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《第十六届中国体视学与图像分析学术会议论文集——交叉、融合、创新》2019年
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Application of stereological methods to investigate the healthy or injured brain:Studies on the healthy basal ganglia and on three different animal models that are relevant to either cerebral palsy,ADHD-like hyperactivity or schizophrenia

DOROTHY E.OORSCHOT  
【摘要】:Purpose:The application of stereological methods is critical to advancing knowledge on the healthy or the injured brain.In this plenary presentation,examples from my laboratory of the scientific value of stereological methods will be presented.Specifically,examples will be presented of:1.Why the definitive measure of the absolute number of neurons is preferable to using indirect measures such as neuronal density or the volume of a brain region.This topic is relevant to the following scientific questions:Is there neuronal loss after injury to the brain?Is a specific treatment effective at rescuing neurons in an animal model of cerebral palsy?Is anatomical convergence at the gross anatomical level reflected in the absolute number of neurons within the normal,healthy basal ganglia?2.Stereological measurement of the amount of myelin in the brain and how this can be correlated with behavioural measures,such as the hyperactivity that is relevant to attention deficit hyperactivity disorder(ADHD),in the same animal.3.Stereological measurement,at the nanosale level,of identified inhibitory synapses in the brain and how this reveals differences in an animal model of ADHD-like hyperactivity versus an animal model of schizophrenia.Materials and Methods:For the first topic,serial 40 μm Technovit sections were cut through the entire striatum,or basal ganglia,of Sprague-Dawley rats.A systematic series of sections,after a random start,were stained with cresyl violet.The Cavalieri and optical disector stereological methods were used to measure the absolute number of neurons,N,within these brain structures.Specifically,the Cavalieri method was used to measure the total(absolute) reference volume(Vref) of the brain structure of interest.The optical disector method was used to measure the number of neurons in a known subvolume(Nv,i.e.the neuronal density).To obtain N,the Vref was multiplied by the Nv.For the second topic,Cavalieri's method was combined with stereological measurement of the surface density(Sv) of myelin.This yielded the absolute surface area of myelin in the cerebrum.For this experiment,serial 50 μm cryostat sections were cut through the entire cerebrum of Sprague-Dawley rats.A systematic series of sections,after a random start,were immunostained for myelin basic protein.The rats had been exposed to either repeated normoxia or repeated hypoxia,with the latter condition leading to ADHD-like hyperactivity.The hyperactivity had been measured using a well characterised fixed-interval schedule using operant chambers.The anatomical and behavioural results were compared using Pearson's correlation.For the third topic,Cavalieri's method was combined with measurement of the shrinkage/swelling during processing to yield the absolute volume of presynaptic terminals and their postsynaptic densities.The overall aim was to investigate nanoscale synaptic changes on dopaminergic neurons in the ventral tegmental area of the brain.To achieve this,serial 80 nm sections throughout identified synapses were viewed in a transmission electron microscope and analysed stereologically.This brain region was examined in repeated hypoxic ADHDlike Sprague-Dawley rats and their control group,and in schizophrenic-like Sprague-Dawley rats that had been exposed to maternal immune activation during the second trimester of pregnancy and their control group.For statistical comparisons,either the Mann-Whitney U test or the Student's t-test was used as appropriate.Results:For the first topic,we investigated the long-term neuroprotective potential of antioxidant/hypothermia combinatorial treatment on striatal medium-spiny neurons in an animal model of cerebral palsy.If the Nv or brain weight had been used as the sole measure of neuroprotection,the efficacy of this treatment would have been undetected.Measurement of the absolute number of neurons revealed neuroprotection.In another study,measurement of the absolute number of neurons revealed anatomical convergence within the healthy rat basal ganglia.For example,there are 2.79 million neurons(unilaterally) in the rat caudateputamen,an input ganglion,and 3,200 neurons in the entopeduncular nucleus,an output ganglion.For the second topic,a decreased amount(i.e.absolute surface density) of cerebral myelin is correlated with increased ADHD-like hyperactivity.This is the first evidence in animals that myelin deficits are associated with ADHD-like hyperactivity.For the third topic,identified inhibitory synaptic structures onto dopaminergic neurons are larger in ADHD-like hyperactive rats,compared to their controls,and smaller in schizophrenic-like rats compared to their controls.These anatomical findings are consistent with a well characterised hypodopaminergic state in the cerebrum in ADHD and a well characterised hyperdopaminergic state in the cerebrum in schizophrenia.Conclusion:Understanding the brain in health and disease has been advanced by the application of stereological methods that measure absolute quantities.

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