrogen have been demonstrated in rats with arthritis. In these rats, physiological levels of both hormones have been found to reduce damage to reticulin organization and loss of collagen and glycosaminoglycan in cartilage. These findings have been associated with a reduction in TNF-α and matrix metalloproteinase 2 (MMP-2) levels and restoration of the antioxidant activity.

In the pathophysiology of joint inflammation, oestrogens perform specialized functions. For example, in rheumatoid arthritis, oestradiol has been found to enhance IL-1β and to increase the secretion of IL-6 in synoviocytes. 17β-Oestradiol has been shown to increase both expression and concentration of IL-1β, IL-6, and IL-8 in mouse TMJ cartilage. Oestradiol has been found to aggravate TMJ inflammation through the nuclear factor kappa B (NF-κB) pathway, leading to the induction of pro-inflammatory cytokines.

In men, high serum levels of testosterone and other androgens have been shown to decrease pain. In male rats, pain associated with TMD has been assessed to be lower due to the protective action of testosterone.

Ovariectomy (OVX) in rats has been shown to cause cartilage thickening and increased turnover in the subchondral bone of the mandibular condyle. However, cartilage thickening caused by OVX cannot be considered a typical characteristic of osteoarthritis.

The hypothesis of this study was that oestrogens play an important role in the maintenance of condylar cartilage and normal subchondral bone volume. The aim of the present study was to quantify the action of gonadal steroid hormones on TMJ cartilage after castration.

Materials and methods

This study was approved by the ethics committee for animal research of the study institution in Piracicaba, Brazil.

Fifteen female (200–300 g) and 15 male (300–400 g) 3-month-old Wistar rats ( Rattus norvegicus albinus ) were assigned randomly to six groups, as follows ( Fig. 1 ): (1) control-male, sham orchidectomy; (2) ORX, orchidectomized animals; (3) ORX+E2, orchidectomized animals + 17β-oestradiol; (4) control-female, sham ovariectomy; (5) OVX, ovariectomized animals; and (6) OVX+E2, ovariectomized + 17β-oestradiol.

Fig. 1
Experimental design and animal distribution into groups.

One millilitre of saline solution (oral) was administered daily to control, ORX, and OVX animals. The ORX+E2 and OVX+E2 groups received 50 μg of 17β-oestradiol intramuscularly (IM) daily (Sigma Chemicals, St. Louis, MO, USA) (modified from Yasuoka et al. ). All treatments were administered for 21 days. Rats were given free access to food and water and were kept in a 12-h light–dark environment at 23 °C.

Vaginal secretions from the control-female group were sampled daily for 21 days to evaluate ovarian function using the Shorr method. Only females presenting two or more consecutive oestrous cycles of 4 days were included in the control-female group.

For female rats, ovariectomy was performed by incision of the bilateral flanks, and ovarian bundles were ligated with 4–0 silk suture and removed, following the procedure detailed by Green et al. Both the fascia and skin were sutured with 5–0 silk suture. For male rats, orchidectomy was performed after a single incision in the scrotum. Testicular bundles were ligated with 4–0 silk suture and removed. The skin was sutured with 5–0 silk suture. A sham operation procedure was performed for both the female-control and male-control animals. Postoperative pain was controlled with pethidine chloride 2 mg/kg IM. In order to verify the efficacy of the ovariectomy, both colpocytology (performed daily for 15 days after surgery) and a post-mortem examination of uterine atrophy were performed. The efficacy of orchidectomy was verified by examination of the post-mortem prostate gland and seminal vesicles.

After 21 days of treatment, all animals were anaesthetized using 2% xylazine at a dose of 10 mg/kg and 10% ketamine at a dose of 90 mg/kg IM. The animals were killed by asphyxiation with carbon dioxide, and the TMJs of both sides were removed and selected randomly for either haematoxylin–eosin (HE) staining or immunoassays. The body weights of all animals were measured daily until euthanasia.

A histomorphological analysis was performed on the TMJs. Tissues were stored in 10% paraformaldehyde for 24 h at 4 °C and decalcified in 6% EDTA (ethylenediaminetetraacetic acid) for 3 months, using 0.1 M phosphate buffer (pH 7.4). After dehydration with ethanol, TMJs were embedded in paraffin blocks. Nine serial coronal 6-μm-thick sections (nine sections for each animal) were stained with HE. The apical region of the condyle was selected for the measurements. The thickness of each condylar cartilage layer (fibrous, proliferative, maturation, and hypertrophic) was assessed using Image-Pro Plus 4.5 software (Media Cybernetics, Inc., Rockville, MD, USA). Each layer measurement was performed in three different areas and the results expressed in millimetres.

Pro-inflammatory cytokines were evaluated in the TMJ, which was macerated and centrifuged in 50 mmol Tris HCl (pH 7.5). The levels of IL-1α, IL-1β, IL-6, and TNF-α cytokines in the supernatants were analysed using ELISA kits (PeproTech Inc., Rocky Hill, NJ, USA). The plates were coated with individual cytokine-specific capture antibodies. Samples, detection antibodies, and enzyme-linked secondary antibodies were then added. The colour change following the addition of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) was assessed by measuring optical density at 405 nm with the wavelength correction set to 650 nm.

Statistical analysis

Levene and Shapiro–Wilk tests were used to verify homogeneity of variance and data distribution, respectively. The body weight gain and measurements of articular cartilage layers and cytokines were analysed by Kruskal–Wallis test followed by the Dunn (post hoc) test. Statistical analyses were performed using Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA), with the significance level set at 5%.

Results

Body weight

Body weight was found to be significantly increased at the end of the study in all animals (Kruskal–Wallis, H = 21.01; df = 5; P = 0.0008) ( Fig. 2 ). OVX gained significantly more weight than ORX ( P = 0.0008), and both OVX and ORX gained more body weight than the other groups (male and female). Treatment with 17β-oestradiol reduced body weight in castrated females ( P = 0.0049) and males ( P = 0.0139).

Fig. 2
Percentage change in weight (difference between the weight at the beginning of the study and at the end of the study) according to the study group. Central bar = median; boxes = 1st and 3rd quartiles; whiskers = maximum and minimum values. Lowercase letters indicate significant differences ( P < 0.05) among the groups.

Cartilage layer thickness

The morphology of fibrous, hypertrophic, proliferative, and maturation layers of TMJ cartilage in female and male rats are shown in Figs. 3 and 4 , respectively. Morphological changes were observed in OVX females in all layers of the condylar cartilage ( Fig. 3 B, E, and H). Cellular derangement and a decrease in fibrous layer thickness were observed in the proliferative, maturation, and hypertrophic layers ( Fig. 3 E). An increased number of chondrocytes was observed in the hypertrophic layer, which also appeared flattened ( Fig. 3 H). In OVX+E2 animals, the thickness of the condylar cartilage was reduced. The thickness of subchondral bone was increased and cellular reorganization was more pronounced relative to sham-female and OVX animals ( Fig. 3 C, F, and I). Many chondrocytes appeared confined between the newly mineralized subchondral bone tissue and the cartilaginous tissue ( Fig. 3 I).

Fig. 3
Coronal sections of female TMJs (HE staining). A–C: 10× magnification, 100 μm scale bar; D–F: 20× magnification, 50 μm scale bar; G–I: 40× magnification, 20 μm scale bar. Condyle region: AD, articular disk; AC, articular cartilage; SB, subchondral bone. The dashed yellow lines divide the cartilage and subchondral bone. The red arrow (image E) indicates morphological changes in the fibrous layer. The yellow arrow (image I) shows a chondroblast cell confined inside the bone matrix. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Fig. 4
Coronal sections of male TMJs (HE staining). A–C: 10× magnification, 100 μm scale bar; D–F: 20× magnification, 50 μm scale bar; G–I: 40× magnification, 20 μm scale bar. Condyle region: AC, articular cartilage; SB, subchondral bone. The dashed yellow lines divide the cartilage and subchondral bone. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Changes in morphology were observed in all cartilage layers in the ORX group. Fibrous layer thickness was decreased, but was normal in appearance and similar to the sham-operated male group ( Fig. 4 B, E). Structural disorganization and increased spacing among cells were observed in the proliferative, hypertrophic, and maturation layers ( Fig. 4 E, H). In the 17β-oestradiol group, the thickness of condylar cartilage was decreased and the mineralization process was accelerated, which enhanced differentiation of the cartilaginous tissue into subchondral bone ( Fig. 4 C, F).

Fig. 5 shows the measurements of all layers. In the OVX+E2 group, total cartilage thickness was lower than in both sham and OVX females (Kruskal–Wallis, H = 154.3; df = 5; P < 0.0001). Cartilage layer thickness in females was greater in the sham group than in the OVX group ( P < 0.0001), with the exception of the hypertrophic layer, which was thicker in OVX than in sham-operated animals ( P = 0.0366).

Fig. 5
Thickness of fibrous (A), maturation (B), proliferative (C), and hypertrophic (D) layers of male and female animals. Central bar = median; boxes = 1st and 3rd quartiles; whiskers = maximum and minimum values. The different lowercase letters show significant differences ( P < 0.05) among groups of the same gender.

In males, no significant differences in cartilage layer thickness were found between sham-operated and ORX, with the exception of the maturation layer ( P < 0.0001). Sham-operated males showed thicker cartilage layers than ORX+E2. In the 17β-oestradiol (ORX+E2) group, decreased thickness in both hypertrophic ( P < 0.0001) and proliferative layers ( P < 0.0001) was measured, but the maturation layer was increased in thickness ( P = 0.0052) when compared to ORX.

Table 1 shows the measurements of total cartilage thickness. No significant differences were observed between the sham-operated and OVX groups ( P = 0.72), but the total thickness of the cartilage in OVX-E2 animals was lower than both sham-operated and OVX animals ( P < 0.0001). In males, the thickness of the cartilage was decreased in the ORX-E2 group when compared to both the ORX and sham-operated groups ( P < 0.0001). Sham-operated males had a thicker cartilage than the ORX ( P < 0.0001).

Table 1
Combined thickness of all cartilage layers according to the study group.
Group Total cartilage layer thickness (μm)
Median; interquartile deviation
(95% CI)
Female Male
Sham 238.4; 22.3 a
(227.3–245.4)
250.1; 6.8 a
(247.0–251.5)
Castrated 231.3; 6.7 a
(229.2–233.7)
229.6; 35.4 b
(217.5–246.8)
Castrated + 17β-oestradiol 85.8; 14.2 b
(81.7–91.2)
201.6; 15.1 a
(196.5–205.7)
a,b Different letters indicate significant differences (Kruskal–Wallis and Dunn’s test, P < 0.05) among groups, considering males and females separately.

Cytokine levels

In both males and females, IL-1β levels were higher in castrated animals (with or without 17β-oestradiol) (Kruskal–Wallis, H = 19.8; df = 5; P < 0.0001; Fig. 6 ). No changes in IL-1β levels were found in the 17β-oestradiol groups. IL-1α levels in males were higher (Kruskal–Wallis, H = 30.9; df = 5; P < 0.0001) in both ORX ( P = 0.0010) and ORX+E2 ( P = 0.0053) than in the sham-operated group. IL-1α levels were decreased in the 17β-oestradiol group relative to OVX females ( P = 0.0129), but no changes in IL-1α were observed in castrated males ( P = 0.9989).

Jan 17, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on Dependence of cytokine levels on the sex of experimental animals: a pilot study on the effect of oestrogen in the temporomandibular joint synovial tissues
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