噪声损伤和苯扎贝特干预对豚鼠耳蜗脂肪酸转运蛋白1和4表达的影响

目的观察豚鼠耳蜗脂肪酸转运蛋白1和4(fatty acid transport protein,FATP1,FATP 4)在噪声损伤和促脂肪酸代谢药物苯扎贝特干预前后的变化,探讨噪声性听力损失与FATP1、FATP4的关系。方法正常听力健康成年雄性Dunkin Hartley豚鼠21只随机分为对照组(15只)和干预组(6只),对照组在噪声暴露前随机选取3只(6耳)动物行耳蜗取材作为正常对照组,其余12只(24耳)与干预组(6只,12耳)一起予以白噪声(110 dB SPL,每天4小时,连续12天)暴露,干预组在噪声暴露的同时给予苯扎贝特(5 mg·kg-1·d-1)灌胃干预;在开始噪声暴露第6、12、24天行听性脑干反应(ABR)检测,采用脂肪酸特异性探针荧光标记技术检测脂类物质在豚鼠耳蜗的分布,采用免疫荧光染色技术、Western blot技术检测噪声暴露前后FATP1、PATP4在两组豚鼠耳蜗的表达变化。结果噪声暴露后对照组和干预组click声及4、8、16 kHz短纯音(tone burst)ABR(tb-ABR)反应阈较噪声暴露前显著升高,在噪声暴露的第12天,干预组4 kHz tb-ABR反应阈(16.25±5.82 dB SPL)明显低于对照组(32.27±5.92 dB SPL)(P<0.05);特异性脂肪酸探针荧光染色提示正常对照组豚鼠耳蜗脂类物质主要分布于基底膜外侧的Hensen细胞、螺旋神经节、血管纹,噪声暴露前免疫荧光染色显示FATP1主要表达于Corti器、螺旋唇,在螺旋神经节细胞、螺旋韧带、血管纹等少量表达;FATP4主要表达于耳蜗Corti器及螺旋韧带,在Corti器外侧的Hensen细胞、螺旋神经节、血管纹极少表达或不表达。噪声暴露后对照组(第12天)FATP1在Corti器、Hensen细胞、螺旋突、螺旋韧带的表达较噪声暴露前明显增加(P<0.01),对照组(第12天)和干预组(第24天)FATP4在Hensen细胞、螺旋唇、螺旋神经节的表达均较噪声暴露前明显增加(P<0.01),干预组(第24天)FATP4在血管纹的表达均较噪声暴露前增加(P<0.05),但对照组(第12天)FATP4在血管纹的表达均较噪声暴露前及干预组(第24天)明显降低(P<0.05);Western blot结果则提示对照组和干预组FATP1和FATP4在全耳蜗的表达均较噪声暴露前增加(P<0.05)。结论噪声损伤可提高FATP1和FATP4在豚鼠耳蜗部分组织的表达水平,促进耳蜗内脂肪酸转运;促脂肪酸代谢药物(苯扎贝特)干预可进一步改变FATP4在耳蜗的表达分布,从而在一定程度上防止噪声性听力损失或促进噪声性听力损失恢复;提示耳蜗内脂肪酸代谢水平可能是改善噪声性听力损失的重要因素。     

肌醇需求酶1对顺铂所致小鼠耳蜗毛细胞凋亡的作用研究

目的 研究肌醇需求酶1(inositol-requiring enzyme 1,IRE1)对顺铂所致小鼠耳蜗毛细胞凋亡的作用。方法 分离生后3～5 d的健康昆明小鼠(500只)耳蜗基底膜并进行体外培养,24 h后随机分为对照组和不同浓度顺铂组(4、8、16和32μg/ml)(每组200条),再培养24 h。应用异硫氰酸荧光素(FITC)染色观察小鼠耳蜗毛细胞的缺失;应用Hoechst 33258和四甲基异硫氰酸罗丹明(TRITC)标记的鬼笔环肽双重荧光染色观察小鼠耳蜗毛细胞的凋亡;并用Western blot检测小鼠耳蜗中p-IRE1α、p-JNK、Bax及caspase-3的蛋白水平。结果 与对照组比较,顺铂组小鼠耳蜗毛细胞缺失、细胞凋亡率以及p-IRE1α、p-JNK、Bax、caspase-3等蛋白水平显著增高(P<0.01),并且p-IRE1α与Bax、caspase-3表达和细胞凋亡率呈正相关(P<0.05)。结论 IRE1可能参与调控顺铂所致小鼠耳蜗毛细胞凋亡。     

c-Maf基因在小鼠耳蜗内外毛细胞中的表达及意义

目的研究c-Maf基因在小鼠耳蜗内、外毛细胞中的表达、分布特点及意义。方法 1)根据形态上的差异分离收集成年小鼠内、外毛细胞,利用Microarray基因芯片技术分别检测内、外毛细胞c-Maf基因RNA水平的表达;2)全耳蜗基底膜铺片,免疫组织化学染色后激光共聚焦显微镜观察c-Maf蛋白在不同发育阶段小鼠耳蜗内、外毛细胞中的分布特点及表达,并使用软件Image Pro Plus进行c-Maf蛋白定量分析。结果 1)耳蜗内、外毛细胞c-Maf基因RNA水平相近;2)c-Maf蛋白主要表达于成年小鼠的耳蜗内外毛细胞胞质,在内毛细胞的核下区表达强于核上区,且在内、外毛细胞的表达强度相近;3)c-Maf蛋白在小鼠出生后0天、7天、12天及成年小鼠耳蜗内、外毛细胞稳定表达。结论 c-Maf蛋白从出生开始在小鼠耳蜗内、外毛细胞上稳定表达,且在成年小鼠内毛细胞的核下区表达较强,表明其可能在毛细胞生长发育以及功能维持中发挥重要的作用。     

内质网应激在顺铂诱导离体小鼠耳蜗毛细胞凋亡中的作用

目的 研究内质网应激(endoplasmic reticulum stress,ERS)在顺铂诱导离体培养小鼠耳蜗毛细胞凋亡中的作用.方法 选取出生后3d的健康昆明小鼠380只(760耳),分离出耳蜗基底膜760条,体外培养24 h后,随机分为对照组和4、8、16 μg/ml顺铂组,每组190条,对照组加入2 ml新鲜培养基,顺铂组分别加入2 ml含不同浓度顺铂(4、8、16 μg/ml)的新鲜培养基,再继续培养24 h后,应用Hoechst 33258荧光染色观察耳蜗毛细胞凋亡情况,并应用Western blot检测ERS标志物葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)和内质网凋亡途径标志物半胱氨酸天冬氨酸蛋白酶-12(cysteinyl aspartate specific proteinase 12,caspase-12)在耳蜗毛细胞中的表达.结果 4、8、16 μg/ml顺铂组耳蜗毛细胞凋亡率分别为10.53％±0.50％、27.12％±2.64％和60.01％±2.75％,均高于对照组(3.67％±0.76％),呈现明显的量效关系;不同浓度顺铂组耳蜗GRP 78和caspase-12的蛋白表达水平亦较对照组明显增高(P＜0.01),并随顺铂浓度增大而显著增强(P＜0.01).结论 顺铂可诱发小鼠耳蜗毛细胞ERS,ERS可能在其诱导的小鼠耳蜗毛细胞凋亡中发挥了作用.     

噪声对小鼠耳蜗外侧壁缝隙连接蛋白Connexin31表达的影响

目的观察噪声性聋小鼠耳蜗外侧壁缝隙连接蛋白Connexin31(Cx31)表达的变化。方法选用成年雄性Balb/c小鼠44只,随机分为噪声组和对照组,每组22只。两组小鼠均在实验前检测听性脑干反应(ABR),随后应用声刺激器给予噪声组小鼠高强度白噪声(115dB SPL,6h/d,共2天),并在噪声暴露结束后1h再检测噪声组小鼠ABR。对照组不予噪声刺激。于噪声暴露后4h,每组各取4只小鼠耳蜗做冰冻切片,其余18只小鼠提取耳蜗外侧壁组织总RNA,通过免疫荧光染色法观察小鼠耳蜗外侧壁Cx31蛋白的表达,荧光实时定量PCR检测小鼠耳蜗外侧壁Cx31mRNA的表达。结果对照组小鼠耳蜗螺旋韧带可见Cx31特异性荧光,血管纹处未见阳性荧光;噪声组小鼠耳蜗外侧壁免疫荧光染色阳性反应较对照组减弱,Cx31mRNA表达量明显低于对照组。结论噪声能下调Cx31在小鼠耳蜗外侧壁组织的表达,Cx31可能参与了噪声性聋的发病机制。     

顺铂对离体培养小鼠耳蜗毛细胞钙蛋白酶表达的影响

目的：观察顺铂对离体培养小鼠耳蜗毛细胞钙蛋白酶（calpain ）表达的影响,探讨顺铂致耳蜗毛细胞凋亡的机制。方法取出生后3 d的昆明小鼠300只（600耳）,分离出耳蜗基底膜600条,体外培养24 h后,随机分为对照组和4、8、16μg／ml顺铂组,每组150条;对照组加入2ml新鲜培养基,顺铂组分别加入2 ml含不同浓度顺铂（4、8、16μg／ml）的新鲜培养基,再继续培养24 h后,应用Hoechst 33258荧光染色观察耳蜗毛细胞凋亡情况,并应用免疫荧光染色和免疫印迹（Western blot）技术检测calpain 1（μ－calpain）和calpain 2（m －calpain）在耳蜗毛细胞的表达。结果4、8、16μg／m l顺铂组耳蜗毛细胞凋亡率分别为15．63％±0．20％、38．40％±2．64％和64．24％±0．05％,均高于对照组（5．55％±0．12％）,呈现明显的量效关系;不同浓度顺铂组μ－calpain和m －cal‐pain的表达均较对照组明显增强（ P＜0．01）,且m －calpain的表达随顺铂浓度的增高而明显增强（ P＜0．01）。结论顺铂可通过calpain通路诱导小鼠耳蜗毛细胞凋亡,从而发挥毒性效应。     

蛋白磷酸酶1在D-半乳糖诱导老化小鼠耳蜗中的表达

目的 观察D-半乳糖诱导老化小鼠耳蜗中蛋白磷酸酶1(protein phosphatase 1,PP1)的定位表达及作用.方法 40只昆明小鼠随机分为对照组和D-半乳糖组,每组20只.D-半乳糖组颈背部皮下注射D-半乳糖(800mg·kg-1·d-1),对照组每日皮下注射等量生理盐水,连续给药8w;停药后检测血浆总超氧化物歧化酶(superoxide dismutase,SOD)活力和丙二醛(Malondialdehyde,MDA)水平;免疫荧光法定位PP1在耳蜗的表达,re-al-time PCR法检测耳蜗中PP1 mRNA水平,Western blot法检测小鼠蛋白磷酸酶1核目标亚基(protein phos-phates 1 nuclear targeting subunit,PNUTS)、PP1及caspase-3的表达.结果 与对照组比较,D-半乳糖组小鼠总SOD活力降低而MDA水平升高(均为P<0.01);PP1主要定位表达于小鼠耳蜗毛细胞、螺旋神经节细胞及血管纹细胞的胞核和胞浆中,且给予D-半乳糖后,小鼠耳蜗中PP1 mRNA及其蛋白表达水平显著升高,而PNUTS表达减少,caspase-3表达增加(均为P<0.05).结论 PP1在D-半乳糖诱导老化小鼠耳蜗毛细胞、螺旋神经节及血管纹细胞中表达,且参与了其耳蜗老化过程.     

突触内吞关键蛋白Dynamin在小鼠耳蜗内毛细胞中的表达研究

目的研究生理情况下Dynamin各亚型在小鼠耳蜗内毛细胞(IHC)的表达分布特点,为探讨其在毛细胞内可能发挥的生理功能以及与听信号传导的关系做铺垫。方法取成年小鼠耳蜗基底膜行免疫荧光染色,激光共聚焦显微镜观察Dynamin各亚型表达及定位分布情况。结果 Dynamin各亚型在小鼠耳蜗内毛细胞中均有表达。其中,Dyna-min-1表达于整个内毛细胞内,Dynamin-2点状分布在内毛细胞胞质,Dynamin-3高表达于内毛细胞核下近螺旋神经节区域。结论网格蛋白介导内吞过程是毛细胞内吞作用的重要机制,作为该过程中的关键蛋白,Dynamin各亚型在耳蜗内毛细胞中均有表达,提示Dynamin在内毛细胞中可能发挥着重要的生理功能。     

衔接蛋白AP-2蛋白在小鼠耳蜗中的表达及意义

目的:研究衔接蛋白AP-2在小鼠耳蜗中的定位与表达,探讨AP-2蛋白与内毛细胞胞吞的相关性。方法:选择健康成年C57BL/6J小鼠,应用免疫荧光标记技术结合激光共聚焦显微镜观察AP-2蛋白在其耳蜗毛细胞的定位与表达。结果:AP-2蛋白主要定位于小鼠耳蜗内毛细胞细胞质,集中表达于内毛细胞基底部与带状突触靠近传入神经元区域。结论:AP-2蛋白在成年小鼠耳蜗中主要表达于内毛细胞突触活化部位,可能与内毛细胞突触囊泡的内吞作用相关,为深入研究AP-2蛋白在内耳听觉生理和病理机制中的作用奠定了基础。     

One‐stop neck lump clinic: phase 2 of audit. How are we doing?

One‐stop neck lump clinic: phase 2 of audit. How are we doing? Regular monitoring and audit of a service are integral to ensuring maintenance of efficiency and standards. This is particularly important where the quality of the service is operator dependent, as is the case in the clinical diagnosis of neck lumps and fine needle aspiration cytology. The one‐stop neck lump clinic has now been running in the department for more than 20 months. A previous article described the results of the first phase audit carried out at 6 months and had identified a waiting time to be seen that was longer than that recommended by the British Association of Otorhinolaryngologists, Head and Neck Surgeons. Measures were implemented to reduce this waiting time and a second audit was carried out after another 10 months with the aims of assessing if modification of the means of referral reduces waiting time and if the outcomes of clinical performance in phase 1 could be maintained or improved. We discuss the results of phase 2 in the audit spiral.     

A Decrease in Maxillary Sinus Pressure,as Seen in Upper Airway Allergy or Infection,Results in an Increase in Upper Airway Nitric Oxide Levels

The paranasal sinuses are connected to the nasal cavity via small osties. Ostial occlusion, caused by mucosal swelling, will result in a slowly increasing negative pressure inside the sinus cavity. In parallel, the oxygen content in the sinus will decrease, resulting in the development of relative hypoxia. Hypoxia is a powerful inducer of nitric oxide (NO) synthase, and inducible NO synthase has been shown to be present in considerable amounts in the upper airways, including the sinuses. The present study was designed to investigate whether a reduction in sinus pressure would affect upper airway NO production. Thirteen healthy volunteers were investigated. A pressure chamber was used to lower the ambient pressure to-4.9 kPa. NO was sampled from one nostril or via a drainage tube inserted into the maxillary sinus before, during and after the hypobaric exposure. When the pressure was decreased, NO levels increased from 256 &#45 15 to 316 &#45 19 ppb ( n =13, p <0.001). The NO levels remained elevated (282 &#45 21 ppb; p <0.05) when measurements were repeated 20 min after leaving the chamber. The nasal airway resistance (V2 tot ) also increased as a result of the chamber session (from 16 &#45 2° before to 21 &#45 3° after; p <0.05). An increase in NO levels was also found when the experiments were repeated with NO sampled directly from the maxillary sinus (225 &#45 6 before and 265 &#45 9 ppb after; n =6, p <0.001). For control purposes the nasal analyses were repeated again, this time under hyperbaric conditions (+4.9 kPa). This resulted in a slight decrease in the NO levels (from 273 &#45 22 to 241 &#45 17 ppb; n =10, p <0.001), but there was no change in the nasal airway resistance. We conclude that a reduction in sinus pressure, as seen in upper airway allergy or infection, may result in an increase in upper airway NO production.     

A case of cortical deafness and anarthria

The nasal epithelium protects the underlying tissue from damage. Epithelial cell growth is controlled by epidermal growth factor (EGF) and is possibly affected by toxic proteins, e.g. eosinophil cationic protein (ECP). The aims of this study were to examine nasal fluid epithelial cell counts and their relations to EGF, eosinophils and ECP in 23 patients with seasonal allergic rhinitis and 20 healthy controls. Nasal fluid epithelial cell counts were lower in patients than in controls. EGF levels did not differ between patients and controls, and correlated with epithelial cell counts in controls but not in patients. Eosinophils and ECP were higher in patients than in controls, but did not correlate with epithelial cell counts. The role of growth factors, such as EGF, in regulating epithelial cells merits further study.     

A simple objective evaluation and grading for facial paralysis outcomes

Matrix metalloproteinase (MMP)-2 and -9 degrade type IV collagen, which is one of the major components of the basement membrane in normal tissue and expressed in the surroundings of the cancer nest in squamous cell carinoma. The degeneration of type IV collagen is an essential step in the metastasis to lymph nodes and distant organs. In this study, we examined MMP-2 and -9 levels of cancer tissue and serum obtained from patients with head and neck squamous cell carcinoma (HNSCC) in order to evaluate the relationship between the clinicopathologic features and MMPs. We examined the production of MMP-2 and -9 in cancer tissue homogenates of 73 patients who had HNSCC and the serum MMP levels of 16 patients with HNSCC and 8 healthy volunteers. We also studied the localization of MMP-2 in the carcinoma using an immunohistochemical approach. The concentrations of MMP-2 and -9 in the tissue homogenates and serum were measured by means of a sandwich enzyme immunoassay using a monoclonal antibody. Immunohistochemical analyses were performed with monoclonal antibody to MMP-2. The concentration of MMP-2 in the tumor tissue homogenates was unrelated to tumor size, but that in patients with lymph node metastases was significantly higher than in those without lymph node metastases. The concentration of MMP-9 was unrelated to lymph node metastasis and tumor size. The levels of both MMP-2 and -9 in serum were unrelated to lymph node metastasis. Immunohistochemistry indicated that MMP-2 was mainly expressed in cancer cells. Because MMP-2 degrades type IV collagen, the level of MMP-2 in carcinomas may be a useful indicator of the degree of invasion and metastasis.     

Speech production in deaf implanted children with additional disabilities and comparison with age-equivalent implanted children without such disorders

OBJECTIVE: To assess the long-term speech intelligibility in implanted children with additional disorders and compare them with age-equivalent implanted children without such disorders. PATIENTS: 175 profoundly deaf children 5 years following cochlear implantation; 67 children with additional difficulties and 108 children without such difficulties. All children were implanted under 5-year-old. MAIN OUTCOME MEASURE: Speech intelligibility rating (SIR) scale that can be readily applied to young deaf children irrespective of their performance and is reliable between observers. RESULTS: Five years following implantation, 47 (70%) children with additional difficulties developed connected intelligible speech versus 104 (96%) in the control group. However, the quality of speech was quite different between the two groups, as only 11 (16%) children with additional difficulties achieved the two higher categories (intelligible to all or to people with little experience), whereas 66 (61%) children in the control group did (P<0.000001). The total number of additional disorders had the strongest correlation with the outcome. Language and communication disorders were the most important contributing factor, followed by physical, cognitive, and autistic spectrum disorders. CONCLUSION: The majority of deaf children with additional disorders develop connected intelligible speech 5 years following implantation; however, a significant proportion do not develop any speech at all. Thus a third of this group did not realise one of the most important objectives for parents of implantation. Benefit from implantation should not be restricted to speech production alone in this specific population.     

Suction cautery and electrosurgical risks in otolaryngology

BACKGROUND: Flash fires, mucosal injuries and commissure burns during otolaryngology procedures have been largely attributed to anesthetic and surgical errors. Reports of direct electrosurgical device related events are rare. The discovery of thermal damage to the oral commissure during routine suction cautery adenoidectomy at our institution prompted a detailed investigation of the device's thermal properties. We complement this analysis with a review of electrocautery device related injuries reported in otolaryngology literature. METHODS: FLIR Systems Thermovision A40 infrared camera was used to evaluate temperature changes along the electrosurgical wand of suction cautery devices. Shaft temperatures were measured at specific times of continuous use, distances along the shaft, and cautery settings. A literature search of electrocautery-associated injuries during upper aerodigestive procedure was then performed. Nine pediatric otolaryngologists were then interviewed for historical experience with electrocautery injuries. RESULTS: Temperatures exceeding 60 degrees C, and sufficient to cause thermal soft tissue damage, occurred along the suction cautery wand at a setting of 40 Watts (W). These temperatures traveled far enough to appose the oral commissure when the device was simultaneously in continuous use, in the fulgurate mode, and with the suction turned off. Literature review identified eleven articles specifically pertaining to electrosurgical injuries during routine oropharyngeal procedures. Flash fires and their associated burns were the most frequently reported complication. Conversely, seven of ten cases elicited from peer interviews were oral or commissure burns attributed to improper insulation of electrocautery devices. CONCLUSIONS: Inadvertent electrosurgical injuries during routine otolaryngology procedures can result from inadequate equipment insulation. Techniques to reduce the likelihood of these events are discussed.     

Bilateral versus unilateral cochlear implantation in young children

Objectives

To compare the preverbal communication skills of two groups of young implanted children: those with unilateral implantation and those with bilateral implantation.

Material and methods

The study assessed 69 children: 42 unilaterally and 27 bilaterally implanted with age at implantation less than 3 years. The preverbal skills of these children were measured before and 1 year after implantation, using Tait Video Analysis that has been found able to predict later speech outcomes in young implanted children.

Results

Before implantation there was no significant difference between the unilateral group and the bilateral group. There was still no difference at 12 months following implantation where vocal autonomy is concerned, but a strongly significant difference between the groups for vocal turn-taking and non-looking vocal turns, the bilateral group outperforming the unilateral group. Regarding gestural turn-taking and gestural autonomy, there was a strongly significant difference between the two groups at the 12 month interval, and also a difference before implantation for gestural autonomy, the unilateral group having the higher scores. Multiple regression of non-looking vocal turns revealed that 1 year following implantation, bilateral implantation contributed to 51% of the variance (p < 0.0001), after controlling for the influence of age at implantation and length of deafness which did not reach statistical significance.

Conclusions

Profoundly deaf bilaterally implanted children are significantly more likely to use vocalisation to communicate, and to use audition when interacting vocally with an adult, compared with unilaterally implanted children. These results are independent of age at implantation and length of deafness.     

Autoimmunity in Sudden Sensorineural Hearing Loss: Possible Role of Anti-endothelial Cell Autoantibodies

In order to verify whether anti-endothelial cell autoantibodies (AECAs) can be used as serological markers of inner ear vasculitis in sudden sensorineural hearing loss (SSHL), 32 patients affected by idiopathic SSHL were investigated. All patients underwent a routine general physical examination and extensive audiovestibular, microbiological and immunological investigations. Fourteen normal subjects without a history of HL, autoimmune or metabolic disease served as controls. Detection of AECAs was performed using an indirect immunofluorescence technique. AECA-positive patients were treated with methylprednisone, while AECA-negative patients were treated with a combined regimen of steroids, plasma expander and aspirin. The average hearing recovery for 5 frequencies (0.25-4 kHz) was analyzed in each subject 1 month after treatment and every 3 months thereafter; median follow-up was 12 months (range 9-18 months). A total of 15/32 patients (46.8%; 11/19 females, 4/13 males) were AECA-positive and thus differed significantly from the normal population in whom only 2/14 tested cases were positive ( p =0.03). Severe hearing loss was associated with being AECA-positive in 8/11 cases. During follow-up, 25/32 patients improved their hearing and 17 of these patients were AECA-negative. The seven cases without hearing improvement were all AECA-positive. In patients with SSHL, immune-mediated vascular damage may have a pathogenetic role and AECAs may represent a serological marker of vasculitis even if they are not inner ear-specific and even if they represent an epi-phenomenon rather than the only cause of SSHL.     

Correction of the Glosso-Larynx and Resultant Positional Changes of the Hyoid Bone and Cranium

In ankyloglossia with deviation of the epiglottis and larynx (ADEL) the tongue is located forward and as a result the epiglottis is elevated and leans towards the mouth. The larynx is also raised and curves ventrally. Various symptoms have been observed as a result of this condition. Correction of the glosso-larynx (CGL) is the operation performed to treat ADEL. The CGL procedure and the results obtained with it are reported in this paper. In addition, we studied the following six parameters using head and neck X-rays before and after CGL (the changes in these parameters as a result of CGL are shown in parentheses): ( i ) the shortest vertical length between the hyoid bone and mandible (+10.3 mm); ( ii ) the vertical length between the hyoid bone and the tangent line of C2-4 (+4.6 mm); ( iii ) the shortest length between the hyoid bone and the chin (+2.9 mm); ( iv ) the angle between the hyoid bone and the tangent line of C2-4 (+3.3°); ( v ) the length of H-M, where H is the intersection of a tangent line of C2-4 and a vertical line from the hyoid bone and M is the intersection of a tangent line of C2-4 and the mandible (+7.4 mm); and ( vi ) the width of the narrowest part of the hypopharynx (+3.0 mm). The changes in all the measured parameters after CGL were significantly different ( p <0.05).