Introduction
Complete lateral rectus paralysis is often due to disorders of the sixth cranial nerve, resulting in paralytic esotropia, absence of abduction and a compensatory head position directed toward the affected side. In the early stages of this disorder, local injection of botulinum toxin A, wearing corrective prisms or other conservative treatments can be applied. At later stages, treatments often require surgical correction involving transposition of the superior and inferior rectus muscles within the affected eyes, which may be combined with medial rectus recession [
1]. However, restoration of abduction after such surgery is problematic [
2,
3]. Recently, we have found that a complete medial rectus recession combined with lateral rectus resection not only achieved orthotopic eye position, but also restored abduction function in some patients. While the underlying mechanisms for this effect remain unknown, it has been reported that increased satellite cell proliferation as well as rapid integration of these cells into myofibers is observed after either stretching or shortening of rectus muscles in rabbits [
4]. Such findings raise the issue as to whether the stimulation resulting from this surgery induces proliferation of extraocular muscle satellite cells to restore abduction within these affected eyes.
To address this issue, an assessment of the molecular background of the affected lateral rectus, especially as related to the proliferation and differentiation of extraocular muscle satellite cells is essential. Currently, no such studies exist on extraocular muscle satellite cell gene expression in these subjects. Therefore, we investigated the gene expression profile with use of microarray analysis and reverse-transcription PCR (RT-PCR) in lateral recti resected from patients with complete sixth cranial nerve paralysis. The lateral rectus of concomitant esotropia patients with normal extraocular muscles [
5,
6] served as controls. Thus, the purpose of this study was to examine potential differences in gene expression within lateral rectus muscles between these two groups. Such information will significantly aid in the understanding of possible molecular mechanisms involved with functional recovery of lateral rectus muscles after horizontal rectus recession and resection in patients with complete lateral rectus paralysis.
Discussion
It is well known that abduction in patients with complete lateral rectus palsy will not recover following routine medial rectus recession and lateral rectus resection. Somewhat surprisingly, we found that maximal medial rectus recession and lateral rectus resection on the affected eye does restore abduction in some of these patients. In this report, we present one such eventuality as shown in a 28-year-old female that experienced complete sixth cranial nerve palsy for 20 years following a traumatic injury. Forced duction test in this case was negative. However, after a 7 mm recession of the medial rectus and 13 mm resection of the lateral rectus in the affected eye, not only was a normal eye position restored, but a nearly normal abduction was also obtained. These findings motivated us to study the possible mechanisms of lateral rectus function recovery in these patients. As surgery on the muscle can contribute to the restoration of abduction in some patients with sixth cranial nerve palsy, we focussed on the muscle and not nerve in this report. A fundamental component for such investigations involves an identification of the molecular background for this effect. Accordingly, we analyzed the gene expression chips in surgically removed lateral rectus specimens of these patients, with special focus on genes related to the activation and inhibition of muscle satellite cells (MSCs).
Muscle differentiation is a complex process which depends on the activation of MSCs [
11]. An initial step in this process involves a high expression of the Six1/4 gene, which then promotes expression of the pax7 gene [
12]. PAX7, a marker of MSCs [
13,
14], mirrors the differentiation potential of MSCs and can also advance the expressions of MYF5 and MyoD, thereby inducing MSCs to develop into myoblasts and triggering initial myoblasts [
15]. Myoblasts express myoG, which then promotes cell differentiation processes [
9,
10]. Meanwhile, myosin, which is expressed in myocytes, fuses into myotubes to produce contractile functions [
16]. Unlike other skeletal muscles, extraocular muscles embody higher concentrations MSCs [
17] which, not only possess a strong differentiation ability [
9], but also share similar differentiation processes to skeletal muscles.
MyoD can function as a switch for inducing processes involved with MSCs differentiation [
18]. In our study, the low expression of MYOD in the paralytic esotropia group suggested that the process of differentiation in the lateral rectus muscle was impaired. Among genes associated with the differentiation process, SIX1 regulates the entire process of differentiation, which includes the expression of PAX7 [
19], along with the myogenic regulatory factors (MRF), MYOD and Myogenin [
12]. In this report, we observed high expression levels of SIX1/4, PAX7 and MYOG. These findings demonstrate that the most genes critically involved in the process of differentiation (except for the suppression of MYOD) in EOMSCs of patients with paralytic esotropia are active, suggesting that EOMSCs in patients with paralytic esotropia exhibit a relatively effective potential for differentiation.
Although SIX1 regulates the expressions of MYOD, PAX7 and MyoG during differentiation, this gene is, in turn, also regulated by MYOD, PAX7, and MyoG. MYOD and PAX7 may even exert a negative feedback upon SIX1 [
20]. Such a mechanism would explain the low expression of MYOD in the lateral rectus muscle of the paralytic esotropia group, while SIX1, PAX7 and MYOG are up-regulated.
PITX1 inhibits muscle regeneration via the p53 pathway and maintains satellite cells in a state of suspension [
21]. In this way, an up-regulation of PITX1 has the potential of inhibiting differentiation within the lateral rectus muscle of the paralytic esotropia group. In contrast, Integrin α7 (ITGA7), which facilitates the adsorption of basement membranes in cells, promotes cell proliferation to activate differentiation and regeneration of MSCs [
22,
23]. Therefore, an up-regulation of ITGA7 indicates the potential for an effective differentiation process of EOMSCs in patients with paralytic esotropia.
We propose the following series of events as a possible mechanism or the findings that an excessive medial rectus recession plus lateral rectus resection on the affected eyes could restore abduction in some patients. Similar to effects resulting from lateral rectus denervation, the differentiation of MSCs subjected to the local anesthetic, bupivacaine, can be inhibited, thus inactivating the MSCs [
24]. However, MSCs in this paralyzed state can be activated and differentiated when damaged by neutrophil infiltration, thereby restoring paralyzed muscle structures to normal [
25]. Direct injection of local bupivacaine into EOMs produces significant myonecrosis, which is followed by a relatively rapid regeneration, eventually resulting in myofiber repair and regeneration and a return to normal function [
26,
27]. Similarly, we suggest that the loss of function within a completely paralyzed lateral rectus may be due to cessation of the differentiation process and low levels of MYOD expression. However, this completely paralyzed lateral rectus retains the capacity to regenerate and restore partial function as a result of an up regulation in the expressions of SIX1, PAX7 and MYOG and increased differentiation potential induced by stimulation resulting from the surgery. To the best of our knowledge, no research has been directed toward understanding the repair mechanisms of lateral rectus function in patients with paralytic esotropia who underwent surgical correction.
As with any study, there are limitations in this report. First, gene expression within the lateral rectus of patients with paralytic esotropia indicates that MSCs are in an inactive state and that differentiated genes are highly expressed. However, the actual molecular mechanisms and interactions among these factors remain unclear. Second, gene expression levels were not confirmed by tissue staining methods. Third, the actual processes of activation and differentiation of MSCs in vivo after corrective surgery were not determined. Finally, as it was not possible to aquire samples from the nerves of these patients, potential effects upon nerves following surgery that may contribute to these mechaisms remain unknown.
In summary, the gene expression profile of paralytic lateral rectus muscles in patients with complete sixth cranial nerve palsy was investigated. Our findings indicate that under these conditions there is an intiation of processes involved with the regulation of MSCs differentiation, while MSCs remain inactive. At the present time, the reason for this inhibition of MSCs is unknown. The possibility that the functional recovery of the lateral rectus after strabismus surgery is due to the activation of MSCs by surgical stimulation warrants further study in animal models.
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