Evaluation of inflammatory processes in temporomandibular joint employing technetium-99m-labelled autologous leukocytes in an animal model

Abstracts

OBJECTIVE: The present study was aimed at identifying temporomandibular joint inflammatory processes employing technetium-99m hexamethylpropylene amine oxime (99mTc-HMPAO)-labelled autonomous leukocytes. MATERIALS AND METHODS: We have utilized an experimental model of arthritis induction in ten adult male New Zealand rabbits by means of ovalbumin intra-articular injection into each left temporomandibular joint. For control purposes, saline solution was injected. After leukocytes radiolabelling with 99mTc-HMPAO and injection into rabbits, scintigraphic images were obtained. RESULTS: A higher 99mTcHMPAO-leukocytes uptake was observed in left temporomandibular joint in comparison with the contralateral joint. Wilcoxon non-parametric test was applied for statistical analysis. There was a statistically significant difference between counts of radioactivity per minute in the inflammed joint and the contralateral one (p = 0.0073). CONCLUSION: The method employing 99mTc-HMPAO-labelled autologous leukocytes allows an early and accurate detection of inflammatory processes, contributing to the adoption of a therapeutic conduct for patients before structural alterations have occurred.

Temporomandibular joint disorder; Arthritis; 99mTc-HMPAO

OBJETIVO: O objetivo deste estudo foi identificar processos inflamatórios na articulação temporomandibular empregando leucócitos autólogos marcados com tecnécio-99m hexametilpropilenoaminooxima (99mTc-HMPAO). MATERIAIS E MÉTODOS: Foi utilizado um modelo experimental de indução de artrite na articulação temporomandibular de dez coelhos machos da raça Nova Zelândia, por meio da injeção intra-articular de ovalbumina na articulação temporomandibular esquerda de cada animal. Para controle, na articulação contralateral foi injetada solução salina. Após a marcação dos leucócitos com 99mTc-HMPAO e injeção endovenosa deste complexo nos coelhos, imagens cintilográficas foram obtidas. RESULTADOS: Observou-se captação aumentada dos 99mTc-HMPAO-leucócitos na articulação temporomandibular esquerda quando comparada à direita. A análise estatística foi realizada utilizando-se o teste não-paramétrico de Wilcoxon. Houve diferença estatisticamente significativa dos valores das contagens por minuto de radioatividade, relativos à articulação inflamada quando comparados aos valores obtidos na articulação contralateral (p = 0,0073). CONCLUSÃO: O método empregando leucócitos autólogos marcados com 99mTc-HMPAO é capaz de identificar focos inflamatórios de forma precoce e precisa, o que poderá contribuir na conduta terapêutica dos pacientes, antes que alterações estruturais sejam instaladas.

Desordem temporomandibular; Artrite; 99mTc-HMPAO

ORIGINAL ARTICLE

Evaluation of inflammatory processes in temporomandilar joint employing technetium-99m-labelled autologous leukocytes in an animal model^*

Cláudia Borges Brasileiro^I; Valbert Nascimento Cardoso^II; Bianca Ruckert^III; Tarcísio Passos Ribeiro de Campos^IV

^IMaster's Degree in Sciences and Nuclear Techniques at Universidade Federal de Minas Gerais School of Engineering, Professor for Course of Odontology at Centro Universitário Newton Paiva

^IIDoctor of Nuclear Sciences by Instituto de Pesquisas Energéticas e Nucleares/Universidade de São Paulo, Adjunct Professor at Universidade Federal de Minas Gerais Faculty of Pharmacy

^IIIGraduating Student – Course of Pharmacy at Universidade Federal de Minas Gerais

^IVPhD in Nuclear Sciences by University of Illinois, Urban Champaign, EUA, Adjunct Professor at Universidade Federal de Minas Gerais Department of Nuclear Engineering

^{Mailing Address}

ABSTRACT

OBJECTIVE: The present study was aimed at identifying temporomandibular joint inflammatory processes employing technetium-99m hexamethyl propylene amine oxime (^99mTc-HMPAO)-labeled autonomous leukocytes.

MATERIALS AND METHODS: We have utilized an experimental model of arthritis induction in ten adult male New Zealand rabbits by means of ovalbumin intra-articular injection into each left temporomandibular joint. For control purposes, saline solution was injected. After leukocytes radiolabeling with ^99mTc-HMPAO and injection into rabbits, scintigraphic images were obtained.

RESULTS: A higher ^99mTc-HMPAO-leukocytes uptake was observed in left temporomandibular joint in comparison with the contralateral joint. Wilcoxon non-parametric test was applied for statistical analysis. There was a statistically significant difference between counts of radioactivity per minute in the inflammed joint and the contralateral one (p = 0.0073).

CONCLUSION: The method employing ^99mTc-HMPAO-labelled autologous leukocytes allows an early and accurate detection of inflammatory processes, contributing to the adoption of a therapeutic conduct for patients before structural alterations have occurred.

Keywords: Temporomandibular joint disorder; Arthritis; ^99mTc-HMPAO.

INTRODUCTION

Temporomandibular joint disorders (TMJD) are a group of complex clinical alterations involving the temporomandibular joint (TMJ), mastication musculature and associated structures. Linear tomography, computed tomography (CT) and magnetic resonance imaging (MRI) are highly sensitive imaging methods widely employed for evaluation of TMJ inflammatory disorders, notwithstanding their low accuracy for determining early stages of such processes. These and other radiographic methods routinely utilized for TMJD evaluation, such as panoramic X-ray, transcranial X-ray and arthrography, allow visualization of already occurred anatomic alterations. Scintigraphy, for being based on physiological and biochemical alterations like increase in vascular patency, increase in local blood flow and cellular extravasation, allows an early detection of inflammatory alterations, differently from other imaging methods. The utilization of indium-111 (¹¹¹In)- and technetium-99m (^99mTc)-labeled autonomous leukocytes is considered as "gold-standard" in inflammatory processes diagnosis. This study objective was to identify inflammatory foci in TMJ employing ^99mTc-hexamethylpropylene amine oxime (^99mTc-HMPAO)-labeled autonomous leukocytes in an animal model.

MATERIALS AND METHODS

Ten adult male New Zealand rabbits have been utilized in this study. For arthritis induction purposes, the animals were sensitized to 4 mg ovalbumin (Sigma) combined with 1 ml Freund's complete adjuvant intradermically injected into five sites (0.2 ml each). Two weeks later, a booster dose in the same concentration was applied. Five days after booster dose, the animals were tested for sensitization by means of intradermically injected 100 µl ovalbumine solution 400 µg/ml in saline solution. After confirming the signs associated with an immunological response like edema and principally erythema, arthritis was unilaterally induced at left TMJ by intra-articular injection of 0.2 ml (20 mg/ml) ovalbumine in saline solution (joint-of-interest). For control purposes, the contralateral joint was injected with 0.2 ml saline solution (control-joint)⁽¹⁾. During the arthritis-induction process, the animals received intramuscular anesthesia with xylazine (Rompun^⠖ 20 mg/ml, 5 mg/kg) associated with ketamine (Dopalen^⠖ 100 mg/ml, 40mg/kg).

The animals´ blood was collected (30 ml) five days after the inflammatory process induction and leukocytes were separated and ^99mTc-HMPAO-labelled⁽²⁾ . The labeling yielding was calculated and HMPAO lipophilia was determined.

Scintigraphic images were acquired one hour after ^99mTc-HMPAO-leukocytes intravenous injection. Static images were obtained in dorsal and ventral views of the target region and whole body images afterwards, aiming at investigating the labeled-leukocytes biodistribution. The Wilcoxon non-parametric test (signal test for paired values) was applied for comparison between values of count per minute (cpm) regarding the inflammed joint (joint-of-interest) and the contralateral side (control joint). The adopted significance level was 1% (p < 0,01).

One week after scintigraphic images had been obtained two animals were randomly selected to undergo CT scans. Several oblique 10 mm-thick slices in the region of TMJ at 130 kV and 340 mAs were obtained.

This study was approved by the Committee for Ethics in Animal Experimentation of Universidade Federal de Minas Gerais (Cetea/UFMG), according to Protocol No. 033/03.

RESULTS

Total mean labeling yielding was 51%. As regards lipophilia, the radioactivity rate (in mCi) present during the oily phase was always higher than that in the aqueous phase. Mean rate found in the organic phase was 81%, while in the inorganic phase was 19%.

The static scintigraphic image (ventral view) obtained one hour following intravenous ^99mTc-HMPAO-leukocytes injection, has shown higher uptake of radiolabeled leukocytes in the left TMJ (inflammatory focus) and lower radiation uptake in the right TMJ (control) (Figure 1). All the experiments have always demonstrated highest uptake in the joint-of-interest. The inflamed joint (joint-of-interest) presented higher values of activity in the region of interest (ROI) in statistically significant cpm when compared with activity values obtained in the contralateral joint (control) (p = 0.0073). Dynamic scintigraphic images (ventral and dorsal views) obtained one hour after intravenous ^99mTc-HMPAO-leukocytes injection, showed radiolabeled leukocytes physiological biodistribution, with uptake through liver, spleen and lung (Figure 2).

Structural alterations were not observed on articular surfaces of the joint-of0interest on CT images obtained 12 days after the inflammatory process induction (Figure 3).

DISCUSSION

TMJ functional disorders are related to a condyle-disk complex disorder, a structural incompatibility of articular surfaces and the occurrence of articular degenerative and inflammatory processes which integrate a group of conditions affecting the masticating system denominated TMJD⁽³⁾. The data collected through anamnesis and patients clinical examination constitute the basis for a correct diagnosis of TMJD. In some situations, additional information obtained through supplementary studies are necessary⁽⁴⁾. Panoramic and transcranial X-ray, linear and computed tomography, arthrography, ultrasound and magnetic resonance imaging are diagnosis methods utilized for evaluating the TMJ anatomy and disorders⁽⁵⁾. However, all of these diagnostic methods depend on the existence of structural alterations to indicate the presence of an inflammatory process, so the early detection of an inflammatory focus is not feasible with such methods.

The early detection of a cellular response to inflammatory processes affecting the TMJ is decisive for determining a definite diagnosis before a structural compromising of articular surfaces has occurred⁽⁶⁾. Scintigraphic images obtained with the use of ^99mTc-HMPAO-labeled leukocytes allow the diagnosis of inflammatory foci in early stages of the disease. Several studies in the literature have effectively evaluated different types of inflammatory conditions employing ^99mTc-HMPAO-labeled leukocytes^(7–9). However, no research utilizing this method for studying inflammatory alterations involving specifically the TMJ has been found in the literature. Therefore our proposal was to utilize scintigraphy with radiolabeled leukocytes for the early diagnosis of inflammatory disorders affecting the TMJ on an animal model.

As regards the leukocytes labeling, the results achieved have shown a mean yielding of 51%, i.e. about half of the ^99mTc-HMPAO complex molecules incubated with leukocytes have penetrated and labeled them. These results are compatible with those described by Martin-Comin et al.⁽¹⁰⁾, who have found a mean rate between 55.3% and 75.4% of yielding in human leukocytes labeling.

For achieving a reasonable yielding of leukocytes labeling (about 55%), it is essential to determine the HMPAO lipophilia. In this study, the mean rate obtained for HMPAO was 81%. Studies in the literature have demonstrated that, at the moment of leukocytes labeling, the HMPAO lipophilia was of 85%⁽⁸⁾ and 78%⁽¹⁰⁾.

In the present study, one hour after intravenous injection into rabbits, the labeled leukocytes presented physiological uptake by lung, liver and spleen, in compliance with several studies described in the literature^(8,10–12). This data suggests that the destruction of these cells during the labeling process has not occurred, highlighting their integrity. The radioactivity uptake observed in the bladder is due to ^99mTc-HMPAO complex renal elimination. A part of the ^99mTc-HMPAO complex may leave the interior of leukocytes, starting a renal elimination process minutes after the radiolabeled leukocytes injection^(13,14).

The interpretation of scintigraphic images employing labeled leukocytes demands comparison between ROI radioactivity and the radioactivity in a predetermined normal region of reference⁽¹⁵⁾. In the present study, the contralateral joint treated with saline solution was utilized as a control joint. In all the animals, the rate of activity demonstrated in the joint-of-interest (inflammed TMJ) was statistically higher than that in the control-joint (p = 0.0073). Therefore, a higher and statistically significant radiolabeled leukocytes uptake in the inflammation site is evidenced five days after the inflammatory process induction. This opens a prospect for utilization of this method for early diagnosis of inflammatory TMJ disorders.

Computed tomography is the most precise method for evaluation of osseous components and possible structural alterations of articular surfaces of the TMJ^(15,16). However, the CT images obtained seven days after scintigraphic images could not reveal any abnormality in the TMJ region. This result comes to confirm the hypothesis that radiolabeled leukocytes may be of help in the early diagnosis of inflammatory foci in TMJ, since this method is based on physiological alterations rather than on anatomical alterations. Vascular events (vasodilatation and increase in the vascular patency) and cellular events (leukocytes exudation) in inflammatory processes constitute significant physiological aspects of acute inflammation. Although the radiolabeled leukocytes method is considered by some researchers^(12,13) as a "gold standard" for the early diagnosis of inflammation, independently from the region affected, there is no record in the literature on the application of this method for evaluation of inflammatory processes involving the TMJ. So, this study has demonstrated that the method of ^99mTc-HMPAO-labeled autologous leukocytes can identify inflammatory foci in the TMJ region, opening a new prospect for the utilization of this technique for diagnosis in the field of Odontology.

CONCLUSION

The method of ^99mTc-HMPAO-labeled leukocytes has demonstrated effectiveness in the early identification of inflammatory foci in TMJ in animal models.

Acknowledgements

We would like to thank Fapemig and Capes for the financial support, Fundação Ezequiel Dias and Dr. Carla Flávia de Lima (Ecograf), responsible for the scintigraphic imaging in this study.

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*

Study developed at Universidade Federal de Minas Gerais Department of Nuclear Engineering and Faculty of Pharmacy, Belo Horizonte, MG, Brazil.

Publication Dates

• Publication in this collection
  26 Sept 2006
• Date of issue
  Aug 2006

History

• Accepted
  17 Oct 2005
• Received
  20 Dec 2004