支气管肺发育不良和慢性亚致死性缺氧对未成熟脑干的影响
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摘要
支气管肺发育不良(Bronchopulmonary Dysplasia,BPD)是早产儿尤其是极早产儿最常见的肺部疾病。BPD存活儿神经损伤和发育缺陷的发生率高,BPD已成为极早产儿神经发育异常的最重要的危险因素之一。早期发现BPD患儿的神经损伤,可为临床早期干预提供重要信息。
BPD患儿形成神经损伤和神经发育缺陷的病理生理机制尚不完全清楚。动物实验证实BPD病程中发生的慢性亚致死性缺氧可导致未成熟脑皮质生成损伤和皮层下白质量的显著减少。缺氧影响未成熟脑功能的完整性和神经发育,脑干听觉神经元对严重的缺氧尤其敏感。BPD患儿合并频繁的缺氧或是持续的低氧血症,可能损伤未成熟脑干的听觉功能和发育。
脑干听觉诱发反应(Brainstem auditory evoked response,BAER)是一种客观无创的电生理技术,反应脑干听觉功能和发育。BAER对动脉血氧饱和度和缺氧或缺血缺氧敏感,已广泛应用于检查围生期缺氧或缺血缺氧后的脑干听觉功能。最大长度序列(Maximum length sequence,MLS)BAER是一种较新的BAER技术,它可以将声音刺激的速率提高到1000/s以上或更高,从而比常规的BAER技术更易发现脑干听觉功能的异常。
本实验主要应用MLS BAER电生理技术研究支气管肺发育不良和慢性亚致死性缺氧对未成熟脑干的影响,主要分为四部分内容:第一部分分析外周听阈和中枢脑干功能的相关性,排除外周听阈对中枢脑干听觉功能的影响;第二部分应用MLS BAER技术分析早产儿至矫正胎龄足月时脑干听觉功能损伤的高危因素,评价BPD是否是脑干听觉功能异常的独立危险因素;第三部分随访BPD患儿至生后两年,研究BPD对早产儿脑干听觉功能发育的影响;第四部分建立慢性亚致死性缺氧的未成熟大鼠模型,初步探讨慢性亚致死性缺氧对听觉脑干影响生物学机制。
第一部分早产儿至矫正胎龄足月时外周听阈和脑干听觉功能相关性研究
在分析早产儿至矫正胎龄足月时脑干听觉功能的高危因素的过程中,我们发现外周BAER听阈是MLS BAER检测的一项重要参数,所以我们首先单独分析外周听阈和中枢脑干功能的相关性。
目的:研究早产儿外周听阈是否和中枢听觉功能相关以及外周听阈升高是否影响中枢听觉功能。
方法:本研究入选133名早产儿(胎龄28-36周),在矫正胎龄足月时行最大长度序列脑干听觉诱发反应(MLS BAER)检测。分析MLS BAER参数和BAER听阈的相关性。
结果:所有MLS BAER波形的潜伏期和振幅和BAER听阈显著相关。但是MLSBAER峰间期和BAER阈值在任何声刺激速率下均无相关性。MLS BAER波形的潜伏期在阈值＞20dB nHL的早产儿(n=30)较阈值≤20 dB nHL(n=103)明显延长(P＜0.01)。比较阈值≤20 dB nHL患儿,MLS BAER波振幅在阈值增高组也明显降低(P＜0.05)。但是,两组之间峰间期没有明显差异。Ⅴ/Ⅰ振幅比值在两组之间相似。在所有声刺激速率都有相似趋势。BAER阈值增高的早产儿和阈值正常组,MLS BAER短声刺激依赖的改变在两组相似。
结论:早产儿矫正胎龄足月后脑干听觉功能和外周听阈无显著相关。中耳疾病所致的外周听阈的升高对脑干听觉功能无明显影响。
第二部分早产儿至矫正胎龄足月时脑干听觉功能损伤高危因素分析
目的:早产儿特别是小早产儿是神经损伤和发育不全的高危人群。本研究应用MLS BAER检测方法分析胎龄≤30周早产儿至矫正胎龄足月时脑干听觉功能损伤的高危因素。
方法:本研究入选胎龄≤30周的早产儿,在矫正胎龄足月时行最大长度序列脑干听觉诱发反应(MLS BAER)检测。分析MLS BAER参数和围产期高危因素的相关性。
结果:共有182名胎龄≤30周的患儿纳入本研究,胎龄在23-30周,出生体重为559-1700g。应用重复测量数据的多元回归分析检测MLS BAER各参数和围产期高危因素的相关性。结果示1)BPD是MLS BAER代表脑干听觉功能近中枢段的Ⅴ波潜伏期、Ⅰ-Ⅴ波、Ⅲ-Ⅴ波峰间期和Ⅲ-Ⅴ/Ⅰ-Ⅲ峰间期比值的独立相关的危险因素,比较其他的相关因素BPD的标准偏回归系数最大;同时随着声刺激速率的提高,标准偏回归系数也随着增大;2)其他和MLS BAER参数相关的危险因素还包括动脉导管未闭、胎膜早破、低血糖、胎龄和小于胎龄儿。
结论:早产儿矫正胎龄足月时脑干听觉功能异常的主要危险因素是BPD,其他相关的危险因素为动脉导管未闭、胎膜早破、低血糖、胎龄和小于胎龄儿。
第三部分:支气管肺发育不良患儿脑干听觉功能发育随访研究
目的:支气管肺发育不良(BPD)是早产儿神经发育不全的高危因素。本文探讨BPD对早产儿脑干听觉功能发育的影响。
方法:共随访32名BPD早产儿,在矫正胎龄34周,36周,38周,40周,44周,52周,66周,92周,118周和144周分别行MLS BAER分析。MLS BAER记录的速率为91、227、455和910/s,记录的强度为阈值上40dB。
结果:1)在矫正胎龄34周和36周,比较健康的早产儿对照组,BPD组患儿Ⅴ波延长,但是并未达到统计学差异。Ⅲ-Ⅴ和Ⅰ-Ⅴ的峰间期以及Ⅲ-Ⅴ/Ⅰ-Ⅲ的比值,在BPD组较对照组有显著延长,考虑在矫正胎龄足月前脑干听觉通路近中枢部更易受累。2)在矫正胎龄足月时,和足月儿以及早产儿的对照组比较,BPD患儿Ⅴ波潜伏期,Ⅲ-Ⅴ波和Ⅰ-Ⅴ波的峰间期和Ⅲ-Ⅴ/Ⅰ-Ⅲ峰间期的比值在各刺激速率都有明显延长。3)在随访研究中,BPD组和早产儿对照组MLS BAER检测各参数迅速追赶足月儿,但是在BPD患儿Ⅲ-Ⅴ波峰间期和Ⅲ-Ⅴ/Ⅰ-Ⅲ的峰间期比值恢复相对较慢。在出生后矫正胎龄两年时,它们仍较足月儿延长。Ⅲ-Ⅴ/Ⅰ-Ⅲ峰间期的比值,反应中枢听觉功能,直到矫正胎龄两年仍较足月儿有明显差异,提高刺激的速率差异更加明显;4)各年龄阶段MLS BAER的参数可以拟合成两个衰减指数相加的方程,方程得出的时间参数在BPD组也较足月儿对照组有明显延长,说明脑干听觉功能成熟时间在BPD患儿是较对照组延长。
结论:BPD早产儿脑干听觉功能存在长期损伤,脑干听觉功能成熟延迟。MLSBAER是早期发现BPD患儿神经损伤和长期预后的有效工具。
第四部分慢性亚致死性缺氧对未成熟大鼠脑干髓鞘化研究
目的:支气管肺发育不良(BPD)是早产儿神经发育不全的高危因素。慢性亚致死性缺氧是BPD患儿脑损伤的重要机制。本研究采用慢性亚致死缺氧的未成熟大鼠模型,研究慢性缺氧对未成熟脑干髓鞘化的影响。
方法:研究组将SD(Sprague-Dawley)幼鼠在出生后第3天放入缺氧箱中(氧气浓度为9.5%)。对照组在正常的空气中。两组在生后13天处死,行电镜和焦油紫染色形态学分析,免疫组化分析少突胶质细胞及其前体。将脑干分为中脑、脑桥和延髓三部分行髓鞘碱性蛋白(Myelin Basic Protein,MBP)免疫印迹分析。
结果:慢性缺氧组幼鼠脑干髓鞘鞘膜的厚度较对照组薄。MBP染色示慢性缺氧组脑干染色较对照组明显减少,分布稀疏,呈斑片样分布。O4和O1是少突胶质细胞前体和未成熟少突胶质细胞的标记,在慢性缺氧组表达明显减少。为了进一步分析慢性缺氧对脑干髓鞘化的影响,我们将脑干分为中脑,脑桥和延髓三部分,分别研究MBP的表达。研究示不论在研究组还是在对照组MBP表达在延髓最高,在中脑最低,同时研究组MBP的表达较对照组减少。
结论:1)新生儿慢性缺氧延迟脑干的髓鞘化;2)慢性缺氧致髓鞘化损伤的机制和少突胶质细胞前体成熟延迟相关。
Bronchopulmonary Dysplasia(BPD) is a major lung disease in infants born very preterm.Increasing evidence suggests that the survivors of BPD have high incidence rates of neurologic impairment and developmental deficits,such that BPD has become one of the greatest risk factors of neurodevelopmental problems in infants.Early detection of neurologic impairment,can provide important information for clinical management of infants with BPD.
The pathophysiological processes underlying neurologic impairment and developmental deficits after BPD remain poorly understood.Animal experiments with BPD revealed that prolonged or chronic sublethal hypoxia,which occurs during the course of BPD,may result in severe impairments in corticogenesis in the developing brain and a significant decrease in subcortical white matter.Hypoxemia affects the functional integrity and development of the immature brain,and brainstem auditory neurons are particularly sensitive to severe hypoxemia.Infants who suffer BPD often experience frequent episodes of hypoxemia or prolonged hypoxemia.It is possible that frequent episodes of hypoxemia or prolonged hypoxemia impair the functional integrity of the neonatal auditory brainstem.
The neonatal brainstem auditory evoked response(BAER) reflects the functional integrity and development of the auditory brainstem.This response is very sensitive to arterial blood oxygen levels and hypoxia.Maximal length sequence(MLS) BAER is a relatively new technique,which can increase acoustic stimuli at much higher repetition rates(up to 1000 clicks per second or even higher),providing a much stronger physiological challenge to brainstem.
To investigate the effect of BPD and chronic sublethal hypoxia on neonatal immature brainstem,we firstly studied risk factors for functional impairment of auditory brainstem in very preterm babies at corrected term age to find whether BPD is one of the greatest risk factor of neurologic impairment;meanwhile,for peripheral auditory threshold may be associated with brainstem auditory function,we also determined the correlation between them beforehand.Then we further explored the effect of BPD on auditory braistem function in very preterm babies at term age and also in a two year follow up study;Finally,to further elucidate some adaptive changes during postnatal chronic sublethal hypoxia,we employed an animal model of chronic sublethal hypoxia to test whether chronic sublethal hypoxia results in brainstem myelination arrest in the developing rat model.
Part 1 Correlation between brainstem auditory function and peripheral auditory threshold in preterm infants at term age
Objective:To determine whether central auditory function in preterm infants correlates with peripheral auditory threshold and whether threshold elevation affects central auditory function.
Methods:Brainstem auditory evoked response(BAER) was recorded at term age using maximum length sequence(MLS) with 91-910/s clicks in 133 preterm infants (gestation 28-36 weeks).
Results:The latencies and amplitudes of all MLS BAER waves correlated significantly with BAER threshold.However,no correlation was found between MLS BAER interpeak intervals and BAER threshold at any rates.In preterm infants with a threshold＞20 dB nHL(n=30),MLS BAER wave latencies were all significantly longer than in those with a threshold≤20 dB nHL(n=103)(P＜0.01-0.001).MLS BAER wave amplitudes were significantly smaller than in those≤20 dB nHL(P＜0.05-0.001).However,no interpeak intervals differed significantly between the two groups of infants.Ⅴ/Ⅰamplitude ratio was similar in the two groups.These findings were true of all click rates.Click rate-dependent changes in MLS BAER of the preterm infants with an elevated BAER threshold are generally similar to those with a normal threshold.
Conclusions:Brainstem auditory function does not closely correlate with peripheral auditory threshold at term in preterm infants.
Part.2 Risk factors for functional impairment of auditory brainstem in very preterm babies at term age
Objectives:Very preterm infants are at a high-risk of developing neurological impairment and later disabilities.To gain deeper understanding of neurologic impairment mechanism,we employed MLS BAER to determine which risk factors associated with neurological impairment in very preterm babies at term age.
Methods:Brainstem auditory evoked response(BAER) was recorded at term age using maximum length sequence(MLS) with 91-910/s clicks in very preterm infants (gestation≤30weeks).The relationship between MLS BAER variables and risk factors was analyzed with multiple regression analysis of repeat measure data.
Results:There were 182 very preterm infants with gestational age＜30 weeks who were included into this study.Multiple regression analysis of repeated measure data showed that BPD was independent risk factor of waveⅤlatency,Ⅰ-Ⅴinterpeak interval,Ⅲ-Ⅴinterpeak interval andⅢ-Ⅴ/Ⅰ-Ⅲinterpeak interval ratio,which represent central function of auditory brainstem;At any acoustic rate of MLS BAER, the stardard partial regression coefficient of BPD was the largest among all risk factors.Other risk factors associated with MLS BAER variables included PDA, PROM,hypoglycemia,GA and SGA.
Conclusions:The most important risk factor for functional impairment of auditory brainstem in very preterm babies at term age is PDA.Other relative risk factors are, PDA,PROM,hypoglycemia,GA and SGA.
Part 3.Auditory Brainstem Maturation in Very Preterm Babies with Bronchopulmonary Dysplasia:a two-year follow up study
Objectives:Bronchopulmonary dysplasia(BPD) is proved to be independent risk factor of neurodevelopmental disorders in very preterm infants.We hypothesize that central auditory brainstem maturation and neurodevelopment are delayed in very preterm infants with BPD
Methods:To assess the effect of BPD on neonatal auditory brainstem maturation,we studied MLS BAER in 32 very preterm infants who suffered BPD but no other major perinatal complications or problems at postconceptional ages of 34,36,38,40,44,52, 66,72,118,144weeks.BAER was recorded with clicks,delivered at 91,227,455 and 910/s and≥40db above BAER threshold of each subject.
Results:1) At postconceptional ages of 34 and 36weeks,compared with healthy,very preterm control subjects,waveⅤlatency in very preterm infants with BPD tended to be longer,though no significant difference was detected.However,Ⅲ-Ⅴ,Ⅰ-Ⅴintervals andⅢ-Ⅴ/Ⅰ-Ⅲratio were significant longer in BPD infants than that in control group, indicating more central components of auditory brainstem were affected before term.2) At term,compared with term control subjects and also very preterm control babies, waveⅤ,Ⅲ-Ⅴintervals andⅠ-Ⅴintervals in BPD infants increased significantly at all click rates 3) In the follow-up study,the wave latency in BPD and very preterm control infants caught up with that in term babies rapidly.However,Ⅲ-ⅤandⅠ-Ⅴintervals in BPD babies recovered relatively slowly to normal value.They still tended to be longer than those variables in term control babies at postconceptional age of two years.Ⅲ-Ⅴ/Ⅰ-Ⅲintervals ratio,reflecting more central function,significantly increased at all occasions of age in BPD group in comparison of term control group.Increasing the stimulus rates improved the detection of neuropathology.4) The decaying exponential functions model fit sufficiently robust both applied to the age-group means and to all data in term control babies and very preterm babies with BPD.The time constants of waveⅤ,Ⅲ-ⅤandⅠ-Ⅴ,tended to be longer in BPD group than the other group.
Conclusions:The results suggest long-term impairment of brainstem function in very preterm infants with BPD,resulting in delayed auditory brainstem maturation.MLS BAER provides a valuable tool to early detection of the impairment and prediction of later outcome.
Part 4.Brainstem Myelination Maturation in the Developing Rats with Chronic Sublethal Hypoxia Injury
Objectives:To further elucidate some adaptive changes during postnatal chronic hypoxia,we employed an animal model of chronic sublethal hypoxia to test the hypothesis that chronic sublethal hypoxia results in brainstem myelination maturation arrest in the developing rat model.
Methods:Litters of SD rats were put into hypoxic chamber(oxygen level maintained at 9.5%) with their dams starting on day 3 postnatal(P3).Age matched normoxic rats were used as control animals.Two groups were sacrificed at P13 and prepared for further analysis in four sections of brainstem(cochlear nucleus,superior olivary nucleus, lateral lemniscus,and inferior colliculus).We performed electron microscope and cresyl violet staining for morphometric analysis,immunohistochemical assays to visualize specific stages in the oligodendrocyte(OL) lineage.Western analyses of myelin basic protein(MBP) were also determined in three parts of brainstern,the midbrain,pons and medulla.
Results:Measurement of axonal sheath size at P13 revealed that axonal sheath of brainstem was significantly smaller in hypoxia compared with control rats. Neuropathological analysis of the brainstem of the developing rats in chronic hypoxia demonstrated a marked reduction in staining for MBP with residual myelination was patchy in distribution.Staining with the O1 and O4 antibodies,markers of premyelinating and early myelinating OLs,also showed a significant reduction of axon sheaths in brainstem.To determine the effect of chronic hypoxia on the changes in brainstem expression of MBP,we also performed Western blotting in whole and three regions of brainstem:midbrain,pons and medulla.We observed that MBP expression was reduced in overall brainstem in hypoxia-reared rots.Among medulla,pons and midbrain,MBP expression was highest in medulla and lowest in midbrain,whether in hypoxia or normoxic group.
Conclusions:We conclude that neonatal chronic hypoxia 1) reduces the extent of myelination in the brainstem,whose process of maturation is sequential from lower to higher brainstem centers of immature animal;2) the mechanism of myelination failure in chronic hypoxia is related to delayed preOLs maturation.
BPD患儿形成神经损伤和神经发育缺陷的病理生理机制尚不完全清楚。动物实验证实BPD病程中发生的慢性亚致死性缺氧可导致未成熟脑皮质生成损伤和皮层下白质量的显著减少。缺氧影响未成熟脑功能的完整性和神经发育,脑干听觉神经元对严重的缺氧尤其敏感。BPD患儿合并频繁的缺氧或是持续的低氧血症,可能损伤未成熟脑干的听觉功能和发育。
脑干听觉诱发反应(Brainstem auditory evoked response,BAER)是一种客观无创的电生理技术,反应脑干听觉功能和发育。BAER对动脉血氧饱和度和缺氧或缺血缺氧敏感,已广泛应用于检查围生期缺氧或缺血缺氧后的脑干听觉功能。最大长度序列(Maximum length sequence,MLS)BAER是一种较新的BAER技术,它可以将声音刺激的速率提高到1000/s以上或更高,从而比常规的BAER技术更易发现脑干听觉功能的异常。
本实验主要应用MLS BAER电生理技术研究支气管肺发育不良和慢性亚致死性缺氧对未成熟脑干的影响,主要分为四部分内容:第一部分分析外周听阈和中枢脑干功能的相关性,排除外周听阈对中枢脑干听觉功能的影响;第二部分应用MLS BAER技术分析早产儿至矫正胎龄足月时脑干听觉功能损伤的高危因素,评价BPD是否是脑干听觉功能异常的独立危险因素;第三部分随访BPD患儿至生后两年,研究BPD对早产儿脑干听觉功能发育的影响;第四部分建立慢性亚致死性缺氧的未成熟大鼠模型,初步探讨慢性亚致死性缺氧对听觉脑干影响生物学机制。
第一部分早产儿至矫正胎龄足月时外周听阈和脑干听觉功能相关性研究
在分析早产儿至矫正胎龄足月时脑干听觉功能的高危因素的过程中,我们发现外周BAER听阈是MLS BAER检测的一项重要参数,所以我们首先单独分析外周听阈和中枢脑干功能的相关性。
目的:研究早产儿外周听阈是否和中枢听觉功能相关以及外周听阈升高是否影响中枢听觉功能。
方法:本研究入选133名早产儿(胎龄28-36周),在矫正胎龄足月时行最大长度序列脑干听觉诱发反应(MLS BAER)检测。分析MLS BAER参数和BAER听阈的相关性。
结果:所有MLS BAER波形的潜伏期和振幅和BAER听阈显著相关。但是MLSBAER峰间期和BAER阈值在任何声刺激速率下均无相关性。MLS BAER波形的潜伏期在阈值＞20dB nHL的早产儿(n=30)较阈值≤20 dB nHL(n=103)明显延长(P＜0.01)。比较阈值≤20 dB nHL患儿,MLS BAER波振幅在阈值增高组也明显降低(P＜0.05)。但是,两组之间峰间期没有明显差异。Ⅴ/Ⅰ振幅比值在两组之间相似。在所有声刺激速率都有相似趋势。BAER阈值增高的早产儿和阈值正常组,MLS BAER短声刺激依赖的改变在两组相似。
结论:早产儿矫正胎龄足月后脑干听觉功能和外周听阈无显著相关。中耳疾病所致的外周听阈的升高对脑干听觉功能无明显影响。
第二部分早产儿至矫正胎龄足月时脑干听觉功能损伤高危因素分析
目的:早产儿特别是小早产儿是神经损伤和发育不全的高危人群。本研究应用MLS BAER检测方法分析胎龄≤30周早产儿至矫正胎龄足月时脑干听觉功能损伤的高危因素。
方法:本研究入选胎龄≤30周的早产儿,在矫正胎龄足月时行最大长度序列脑干听觉诱发反应(MLS BAER)检测。分析MLS BAER参数和围产期高危因素的相关性。
结果:共有182名胎龄≤30周的患儿纳入本研究,胎龄在23-30周,出生体重为559-1700g。应用重复测量数据的多元回归分析检测MLS BAER各参数和围产期高危因素的相关性。结果示1)BPD是MLS BAER代表脑干听觉功能近中枢段的Ⅴ波潜伏期、Ⅰ-Ⅴ波、Ⅲ-Ⅴ波峰间期和Ⅲ-Ⅴ/Ⅰ-Ⅲ峰间期比值的独立相关的危险因素,比较其他的相关因素BPD的标准偏回归系数最大;同时随着声刺激速率的提高,标准偏回归系数也随着增大;2)其他和MLS BAER参数相关的危险因素还包括动脉导管未闭、胎膜早破、低血糖、胎龄和小于胎龄儿。
结论:早产儿矫正胎龄足月时脑干听觉功能异常的主要危险因素是BPD,其他相关的危险因素为动脉导管未闭、胎膜早破、低血糖、胎龄和小于胎龄儿。
第三部分:支气管肺发育不良患儿脑干听觉功能发育随访研究
目的:支气管肺发育不良(BPD)是早产儿神经发育不全的高危因素。本文探讨BPD对早产儿脑干听觉功能发育的影响。
方法:共随访32名BPD早产儿,在矫正胎龄34周,36周,38周,40周,44周,52周,66周,92周,118周和144周分别行MLS BAER分析。MLS BAER记录的速率为91、227、455和910/s,记录的强度为阈值上40dB。
结果:1)在矫正胎龄34周和36周,比较健康的早产儿对照组,BPD组患儿Ⅴ波延长,但是并未达到统计学差异。Ⅲ-Ⅴ和Ⅰ-Ⅴ的峰间期以及Ⅲ-Ⅴ/Ⅰ-Ⅲ的比值,在BPD组较对照组有显著延长,考虑在矫正胎龄足月前脑干听觉通路近中枢部更易受累。2)在矫正胎龄足月时,和足月儿以及早产儿的对照组比较,BPD患儿Ⅴ波潜伏期,Ⅲ-Ⅴ波和Ⅰ-Ⅴ波的峰间期和Ⅲ-Ⅴ/Ⅰ-Ⅲ峰间期的比值在各刺激速率都有明显延长。3)在随访研究中,BPD组和早产儿对照组MLS BAER检测各参数迅速追赶足月儿,但是在BPD患儿Ⅲ-Ⅴ波峰间期和Ⅲ-Ⅴ/Ⅰ-Ⅲ的峰间期比值恢复相对较慢。在出生后矫正胎龄两年时,它们仍较足月儿延长。Ⅲ-Ⅴ/Ⅰ-Ⅲ峰间期的比值,反应中枢听觉功能,直到矫正胎龄两年仍较足月儿有明显差异,提高刺激的速率差异更加明显;4)各年龄阶段MLS BAER的参数可以拟合成两个衰减指数相加的方程,方程得出的时间参数在BPD组也较足月儿对照组有明显延长,说明脑干听觉功能成熟时间在BPD患儿是较对照组延长。
结论:BPD早产儿脑干听觉功能存在长期损伤,脑干听觉功能成熟延迟。MLSBAER是早期发现BPD患儿神经损伤和长期预后的有效工具。
第四部分慢性亚致死性缺氧对未成熟大鼠脑干髓鞘化研究
目的:支气管肺发育不良(BPD)是早产儿神经发育不全的高危因素。慢性亚致死性缺氧是BPD患儿脑损伤的重要机制。本研究采用慢性亚致死缺氧的未成熟大鼠模型,研究慢性缺氧对未成熟脑干髓鞘化的影响。
方法:研究组将SD(Sprague-Dawley)幼鼠在出生后第3天放入缺氧箱中(氧气浓度为9.5%)。对照组在正常的空气中。两组在生后13天处死,行电镜和焦油紫染色形态学分析,免疫组化分析少突胶质细胞及其前体。将脑干分为中脑、脑桥和延髓三部分行髓鞘碱性蛋白(Myelin Basic Protein,MBP)免疫印迹分析。
结果:慢性缺氧组幼鼠脑干髓鞘鞘膜的厚度较对照组薄。MBP染色示慢性缺氧组脑干染色较对照组明显减少,分布稀疏,呈斑片样分布。O4和O1是少突胶质细胞前体和未成熟少突胶质细胞的标记,在慢性缺氧组表达明显减少。为了进一步分析慢性缺氧对脑干髓鞘化的影响,我们将脑干分为中脑,脑桥和延髓三部分,分别研究MBP的表达。研究示不论在研究组还是在对照组MBP表达在延髓最高,在中脑最低,同时研究组MBP的表达较对照组减少。
结论:1)新生儿慢性缺氧延迟脑干的髓鞘化;2)慢性缺氧致髓鞘化损伤的机制和少突胶质细胞前体成熟延迟相关。
Bronchopulmonary Dysplasia(BPD) is a major lung disease in infants born very preterm.Increasing evidence suggests that the survivors of BPD have high incidence rates of neurologic impairment and developmental deficits,such that BPD has become one of the greatest risk factors of neurodevelopmental problems in infants.Early detection of neurologic impairment,can provide important information for clinical management of infants with BPD.
The pathophysiological processes underlying neurologic impairment and developmental deficits after BPD remain poorly understood.Animal experiments with BPD revealed that prolonged or chronic sublethal hypoxia,which occurs during the course of BPD,may result in severe impairments in corticogenesis in the developing brain and a significant decrease in subcortical white matter.Hypoxemia affects the functional integrity and development of the immature brain,and brainstem auditory neurons are particularly sensitive to severe hypoxemia.Infants who suffer BPD often experience frequent episodes of hypoxemia or prolonged hypoxemia.It is possible that frequent episodes of hypoxemia or prolonged hypoxemia impair the functional integrity of the neonatal auditory brainstem.
The neonatal brainstem auditory evoked response(BAER) reflects the functional integrity and development of the auditory brainstem.This response is very sensitive to arterial blood oxygen levels and hypoxia.Maximal length sequence(MLS) BAER is a relatively new technique,which can increase acoustic stimuli at much higher repetition rates(up to 1000 clicks per second or even higher),providing a much stronger physiological challenge to brainstem.
To investigate the effect of BPD and chronic sublethal hypoxia on neonatal immature brainstem,we firstly studied risk factors for functional impairment of auditory brainstem in very preterm babies at corrected term age to find whether BPD is one of the greatest risk factor of neurologic impairment;meanwhile,for peripheral auditory threshold may be associated with brainstem auditory function,we also determined the correlation between them beforehand.Then we further explored the effect of BPD on auditory braistem function in very preterm babies at term age and also in a two year follow up study;Finally,to further elucidate some adaptive changes during postnatal chronic sublethal hypoxia,we employed an animal model of chronic sublethal hypoxia to test whether chronic sublethal hypoxia results in brainstem myelination arrest in the developing rat model.
Part 1 Correlation between brainstem auditory function and peripheral auditory threshold in preterm infants at term age
Objective:To determine whether central auditory function in preterm infants correlates with peripheral auditory threshold and whether threshold elevation affects central auditory function.
Methods:Brainstem auditory evoked response(BAER) was recorded at term age using maximum length sequence(MLS) with 91-910/s clicks in 133 preterm infants (gestation 28-36 weeks).
Results:The latencies and amplitudes of all MLS BAER waves correlated significantly with BAER threshold.However,no correlation was found between MLS BAER interpeak intervals and BAER threshold at any rates.In preterm infants with a threshold＞20 dB nHL(n=30),MLS BAER wave latencies were all significantly longer than in those with a threshold≤20 dB nHL(n=103)(P＜0.01-0.001).MLS BAER wave amplitudes were significantly smaller than in those≤20 dB nHL(P＜0.05-0.001).However,no interpeak intervals differed significantly between the two groups of infants.Ⅴ/Ⅰamplitude ratio was similar in the two groups.These findings were true of all click rates.Click rate-dependent changes in MLS BAER of the preterm infants with an elevated BAER threshold are generally similar to those with a normal threshold.
Conclusions:Brainstem auditory function does not closely correlate with peripheral auditory threshold at term in preterm infants.
Part.2 Risk factors for functional impairment of auditory brainstem in very preterm babies at term age
Objectives:Very preterm infants are at a high-risk of developing neurological impairment and later disabilities.To gain deeper understanding of neurologic impairment mechanism,we employed MLS BAER to determine which risk factors associated with neurological impairment in very preterm babies at term age.
Methods:Brainstem auditory evoked response(BAER) was recorded at term age using maximum length sequence(MLS) with 91-910/s clicks in very preterm infants (gestation≤30weeks).The relationship between MLS BAER variables and risk factors was analyzed with multiple regression analysis of repeat measure data.
Results:There were 182 very preterm infants with gestational age＜30 weeks who were included into this study.Multiple regression analysis of repeated measure data showed that BPD was independent risk factor of waveⅤlatency,Ⅰ-Ⅴinterpeak interval,Ⅲ-Ⅴinterpeak interval andⅢ-Ⅴ/Ⅰ-Ⅲinterpeak interval ratio,which represent central function of auditory brainstem;At any acoustic rate of MLS BAER, the stardard partial regression coefficient of BPD was the largest among all risk factors.Other risk factors associated with MLS BAER variables included PDA, PROM,hypoglycemia,GA and SGA.
Conclusions:The most important risk factor for functional impairment of auditory brainstem in very preterm babies at term age is PDA.Other relative risk factors are, PDA,PROM,hypoglycemia,GA and SGA.
Part 3.Auditory Brainstem Maturation in Very Preterm Babies with Bronchopulmonary Dysplasia:a two-year follow up study
Objectives:Bronchopulmonary dysplasia(BPD) is proved to be independent risk factor of neurodevelopmental disorders in very preterm infants.We hypothesize that central auditory brainstem maturation and neurodevelopment are delayed in very preterm infants with BPD
Methods:To assess the effect of BPD on neonatal auditory brainstem maturation,we studied MLS BAER in 32 very preterm infants who suffered BPD but no other major perinatal complications or problems at postconceptional ages of 34,36,38,40,44,52, 66,72,118,144weeks.BAER was recorded with clicks,delivered at 91,227,455 and 910/s and≥40db above BAER threshold of each subject.
Results:1) At postconceptional ages of 34 and 36weeks,compared with healthy,very preterm control subjects,waveⅤlatency in very preterm infants with BPD tended to be longer,though no significant difference was detected.However,Ⅲ-Ⅴ,Ⅰ-Ⅴintervals andⅢ-Ⅴ/Ⅰ-Ⅲratio were significant longer in BPD infants than that in control group, indicating more central components of auditory brainstem were affected before term.2) At term,compared with term control subjects and also very preterm control babies, waveⅤ,Ⅲ-Ⅴintervals andⅠ-Ⅴintervals in BPD infants increased significantly at all click rates 3) In the follow-up study,the wave latency in BPD and very preterm control infants caught up with that in term babies rapidly.However,Ⅲ-ⅤandⅠ-Ⅴintervals in BPD babies recovered relatively slowly to normal value.They still tended to be longer than those variables in term control babies at postconceptional age of two years.Ⅲ-Ⅴ/Ⅰ-Ⅲintervals ratio,reflecting more central function,significantly increased at all occasions of age in BPD group in comparison of term control group.Increasing the stimulus rates improved the detection of neuropathology.4) The decaying exponential functions model fit sufficiently robust both applied to the age-group means and to all data in term control babies and very preterm babies with BPD.The time constants of waveⅤ,Ⅲ-ⅤandⅠ-Ⅴ,tended to be longer in BPD group than the other group.
Conclusions:The results suggest long-term impairment of brainstem function in very preterm infants with BPD,resulting in delayed auditory brainstem maturation.MLS BAER provides a valuable tool to early detection of the impairment and prediction of later outcome.
Part 4.Brainstem Myelination Maturation in the Developing Rats with Chronic Sublethal Hypoxia Injury
Objectives:To further elucidate some adaptive changes during postnatal chronic hypoxia,we employed an animal model of chronic sublethal hypoxia to test the hypothesis that chronic sublethal hypoxia results in brainstem myelination maturation arrest in the developing rat model.
Methods:Litters of SD rats were put into hypoxic chamber(oxygen level maintained at 9.5%) with their dams starting on day 3 postnatal(P3).Age matched normoxic rats were used as control animals.Two groups were sacrificed at P13 and prepared for further analysis in four sections of brainstem(cochlear nucleus,superior olivary nucleus, lateral lemniscus,and inferior colliculus).We performed electron microscope and cresyl violet staining for morphometric analysis,immunohistochemical assays to visualize specific stages in the oligodendrocyte(OL) lineage.Western analyses of myelin basic protein(MBP) were also determined in three parts of brainstern,the midbrain,pons and medulla.
Results:Measurement of axonal sheath size at P13 revealed that axonal sheath of brainstem was significantly smaller in hypoxia compared with control rats. Neuropathological analysis of the brainstem of the developing rats in chronic hypoxia demonstrated a marked reduction in staining for MBP with residual myelination was patchy in distribution.Staining with the O1 and O4 antibodies,markers of premyelinating and early myelinating OLs,also showed a significant reduction of axon sheaths in brainstem.To determine the effect of chronic hypoxia on the changes in brainstem expression of MBP,we also performed Western blotting in whole and three regions of brainstem:midbrain,pons and medulla.We observed that MBP expression was reduced in overall brainstem in hypoxia-reared rots.Among medulla,pons and midbrain,MBP expression was highest in medulla and lowest in midbrain,whether in hypoxia or normoxic group.
Conclusions:We conclude that neonatal chronic hypoxia 1) reduces the extent of myelination in the brainstem,whose process of maturation is sequential from lower to higher brainstem centers of immature animal;2) the mechanism of myelination failure in chronic hypoxia is related to delayed preOLs maturation.
引文
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66.Eggermont JJ.Development of auditory evoked potentials[J].Acta Otolaryngol 1992;112(2):197-200.
67 Jiang ZD.Damage of chronic sublethal hypoxia to the immature auditory brainstem.In:Auditory System:Anatomy,Cognition,Cells and Circuits.New York:Nova Science Publishers;2009(in press).
1 Sarda P,Dupuy RP,Boulot P,Rieu D.Brainstem conduction time abnormalities in small for gestational age infants.J Perinat Med 1992;20:57-63
2 Kohelet D,Arbel E,Goldberg M,Arlazzoroff A.Intrauterine growth retardation and brainstem auditory-evoked response in preterm infants.Acta-Paediatr.2000;89:73-6
3 Jiang ZD,Yin R,Shao XM,Wilkinson AR.Brainstem auditory impairment during the neonatal period in infants after asphyxia:dynamic changes in brainstem auditory evoked responses to different rate clicks.Clin Neurophysiol 2004;115:1605-1615.
4 Pettigrew AG,Edwards DA,Henderson-Smart DJ.The influence of intra-uterine growth retardation on brainstem development of preterm infants.Dev Med Child Neurol.1995;27:467-72
5 Jiang ZD,Brosi DM,Wang J,Wilkinson AR.Brainstem auditory-evoked responses to different rates of clicks in small-for-gestational age preterm infants at term.Acta Paediatr 2004;93:76-81.
6 Fawer C-L,Dubowitz LMS Auditory brain stem response in neurologically normal preterm and fullterm newborn infants.Neuropediatrics 13:200-206
7 Rotteveel JJ,de Graaf R,Colon EJ,Stegeman DF,Visco YM 1986 The maturation of the central auditory conduction in preterm infants until three months post term.Ⅱ:The auditory brainstem responses(ABRs).Hear Res 26:21-35
8 Jiang ZD,Tierney TS.Development of human peripheral hearing revealed by brainstem auditory evoked potentials.Acta Paediatrica 1995;84:1216-1220.
9 Van de Bor M,Guit GL,Schreuder AM,van Bel F,Wondergem J,den Ouden L Vielvoye GJ 1990 Does very preterm birth impair myelination of the central nervous system? Neuropediatrics 21:37-39
10 Jiang ZD,Brosi DM,Wilkinson AR.Auditory neural responses to click stimuli of different rates in the brainstem of very preterm babies at term.Pediatr Res 2002;51:454-459.
11 Jiang ZD,Brosi DM,Wilkinson AR.Brainstem auditory function in very preterm infants with chronic lung disease - delayed neural conduction.Clin Neurophysiol 2006;
12 Hecox K,Cone B.Prognostic importance of brainstem auditory evoked response after asphyxia.Neurology(NY) 1981;31:1429-33
13 Jiang ZD,Tiemey TS.Long-term effect of perinatal and postnatal asphyxia on developing human auditory brainstem responses:brainstem impairment.International Journal of Pediatric Otorhino- laryngology 1996;34:111-127.
14 Jiang ZD.Maturation of peripheral and brainstem auditory function in the first year following perinatal asphyxia - a longitudinal study.J Speech Lang Hear Res 1998;1:83-93.
15 Tomimatsu T,Fukuda H,Endoh M,Mu J,Watanabe N,Kohzuki M,Fujii E,Kanzaki T,Oxhima K,Doi K,Kubo T,Murata Y 2002 Effects of neonatal hypoxic-ischemic brain injury on skilled motor tasks and brainstem function in adult rats.Brain Res 926:108-117
16 Jiang ZD,Woung JM,Shao XM.Bminstem auditory conduction in hypoxiceischaemic piglets treated with selective head cooling.E-PAS 2006;59(3594):441.
17 Tomimatsu T,Fukuda H,Endoh M,Mu J,Kanagawa T,Hosono T,et al.Long-term neuroprotective effects of hypothermia on neonatal hypoxiceischemic brain injury in rats,assessed by auditory brainstem response.Pediatr Res 2003;53:57e61.
18.Freitag C,R(o|¨)hling D,Seifen S,Pukrop R,von Gontard A.Neurophysiology of nocturnal enuresis:evoked potentials and prepulse inhibition of the startle reflex.Dev Med Child Neurol 2006:48:278-84.
19. Chou YH, Wang PJ. Auditory brainstem evoked potentials in early-treated congenital hypothyroidism. J Child Neurol 2002;17:510-4.
20. Jiang ZD, Brosi DM, Wilkinson AR. Brainstem auditory evoked response recorded using maximum length sequences in term neonates. Biol Neonat 1999;76:193-9.
21. Jiang ZD, Brosi DM, Shao XM, Wilkinson AR. Maximum length sequence brainstem auditory evoked response in term infants after perinatal hypoxiaeischaemia. Pediatr Res 2000;48: 639e45.
22. Jiang ZD, Brosi DM, Wang J, Xu X, Chen GQ, Shao XM, et al. Time course of brainstem pathophysiology during first month in term infants after perinatal asphyxia, revealed by MLS BAER latencies and intervals. Pediatr Res 2003;54:680e7.
23. Jiang ZD, Brosi DM, Li ZH, Chen C, Wilkinson AR. Brainstem auditory function at term in preterm babies with and without perinatal complications. Pediatr Res 2005;58:1164-9.
24 . Jiang ZD, Brosi DM, Wilkinson AR. Maximum length sequence BAER at term in low-risk babies born at 30e32 week gestation. Brain Dev 2006;28:l-7.
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66.Eggermont JJ.Development of auditory evoked potentials[J].Acta Otolaryngol 1992;112(2):197-200.
67 Jiang ZD.Damage of chronic sublethal hypoxia to the immature auditory brainstem.In:Auditory System:Anatomy,Cognition,Cells and Circuits.New York:Nova Science Publishers;2009(in press).
1 Sarda P,Dupuy RP,Boulot P,Rieu D.Brainstem conduction time abnormalities in small for gestational age infants.J Perinat Med 1992;20:57-63
2 Kohelet D,Arbel E,Goldberg M,Arlazzoroff A.Intrauterine growth retardation and brainstem auditory-evoked response in preterm infants.Acta-Paediatr.2000;89:73-6
3 Jiang ZD,Yin R,Shao XM,Wilkinson AR.Brainstem auditory impairment during the neonatal period in infants after asphyxia:dynamic changes in brainstem auditory evoked responses to different rate clicks.Clin Neurophysiol 2004;115:1605-1615.
4 Pettigrew AG,Edwards DA,Henderson-Smart DJ.The influence of intra-uterine growth retardation on brainstem development of preterm infants.Dev Med Child Neurol.1995;27:467-72
5 Jiang ZD,Brosi DM,Wang J,Wilkinson AR.Brainstem auditory-evoked responses to different rates of clicks in small-for-gestational age preterm infants at term.Acta Paediatr 2004;93:76-81.
6 Fawer C-L,Dubowitz LMS Auditory brain stem response in neurologically normal preterm and fullterm newborn infants.Neuropediatrics 13:200-206
7 Rotteveel JJ,de Graaf R,Colon EJ,Stegeman DF,Visco YM 1986 The maturation of the central auditory conduction in preterm infants until three months post term.Ⅱ:The auditory brainstem responses(ABRs).Hear Res 26:21-35
8 Jiang ZD,Tierney TS.Development of human peripheral hearing revealed by brainstem auditory evoked potentials.Acta Paediatrica 1995;84:1216-1220.
9 Van de Bor M,Guit GL,Schreuder AM,van Bel F,Wondergem J,den Ouden L Vielvoye GJ 1990 Does very preterm birth impair myelination of the central nervous system? Neuropediatrics 21:37-39
10 Jiang ZD,Brosi DM,Wilkinson AR.Auditory neural responses to click stimuli of different rates in the brainstem of very preterm babies at term.Pediatr Res 2002;51:454-459.
11 Jiang ZD,Brosi DM,Wilkinson AR.Brainstem auditory function in very preterm infants with chronic lung disease - delayed neural conduction.Clin Neurophysiol 2006;
12 Hecox K,Cone B.Prognostic importance of brainstem auditory evoked response after asphyxia.Neurology(NY) 1981;31:1429-33
13 Jiang ZD,Tiemey TS.Long-term effect of perinatal and postnatal asphyxia on developing human auditory brainstem responses:brainstem impairment.International Journal of Pediatric Otorhino- laryngology 1996;34:111-127.
14 Jiang ZD.Maturation of peripheral and brainstem auditory function in the first year following perinatal asphyxia - a longitudinal study.J Speech Lang Hear Res 1998;1:83-93.
15 Tomimatsu T,Fukuda H,Endoh M,Mu J,Watanabe N,Kohzuki M,Fujii E,Kanzaki T,Oxhima K,Doi K,Kubo T,Murata Y 2002 Effects of neonatal hypoxic-ischemic brain injury on skilled motor tasks and brainstem function in adult rats.Brain Res 926:108-117
16 Jiang ZD,Woung JM,Shao XM.Bminstem auditory conduction in hypoxiceischaemic piglets treated with selective head cooling.E-PAS 2006;59(3594):441.
17 Tomimatsu T,Fukuda H,Endoh M,Mu J,Kanagawa T,Hosono T,et al.Long-term neuroprotective effects of hypothermia on neonatal hypoxiceischemic brain injury in rats,assessed by auditory brainstem response.Pediatr Res 2003;53:57e61.
18.Freitag C,R(o|¨)hling D,Seifen S,Pukrop R,von Gontard A.Neurophysiology of nocturnal enuresis:evoked potentials and prepulse inhibition of the startle reflex.Dev Med Child Neurol 2006:48:278-84.
19. Chou YH, Wang PJ. Auditory brainstem evoked potentials in early-treated congenital hypothyroidism. J Child Neurol 2002;17:510-4.
20. Jiang ZD, Brosi DM, Wilkinson AR. Brainstem auditory evoked response recorded using maximum length sequences in term neonates. Biol Neonat 1999;76:193-9.
21. Jiang ZD, Brosi DM, Shao XM, Wilkinson AR. Maximum length sequence brainstem auditory evoked response in term infants after perinatal hypoxiaeischaemia. Pediatr Res 2000;48: 639e45.
22. Jiang ZD, Brosi DM, Wang J, Xu X, Chen GQ, Shao XM, et al. Time course of brainstem pathophysiology during first month in term infants after perinatal asphyxia, revealed by MLS BAER latencies and intervals. Pediatr Res 2003;54:680e7.
23. Jiang ZD, Brosi DM, Li ZH, Chen C, Wilkinson AR. Brainstem auditory function at term in preterm babies with and without perinatal complications. Pediatr Res 2005;58:1164-9.
24 . Jiang ZD, Brosi DM, Wilkinson AR. Maximum length sequence BAER at term in low-risk babies born at 30e32 week gestation. Brain Dev 2006;28:l-7.