American Journal of Respiratory and Critical Care Medicine

Inflammatory cell infiltration and afferent neuropathy have been shown in the upper airway (UA) mucosa of subjects with obstructive sleep apnea (OSA). We hypothesized that inflammatory and denervation changes also involve the muscular layer of the pharynx in OSA. Morphometric analysis was performed on UA tissue from nonsnoring control subjects (n = 7) and patients with OSA (n = 11) following palatal surgery. As compared with control subjects, inflammatory cells were increased in the muscular layer of patients with OSA, with CD4+ and activated CD25+ T cells (both increased ∼ threefold) predominating. Inflammation was also present in UA mucosa, but with a different pattern consisting of CD8+ (2.8-fold increase) and activated CD25+ (3.2-fold increase) T cell predominance. As ascertained by immunoreactivity for the panneuronal marker PGP9.5, there was a dramatic (5.7-fold) increase in intramuscular nerve fibers in OSA patients compared with control subjects, as well as direct evidence of denervation based on positive immunostaining of the muscle fiber sarcolemmal membrane for the neural cell adhesion molecule in patients with OSA. These data suggest that inflammatory cell infiltration and denervation changes affect not only the mucosa, but also the UA muscle of patients with OSA. This may have important implications for the ability to generate adequate muscular dilating forces during sleep.

Upper airway (UA) dilator muscles are critically important for maintaining pharyngeal patency. In obstructive sleep apnea (OSA), there is an imbalance between UA muscular dilating forces and negative suction forces within the pharynx, which permits occlusion of the UA during sleep (1). Under these conditions, UA tissue is subjected to mechanical trauma induced by dramatic pharyngeal pressure swings and violent muscle contractions against an occluded airway (2). Such mechanical trauma of the UA has the potential to cause structural damage to nerves and muscle fibers (24) as well as inflammation within the pharyngeal tissues (5, 6).

In support of the above scenario, several recent investigations have revealed evidence for sensory nerve damage in the UA of patients with OSA (710). Hence, an impaired ability to detect UA temperature changes, vibratory sensation, and two-point discrimination, as well as abnormal vascular responses to afferent stimulation, have all been reported (710). Moreover, the fact that such abnormalities were only partly reversed by nasal continuous positive airway pressure therapy (9) suggests that structural damage to afferent nerves may be at least partly responsible for these changes in sensory function. This theory is further supported by superficial mucosal biopsy specimens showing an increase in the density of sensory nerve fibers in OSA, a finding that is consistent with postdenervation nerve regeneration or sprouting (8). Some studies have also suggested the possibility of an efferent neuropathy affecting the UA dilator muscles in OSA (1114), although others failed to find evidence of UA muscle denervation (1517).

In addition to evidence of afferent nerve injury or dysfunction in OSA, inflammatory cell infiltration and edema have been found within the UA mucosa (5, 1819). Increased levels of polymorphonuclear leukocytes and inflammatory mediators in nasal lavage fluid have also been reported (6). However, whereas these previous investigations point to the existence of inflammation within the UA mucosa of patients with OSA, it is not known whether this is also the case within the UA muscles. Because inflammatory cells and their mediators are able to interfere directly with both peripheral nerve and muscle function (2024), inflammation within UA dilator muscles could greatly alter their function.

The presence of either inflammation or denervation changes within UA dilator muscles would have important implications for the pathophysiologic mechanisms underlying UA closure in OSA. Therefore, the principal objectives of this investigation were threefold. First, we sought to determine whether there is increased inflammation not only within the UA mucosa of patients with OSA, but also of the UA musculature. Second, we wished to ascertain the specific immunophenotype of inflammatory cells within the UA muscles, and whether the nature of the inflammatory cells within this tissue compartment differs from that within the mucosa. Our final major objective was to look for morphologic evidence of denervation of UA muscles in OSA by using combined immunohistochemical detection of intramuscular axons and a molecular marker of muscle fiber denervation, neural cell adhesion molecule (N-CAM). Some of the results of this study have previously been published as an abstract (25).

Subjects

The study was approved by the institutional research ethics board, and written informed consent was obtained from all subjects. The study consisted of 18 retrospectively identified patients who had undergone UA surgery within the past 6 years. Potential subjects completed a questionnaire relating to exclusion criteria. Inclusion criteria for patients with OSA included a full overnight polysomnogram with an apnea–hypopnea index (AHI) greater than 15 events per hour. Patients with OSA (n = 11) had undergone uvulopalatopharyngoplasty and tonsillectomy for treatment of OSA. Nonsnoring control subjects (n = 7) were screened to exclude OSA using a validated clinical prediction score (26). Further subject data and exclusion criteria can be found in the online data supplement.

Tissue

Surgical specimens had been obtained from the soft palate and/or tonsillar pillars, formalin-fixed and embedded in paraffin. Slides were first screened using hematoxylin and eosin (HandE) staining to ensure that adequate muscle tissue (encompassing at least three 20X fields) was present in the specimens prior to study enrollment.

Immunohistochemical Procedures

Immunostaining with antibodies directed against inflammatory cells and nerve tissue was performed using standard techniques. Deparaffinization with methanol and antigen retrieval with EDTA or citrate buffer was performed in all surgical specimens. Tissue sections (3 μm thick) were labeled with primary antibodies against a panleukocyte marker (CD45 Clones 2B11 + PD7/26; Dakocytomation, Carpinteria, CA), a monocyte/macrophage marker (CD68 clone KP1; Dakocytomation) and a marker of activated T cells (CD25 ab9496; Abcam Ltd, Cambridge, UK). To further delineate the nature of the T cell response, markers for both helper (CD4 Clone 1F6; Ventana Medical Systems, Tucson, AZ) and cytotoxic T cells (CD8 Clone 1A5; Ventana Medical Systems) were also employed. To examine neural changes, we performed immunolocalization of protein gene product 9.5 (PGP 9.5), a panneuronal marker, which stains all types of afferent and efferent nerve fibers (NCL-PGP9.5, Novocastra Laboratories, Newcastle-Upon-Tyne, UK) (27). In addition, UA muscle tissue was immunostained to detect N-CAM CD56, a cell surface marker expressed on denervated muscle fibers but which disappears with reinnervation (NCL-CD56–1B6; Novocastra Laboratories) (28, 29). The secondary antibody was a biotinylated Ig against mouse or rabbit IgG, detected by means of an avidin–biotin complex and enzymatic (peroxidase) reaction using diaminobenzidene as substrate (Ventana Medical Systems). Slides were subsequently counterstained with hematoxylin.

Quantitative Image Analysis

All specimens were labeled by randomly assigned numeric code to achieve blinding as to group designation. Microscopic images were captured to computer using a digital camera (UTVO-5K; Olympus, Melville, NY). Slides were initially scanned at low power to identify areas of muscle or mucosa. Three randomly selected fields of muscle and mucosa were then used for the analysis of each experimental subject. Analysis of the individual inflammatory cells per unit area was performed by manual tag using a commercial software package (Image-Pro Plus; Media Cybernetics, Silver Springs, MD). All data (counts per unit area) were then compared between the OSA and control groups using Student's t test.

Inflammatory Cell Markers
UA Mucosa.

As shown in Figure 1

(upper panel), the panleukocyte marker CD45 indicated a significant increase in mucosal inflammatory cell infiltration in the OSA group. This was due to an increase in both CD8+ and CD4+ T cells in the OSA group relative to control group (2.8-fold and 2.3-fold, respectively). Moreover, these T cells appeared to be activated, as evidenced by a substantial increase in CD25+ cells (relative increase of 3.2-fold). There was no significant increase in the monocyte/macrophage marker CD68 in the mucosa of patients with OSA, which actually tended to be reduced in comparison to that in control subjects.

UA Muscle.

Results for all inflammatory cell markers are presented in Figure 1 (lower panel), whereas representative photomicrographic images are shown in Figure 2

. The absolute number of inflammatory cells per unit area in UA muscle was, on average, approximately one-fifth of that found in the mucosa. However, as with the mucosa, a significant increase in T cell infiltration was observed in the OSA group (twofold) in comparison with the control tissue samples. It should be noted that whereas mucosal inflammation was characterized by both CD8+ and CD4+ T cells, only the latter were significantly elevated in the UA muscle (threefold vs. control). Once again, these cells appeared to be activated, as indicated by significantly increased values for CD25 (threefold vs. control). There was also a trend toward increased monocytes/macrophages within the muscle, but this did not achieve statistical significance. The inflammatory cells were generally single and scattered in an even fashion throughout the muscle.

Neural Markers
UA Mucosa.

The number of PGP9.5-immunoreactive nerves was small, and no significant differences were observed between the OSA and control samples (Figure 3

, upper panel). Although occasional N-CAM staining of neural structures was found in both groups, there were no significant differences between OSA and control samples with respect to mucosal N-CAM staining (data not shown).

UA Muscle.

Patients with OSA demonstrated a striking increase (5.7-fold) in PGP9.5-immunoreactive nerves in comparison with control subjects, as shown in Figure 3 (upper panel). Because PGP9.5 stains both afferent and efferent nerve fibers, more direct evidence of motor denervation was sought by immunostaining muscle fibers for N-CAM. In the UA muscle of patients with OSA, N-CAM–immunoreactivity was present in a pattern of peripheral sarcolemmal staining, which is also seen after efferent denervation of muscle fibers (28, 29). Such N-CAM–positive fibers were found mostly in clusters rather than being evenly dispersed throughout the UA muscle. Accordingly, the percentage of 20× fields containing N-CAM–positive fibers was quantified. As can be seen in Figure 3 (lower panel), the percentage of fields containing N-CAM–positive fibers was 16% in patients with OSA versus only 1% in the control subjects. Representative photomicrographic images of PGP9.5 and N-CAM immunostaining are shown in Figure 4

.

As previously suggested by ourselves (2, 4, 9) and others (3, 10), UA-obstructive events have the potential to cause muscle and nerve injury due to the mechanical trauma induced by violent intraluminal pharyngeal pressure swings, tissue vibration, and eccentric muscle contractions. The main findings of our study support this scenario, and can be summarized as follows: (1) an abnormally elevated level of UA inflammation is present in OSA, which involves the UA musculature as well as mucosal tissue; (2) UA inflammation in both the muscular and mucosal tissue compartments is characterized by infiltration of activated T lymphocytes, with the muscular (but not mucosal) T cells showing a predominantly CD4+ T helper phenotype; (3) there is a dramatic increase in axonal density within the UA muscle of patients with OSA, which is consistent with nerve regeneration and/or axonal sprouting; and (4) sarcolemmal staining for N-CAM, a known stimulator of neurite outgrowth which is expressed in adult muscle fibers after denervation (28, 29), was found to be increased in the UA muscle of patients with OSA.

Inflammatory Changes within the UA

Previous studies of inflammation in the UA of patients with OSA have focused on the mucosal layer, most likely due to the relative ease and noninvasiveness of obtaining biopsies in comparison to the deeper muscular tissue. Measurements of inflammatory mediators (e.g., bradykinin and vasoactive intestinal peptide) in nasal lavage fluid support an abnormal inflammatory response in the UA of patients with OSA (6). In addition, several authors (5, 18, 19) have reported increased inflammatory cell infiltration within the UA mucosa of patients with OSA in comparison with control subjects. However, to our knowledge, the specific inflammatory cell immunophenotypes present in the UA mucosa have not been precisely defined, nor has the UA muscle been investigated for evidence of inflammation in previous studies.

In the present study, we demonstrate a significant increase in inflammatory cell infiltration of the UA in patients with OSA, which encompasses both the mucosal and muscular layers. In both cases, the inflammatory infiltrate consisted primarily of T lymphocytes. However, an intriguing finding was the difference in T cell subsets between the mucosal (both CD4+ and CD8+) and muscular (CD4+ predominance) layers. This suggests that inflammation present within the muscle is not simply spillover from the adjacent mucosa, but rather points to a certain degree of compartmentalization of the inflammatory process between mucosa and muscle. Indeed, it is well established that skeletal muscle itself can produce proinflammatory cytokines in response to a number of stimuli, including damage or overuse (24, 3031). Cytokines expressed by muscle fibers may in turn trigger inflammatory cell activation and migration into affected areas of the muscle (30, 32). It is also important to recognize that inflammatory cell infiltration of skeletal muscle, together with production of proinflammatory mediators, such as cytokines and oxygen free radicals, can cause significant muscle weakness. For instance, tumor necrosis factor–α and nitric oxide are both known to have direct inhibitory effects on the force-generating capacity of muscle fibers (23, 24, 33). In addition, it has been established in models of peripheral neuropathy that the presence of non–neural-specific activated CD8+ T cells can induce or worsen neuropathy (2022). Under these conditions, the neural toxicity appears to be mediated via direct cytotoxic T cell–induced axonal injury as well as by cytokines, such as tumor necrosis factor–α, which can induce Wallerian degeneration (21). Therefore, inflammation within the UA muscles of patients with OSA has the potential to produce contractile dysfunction and weakness of UA dilator muscles via at least two distinct but related mechanisms: (1) direct effects on the UA muscle fibers per se; and (2) effects on the nerves through which these UA muscle fibers are normally stimulated.

Denervation Changes within the UA

There is morphologic as well as physiologic evidence that a UA afferent sensory deficit is present at the mucosal level in patients with OSA (710). Moreover, topical anesthesia of the UA induces apneas in previously normal control subjects and increases obstructive events in snorers (3437), indicating that altered UA sensory function may play a role in exacerbating the disease process. Some, although not all, previous studies have provided evidence of UA muscle denervation in OSA. Woodson and colleagues (18) reported demyelination in UA tissue specimens. Indirect signs of motor denervation, such as fiber-type grouping and grouped atrophy, have also been reported in several recent studies (1213). On the other hand, Series and colleagues (17) failed to show any alteration in muscle fiber area frequency distribution in OSA. Functionally, however, Carrera and coworkers (14) showed increased fatigability of genioglossus from patients with OSA.

In this study, we addressed the issue of possible UA muscle denervation in OSA by seeking characteristic morphologic changes of both the nerves and muscle fibers. To study neural changes, we employed the panneuronal marker PGP9.5, whereas N-CAM expression on the sarcolemma was used to indicate the presence of denervation effects on the target muscle fibers themselves. The presence of denervated muscle fibers scattered throughout muscle has long been known to be a powerful stimulus for outgrowth of new nerve sprouts from the adjacent surviving nerves (3841), leading to increased axonal density within the affected muscle. Accordingly, our findings of an almost sixfold increase in PGP9.5-immunoreactive nerves is consistent with such denervation-related phenomena in the UA muscle of patients with OSA.

Additional evidence for ongoing denervation of UA muscle in OSA is provided by the significant increase in sarcolemmal N-CAM staining. N-CAM is a cell surface sialoglycoprotein, which plays an important role in directing axonal growth to muscle fibers (28, 29, 42). Neutralizing antibodies against N-CAM greatly impair nerve growth to muscle fibers during embryonic development, and also interfere with axonal sprouting after denervation (43). In denervated adult muscle fibers, N-CAM is expressed extrajunctionally along the sarcolemma (28, 29), where it is believed to act as a signal to guide the approaching axons to their targets. Upon reinnervation of denervated skeletal muscle, N-CAM expression is downregulated and becomes restricted to the neuromuscular junction. Thus, the presence of increased PGP 9.5 staining together with areas of N-CAM positivity implies an active process of denervation and reinnervation in the UA muscles of patients with OSA.

Axonal sprouting after partial denervation is a compensatory mechanism, which helps to preserve the force-generating capacity of the muscle by expanding the territory of surviving motor units. Such compensation may largely mitigate the physiologic impact of UA muscle denervation, especially in mild cases or early in the disease. However, it is important to recognize that expansion of the motor unit also places an increased physiologic burden on the corresponding motor neurons. Thus, each surviving motor nerve must now stimulate a greater number of muscle fibers. Interestingly, chronic overstimulation and “exhaustion” of surviving motor units is believed to play a role in the age-related loss of motor neuron function and muscle weakness found in patients with postpolio syndrome (44). Therefore, it is conceivable that a similar phenomenon could lead to progressive motor neuron dysfunction and weakness over the long term in UA muscles of patients with OSA.

In conclusion, the findings of this study provide evidence for inflammatory and denervation changes within the UA of patients with OSA which may have important implications for neuromuscular function and the pathophysiology of OSA. Further studies will be required to elucidate more precisely the mechanisms involved in these processes. It should also be noted that there is emerging data indicating an increase in systemic inflammation in patients with OSA (45), which may also play a role in the increased risk of cardiovascular events associated with OSA (46). Further investigations will be required to determine whether local activation of inflammatory cells and proinflammatory mediator production within the UA tissues contributes to a state of heightened systemic inflammation in OSA.

The authors gratefully acknowledge the expert statistical advice of Dr. Heberto Ghezzo of the Meakins-Christie Laboratories, the technical contribution of Ms. Carmen Loiselle of the Pathology Department for the immunohistochemical staining work, and the contribution of Dr. Marie-Christine Guiot and her staff in the Neuropathology Department for the N-CAM staining.

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Correspondence and requests for reprints should be addressed to R. John Kimoff, M.D., Room L4.08, 687 Pine Avenue West, McGill University Health Center, Montreal, PQ, Canada H3A 1A1. E-mail:

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