Cisplatin-induced apoptotic cell death in spiral ganglion and organ of Corti of mongolian gerbil cochlear]

OBJECTIVE: To investigate whether apoptosis is one of the mechanism for cell death in spiral ganglion(SG) and organ of Corti of Mongolian gerbils cochlea induced by cisplatin. METHODS: Mongolian gerbils were administered 4 mg.kg-1.d-1 cisplatin consecutively for 4 to 7 days. The apoptotic cell death in spiral ganglion and organ of Corti of cochlea basal turn was detected via transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. RESULTS: The observation of electron microscopy shown special morphological change consistent with the feature of cell apoptosis in some spiral ganglion cells and outer hair cells after 5-7 days' cisplatin treatment. The apoptotic labeling by TUNEL in spiral ganglion and organ of Corti was detected after 5 days' treatment and increased after 6 and 7 days' treatment whereas almost negative result were obtained in the normal control animals. CONCLUSION: Apoptosis is one of the important mechanisms for cell injury of spiral ganglion and organ of Corti induced by cisplatin.     

Role of reactive radicals in degeneration of the auditory system of mice following cisplatin treatment

OBJECTIVE: It has been suggested that reactive radical species are involved in the mechanism of cisplatin-induced hearing loss. However, the nature of the free radicals involved is not fully understood. We examined the effects of two highly reactive species, hydroxyl radicals and peroxynitrite, on the auditory system of mice following cisplatin treatment. MATERIAL AND METHODS: Expression of 4-hydroxynonenal (HNE), a marker of lipid peroxidation by the hydroxyl radical, and nitrotyrosine (NT), a marker for protein peroxidation by peroxynitrite, was examined immunohistochemically in mouse cochleae injured by means of local application of cisplatin. RESULTS: Loss of outer hair cells (OHCs) and spiral ganglia was found in cochleae affected by cisplatin. Both HNE and NT were detected in auditory epithelia and neurons damaged by cisplatin. Interestingly, auditory hair cells produced HNE, but not NT. Our findings indicate contributions by both HNE and NT to the degeneration of the auditory system due to cisplatin, and a crucial role of the hydroxyl radical in degeneration of OHCs. CONCLUSION: The hydroxyl radical may be a critical target for a strategy aimed at protecting auditory function from cisplatin toxicity.     

Mechanisms of apoptosis induced by cisplatin in marginal cells in mouse stria vascularis

Degeneration of the stria vascularis (SV) is amongst the major causes of cisplatin (CDDP)-induced hearing impairment. The pathways of apoptosis occurring in the SV due to CDDP were examined using a mouse experimental model. Temporal bones of adult C57BL/6 mice were collected on days 3, 7 and 14 after the local application of CDDP. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and immunostaining for apoptosis-related proteins or reactive radical species were employed for analysis. Local application of CDDP caused apoptotic cell death of marginal cells 3 days after CDDP treatment. Immunohistochemical analyses demonstrated activation of caspase-3 and -9, but not -8, and redistribution of cytochrome c in affected marginal cells, indicating a caspase-dependent, mitochondrion-mediated apoptotic pathway in marginal cells. Temporary expression of hydroxynonenal, nitrotyrosine and inducible nitric oxide synthase in the SV was observed at the induction of apoptosis in marginal cells. CDDP toxicity generates reactive radical species in the SV, which causes mitochondrial membrane permeabilization leading to apoptosis of marginal cells.     

Detection of apoptosis by RT-PCR array in mefloquine-induced cochlear damage

Objective To investigate the occurrence and possible mechanisms of apoptosis in cochlear epithelium and spiral ganglion neurons after mefloquine treatment.Methods We used quantitative RT-PCR apoptosis-...     

内耳免疫反应中细胞凋亡的研究

目的探讨内耳免疫反应过程是否引起细胞凋亡以及Fas和FasL、Bcl-2和Bax的表达情况。方法选用雌性白色豚鼠16只，随机分为实验组和对照组各8只，以钥孔蛾血蓝蛋白全身免疫后，实验组以相同抗原进行内耳免疫，对照组内耳注射等量的磷酸盐缓冲生理盐水，在内耳免疫7d后处死动物，取内耳免疫侧耳蜗做石蜡切片。通过电镜和脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(terminal-deoxynucleotidyl transferase mediated nick end labeling，TUNEL)检测内耳凋亡细胞，免疫组化检测内耳Fas和FasL以及Bcl-2和Bax的表达。结果透射电镜观察发现实验组术后7d内耳外毛细胞、血管纹细胞及螺旋神经节细胞都出现了凋亡细胞的特征性改变，而对照组未发现具有上述特征的细胞。实验组内耳Corti器毛细胞，血管纹的缘细胞和螺旋神经节细胞存在TUNEL染色阳性细胞，TUNEL染色阳性细胞具有凋亡细胞的典型形态学特征，对照组内耳的任何结构中都没发现TUNEL染色阳性细胞。免疫组化染色实验组Corti器、螺旋神经节细胞、血管纹和螺旋韧带Fas和FasL蛋白表达阳性，而对照组只有螺旋神经节细胞和血管纹有较弱的Fas蛋白表达，FasL蛋白表达阴性。实验组Corti器、螺旋神经节细胞、侧壁Bcl-2蛋白表达阴性，对照组的Corti器、侧壁和螺旋神经节细胞Bcl-2蛋白表达阳性。实验组Corti器、侧壁和螺旋神经节细胞Bax蛋白表达阳性，对照组只有螺旋神经节细胞Bax蛋白表达弱阳性，Corti器、侧壁表达阴性。结论内耳免疫反应可诱导细胞凋亡发生，Fas-FasL是此过程的信号转导途径之一，Bcl-2和Bax蛋白在其中起了重要调节作用。     

Gene expression in cisplatin ototoxicity and protection with p53 inhibitor

Cisplatin damages cochlear hair cells and spiral ganglion neurons through cell death signaling pathways that are not fully understood. We used focused apoptosis gene microarrays to study early changes in gene expres-sion in cochlear cultures from P3 neonatal rats treated with cisplatin (0.2 mM). After 12 hours of cisplatin treat-ment, more than 50% of the 96 genes on the array showed a significant decrease in expression, consistent with widespread cell death. However, after 3 hours of cisplatin treatment, 10 genes showed significant increase in ex-pression in total cochlear tissue. In experiments with subsets of cochlear tissues, at 3h, cisplatin induced increased expression of 12 genes in the cochlear sensory epithelium (basilar membrane) and 11 genes in the spiral ganglion (tissue of Rosenthal's canal, containing the spiral ganglion). These included pro- and anti-apoptotic genes in-volved in the p53 signaling pathway, TNF receptor family, NF-kappaB pathway, death domain family, death effec-tor domain family, Bcl-2 family, CARD. family, TRAF family, and GTP signal transduction. Although the changes in gene expression showed an overlap between basilar membrane and spiral ganglion, other changes, which may reflect the unique response of each tissue, were also observed. Pifithrin-ot blocked cisplatin-induced up-regulation of genes in the p53 signaling pathway when assayed by both superarray and real time PCR. The data add to our understanding of the involvement of p53 in cisplatin-induced ototoxicity and otoprotection, conferred by the p53 inhibitor Pifithrin-α.     

Activation of caspase-3 is associated with oxidative stress in the hydropic guinea pig cochlea

The aim of this study was to investigate the involvement of oxidative stress and apoptosis in an animal model of Meniere's disease. Endolymphatic hydrops (ELH) is generally accepted as the decisive histological characteristic of Meniere's disease. Closure of the endolymphatic duct (Kimura's method) was used to induce endolymphatic hydrops in guinea pigs. Sham-operated animals served as controls. After 4 weeks the animals operated showed a significant elevation of the hearing thresholds as measured by audiometric brainstem responses (ABR) pre- and postoperatively. Immediately after the second ABR measurement, the animals were sacrificed for further immunohistological examinations of the inner ear with specific antibodies to active caspase-3 (cas-3) as a marker for apoptosis and antibodies to 8-isoprostane (8-iso) and nitrotyrosine (NT) as indicators of oxidative stress. Compared with the sham-operated controls, hydropic cochleae showed strong immunostaining for both oxidative stress markers in spiral ganglion cells, in the blood-vessels and fibrocytes of the lateral wall, as well as in supporting cells of the organ of Corti. Activation of cas-3 in spiral ganglion cells and the lateral wall was found exclusively in hydropic cochleae. Our findings suggest that oxidative stress is involved in the development of endolymphatic hydrops and may lead to cellular damage which induces apoptosis by activation of cas-3. Apoptotic cell death might contribute to the sensorineural hearing loss found in later stages of Meniere's disease.     

快速老化小鼠的听功能和耳蜗螺旋神经元的增龄性变化

目的探讨快速老化小鼠听觉功能和耳蜗螺旋神经元的增龄性变化。方法选取1、3、5、7、9月龄的快速老化小鼠亚系1( Senescence accelerated mouse/prone 1, SAMP 1)作为实验组，而同龄抗快速老化小鼠亚系1(Senescence accelerated mouse/resistance 1, SAMR 1) 作为SAMP 1的正常对照组。分别观察其8kHz短纯音听觉脑干反应阈值、耳蜗螺旋神经节细胞透射电镜形态学、末端脱氧核苷酸转移酶介导的dUTP原位切口末端标记［terminal deoxynucleotidyl transferase(TdT) dUTP nick end labeling］ TUNEL免疫组化染色等方面的增龄性变化。结果① 听功能检测。第7、9个月龄SAMP 1小鼠跟同龄SAMR 1小鼠比较听觉脑干反应阈值差异有统计学意义；② 耳蜗螺旋神经节细胞形态学检测。第7、9个月龄SAMP 1小鼠可见螺旋神经元凋亡，而同龄SAMR 1小鼠罕见螺旋神经元凋亡；③ 耳蜗中轴位切片TUNEL染色。第7个月龄SAMR 1小鼠SGN细胞核基本上不着色［TUNEL染色阳性率为（2.27±2.43）%］，第7个月龄SAMP 1小鼠部分SGN胞核着色［染色阳性率为（11.36±4.96）%］，两者的染色阳性率差异有统计学意义。结论SAMP 1小鼠随月龄增长耳蜗螺旋神经元凋亡、听功能减退，7月龄SAMP 1小鼠即出现明显的听功能老化特征，可作为老年聋动物模型用于耳聋的相关研究。     

大鼠出生后发育中螺旋神经节神经元凋亡的观察

目的：研究大鼠出生后发育时螺旋神经节（spiral ganglion,SG）神经元凋亡和分化的关系。方法：应用透射电镜观察1、3、5、7、10、14和30天龄大鼠螺旋神经节神经元超微结构的变化及细胞凋亡现象。结果：大鼠出生后5和7天，在透射电镜下观察到SG神经元凋亡，其典型的超微结构改变为染色质凝聚和着边形成的新月样结构、凋亡小体和细胞皱缩；随后的发育阶段细胞凋亡现象消失；最早可识别的富含神经丝的Ⅱ型神经元（neuron2,N2）出现在出生后7天，新出现的N2有明显的细胞皱缩现象；在随后发育的各阶段，SG中I型神经元（neuron1,N1）和N2的结构特征更明显，逐渐发育成熟。结论：神经元凋亡是大鼠螺旋神经节出现后发育过程中的一个重要事件，发生在N2分化过程中，可能与SG中N2发育有关。     

对灰鼠延迟性神经元死亡模型中螺旋神经节细胞缺损的评估

目的探索观察耳蜗螺旋神经节细胞的简便定量研究方法 ,并在灰鼠延迟性螺旋神经节细胞死亡动物模型中验证本方法的实用性和可靠性。方法 15只成年灰鼠平均分为3组,第1组用于正常对照;第2组一次性同时肌肉注射庆大霉素（125mg/kg）和静脉注射利尿酸钠（40mg/kg）,并在用药后2个月处死;第3组接受与第2组同样的药物注射,但在用药后4个月处死。耳蜗样品被常规应用环氧树脂包埋并制作成耳蜗中轴半薄切片,计数耳蜗各回蜗轴螺旋管腔切片截面内的螺旋神经节数量并进行统计分析。结果灰鼠耳蜗底回起始端的蜗轴螺旋管腔比耳蜗底回中部和耳蜗中回大,因此耳蜗底回起始端蜗轴螺旋管内的螺旋神经节细胞数量多于耳蜗底回中部和耳蜗中回。耳蜗毛细胞被彻底破坏后2个月,与正常灰鼠耳蜗各回蜗轴螺旋管腔切片截面内的螺旋神经节细胞数量相比,耳蜗底回蜗轴螺旋管切片截面内的螺旋神经节细胞减少数量比耳蜗中回严重,提示延迟性螺旋神经节细胞死亡可能遵循着从耳蜗底回向顶回扩展的规律。耳蜗毛细胞被彻底破坏后4个月,全耳蜗蜗轴螺旋管内的螺旋神经节细胞基本上丧失殆尽。结论计数耳蜗中轴切片各回蜗轴螺旋管腔切片截面内的螺旋神经节细胞数量是一种简便可靠的定量分析方法 。     

Role of reactive radicals in degeneration of the auditory system of mice following cisplatin treatment

Objective—It has been suggested that reactive radical species are involved in the mechanism of cisplatin-induced hearing loss. However, the nature of the free radicals involved is not fully understood. We examined the effects of two highly reactive species, hydroxyl radicals and peroxynitrite, on the auditory system of mice following cisplatin treatment.

Material and Methods—Expression of 4-hydroxynonenal (HNE), a marker of lipid peroxidation by the hydroxyl radical, and nitrotyrosine (NT), a marker for protein peroxidation by peroxynitrite, was examined immunohistochemically in mouse cochleae injured by means of local application of cisplatin.

Results—Loss of outer hair cells (OHCs) and spiral ganglia was found in cochleae affected by cisplatin. Both HNE and NT were detected in auditory epithelia and neurons damaged by cisplatin. Interestingly, auditory hair cells produced HNE, but not NT. Our findings indicate contributions by both HNE and NT to the degeneration of the auditory system due to cisplatin, and a crucial role of the hydroxyl radical in degeneration of OHCs.

Conclusion—The hydroxyl radical may be a critical target for a strategy aimed at protecting auditory function from cisplatin toxicity.     

Inhibition of the c-Jun N-terminal kinase signaling pathway influences neurite outgrowth of spiral ganglion neurons in vitro

OBJECTIVES: Inhibitors of the c-Jun N-terminal kinase (JNK) signaling pathway have been demonstrated to protect hair cells of the auditory system and different types of neurons from various insults, and their use for future therapeutic applications has been proposed. In the study, we evaluated the effects of inhibition of the JNK pathway on process outgrowth from spiral ganglion neurons. METHODS: Spiral ganglion explants from rats (postnatal days 3-5) that were cultured on laminin were treated with neurotrophin-3 and/or the JNK signaling pathway inhibitor CEP-11004. Both neurite length and number of the explants were evaluated and statistically analyzed by analysis of variance. RESULTS: Inhibition of the JNK signaling pathway reduced process outgrowth from spiral ganglion explants. The reduction, both in length and number of neurites, was reversed by the application of neurotrophin-3. CONCLUSIONS: The results indicate that an intact JNK signaling pathway is important for process outgrowth of spiral ganglion neurons. However, neurotrophin-3 stimulates process extension by a JNK independent pathway. Our results demonstrate that inhibition of the JNK pathway can have adverse effects on the extension of spiral ganglion neurons, but that the negative effects can be ameliorated by appropriate treatment.     

顺铂致豚鼠螺旋神经节细胞凋亡及Caspase-3活化的研究

目的：探讨顺铂的耳毒性作用与螺旋神经节细胞（sprial glanglion cell,SGC)凋亡的关系，及SGC的凋亡是否与凋亡蛋白酶Caspase-3信号转导有关。方法：取40只听力正常的豚鼠，随机分为顺铂1、2、4天组和对照组，每天检测听性脑干反应（ABR）和40Hz相关电位以观察豚鼠高，低频的听阈变化，并用TNEL法检测凋亡细胞数量变化，免疫组化检测SGC中Caspase-3p20活性片段的表达。结果：随着注射顺铂时间的延长，豚鼠听力逐渐下降，同时SGC的TUNEL染色明显增强，Caspase-3p20活性片段的表达增高，与对照组比较均有显著性差异（P＜0．01），结论：顺铂的耳毒性损害机制与SGC的凋亡有关；顺铂所致的SGC凋亡过程中有Caspase-3p20的激活。     

低剂量哇巴因对螺旋神经节细胞损伤的保护作用

目的:探讨低剂最哇巴因对B27剥夺诱发的螺旋神经节细胞损伤的保护作用,并初步探讨其保护机制.方法:螺旋神经节细胞培养第7天开始分组实验,设B27剥夺损伤组(损伤组)、B27剥夺加10 nmol/L哇巴因保护组(保护组)和正常培养对照组(对照组).各组继续培养48 h后用FITC标记的Annexin-V和碘化丙啶双染试剂盒染色,用流式细胞仪检测各组螺旋神经节细胞损伤情况.各组螺旋神经节细胞分别于加药后6、12 h两个时间点用免疫细胞化学方法检测细胞内Bel-2表达水平.另外,应用螺旋神经节组织块分组培养48h后光学显微镜下观察轴(树)突生长情况.结果:流式细胞仪检测结果显示,保护组螺旋神经节细胞损伤率为(9.0±1.3)%,显著低于损伤组(32.8±2.4)%,与对照组(6.3±0.3)%相比无明显差异;免疫细胞化学结果显示,保护组螺旋神经节细胞6h点Bcl-2水平较损伤组和对照组增加.螺旋神经节组织块培养显示,保护组轴(树)突生长明显优于损伤组.结论:低浓度哇巴因对B27剥夺诱发的螺旋神经节细胞损伤具有保护作用.此作用可能通过上调螺旋神经节细胞内Bcl-2表达水平来实现.     

Cisplatin induces cytoplasmic to nuclear translocation of nucleotide excision repair factors among spiral ganglion neurons

Genomic DNA is a high-affinity target for the antineoplastic molecule cisplatin. Cell survival from cisplatin DNA damage is dependent on removal of cisplatin-DNA adducts by nucleotide excision repair (NER) pathways. The rate-limiting steps in the NER pathways are DNA damage identification and verification. These steps are accomplished by xeroderma pigmentosum complementation group C and A (XPC and XPA) and RNA polymerase II. Unlike RNA polymerase II, XPC and XPA have no known cellular function beyond DNA repair. Cisplatin is known to damage spiral ganglion neurons at the basal coil of the cochlea therefore it was posited that cisplatin may target their DNA and mobilize XPC and XPA. Female Fisher344 rats were given two, four day cycles of cisplatin (2 mg/kg) or saline, separated by a 10 day rest period. A 2 × 3 × 2 factorial design, consisting of two treatment conditions (cisplatin and saline treatment), three survival times (5, 19 and 22 days) and two analysis methods (quantitative RT-PCR and immunohistochemistry) was employed to evaluate the expression and distribution of XPC and XPA. Quantitative RT-PCR revealed statistically significant differences in cochlear XPC and XPA mRNA levels after cisplatin treatment at all times except day 22 for XPA. Immunohistochemistry revealed that a proportion (50%) of spiral ganglion neurons in control rats showed cytoplasmic expression of XPC and XPA. After cisplatin treatment, a similar proportion (50%) of spiral ganglion neurons showed increased nuclear expression of XPC and XPA, which appears to represent translocation from the cytoplasm. Basal coil spiral ganglion neurons translocated XPC and XPA at later treatment cycles and with less magnitude than apical coil neurons after cisplatin treatment. Therefore, it is suggested that cisplatin treatment induces nuclear translocation of NER proteins among spiral ganglion neurons and that this nuclear translocation is less efficient at the base relative to the apex.     

Cisplatin effects on guinea pigs: cochlear histology and genotoxicity

To understand how the DNA answers to external agents such as cisplatin may be relevant to the diagnosis and treatment of hearing disorders caused by the administration of such drug.ObjectivesTo investigate the cisplatin influence on the cochlea and DNA of guinea pigs.Material and MethodsExperimental study carried out with 12 guinea pigs (Cavia porcellus). The inclusion criterion was the presence of Preyer's reflex and distortion-product otoacoustic emissions. Guinea pigs were divided into two groups: Control Group (CG) - made up of six guinea pigs, to which we administrated saline solution during six consecutive days, intraperitoneally; and a Study Group (SG) - made up of six guinea pigs, to which we administrated cisplatin during six consecutive doses of 3mg/kg/day intraperitoneally. Twenty-four hours after the last administration of cisplatin the guinea pigs were slaughtered, blood samples were collected and the cochleae were removed.ResultsThe administration of cisplatin did not cause identifiable changes to the DNA. Histological analysis showed changes in the organ of Corti and spiral ganglion.ConclusionCisplatin causes changes in cochlear histology, such as the loss of the normal microcytoarchitecture of the organ of Corti, and reduction of neurons of the spiral ganglion with cell alterations, however, DNA damage was not detected.     

Peripherin as a marker for degeneration of spiral ganglion neurons after aminoglycoside ototoxicity

Conclusion. Our data show that temporary appearance of atypical type 1 neurons, like type 3 neurons, might be another degenerating form of spiral ganglion neurons (SGNs); peripherin might be a marker of degenerating neurons. Objectives. Further morphological and biochemical studies on surviving SGNs after loss of hair cells might offer clues for preventing their degeneration. Materials and methods. We observed the ultrastructural features of surviving SGNs and analyzed the peripherin immunoreactivity at 4, 10, or 20 weeks after systemic injection of neomycin in rats. Results. Type 3 neurons, similar to type 1 neurons but unmyelinated, appeared in the spiral ganglion by 4-week survival, and showed a survival advantage in remaining SGNs by longer surviving periods. We observed neurons packed with dense intermediate filament and with multiple layers of dense myelin sheath (atypical type 1 neurons) in the degenerating neurons. Atypical type 1 neurons were observed among the degenerating neurons in the 4- and 10-week survival groups, but disappeared in longer surviving animals. By means of immunohistochemistry, only smaller SGNs of normal rats were strongly stained by anti-peripherin antibody, whereas increased immunoreactivity was observed in both large and small remaining neurons after neomycin treatment, especially in 10- and 20-week survival animals.     

Localization of substance P in middle ear mucosa and peripheral auditory pathways in guinea pigs]

The distribution and intracellular localization of substance P (SP) in middle ear mucosa (MEM), cochlea and spiral ganglion (SG) were studied by immunohistochemical technique and immunoelectron microscopy. There was a widespread distribution of SP positive nerve fibers (NF) along the median and small vessels of MEM. SP-immunoreactivity (SP-IR) positive cells could be seen in the MEM near the promontorium tympani. In the Corti's organ, SP-IR positive products were located at the base of inner hair cells. The majority of positive NF emerged like strings of beads and were radially distributed from osseous spiral laminal to the Corti's organ. About 50% of the SG cells were SP-IR positive. Two types of SP-IR positive NF were found in the VIII cranial nerve by light microscopy. Small clear vesicles with a diameter of 50-70nm were localized in the cytoplasm of the type-I SG cells by immunoelectron microscopy. In the outer membrane and inside the mitochondria, SP-IR positive substances could be distinguished as an electron dense matter. The possibility of SP as an afferent neurotransmitter or modulator in cochlea and the significance of its presence in the MEM were discussed.     

Immunocytochemical localization of neurofilament subunits in the spiral ganglion of normal and neomycin-treated guinea pigs

Neurofilaments are a major component of the neuronal cytoskeleton and present in all neurons. The expression of subunits of neurofilaments has been shown to be altered by conditions such as development, aging, degeneration and regeneration of the neuron. In the present study, we determined 1) the distribution of neurofilament subunits in spiral ganglion cells of normal guinea pigs and 2) if this distribution is altered by hair cell degeneration. Immunocytochemical analyses were done with monoclonal antibodies to the 200,000 (NF 200), 160,000 (NF 160), and 68,000 (NF 68) daltons neurofilament subunits. In the normal guinea pig, type II spiral ganglion cells were intensely labeled with NF 200, NF 160, NF 68 antibodies, whereas type I cells were significantly labeled only with NF 200 antibody. Neurofilament subunit immunoreactivity was also localized in the auditory nerve and afferent and efferent fibers to the hair cells. To determine the effects of hair cell loss on neurofilament expression in spiral ganglion cells, guinea pigs were treated with neomycin at doses known to cause extensive hair cell damage. Type I and type II spiral ganglion cells responded differently to this treatment. Type II cells remained strongly immunoreactive after treatment although the number of such cells was reduced, especially in the longer surviving animals. NF 160 and NF 68 immunoreactivities increased gradually from base to apex in type I cells after neomycin treatment, while NF 200 immunoreactivity decreased in all turns.