The present study was undertaken to determine molecular mechanism responsible for the effects of corticosteroid on the neuropeptide and cytokines in lateral epicondylitis. In addition, we examined tenocyte viabilities to investigate the detrimental effects corticosteroid has on tendon integrity.
The previous studies about neurogenic inflammation and lateral epicondylitis have used an immunohistochemical approach. However, immunohistochemical studies have some disadvantages, such as, photobleaching, marker infidelity, and lack of specificity. Furthermore, they rely on qualitative rather than quantitative assessments of effects, and even when results are scored, intra observer variability is problematic. Therefore, in the present study, the mRNA expressions of SP, CGRP, IL-1α, and TGF-β were determined by quantitative real-time PCR.
Before operations, 20 patients were treated with local TAA injection. Five patients were injected once, 7 patients twice, and 8 patients three times. These local injections may have influenced outcomes because corticosteroid infiltration has been reported to cause the necrosis of tendon collagen . However, surgery was performed a minimum of six months after last injections when the patient had sustained or recurred tenderness over the lateral side of the elbow. Thus, it appears reasonable to assume that the effects of previous local injections would have been minimal, as has mentioned previously [8, 11].
To decide on the concentration of TAA to use in the present study, we examined the effects of TAA at 25, 50, and 100 mM on tenocytes in small number of cases, and attempted to assess its dose-dependent effect on neuropeptides, cytokines and tenocyte viabilities. However, no dose-dependent effect was observed due to the poor solubility of TAA in culture medium, as described by Herbert et al.  Wong et al.  reported medical preparations of TAA used for local injections have concentrations over 10 mM, and added that tissue levels immediately after a local glucocorticoid injection would be near that in preparations, and thus, we treated tenocytes with TAA at 100 mM (the same concentration used for injection at outpatient clinics). Furthermore we did not add any solvent except saline. In outpatient department (in vivo), TAA was injected only with normal saline or lidocain.
The time points used in the present study were obtained by reviewing the literature. In several studies, tenocyte viability was analyzed after 2 to 14 days of incubation with corticosteroid [14, 15]. However, we considered that neuropeptides and cytokines would response to corticosteroid rapidly because steroid injection in outpatient clinics sometimes improves pain within 24 hours. Accordingly, we performed analysis at 7 time points, at 0 hours (baseline), and after 1, 3, 5, 24, 48, and 72 hours TAA treatment to detect the early effects of corticosteroid.
The peripheral nervous system, acting through neuropeptides, not only relays sensory information to the central nervous system, but also plays an effector role in inflammatory, proliferative, and reparative processes after injury. Of these neuropeptides, SP and CGRP are known to be mainly involved in neurogenic inflammation. Not only do SP and CGRP transmit nociceptive information to the spinal cord, but they are also involved in vasodilation and plasma extravasation .
It was previously postulated that the level of SP innervation within tendon tissue is limited. However, recent studies have investigated the expressions of SP and neurokinin-1 receptor in tenocytes, and found that their expression levels are higher in tenocytes in human Achilles tendinosis . In 2010, Lui et al.  also reported that the expressions of SP and CGRP are increased in tendon fibroblasts, chondrocyte-like cells, and calcific deposits after collagenase-induced tendon injury. Therefore, we assumed that the cultured tenocyte from lateral epicondylitis has the abundant expression of neuropeptide mRNA in contrast to the normal or unaffected tenocyte which express the neuropeptide mRNA minimally. We also hypothesized that the mRNAs of SP and CGRP might be over-expressed in the tenocytes of lateral epicondylitis in vitro, and preliminarily performed the analysis of mRNA expression from healthy tenocyte, and compared the results with the diseased group. Actually it is not easy to harvest normal healthy tenocyte from human being, consequently the sample number of control was limited to only 5 tendons. However, we found the expressions of neuropeptide and cytokine in control were significantly lower than that of the diseased group (Figure 1). Furthermore several reports have been already demonstrated the increased expression of neuropeptide in the tenocyte of lateral epicondylitis and concluded that the neuropeptide might be the important factor in the pathogenesis of lateral epicondylitis [7, 20]. We also did determine the increased expressions of neuropeptides in diseased tenocytes, and we found that the expression of CGRP was significantly inhibited by corticosteroid.
Although the reaction mechanisms responsible for the effects of corticosteroids are not fully understood, it has been reported that corticosteroids can modulate cytokines and other inflammatory mediators . Corticosteroids bind to specific intracellular receptors that function as ligand-induced transcription factors, and thus, modulate the expressions of particular target genes. Furthermore, the expressional levels of various cytokines, such as, IL-1, IL-2, IL-6, IL-8, and interferon-γ are decreased by corticosteroids .
Interleukin-1α is an initial phase cytokine responsible for inflammation. We observed that the mRNA expression of IL-1α significantly decreased by 65.8% at 3 hours and then gradually decreased by 94.7% after treatment with TAA for 72 hours. We suppose that these changes are responsible for the symptomatic improvement shown after corticosteroid injection in lateral epicondylitis.
TGF-β is one of the repair-phase cytokines and is involved in the synthesis and deposition of collagen, and scar formation after tendon transection . The effects of corticosteroid on TGF-β are controversial. Some researchers have reported that steroids upregulate TGF-β expression [22, 24] but Tempfer et al.  reported that TAA inhibits the production of TGF-β by human dermal fibroblasts.
In this study, the TGF-β mRNA expression was slightly higher after treatment for 5 hours without statistical significance, and then started to be inhibited at 24. We hypothesize that these responses are reflective of secondary changes associated with compromised tenocyte viability. However, Pearson's correlation analysis did not reveal any significant correlation.
Steroid injections are also known to decrease neurogenic edema, and to influence the effects and levels of SP and CGRP [8, 25]. Ihara et al.  studied the effect of glucocorticoids on the regulation of substance P receptor mRNA in rat pancreatic acinar AR42J cells by northern blot analysis using cloned cDNA. It was found that the expression of substance P receptor mRNA was selectively and negatively regulated by glucocorticoid. In the present study, treatment with TAA for 24 and 48 hours significantly reduced the expression of SP mRNA, and subsequently its expression increased slightly. These results suggest that SP is temporarily inhibited by corticosteroid.
On the other hand, CGRP mRNA expression showed a rapid and significant decrease at 24 hours, and this was not recovered. Thus, we speculate that the inhibitionof CGRP is probably a more significant molecular event than the inhibition of SP in reaction mechanism responsible for the effects of corticosteroid in lateral epicondylitis.
In addition to the mRNA expressions of neuropeptides and cytokines, we examined the effect of TAA on tenocyte viability using an MTT assay. In particular, steroids are known to induce adverse histologic changes and reduce the mechanical strengths of tendons [15, 27]. In the present study, TAA was found to gradually reduce tenocyte viability by 39.3% at 48 hours and by 47.4% at 72 hours versus baseline.
It could be questioned that the changes of tenocyte viability could affect to the expression of neuropeptide and cytokine. However, statistically the tenocyte viability was decreased significantly 24 hours after TAA treatment. But, although the CGRP was decreased significantly 24 hours after TAA treatment, substance P and interleukin was decreased significantly 1 and 3 hours after TAA treatment (before 24 hours). So we hypothesized that it would be more reasonable that the changes of neuropeptide and cytokine were not affected by the tenocyte viability.
Several studies have addressed the relationship between neuropeptides and cytokines because neuropeptides are probably potent stimulators of proinflammatory cytokine generation . Our study demonstrates a significant positive correlation between CGRP and IL-1α (p = 0.0184 and r = 0.45) after 72 hours of TAA treatment. These results support the idea that the neuropeptide, CGRP might have effect on the synthesis of IL-1α without inflammatory cell infiltration in this lesion and that neurogenic inflammation underlies the pathogenesis of lateral epicondylitis.
Summarizing, the mRNA expressions of neuropeptides were detected in the lesions of lateral epicondylitis, and TAA treatment was found to inhibit the expressions of the mRNAs of neuropeptides, especially of CGRP, in 24 hours, and the expressions of cytokine mRNAs, especially that of IL-1α, in 3 hours. Furthermore, the expression of SP mRNA was maximally inhibited by TAA at 24 hours and recovered at 72 hours, whereas the expression of TGF-β mRNA was slightly increased versus baseline until 5 hours, and subsequently decreased. In addition, MTT assays showed that tenocyte viability was significantly decreased by TAA at 24 hours, and a significant positive correlation was found between the relative mRNA expressions of CGRP and IL-1α after 72 hours of TAA treatment.