Sensory innervation of soft tissues of the lumbar spine in the rat

The purpose of this study was to investigate neurophysiologically and anatomically the soft tissues of the dorsal compartment of the lumbar spine in order to understand better their possible role in low back pain. The focus was primarily on the lumbar facet joint and supraspinous ligament of the Sprague-Dawley rat. Microdissection of the dorsal ramus and electrophysiological and neuroanatomical studies of the dorsal ramus and its terminations in paravertebral tissue revealed that (a) there are mechanosensitive, slowly adapting fibers in the rat lumbar facet joint capsule; (b) there are slowly adapting, mechanosensitive units in the rat supraspinous ligaments that respond to tensile loading; both types of mechanosensitive units have high threshold; (c) mechanical stimulation of these tissues sometimes elicits afterdischarges lasting several minutes; (d) many extracellular recordings from the medial branch of the dorsal ramus appear to be reflex activity to mechanical stimulation; (e) silver impregnation of the rat joint capsule reveals individual axons, very few of which were encapsulated, suggesting that they terminate in free nerve endings; and (f) the nerves of the rat facet joint capsule contain 68, 160, and 200 kdalton polypeptide subunits of neurofilament protein (NFP). These results indicate that neurons of lumbar facet joint capsules and ligaments in the back are sensitive to mechanical strain and that the higher threshold neurons may serve a nociceptive (pain) function.     

Tensile stretching of cervical facet joint capsule and related axonal changes

This study examines axonal changes in goat cervical facet joint capsules (FJC) subjected to low rate loading. Left C5–C6 FJC was subjected to a series of tensile tests from 2 mm to failure using a computer-controlled actuator. The FJC strain on the dorsal aspect was monitored by a stereo-imaging system. Stretched (n = 10) and unstretched (n = 7) capsules were harvested and serial sections were processed by a silver impregnation method. The mean peak actuator displacement was 21.3 mm (range: 12–30 mm). The average peak strain encompassing various regions of the capsule was 72.9 ± 7.1%. Complete failure of the capsule was observed in 70% of the stretched capsules. Silver impregnation of the sections revealed nerve fibers and bundles in all the regions of the capsule. A blinded analysis of digital photomicrographs of axons revealed a statistically significant number of swollen axons with non-uniform caliber in stretched FJCs. Axons with terminal retraction balls, with occasional beaded appearance or with vacuolations were also observed. Stretching the FJC beyond physiological range could result in altered axonal morphology that may be related to secondary or delayed axotomy changes similar to those seen in central nervous system injuries where axons are subjected to stretching and shearing. These may contribute to neuropathic pain and are potentially related to neck pain after whiplash events.     

Pain generation in lumbar and cervical facet joints

Facet joints are implicated as a major source of neck and low-back pain. Both cervical and lumbar facet syndromes have been described in the medical literature. Biomechanical studies have shown that lumbar and cervical facet-joint capsules can undergo high strains during spine-loading. Neuroanatomic studies have demonstrated free and encapsulated nerve endings in facet joints as well as nerves containing substance P and calcitonin gene-related peptide. Neurophysiologic studies have shown that facet-joint capsules contain low-threshold mechanoreceptors, mechanically sensitive nociceptors, and silent nociceptors. Inflammation leads to decreased thresholds of nerve endings in facet capsules as well as elevated baseline discharge rates. Recent biomechanical studies suggest that rear-end motor-vehicle impacts give rise to excessive deformation of the capsules of lower cervical facet joints. Still unresolved is whether this stretch is sufficient to activate nociceptors in the joint capsule. To answer this question, recent studies indicate that low stretch levels activate proprioceptors in the facet-joint capsule. Excessive capsule stretch activates nociceptors, leads to prolonged neural afterdischarges, and can cause damage to the capsule and to axons in the capsule. In instances in which a whiplash event is severe enough to injure the joint capsule, facet capsule overstretch is a possible cause of persistent neck pain.     

Immunohistochemical demonstration of nerve fibers in the synovial fold of the human cervical facet joint.

The role of the intra-articular synovial fold as a source of facet joint pain is unclear, because the nature of nociceptive innervation in lumbar synovial folds is controversial, and there have been no such studies in cervical synovial folds. The present study aimed to demonstrate the presence of nerve fibers including nociceptive fibers in synovial folds of human cervical facet joints using immunohistochemistry. Synovial folds of cervical facet joints removed from patients undergoing cervical spine laminoplasty were analyzed immunohistochemically using antibodies to protein gene product 9.5, beta III-tubulin, substance P and calcitonin gene-related peptide. Many nerve fibers immunoreactive for protein gene product 9.5 and beta III-tubulin were demonstrated both around blood vessels and as free fibers in the stroma of the synovial fold. Also. immunostaining showed the presence of free nerve fibers immunoreactive for substance P and calcitonin gene-related peptide in the stroma. The presence of putative nociceptive fibers in cervical synovial folds supports a possible role for these structures as a source of cervical facet joint pain.     

Silver impregnation and immunohistochemical study of nerves in lumbar facet joint plical tissue

Impingement of plical synovial tissue in a facet joint could cause pain. Plical tissue was removed during surgery for recurrent disc herniation or spinal stenosis. The presence of nerves was studied with silver impregnation, immunofluorescence, and avidin-biotin-peroxidase complex (ABC) immunostaining. Heterologous antisera to protein gene product (PGP) 9.5, substance P, calcitonin gene-related peptide (CGRP), and galanin were used to stain nerves. After silver impregnation, nerve-like structures were observed perivascularly. Such nerves located close to blood vessels were also immunoreactive for PGP 9.5, a more general cytoplasmic neural marker, whereas only few perivascular small varicosities were seen with antisera to substance P and galanin and none with antiserum to CGRP. In addition, PGP-9.5-, substance-P-, and galanin-immunoreactive nerves were occasionally seen very near to fat globules. Very few peptide-immunoreactive nerve varicosities were seen with immunofluorescence, and none of the PGP-9.5-immunoreactive nerves that were observed with ABC immunostaining were immunoreactive for neuropeptides as well. One mechanism for pain production could be mechanical compression of fatty tissue, but it is considered more likely that nerves in this particular tissue are mainly involved in local vasoregulation and that they are not sensory nociceptive nerves.     

Morphometry of the lumbar zygapophyseal facet capsule and cartilage with special reference to degenerative osteoarthritic changes: an anatomical study using fresh cadavers of elderly Japanese and Korean subjects

Morphometric data were obtained from fresh cadaver dissections, and observations of degenerative changes in the joint cartilage (DCs) were analyzed to determine whether the morphometric parameters of the lumbar zygapophyseal joint capsule varied according to the presence and severity of DCs. There have been no previous morphometric studies of the facet capsule that describe age-related DCs. Using 23 fresh osteoligamentous lumbar spines from donated cadavers, we performed morphometric investigations of the surface areas of the joints and their capsules and measured the capsular thickness. We hypothesized that the ratio of the inner capsular area to the joint surface area for each facet (the capsule/facet index) could serve as an index showing a functional aspect of a large or small capsule. Our results showed that the joint surface area increased significantly with increasing severity of DCs, according to Grogans classification. Facets with advanced DCs tended to have a small inner capsular surface. The capsule/facet index generally correlated positively with capsular thickness, especially the dorsal portion; however, this was not true for the ventral portion. The index also correlated negatively with DC progression. Thus, lumbar facet DCs were strongly linked to reconstructive alterations in capsular size, thickness, and looseness. The occurrence and progression of DCs narrowed the joint capsule, especially the dorsal portion, and this seemed to decrease the potential looseness of the joint. Conversely, these capsular alterations seemed to accelerate DC progression. A negative adaptation cycle seemed to occur.An erratum to this article can be found at     

大鼠腰椎P物质和降钙素基因相关肽能神经纤维的分布

目的　观察腰椎小关节 ,椎间盘 ,前、后纵韧带 ,棘上、棘间韧带上 P物质 (SP)和降钙素基因相关肽 (CGRP)能神经纤维的分布 ,探讨其意义。方法　采用 2 5例 SD大鼠腰椎组织切片 ,以SP、CGRP多克隆抗体进行免疫组织化学研究。结果　 SP、CGRP能神经纤维在腰椎上有广泛的分布。除滑膜皱襞外 ,小关节的其他部位均观察到上述神经纤维。在椎间盘 ,神经纤维只分布于纤维环的最外层。棘间韧带上仅见 CGRP能神经纤维。结论　腰椎上有广泛的感觉神经分布 ,上述结构参与慢性下腰痛发生有其神经解剖学基础     

Morphological basis for back pain: the demonstration of nerve fibers and neuropeptides in the lumbar facet joint capsule but not in ligamentum flavum.

The innervation of lumbar facet capsule and ligamentum flavum was investigated using antisera to a general neuronal marker protein gene product (PGP) 9.5 and to peptide markers of sensory nerves (calcitonin gene-related peptide [CGRP] and substance P) and autonomic nerves (vasoactive intestinal polypeptide [VIP] and C-flanking peptide of neuropeptide Y [CPON]). In the facet capsule (n = 14), PGP 9.5 and CGRP-immunoreactive nerves occurred in 12 and five specimens, respectively, both around blood vessels and as free fibers in the stroma. Free fibers immunoreactive for substance P or VIP were noted in three and five specimens, whereas in nine specimens there were CPON-immunoreactive nerves located perivascularly. There was no immunoreactivity in the ligamentum flavum. This study provides further evidence that the facet capsule but not the ligamentum flavum has substantial innervation by sensory and autonomic nerve fibers and has a structural basis for pain perception.     

Effects of anterior lumbar spinal fusion on the distribution of nerve endings and mechanoreceptors in the rabbit facet joint: quantitative histological analysis

The purpose of this study was to investigate the density and distribution of neural endings in rabbit lumbar facet joints after anterior spinal fusion and to evaluate the effects of intervertebral immobilization. An extraperitoneal approach was applied, and L5/6 was fixed with a plate and screws. Bilateral L4/5, L5/6, and L6/7 facet joint capsules were harvested from the rabbits 4, 8, and 16 weeks postoperatively. Capsular tissues were processed using a modified gold chloride staining method, and the specimens were sliced into 15-m sections. All sections were analyzed microscopically, and neural ending numbers per unit volume were calculated. Three types of neural ending were identified in each specimen: Pacinian corpuscles, Ruffini corpuscles, and free nerve endings. In the L5/6 fusion segment there was a significant decrease in the number of Pacinian corpuscles at 4 weeks and of Ruffini corpuscles at 4, 8, and 16 weeks after the fusion compared with the control; and in the L4/5 upper adjacent segment there was a significant increase in the number of free nerve endings. The number of Ruffini endings for the L6/7 lower adjacent segment was significantly lower more than 8 weeks after the fusion. These results suggest that immobilization of the intervertebral segment causes a reduction in the number of mechanoreceptors in the facet joint capsules because of the reduction in mechanical stimulation. Moreover, in the upper adjacent facet joint there may be neural sprouting caused by nociceptive stimulation.     

Distribution of A-delta and C-fiber receptors in the cervical facet joint capsule and their response to stretch

BACKGROUND: It has been proposed that cervical facet joint capsules are a major source of whiplash pain. However, there is a paucity of neurophysiologic data to support this hypothesis. The purposes of this study were to determine the distribution of A-delta and C-fiber sensory receptors in the facet joint capsule and to test their patterns of response to stretch and related sensory function. METHODS: Laminectomy from C4 to C7 was performed in seventeen goats, while they were under general anesthesia, to expose the C6 nerve roots. Customized dual bipolar electrodes were used to record neural activity from one of the C6 branches. An 8 or 15-V electrical stimulus was used to provoke receptor activity in nine designated areas on the dorsal part of the C5-C6 facet joint capsule. Receptors were classified on the basis of conduction velocities. The waveform of an identified receptor was set up as a template to determine its neural activity in response to capsular stretch. The characteristics of each single receptor's response to capsular stretch were analyzed to determine its sensory function as a mechanoreceptor or nociceptor. RESULTS: Two hundred and forty-eight receptors on the dorsal part of the C5-C6 facet joint capsule were evoked by electrical stimulation in the seventeen goats. More C-fiber receptors were found on the dorsolateral aspect of the facet joint capsule, where tendons and muscles were attached. The response to stretch of 120 receptors, from twelve goats, were analyzed to classify them into one of four categories (high-threshold mechanoreceptors, non-saturated low-threshold mechanoreceptors, saturated low-threshold mechanoreceptors, and silent receptors) or as unclassified receptors. CONCLUSIONS: The existence of receptors in the facet joint capsule indicates that the capsule has pain and proprioceptive sensory functions.     

Histopathological Evaluation of Mechanoreceptors in the Metatarsophalangeal Joint Capsule in Hallux Valgus

To date, we could find no study concerning the relationship between mechanoreceptors in the joint capsule of the first metatarsophalangeal joint and hallux valgus deformity. We aimed to investigate the presence of mechanoreceptors in samples obtained from the first metatarsophalangeal joint capsules of patients with hallux valgus deformity to improve our understanding of the clinical and histopathological features of the disease. Samples were taken from the first metatarsophalangeal joint capsules of 13 fresh-frozen cadavers with normal anatomy (controls) and 29 patients undergoing surgery for hallux valgus (cases). For light microscopy, excised specimens were fixed in 10% formaldehyde and processed for routine histopathological investigation. All samples were dehydrated in a series of ethanol, cleared in xylene, and embedded in paraffin. Orientation of collagen fibers was determined on Masson's trichrome–stained sections, and mechanoreceptors were evaluated on S-100-immunostained sections. In the sections stained with Masson's trichrome, the orientation of collagen fibers was regular in the control group. However, coarse and disoriented collagen bundles were observed in the hallux valgus cases (P ≤ .05). S-100 immunostaining was positive in the sections of both the cases and controls. Finally, free nerve endings were more abundant in the samples obtained from the capsules of hallux valgus cases than from the control group (P ≤ .05). An increase in the number of free nerve endings within the capsules of the first metatarsophalangeal joints in feet with hallux valgus deformity might have a role in the development of clinically relevant joint pain and instability.     

Innervation of nociceptors in the menisci of the knee joint: an immunohistochemical study

Using histology, we studied the innervation of nociceptors in the medial and lateral menisci of the knee joint. Specimens examined were taken from 16 patients during arthroplasty. The patients were 6 men and 10 women, with ages ranging from 14 to 76 years (mean 56 years). Immunohistochemistry with the unlabeled antibody biotin-streptavidin method was employed to detect protein gene product 9.5 (PGP 9.5) or substance P (SP) in the specimen. The antibody for PGP 9.5 detected nerve tissues in the menisci. Most but not all of the nerve fibers were associated with blood vessels. Nerve fibers and sensory receptors were found mainly in the peripheral, vascular zone, representing the outer one-third of the meniscus, and the innervated area was wider in the anterior and posterior horns. Pacinian and Ruffini corpuscles as well as free nerve endings were identified in these areas. Larger fibers coursed circumferentially in the peripheral zone, with smaller branches of nerve fibers running radially into the meniscus. Nerve fibers positive for SP were also detected in the menisci, but were fewer in number. Their branches also were fewer, oriented radially and paralleling blood vessels. This study showed that some of the pain in cases of meniscal tear could originate in the meniscus itself, especially with peripheral tears that may be accompanied by bleeding. Received: 2 June 1998     

雌激素缺乏脊柱神经肽P物质变化的实验研究

目的阐明雌激素缺乏时椎体、椎间盘及小关节等部位ＳＰ免疫反应阳性神经纤维的变化规律。方法选用健康２月龄雌性ＳＤ大鼠９６只,随机分为卵巢切除（Ｏｖａｒｉｅｃｔｏｍｙ,ＯＶＸ）和假手术（Ｓｈａｍ－ｏｐｅｒａｔｅｄ,Ｓｈａｍ－Ｏ）两组。ＯＶＸ组行双侧卵巢切除术,Ｓｈａｍ－Ｏ组行假手术。分别于术后１５天、１、２、３、６、９月时每组各处死８只动物,切取Ｌ３～５脊柱小关节、棘上韧带、椎体和椎间盘。免疫组织化学Ｓ－Ｐ法观察各部位ＳＰ免疫反应阳性神经纤维分布。结果①ＯＶＸ后骨小梁及小梁膜上ＳＰ免疫反应阳性神经纤维增多,增粗。②软骨组织内ＳＰ反应阳性纤维增多;２月时椎间盘ＳＰ阳性反应物增加,分布至纤维环中带,３月时观察到纤维环内带,６月在纤维环髓核中观察到ＳＰ免疫反应阳性神经纤维。结论雌激素缺乏时大鼠腰椎椎体、椎间盘、小关节ＳＰ阳性反应神经纤维增多,与骨质疏松性腰背疼痛有密切关系。     

慢性小关节源性腰痛与小关节囊神经纤维数量及神经肽Y表达变化的病例对照研究

目的：通过研究临床症状、核磁共振小关节Fujiwara分级与末端神经纤维分布及神经肽Y表达的关系,探索慢性小关节源性腰痛的病理机制.方法：选取无腰痛的腰椎管占位需手术内固定患者为对照组（A组）,共15例.慢性腰痛患者,术前封闭腰椎小关节,注射前后进行VAS测评.根据测评结果将其分为3组：疼痛完全无缓解患者,为慢性非小关节源性腰痛组（B组）,共15例;腰痛基本消失的患者,为慢性小关节源性腰痛组（C组）,共20例;腰痛有改善但不能完全消除的患者,为慢性混合性腰痛组（D组）,共20例.取各组患者的小关节囊标本,分别用氯化金染色及免疫组化法研究小关节囊上的神经末梢及神经肽Y的分布及表达的变化.结果：与对照组相比,慢性小关节源性腰痛组和慢性混合性腰痛组腰椎小关节退变趋势明显,神经纤维分布明显增多,且慢性小关节源性腰痛组神经分布稠密.免疫组化结果表明,慢性小关节源性腰痛组神经肽Y阳性分布稠密.此外,小关节囊神经纤维数量分布及神经肽Y阳性分布具有一定的相关性,但二者与小关节MRI Fujiwara分级无相关性.结论：慢性小关节源性腰痛患者小关节囊中存在丰富的末端神经纤维;当关节囊受压、牵张,组织炎症或化学物质刺激时或者脊柱小关节病变及力学行为改变时,小关节囊神经末梢疼痛介质分布会发生改变,进而参与腰痛的发生发展.     

Nerve distribution to the human knee joint: anatomical and immunohistochemical study

The nerve distribution to the knee joints was analyzed in 5 cadavers and 10 joint capsules specimens were resected during total knee arthroplasty. We found nerve fibers immunoreactive for anti-substance P antibody in the articular capsule. By confocal laser scanning microscopy, we evaluated the three-dimensional structures of the Ruffini's corpuscles and the free nerve endings, both of which were immunoreactive for anti-protein gene product 9.5.     

Nociceptive nerve fibers in the sacroiliac joint in humans

BACKGROUND AND OBJECTIVES: A positive response to sacroiliac joint intra-articular infiltration with local anesthetics is used to confirm sacroiliac joint pain. However, current anatomical and histological knowledge concerning the anatomy of pain perception within the sacroiliac joint intra- and peri-articular structures is insufficient to explain the efficacy of this infiltration, because of the use of unspecific histochemical visualization techniques. METHODS: In this study, immunohistochemistry for calcitonin gene-related peptide (CGRP) and substance P was used to trace nociceptive fibers and receptors in the anterior and interosseous sacroiliac ligaments obtained from 5 human cadavers without history of sacroiliac joint pain. RESULTS: Microscopic analysis of stained slides showed presence of CGRP and substance P immunoreactive fibers. Thick, wavy, formed bundles were observed in dense and loose connective tissue, whereas single, beaded nerve fibers, occasionally ramified, were observed more frequently in the dense connective tissue and next to blood vessels. Based on their morphologic features, these immunoreactive structures were classified as receptors type IV. Additionally, receptors type II were found in anterior and interosseous ligaments, which contained CGRP or substance P immunoreactive free nerve endings. CONCLUSIONS: We conclude that the presence of CGRP and substance P immunoreactive fibers in the normal anterior capsular ligament and interosseous ligament provides a morphological and physiological base for pain signals originating from these ligaments. Therefore, diagnostic infiltration techniques for sacroiliac joint pain should consider extra- as well as intra-articular approaches.     

An autoradiographic study of the distribution of sensory nerve endings in the knee joint of the rat

The distribution of sensory nerve endings of the knee joint in rats has been investigated by autoradiography of 3H-leucine. Sections were made and each section was examined by autoradiography and silver impregnation staining. Free nerve endings were found only in the synovial membrane, the menisci and their synovial junction. In the joint capsule, capsulated sensory nerve endings were sparsely distributed. Numerous sensory nerve endings were found at the insertion of the patellar ligament, the collateral ligament and the cruciate ligament. These resembling Golgi-Mazzoni nerve endings or capsulated sensory nerve endings are thought to be proprioceptors. Radioactivity, which would indicate the presence of sensory nerve endings, was not recognized in the osseous tissue. It is hypothesized that proprioceptor at the insertion of ligaments plays an important role as monitor of joint motion by the reflex pathway.     

An immunohistochemical study of the dorsal capsule of the lumbar and thoracic facet joints

STUDY DESIGN: The molecular composition of the extracellular matrix in the dorsal capsules of lumbar and thoracic facet joints was analyzed immunohistochemically. OBJECTIVES: To determine whether the immunohistochemical profile of the lumbar joint capsule suggests a role of the capsule in limiting axial rotation of the lumbar motion segment. SUMMARY OF BACKGROUND DATA: During axial rotation of the lumbar vertebrae, the axis of rotation shifts toward the facet joints in the direction of rotation. Thus, the capsule of the opposing joint should become tensed and wrap around the inferior articular process. Previous studies suggest that wrap-around ligaments are fibrocartilaginous. However, thoracic joint capsules are largely shielded from such loading and should be purely fibrous. METHODS: Dorsal capsules were removed from lumbar and thoracic facet joints of six adult cadavers. Specimens were immunolabeled with monoclonal antibodies for collagens, chondroitin, dermatan and keratan sulfates, versican, tenascin, aggrecan and link protein. Antibody binding was detected using the Vectastain ABC 'Elite' peroxidase kit (Vector Laboratories, Inc., Burlingame, CA). RESULTS: Both lumbar and thoracic joint capsules immunolabelled for most glycosaminoglycans and for Type I, III and VI collagens. However, labeling for Type II collagen, chondroitin-6-sulfate, aggrecan, and link protein was restricted to lumbar capsules. Such labeling was constantly seen at entheses and occasionally in the midsubstance. CONCLUSIONS: The molecular composition of the lumbar joint capsule suggests that it acts as a fibrocartilaginous, 'wrap-around' ligament that withstands compression in addition to tension during torsional movements of the lumbar spine. It wraps around the inferior articular process as rotation occurs and limits further movement.     

颈椎小关节囊的神经支配及其临床意义

目的：研究颈椎小关节囊的神经支配来源及其通路，为阐明小关节损伤后弥漫性头颈肩部疼痛的机制及其治疗提供依据。方法：39只SD雄性大鼠，分为颈交感神经未切除组（A组，18只）、切除组（B组，18只）及对照组（C组．3只）．A、B组分别于Cl-2、C3—4或C5—6（各6只大鼠）小关节囊内注射5％双雀甲亚胺（Bb）0．6μl．C组注射等量生理盐水。8h后检查各组C1～C8脊神经节（DRG）和交感神经节（SG）的荧光标记细胞，片作统计学分析。结果：A组脊神经节和交感神经节内均可见到荧光标记细胞，其中，C1-2和C3—4关节囊内注射组的C1～C8DRG内有Bb（＋）细胞，C5—6关节囊内注射组的C3～C8DRG内可见Bb（＋）细胞；B组注射节段和相邻节段Bh（＋）细胞无明显减少，但远节段的Bh（＋）细胞明显减少；C组未见Bb（＋）细胞。结论：颈椎小关节囊神经支配来源于感觉和交感神经系统，脊神经节和交感神经节间有神经纤维联系，存临床上阻断脊神经和交感神经有可能缓解患者的头颈肩痛症状。     

Sensory nerve endings in the mucosa of the epiglottis--morphologic investigations with silver impregnation, immunohistochemistry, and electron microscopy

This study was conducted in order to investigate the structure of sensory nerve endings of the human epiglottis and substance P immunoreactive nerve fibers of the canine epiglottis in relationship to physiologic functions of the larynx. The human epiglottis was observed by light microscopy (silver impregnation) and electron microscopy, and the canine epiglottis was studied by peroxidase-anti-peroxidase (PAP) immunohistochemistry. The results are summarized as follows: (1) In the membranes of the epiglottis, we observed free endings of simple or complex tree shape, corpuscle endings with glomerular patterns, and taste-bud-like structures, and (2) electron microscopic studies revealed varicosity of the terminal axon with processes that contained small, clear and large, dense cored vesicles. Substance P was observed in these structures, and it was suggested that substance P was related to perception in the larynx.