Basic flbroblast growth factor protects auditory neurons and hair cells from noise exposure and glutamate neurotoxicity

The purpose of the present study was to determine protectivie effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo. In experiment I, cultured spiral ganglion neurons (SGNs) prepared from P3 mice were exposed to 20mM glutamate for 2 hours before the culture medium was replaced with fresh medium containing 0, 25, 50, and 100 ng/ml bFGF, respectively. Fourteen days later, all cultures were fixed with 4% paraformaldehyde, and stained with 1% toluidine blue. The number of surviving SGNs were counted and the length of SGNs neurites were measured. Exposure to 20 mM glutamate for 24 hours resulted in an inhibition on neurite outgrowth of SGNs and elevated cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and elevated number of surviving SGNs. Effects of bFGF were dose dependent with the highest potency at 100 ng/ml. In experiment II, in vivo studies were carried out with guinea pigs in which bFGF or artificial perilymph was     

Basic fibroblast growth factor protects auditory neurons and hair cells from glutamate neurotoxicity and noise exposure

Objective To determine the protective effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo.

Material and Methods In Experiment I, cultured spiral ganglion neurons (SGNs) prepared from postnatal Day 3 mice were exposed to 20 mM glutamate for 2 h before the culture medium was replaced with fresh medium containing 0, 25, 50 or 100 ng/ml bFGF. Fourteen days later, all cultures were fixed with 4% paraformaldehyde and stained with 1% toluidine blue. The number of surviving SGNs was counted and the length of the neurites of the SGNs was measured. In Experiment II, in vivo studies were carried out with guinea pigs in which bFGF or normal saline was injected intramuscularly to assess possible protective effects of bFGF on cochlear hair cells and to accelerate the recovery of the auditory brainstem response (ABR). The ABRs were measured before, immediately after and 2 and 4 weeks after exposure to noise.

Results Exposure to 20 mM glutamate for 2 h resulted in an inhibition of neurite outgrowth of SGNs and an increase in cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and an increase in the number of surviving SGNs. In Experiment II, significant (p<0.05) differences in ABR thresholds were observed between the groups injected with bFGF and saline (t=2.689) at 4 weeks after noise exposure. Cochleae were removed and hair cell loss analyzed in surface preparations prepared from all experimental animals. Acoustic trauma caused loss of 240 and 2160 inner hair cells in the groups injected with bFGF and saline, respectively. Similarly, more outer hair cells were lost in the normal saline injection group (99 291) than in the group treated with bFGF (70 377).

Conclusions Our results demonstrate that bFGF protects SGNs against glutamate neurotoxicity in vitro. In addition, treatment with bFGF protects hair cells from acoustic trauma.     

Basic fibroblast growth factor protects auditory neurons and hair cells from glutamate neurotoxicity and noise exposure

OBJECTIVE: To determine the protective effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo. MATERIAL AND METHODS: In Experiment I, cultured spiral ganglion neurons (SGNs) prepared from postnatal Day 3 mice were exposed to 20 mM glutamate for 2 h before the culture medium was replaced with fresh medium containing 0, 25, 50 or 100 ng/ml bFGF. Fourteen days later, all cultures were fixed with 4% paraformaldehyde and stained with 1% toluidine blue. The number of surviving SGNs was counted and the length of the neurites of the SGNs was measured. In Experiment II, in vivo studies were carried out with guinea pigs in which bFGF or normal saline was injected intramuscularly to assess possible protective effects of bFGF on cochlear hair cells and to accelerate the recovery of the auditory brainstem response (ABR). The ABRs were measured before, immediately after and 2 and 4 weeks after exposure to noise. RESULTS: Exposure to 20 mM glutamate for 2 h resulted in an inhibition of neurite outgrowth of SGNs and an increase in cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and an increase in the number of surviving SGNs. In Experiment II, significant (p < 0.05) differences in ABR thresholds were observed between the groups injected with bFGF and saline (t = 2.689) at 4 weeks after noise exposure. Cochleae were removed and hair cell loss analyzed in surface preparations prepared from all experimental animals. Acoustic trauma caused loss of 240 and 2160 inner hair cells in the groups injected with bFGF and saline, respectively. Similarly, more outer hair cells were lost in the normal saline injection group (99,291) than in the group treated with bFGF (70,377). CONCLUSIONS: Our results demonstrate that bFGF protects SGNs against glutamate neurotoxicity in vitro. In addition, treatment with bFGF protects hair cells from acoustic trauma.     

碱性成纤维细胞生长因子对耳蜗螺旋神经节细胞谷氨酸钠毒性作用的影响

目的了解谷氨酸钠致单离培养的小鼠耳蜗螺旋神经节细胞损伤后，碱性成纤维细胞生长因子（ｂＦＧＦ）对其生存及生长的影响，以及二者对细胞内游离钙离子浓度的影响。方法在培养液中加入２×１０２ｍｏｌ／Ｌ的谷氨酸钠２小时后，实验组分别换成含ｂＦＧＦ２５、５０、１００μｇ／Ｌ的培养液，对照组加空白培养液。结果２４小时后可见部分细胞裂解、死亡，对照组明显多于实验组。培养１４天后甲苯胺蓝染色证实，实验组的细胞生存数及细胞突起长度明显高于对照组，差异有显著性（Ｆ检验，Ｐ＜０．０１），且随着ｂＦＧＦ浓度增加，细胞生存数及细胞突起长度均增加，不同浓度实验组间差异有显著性（Ｐ＜０．０１）。用Ｆｌｕｏ３染色，激光扫描共聚焦显微镜观察１０个螺旋神经节细胞，培养液中加入谷氨酸钠后细胞内钙离子浓度迅速上升，在１０～１５秒内达峰值，１００秒左右恢复到正常水平；加入ｂＦＧＦ后细胞内钙离子浓度无明显变化（ｔ检验，Ｐ＝０．２０４）。结论谷氨酸钠可致螺旋神经节细胞内钙离子浓度升高，ｂＦＧＦ可以降低谷氨酸钠对螺旋神经节细胞的细胞毒性。     

碱性成纤维细胞生长因子对豚鼠椭圆囊毛细胞再生的影响

目的 利用豚鼠椭圆囊培养的方法观察碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对毛细胞再生的影响。方法 应用新霉素破坏豚鼠离体椭圆囊毛细胞,实验组培养液含有bFGF,对照组培养液则不含有bFGF。培养18d后两组均行扫描电镜检查,并行新生毛细胞计数,比较两组新生毛细胞数目的差异,观察bFGF对椭圆囊毛细胞再生的影响。结果 实验组新生毛细胞数目明显多于对照组(P<0.05)。结论bFGF对椭圆囊毛细胞的再生有促进作用。     

碱性成纤维细胞生长因子对外伤性鼓膜穿孔的治疗观察

目的 观察碱性成纤维细胞生长因子（bFGF）对外伤性鼓膜穿孔的治疗效果。方法 随机将外伤性鼓膜穿孔分为两组：治疗组66例，用浸湿bFGF的压薄无菌棉片贴补，并每日两次用bFGF喷耳；对照组22例仅用贴补治疗。结果治疗4周后，治疗组鼓膜穿孔愈合率为98.3％，平均愈合时间为10．6天；对照组鼓膜穿孔愈合率为63．6％，平均愈合时间为22.2天。结论 bFGF浸湿棉片贴补加喷耳治疗外伤性鼓膜穿孔可促进穿孔愈合。缩短愈合时间。     

鼻咽癌中VEGF、bFGF的表达及其与颈淋巴结转移的关系

目的 探讨鼻咽癌中血管生长因子VEGF、bFGF的表达及其与颈淋巴结转移的关系。方法 采用免疫组化S－P法对35例鼻咽癌（其中15例同时取颈部转移癌组织）,20例鼻咽粘膜不典型增生组织和10例慢性炎症组织中VEGF、bFGF的表达进行了检测。结果（1）35例鼻咽癌中17例（48．6％）VEGF表达阳性,明显高于非癌对照组（6．7％,P＝0．000）,有颈淋巴结转移鼻咽癌中VEGF的表达（73．3％）明显高于无颈淋巴结转移者（30％,P＝0．018）;晚期（Ⅲ、Ⅳ期,70．6％）明显高于早期（Ⅰ、Ⅱ期,27．8％,P＝0．018）：但咽癌原发灶中VEGF的表达与颈部转移灶中的表达无相关性（P＞0．05）;（2）35例鼻咽癌中有8例（22．9％）bFGF表达阳性,明显高于非癌对照组（3．3％,P＝0．031）。但鼻咽癌中bFGF的表达与患者的年龄、性别、肿瘤的组织学类型和分期无关,也与肿瘤的N分期无关（P＞0．05）。结论 （1）VEGF与鼻咽癌的生长及颈淋巴结转移有关,它可能可以作为预测鼻咽癌颈淋巴结转移的的指标;（2）bFGF可能与鼻咽癌的生长有关系,但与肿瘤的颈淋巴结转移可能无关。     

Safety and short-term outcomes of basic fibroblast growth factor injection for sulcus vocalis

Abstract

Background: Sulcus vocalis (SV) is characterized by the appearance of a groove and fibrotic changes in the vocal fold mucosa and an often irrevocable loss of tissue viscoelasticity and vibratory potential. Although several surgical approaches have been proposed, none are ideal treatments. Basic fibroblast growth factor (bFGF) may stimulate fibroblasts in the superficial layer of the lamina propria (SLP) and increase the vibration of vocal fold mucosa.

Aims/objectives: The aim of this study was to evaluate the safety and short-term outcomes of bFGF injection for SV.

Materials and methods: This study was registered with the University Hospital Medical Information Network-Clinical Trials Registry (UMIN000019347). Twelve cases of pathological SV were treated using a method involving bFGF injection. The treatment regimen involved the injection of 50 µg of bFGF into the SLP. More than 3 months after the injection, aerodynamic and acoustic outcomes were examined.

Results: No adverse events were recorded. Significant improvements were observed in the maximum phonation time (MPT) and Voice Handicap Index (VHI) after treatment. Multiple injections achieved additional effects.

Conclusions and significance: bFGF injection may be a safe and suitable office-based surgery for the alleviation of hoarseness caused by SV based on this preliminary short-term study.     

噪声刺激致豚鼠外毛细胞凋亡时半胱氨酸天冬氨酸蛋白酶3激活和凋亡诱导因子转移

目的探讨噪声损伤后豚鼠外毛细胞发生凋亡的通路及其机制。方法分离解剖对照组（12只）和噪声暴露组（12只）豚鼠耳蜗，利用免疫荧光抗体和免疫荧光染料，分别染色细胞核、线粒体、凋亡诱导因子（apoptosis inducing factor，AIF）和半胱氨酸天冬氨酸蛋白酶3（Caspase 3），按表面制备法行耳蜗铺片，观察凋亡和坏死毛细胞内的荧光信号。结果对照组正常耳蜗毛细胞内没有激活的Caspase 3，AIF分布在毛细胞的线粒体内。实验组动物暴露于声压级120dB的白噪声环境中每天4h，连续2d后，耳蜗外毛细胞出现死亡，表现为坏死和凋亡两种方式，但以凋亡为主。在凋亡的外毛细胞中，Caspase 3被激活，AIF从线粒体转移到细胞核中；而在坏死的外毛细胞中，没有Caspase 3的激活，只有AIF自线粒体向细胞核的转移。结论噪声损伤后耳蜗外毛细胞的凋亡以Caspase依赖型的通路为主；线粒体释放的AIF在外毛细胞死亡通路中起重要作用。     

Regenerative potential of basic fibroblast growth factor contained in biodegradable gelatin hydrogel microspheres applied following vocal fold injury: Early effect on tissue repair in a rabbit model

IntroductionPostoperative dysphonia is mostly caused by vocal fold scarring, and careful management of vocal fold surgery has been reported to reduce the risk of scar formation. However, depending on the vocal fold injury, treatment of postoperative dysphonia can be challenging.ObjectiveThe goal of the current study was to develop a novel prophylactic regenerative approach for the treatment of injured vocal folds after surgery, using biodegradable gelatin hydrogel microspheres as a drug delivery system for basic fibroblast growth factor.MethodsVideoendoscopic laryngeal surgery was performed to create vocal fold injury in 14 rabbits. Immediately following this procedure, biodegradable gelatin hydrogel microspheres with basic fibroblast growth factor were injected in the vocal fold. Two weeks after injection, larynges were excised for evaluation of vocal fold histology and mucosal movement.ResultsThe presence of poor vibratory function was confirmed in the injured vocal folds. Histology and digital image analysis demonstrated that the injured vocal folds injected with gelatin hydrogel microspheres with basic fibroblast growth factor showed less scar formation, compared to the injured vocal folds injected with gelatin hydrogel microspheres only, or those without any injection.ConclusionA prophylactic injection of basic fibroblast growth factor -containing biodegradable gelatin hydrogel microspheres demonstrates a regenerative potential for injured vocal folds in a rabbit model.     

新生大鼠耳蜗增殖细胞的培养鉴定及超微结构观察

目的 研究新生大鼠耳蜗内是否存在增殖细胞,以及该增殖细胞能分化为何种细胞.方法 分离新生SD大鼠耳蜗细胞并进行培养,加入5-溴-2-脱氧尿嘧啶(5-bromo-2-demoxyuridine,BrdU)检测细胞的增殖状态,通过免疫荧光鉴定细胞球及分化细胞的性质.分离48个耳蜗Corti器,采用数字表格法随机分成4组进行细胞培养:对照组、表皮生长因子(epidermal growth factor,EGF)组、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)组和EGF+bFGF组,每组12个,定量计数单个耳蜗培养细胞球的数量,结果进行方差分析.采用透射和扫描电镜观察细胞球的超微结构.结果 耳蜗Corti器细胞培养可见细胞球形成,90.1%球内细胞BrdU表达阳性,且大部分细胞巢蛋白表达阳性.分化细胞表达毛细胞标志物肌球蛋白7A、espin及鬼笔环肽阳性,神经元标志物神经丝蛋白(neurofilament M,NF-M)表达阳性,并且可见肌球蛋白7A和BrdU,espin和BrdU,以及NF-M和BedU双标阳性的分化细胞.对照组平均((-x)±s,以下同)每个耳蜗Corti器的增殖细胞数量为(45.3±23.0)个,EGF组(86.2±34.1)个,bFGF组(96.5±33.6)个,EGF+bFGF组(131.2±47.0)个.除EGF组和bFGF组之间P＞0.05外,其余各组之间P值均＜0.05,差异具有统计学意义.扫描和透射电镜下细胞球内细胞呈圆球形,大小均匀一致,表面具有众多微绒毛.胞质内有丰富的内质网、微管、微丝和线粒体等细胞骨架结构和细胞器,相邻细胞之间可见紧密连接、桥粒与缝隙连接.结论 大鼠耳蜗内存在增殖细胞,可分化为具有纤毛样结构的毛细胞及神经元.EGF和bFGF均可诱导增殖细胞分裂增殖.该增殖细胞的超微结构显示其具有早期幼稚细胞发育的特征.     

Restoring vocal fold movement after transection and immediate suturing of the recurrent laryngeal nerve with local application of basic fibroblast growth factor: an experimental study in the rat

OBJECTIVE: To evaluate the effects of basic fibro-blast growth factor (bFGF) on the recovery of vocal fold movement and the attenuation of laryngeal muscle atrophy after transection of the recurrent laryngeal nerve (RLN). STUDY DESIGN: Quantitative assessment of vocal fold movement using the video cassette recorder (VCR) image-analysis method and histologic examination of the laryngeal muscle. METHODS: Fifty-eight Wistar rats underwent RLN transection and one of the following three procedures: 1) transection of the RLN alone (transection group, n = 18), 2) suture of the nerve stumps followed by local administration of phosphate-buffered saline (PBS) solution using an osmotic pump (PBS group, n =20), or 3) suture of the nerve stumps followed by local administration of bFGF (FGF group, n = 20). Vocal fold movements were recorded with VCR by way of a rigid endoscope, and the VCR images were analyzed on a computer. Histologic changes in the thyroarytenoid (TA) muscle were evaluated by measuring the cross-sectional area of the muscle and average size of muscle fibers. RESULTS: In the transection group, vocal fold movement did not recover, and atrophy of the TA muscle gradually progressed after sectioning the nerve. In contrast, vocal fold movement as assessed by VCR image-analysis recovered in some cases in the immediate suturing groups, more markedly in the FGF group (34.1 +/- 29.1%) than in the PBS group (5.5 +/- 7.9%) (P <.05). Histologically, atrophy of the laryngeal muscle was significantly attenuated by the local administration of bFGF. CONCLUSION: bFGF facilitates regeneration of the transected RLN and attenuation of intrinsic laryngeal muscle atrophy, thereby restoring laryngeal function.     

脑源性神经营养因子基因修饰的骨髓间充质干细胞在药物致聋豚鼠内耳的表达及保护作用

目的 探讨脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)基因修饰的骨髓间充质干细胞(mesenchymal stem cell,MSC)在药物致聋豚鼠内耳的表达及对螺旋神经节细胞(spiral ganglion cell,SGC)的保护作用.方法 阿米卡星致聋的豚鼠随机数字表法分为两组,治疗组经鼓阶开窗注射BDNF基因修饰的MSC,对照组注射外淋巴液.各组分别在术后7 d及28 d处死动物,荧光定量反转录聚合酶链反应(RT-PCR)检测耳蜗组织中BDNF mRNA的表达,耳蜗切片计算SGC细胞密度,末端脱氧核苷酸转移酶介导dUTP缺口末端标记法(terminal deoxynucleotidyl transferase mediated dUTP nick end labeling,TUNEL)检测SGC凋亡情况.结果 治疗组在术后7 d和28 d的BDNF mRNA相对表达量均明显高于对照组,差异具有统计学意义(P值均＜0.01).治疗组术后7 d及28 d的SGC密度均高于对照组,并且治疗组术后7 d及28 d的SGC凋亡指数也比对照组明显下降,差异具有统计学意义(P值均＜0.01).结论 BDNF基因修饰的MSC在致聋豚鼠内耳中的表达时间超过28 d,对SGC具有保护作用.