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Gross morphologic and histopathological evaluation of effect of Azadirachta indica (neem) leaf extract on monosodium iodoacetate-induced osteoarthritis of temporomandibular joint in adult Wistar rats
*Corresponding author: Ekaniyere Benlance Edetanlen, FWACS, Department of Oral and Maxillofacial Surgery, University of Benin Teaching Hospital, Benin, Edo State, Nigeria. ehiben2002@yahoo.com
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Received: ,
Accepted: ,
How to cite this article: Edetanlen EB, Innih SO, Eze GI. Gross morphologic and histopathological evaluation of effect of Azadirachta indica (Neem) leaf extract on monosodium iodoacetate-induced osteoarthritis of temporomandibular joint in adult Wistar rats. Am J Pharmacother Pharm Sci 2026:010.
Abstract
Objective:
Osteoarthritis (OA) of the temporomandibular joint (TMJ) is a global health burden as no current treatment is agreed to be optimum. Therefore, the aim of this study is to evaluate the effects of Azadirachta indica aqueous leaf extracts on gross morphology and histopathological changes in monosodium iodoacetate-induced (MIA) OA of TMJ of adult Wistar rats.
Materials and Methods:
Fifty-four male adult Wistar rats were randomly allocated into 6 groups: (1) Control, (2) MIA alone, (3) MIA + Steroid, (4) MIA +250 mg/kg A. indica, (5) 500 mg/kg A. indica, (6) MIA +1000 mg/kg A. indica. Morphological data collected were condylar head shape (CHS), condylar head length (CHL), condylar head width (CHW), and condylar head height (CHH). The histopathological data collected were condylar cartilage thickness (CCT), presence of subchondral cysts (SCC), inflammation score, and Mankin’s score. Both descriptive and inferential statistics were performed.
Results:
The CHS, CHW, CHL, and CHH remained unchanged across the experimental animals (P > 0.05). The CCT was smaller (P = 0.002) in the MIA group compared to the control group. There was a larger (P = 0.01) number of SCCs in the MIA alone group than in the controls. The value of the inflammation score was higher (P = 0.004) in the group that received MIA only compared to the control group. The value of Mankin’s score was higher (P = 0.002) in the group that received MIA alone compared to the control group.
Conclusion:
The protective effect of A. indica against MIA-induced OA of TMJ was demonstrated by histopathological evaluation but not by morphological analysis in adult Wistar rat.
Keywords
Azadirachta indica
Gross morphologic
Histopathological
Monosodium iodoacetate
Osteoarthritis
INTRODUCTION
TMJ osteoarthritis (OA) is a common musculoskeletal condition affecting approximately 10– 17% of patients showing pain.[1] OA is a major cause of morbidity and disability and constitutes a significant burden on healthcare resources and interferes with the ability to perform daily tasks, ability to walk, and ability to exercise.[2] People with OA experience depression, anxiety, fatigue, sleep disturbance, and consequently poor quality of life.[3] Itis the most prevalent and disabling joint disease in the world, and its social and psychological consequences have been reported.[4,5]
OA is defined as a chronic, progressive degenerative pathology, characterized by cartilage degradation, remodeling of the subchondral bone, synovitis, narrowing of the joint space, formation of osteophytes, and chronic pain.[6] As synovial inflammation is one of the major signs of OA, the role of inflammatory cytokines and mediators in the progression of OA is now well recognized.[7] Synovium and chondrocytes produce and release cytokines and inflammatory mediators in the synovial fluid of OA patients.[8]
Although steroidal and non-steroidal anti-inflammatory drugs (NSAIDs) are currently being used for OA pain management in developing countries such as Nigeria, their long-term use is often associated with negative health consequences, including cardiovascular, renal, and gastrointestinal disorders.[9] In addition, while these treatments are used in everyday clinical practice, there is currently little or no high-quality evidence to support their utility, and thus their true efficacy is controversial, and there is no consensus on optimum treatment of OA globally.[10,11] Therefore, the demand for an alternative treatment of OA that is safe and effective is increasing.
Despite the anti-inflammatory role of Azadirachta indica (Neem) has been well documented in the treatment of several inflammatory diseases,[11-14] its role in the treatment of OA of the TMJ is yet to be documented both in human and animal studies. In addition, several researchers[15-17] have investigated traditional herbal medicinal resources in the treatment of OA, but it is unclear if this is so with A. indica. Therefore, this study aimed to evaluate the effect of A. indica aqueous leaf extract on monosodium iodoacetate (MIA)-induced TMJ OA in adult Wistar rats using gross morphological and histopathological analyses.
MATERIALS AND METHODS
Preparation of plant extract
A. indica leaves were obtained from the University of Benin (UNIBEN) environment in Benin City, Edo State, Nigeria. They were identified in the Department of Plant Biology and Biotechnology, Faculty of Life Sciences, UNIBEN, with a voucher number of UBH-A286.
The leaves were air-dried for 7 days before being ground into powder and weighed on an electronic weighing scale (Gullan®, Munich, Germany). Extraction was carried out utilizing proven methods.[18] Preparation of aqueous leaf extract of A. indica was conducted in the Department of Pharmacognosy, Faculty of Pharmacy of the UNIBEN. Before being macerated in distilled water in a jar, the leaf was pulverized in a British milling machine (Miller®, London, UK). About 500 g of powder was soaked in 2 L of cold distilled water in a conical flask. After 24 h, the solution was filtered through Whatman no. 3 filter paper (Sigma Aldrich; Merck KGaA, Darmstadt, Germany) funnel. Before decanting the supernatant, the filtrate was allowed to settle for a time. At 60°C, the supernatant was steamed to dryness in an evaporating dish (Royal Worcester, England) using an H-H Digital Thermometer Water Bath (Mc Donald Scientific International – 22050Hz1.0A). The extracts were kept refrigerated at 4°C in plastic vials until needed.
Experimental animals
A total of 54 male adult Wistar rats were used in this study. They were older than 5 months, weighed between 200 and 250 g, and they were purchased from the animal house of the Department of Anatomy, School of Basic Medical Sciences, UNIBEN. They were housed in standard animal cages under a 12h/12h light and dark cycle, temperature of 21 ± 1°C, and relative humidity of 40–60%, and they were fed with standard pellet animal feed and clean water ad libitum throughout the study. They were acclimatized for 2 weeks before commencement of the study. All rats were weighed before induction and after treatment with a beam balance (Gullan®, Munich, Germany). All procedures were performed in accordance with the Ethical Principles and Guidelines for Experiments on Animals and with the Nigerian regulations on the practical, educational, and scientific use of vivisection.[14] Ethical clearance approval was obtained from the Ethical and Research Committee of the UNIBEN (CMR/REC/01/VOL.2/569).
Experimental design
The rats were randomly divided into 6 groups of 9 each as follows: Group 1 received intra-articular injection of 50 µl of distilled water as a single dose and oral intake of 1 ml distilled water daily for 4 weeks by gavage. Group 2 received intra-articular injection of 50 µl of 0.5 mg/kg of MIA as a single dose and oral intake of 1 ml of distilled water daily for 4 weeks by gavage. Group 3 received intra-articular injection of 50 µl of 0.5 mg/kg of MIA as a single dose and 0.1 mg/kg of dexamethasone intramuscularly thrice weekly for 4 weeks. Group 4 received intra-articular injection of 50µl of 0.5 mg/ kg of MIA as a single dose and oral intake of 1 ml of 250 mg/ kg of aqueous extract of A. indica daily for 4 weeks by gavage. The doses of A. indica, MIA, and dexamethasone were based on previous studies.[19-21] The extract and the steroid were given on the same day as the intra-articular injection of MIA. Group 5 received intra-articular injection of 50 µl of 0.5 mg/ kg of MIA as a single dose and oral intake of 1 ml of 500 mg/kg of aqueous extract of A. indica daily for 4 weeks by gavage.
Group 6 received intra-articular injection of 50 µl of 0.5 mg/ kg of MIA as a single dose and oral intake of 1 ml of 1000 mg/kg of aqueous extract of A. indica daily for 4 weeks by gavage.
The doses of A. indica, MIA and dexamethasone was based on a previous studies.[19-21] The extract and the steroid were given same day of intra-articular injection of MIA.
OA inductions
The procedure for induction of OA of the TMJ has been established as previously reported[22], and this was performed bilaterally. In brief, the animals were anesthetized with an intramuscular injection of a 1:1 combination of ketamine (10 mg/kg) and midazolam (1 mg/kg) at a dosage of 0.5 mg/100g. A 30-gauge 0.5-inch needle was used to inject the MIA solution. The anatomical points essential for performing this procedure are located 5 mm below the zygomatic arch and 5 mm anterior to the external ear. A line was drawn from the external ear to the eyeball, parallel and over the zygomatic arch of the rat. Subsequently, the condyle was located superficially, about 5 mm in front of the external ear. Thereafter, a line was drawn perpendicular to the initial line. The needle was inserted obliquely at an angle of 45°superomedially and 5 mm below the cross-linking of the two lines.
Gross morphology procedure
The rats were sacrificed by cervical dislocation, and articular condylar tissues were harvested.
Gross morphology assessment
Gross morphological analysis [Figure 1] has been an established method that was previously described.[23] This analysis was performed by a blinded research assistant.
- Gross morphological assessment of the condylar head.
Condylar head shape (CHS)
The shapes of the condyles were assessed for any changes. If the shape was convex, it was considered normal or no changes and categorized as “No” and if the shape was flattened, it was considered a change in shape and categorized as “Yes.”
Condylar head width (CHW)
This was measured with a self-retaining digital Vernier caliper (Tresna Limited, Tokyo, Japan) in millimeters. This was the most lateral (left) point of the condyle to the most medial (right) point of the condyle.
Condylar head length (CHL)
This was measured with a self-retaining digital vernier caliper (Tresna Limited, Tokyo, Japan) in millimeters. This was the most anterior point of the condyle to the most posterior point of the condyle.
Condylar head height (CHH)
This was measured with a self-retaining digital Vernier caliper (Tresna Limited, Tokyo, Japan) in millimeters. This was the highest point of the condyle to the neck of the condyle.
Histopathological procedure
Following the rat sacrifice at 4 weeks, joint tissues were resected, fixed in 10% formalin (SigmaAldrich; Merck KGaA) for 24 h at 4°C, and decalcified with 5% hydrochloric acid (SigmaAldrich; Merck KGaA) for 4 days at 4°C. Specimens were then dehydrated in graded acetone and embedded in paraffin. Sections (thickness, 2–3 μm) were stained with 0.2% hematoxylin and 1% eosin (Hematoxylin and Eosin; SigmaAldrich; Merck KGaA) for 5 min and 3 min, respectively.
Histopathological assessment
The histopathological slides were analyzed by a blinded histopathologist using a light microscope (Olympus, Japan) with a camera (Leica CC50).
Condylar cartilage thickness (CCT)
This method of analysis has been previously described.[11] The CCT was estimated using the light microscope as previously reported and recorded in micrometers. With the image analysis software - Image-Pro-Plus 4 (Media Cybernetics, USA), the thicknesses of the condylar cartilage were measured at the central portion of the cartilage.
Prevalence of subchondral cyst (SCC)
The presence of SCC was assessed and recorded. The prevalence of the SCC was estimated from the number counted for all slides. The presence and absence of SCC were categorized as “yes” and “no” respectively. This was used for cross-tabulation in statistical analysis.
Mankin’s score histological
The histological grading of the OA was based on Mankin’s system[24] as shown in Table 1. Lower value indicates absence or mild OA, while higher values indicate severe OA.
| Criteria | Score | Histological finding |
|---|---|---|
| Structure | 0 | Smooth intact surface |
| 1 | Slight surface irregularities | |
| 2 | Pannus/surface fibrillation | |
| Structure | 3 | Clefts into transitional zone |
| 4 | Clefts into radial zone | |
| 5 | Clefts into calcified zone | |
| 6 | Total disorganization | |
| 0 | Uniform cell distribution | |
| Cells | 1 | Diffuse cell proliferation |
| 2 | Cell clustering | |
| 3 | Cell loss | |
| Tide mark integrity | 0 | Intact |
| 1 | Vascularity |
Inflammatory score
The level of inflammation was recorded and scored based on Kristensen’s classification system, as shown in Table 2.[25] Lower value indicates absence or mild inflammation, while higher values indicate severe inflammation.
| Description | Score |
|---|---|
| Absence of inflammatory cells | 0 |
| Mild congestion and edema, few scattered inflammatory cells | 1 |
| Congestion and edema, small number of neutrophils | 2 |
| Multiple inflammatory cells organized in bands | 3 |
| Massive presence of inflammatory cells | 4 |
Reliability analysis
To minimize the risk of false assessment caused by fatigue, no more than 10 tissue specimens were evaluated at a time. To assess intra-observer agreement, the intra-class correlation coefficient (ICC) was calculated. All measurements were made twice at an interval of 2 days, and the mean values were used for the analysis.
Statistical analysis
The collected data were entered and analyzed using the statistical package for the social sciences, version 26 (IBM, Armonk, NY, United States of America). Both descriptive and inferential statistics were performed. In the descriptive statistics, the categorical data were reported as frequency and percentage, while that of continuous data were summarized as range, means ± standard deviation. In the inferential statistic, the normality of data was assessed with Shapiro– Wilk normality test. Chi-square test was used for categorical data, while one-way analysis of variance and post hoc with Tukey was used for the continuous data to assess the statistical differences among groups. A critical probability level (P-value) of <0.05 was used as the cutoff level for statistical significance.
RESULTS
A total number of 54 experimental animals survived throughout the duration of the study. There was excellent intra-observer reliability among the two measurements with an ICC value of 0.96, 95% confidence interval = 0.92– 0.99 for mean measures. This showed that the reliability of the variables chosen to identify the extremities of the measurements was good.
There were no significant differences between initial and final body weights across the groups (P = 0. 63) [Figure 2]. As shown in Figures 3-6, the CHS, CHW, CHL, and CHH remained unchanged across the experimental animals (P > 0.05). Figures 7 and 8 show the effect of A. indica aqueous leaf extract on CCT of adult Wistar rats. The CCT was smaller (P = 0.001) in the MIA group (12.7 ± 1.90 μm) compared to the control group (35.9 ± 1.69 μm). The CCT in the group that receive steroid (31.3 ± 1.45 μm; P = 0.001), 250 mg/kg (30.4 ± 2.85 μm; P = 0.001), 500 mg/kg (29.9 ± 2.95 μm; P = 0.006), and 1000 mg/kg (30.2 ± 3.93 μm; P = 0.004) A. indica therapies were larger than that of MIA alone group. Figure 9 shows the occurrence of SCC in the adult Wistar rats. Using the rats as units of analysis, there was a larger (P = 0.01) number of SCCs in the MIA alone group (45.5%) than in the controls (0.0%). There was reduced number of SCCs among the rats that had steroid (9.1%, P = 0.008) and A. indica therapies (250 mg/kg (9.1%, P = 0.02); 500 mg/kg (18.2%, P = 0.01); 1000 mg/kg (9.1%, P = 0.02) compared to the MIA alone group.
- Effect of Azadirachta indica aqueous leaf extract on body weights. MIA: Monosodium iodoacetate. Each value is the mean ± SD. The error bars point out the standard deviation of the mean.
- Effect of Azadirachta indica aqueous leaf extract on prevalence of condylar head shape. Values were given as percentage. MIA: Monosodium iodoacetate.
- Effect of Azadirachta indica aqueous leaf extract on condylar head width. Each value is the mean ± SD. The error bars point out the standard deviation of the mean. MIA: Monosodium iodoacetate.
- Effect of Azadirachta indica aqueous leaf extracts on condylar head length. Each value is the mean ± SD. The error bars point out the standard deviation of the mean. MIA: Monosodium iodoacetate *p < 0.05.
- Effect of Azadirachta indica aqueous leaf extract on condylar head height. Each value is the mean ± SD. The error bars point out the standard deviation of the mean. MIA: Monosodium iodoacetate.
- Effect of Azadirachta indica aqueous leaf extract on condylar cartilage thickness. Each value is the mean ± SD. The error bars point out the standard deviation of the mean. *Significantly different from the control group, #Significantly different from the MIA group. MIA: Monosodium iodoacetate.
- Representative photomicrographs of the condylar tissue across experimental group. (a) Control. (H&E X100); (b) MIA alone (H&E X100); (c) MIA+ steroid (H&E X100); (d) MIA + 250mg/kg A.indica (H&E X100); (e).MIA + 500mg/kg A.indica (H&E X100); (f) MIA 1000mg/kg A.indica (H&E X100); F: Fibrous layer, M: Maturation layer, P: Proliferative layer, H: Hypertrophic layer, O: Osteochondral junction, MC: Medullary cavity, SC: Subchondral cyst. Black (arrows) indicates pitting in fibrous layer. MIA: Monosodium iodoacetate.
- Effect of Azadirachta indica aqueous leaf extract on prevalence of subchondral cyst. Values are given as percentage. *p<0.05 compared with the control group, #p<0.05 compared with the MIA-alone group. MIA: Monosodium iodoacetate.
Figure 10 shows the effect of A. indica aqueous leaf extract on the inflammation score of adult Wistar rats. The value of the inflammation score was higher (P = 0.004) in the group that received MIA only (5.4 ± 0.5) compared to the control group. The inflammation score reduced in the groups that were treated with steroid (0.8 ± 0.3, P = 0.002), 250 mg/kg (1.3 ± 0.4, P = 0.003), 500 mg/kg (1.1 ± 0.2; P = 0.005) and 1000 mg/kg (1.5 ± 0.06, P = 0.008) A. indica extracts when compared to the group that received MIA alone.
- Effect of Azadirachta indica aqueous leaf extract on inflammation score. Each value is the mean ± SD. The error bars point out the standard deviation of the mean. *Significantly different from control group, #Significantly different from the MIA group. MIA: Monosodium iodoacetate.
The value of Mankin’s score was higher (P = 0.002) in the group that received MIA alone (6.7 ± 0.9) compared to the control group [Figure 11]. The Mankin’s score reduced (P < 0.05) in the steroid (2.6 ± 0.2, P = 0.003), 250 mg/kg A. indica (3.5 ± 0.5, P = 0.03), 500 mg/kg A. indica (3.8 ± 0.3, P = 0.002) and 1000 mg/kg A. indica (2.9 ± 0.1, P = 0.004) when compared to the group that received MIA alone.
- Effect of Azadirachta indica aqueous leaf extract on Mankin’s score. Each value is the mean ± SD. The error bars point out the standard deviation of the mean. Significantly different from control group, #Significantly different from the MIA group. MIA: Monosodium iodoacetate. *p < 0.05.
DISCUSSION
The anti-inflammatory effect of A. indica was evaluated on MIA-induced OA of TMJ in Adult Wistar rats. A. indica, or neem, a widely cultivated pan-tropical plant, is recognized for its diverse applications, including religious, economic, medical, and decorative purposes.[26] It serves as a “wonder” tree with various valuable components (roots, trunk, bark, leaves, flowers, fruits, and seeds) utilized for wood, fuel, pharmaceuticals, insecticides, and oil.[27-31] A. indica has various properties such as antibacterial, antiparasitic, anti-inflammatory, and antioxidant. It has a complex of various pharmacologically active bioconstituents, including nimbin, nimbidin, nimbolide, and limonoids.[13]
Weight loss may result from poor feeding secondary to occlusal disharmony.[32] In our study, bilateral induction of OA was done, and no significant weight loss was observed, which could be because feeding was not hampered. However, Ren et al., (2004) suggested unilateral OA induction to prevent feeding disturbances.[32] It was also observed that administration of A. indica extract for 4 weeks did not affect the body weight of the animals. This corroborated the findings of Innih et al., (2014), who found no difference between initial and final body weight in their experimental rats.[18] Contrary to our finding, Ghimeray et al. (2009) reported weight loss in rats at doses higher than 200 mg/kg.[33]
Cho et al. (2009) and other researchers have reported morphological changes in individuals suffering from OA.[8] In this current study, the morphological parameters evaluated were CHS, CHW, CHL, and CHH. Meanwhile, there was no significant alteration in the gross morphology across the experimental animals in the current study. The possible reason for this variation could be that the duration of the study was short, and the full progression of OA was not observed. Aube and Ramirez-Yanez (2019) reported that OA is a chronic condition with both degenerative and inflammatory components.[34] Another possible reason could be the nature of samples, as most previous studies[35-38] were on humans. In our study, MIA could not cause deformation of the morphology of the mandibular condylar head, and this finding is in accordance with previous studies.[11,14,39,40,41]
In this study, the average total thickness of MCC in control rats was 2.44 μm, which is consistent with 2.41 μm reported by Sa et al. (2017).[22] The mandibular CCT was remarkably reduced in the MIA group, and this was a confirmation that OA was induced in our study. This finding is in agreement with that of Béret et al., (2023), who reported a decrease in CCT following intra-articular injection of MIA.[15] Although the anti-osteoarthritic property of A. indica was weaker than that of dexamethasone, A. indica significantly prevents the reduction of the MCC thickness. The effect of A. indica was not dose-dependent in preventing the alteration of the mandibular condylar cartilage caused by MIA in our study. This finding is contrary to that of Katiyar et al., in 2023, who reported that A. indica has concentration-dependent killing properties against Gram-positive (S. aureus) and Gram-negative (P. aeruginosa) bacteria.[25] Mandibular condylar cartilage, unlike other cartilage, has a unique structure. It plays dual roles as articular cartilage responding to biomechanical stress, such as that produced by jaw movement during mastication, and as the growth plate cartilage of long bones during growth.[42] The MCC is composed of four different zones: The fibrous, proliferative, pre-hypertrophic (mature), and hypertrophic.[7] The MCC is a load-bearing musculoskeletal tissue, which distributes stress.[43] The nature and duration of applied loads determine the biomechanical properties of the MCC. Small changes in the integrity, composition, or organization of cellular components of the cartilage will alter the matrix production and may eventually alter its mechanical properties. Effects of abnormal occlusal loading on MCC are well reported[44,45] but none appear to be reported regarding OA.
SCCs were significantly present in the MIA group in this study, as this was an indication of the induction of OA using MIA. A similar finding was reported by Tanaka et al., in 2020, who reported a reduction of SCC following an interventional study in patients with OA.[43] Both steroid and A. indica were able to reduce the formation of SCCs in the experimental animals. A low dose of A. indica was as effective as the high dose in the prevention or reduction of SCC formation. Although OA was previously considered primarily as an articular cartilage disorder, it is now recognized as a type of pan-arthritis, a condition involving all the structures of a joint, including the calcified cartilage, the subchondral bone, the capsular ligaments, and the synovial fluid.[46,47] In addition to its very important structural support role, the subchondral bone presents an equally important biological crosstalk with the overlying cartilage, making the articular cartilage–subchondral bone system a single inseparable functional unit.[48] A SCC is a fluid-filled space inside a joint that extends to the subchondral bone.[49] Radiological and histological feature of OA of SCCs was described by Kaspiris et al., (2023).[47] They also reported that SCCs can be features of both chronic and acute OA. They develop due to the bone’s attempt to adapt in areas subject to increased loads. SCCs are referred to in the literature under the following terms: Pseudocysts or osteoarthritic cysts.[50] They were first identified by Kapoor et al., (2011) in the load-bearing regions of the femur, patella, and shoulder joints of arthritic patient.[48] Although their pathogenic mechanism has not yet been elucidated, there are two prevalent theories regarding the formation of SCCs in load-bearing bone regions in the context of OA. According to the first theory (synovial breach theory), SBCs are created due to the loss-thinning of the supernatant articular cartilage and repeated microtrauma to the osteochondral junction. In this case, microfractures in the articular surface allow the penetration of synovial fluid and/or tissue into the area of the subchondral bone, leading to an acute inflammatory response and the development of myxomatous tissue within the bone marrow.[51] In contrast, according to the second theory (bone contusion theory), there is no direct communication between the joint and the subchondral bone.[52] The wear and subsequent weakening of the articular cartilage leads to uneven distribution of loads and degradation of the quality of the underlying bone, resulting in microfractures of the subchondral bone and bone marrow oedema, activating the bone reconstruction process. The aggregation of osteoclasts and macrophages causes bone absorption and phagocytosis of necrotic elements, with subsequent recruitment of osteoblasts for the deposition of new bone, which eventually encapsulates a cavity with fibrous content, creating the SBC.[46]
In our study, Mankin’s score was significantly higher in the MIA alone group compared to the control group. This finding is in accord with that of Thomas et al., (2019), who reported a higher Mankin’s score in animals with induced OA and marked lower value in the treated animals.[49] Mankin’s scoring confirms the development and prevention of OA histopathology of the TMJ following A. indica and steroid therapy. For the histopathological classification of the severity of OA or level of cartilage degradation, the Mankin’s score has been frequently used as reported by Pauli et al., (2012).[50] This system was developed originally for the assessment of human hip OA cartilage, and subsequently it has also been used to evaluate cartilage degradation, repair, and regeneration in various animal models of OA. The Mankin’s system assesses three parameters: cartilage structure, cellularity, and tidemark integrity. Each parameter has subcategories, and the scores are summed to provide a total score ranging from 0 (normal) to 14 (most severe OA).[24]
The inflammation score was significantly higher in the MIA alone group compared to the control group and the score was significantly lower in the treated groups compared to the MIA alone group. This is a confirmation of the anti-inflammatory role of steroids and A. indica, and these findings corroborated that reported in previous studies.[16,22] Though in our study, steroid was a stronger anti-inflammatory agent, Patil et al., (2012),[51] contrarily, reported that A. indica was more effective than commercially available NSAID such as ibuprofen. In TMJ-OA pathogenesis, adverse inflammation activates catabolic matrix degradation that induces condyle cartilage degeneration, apoptosis, necroptosis, and chondrocyte death to exacerbate joint damage, pain, and disease progression. Various pro-inflammatory molecules, including interleukin -1β (IL-1β) and tumor necrosis factor-α (TNF-α), have been reported to play crucial roles in TMJ-OA progression. Their levels are significantly elevated in the synovial fluid of patients with TMJ-OA, and they are believed to mediate condyle cartilage degeneration and matrix degradation by inducing matrix metalloproteinase (MMP)-3 and MMP-13 expression.[40] Commonly, the elevated inflammation reduces chondrocyte proliferation and induces alterations in the condyle cartilage matrix due to apoptosis and necroptosis.[40] IL-1β enhances calcium influx in chondrocytes, which inhibits mitochondrial function and leads to chondrocyte apoptosis. A. indica can remarkably reduce the release of monocyte chemotactic protein-1 and TNF-α.[52]
This study has a few limitations. First, the duration of the outcome assessment could have been on a long-term basis. Second, the alcohol extract of A. indica could have been used for better storage, though the aqueous extract was kept in the refrigerator. Finally, larger sample size could have been used. In conclusion, the protective effect of A. indica against MIA- induced OA of TMJ was demonstrated by histopathological evaluation but not with morphological analysis in adult Wistar rats.
CONCLUSION
The protective effect of Azadirachta indica against monosodium iodoacetate- induced osteoarthritis of the temporomandibular joint was demonstrated by histopathological evaluation unlike gross morphologic analysis in adult Wistar rat.
Author contributions:
EBE and GIE conceived and designed the research, EBE collected and analyzed the data. EBE wrote the manuscript, SOI and GIE reviewed the manuscript.
Ethical approval:
The research/study approved by the Institutional Review Board at University of Benin, number CMR/REC/2024/560, dated June 25 2024.
Declaration of patient consent:
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Conflicts of interest:
There are no conflict of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation:
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
Financial support and sponsorship: None.
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