A Comparison of 2D and 3D Neuron Counting

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2009-12-06 1 A Comparison of A Comparison of 2D Image Analysis and Design 2D Image Analysis and Design-based Stereology based Stereology for Morphological and Anatomical Changes for Morphological and Anatomical Changes in the Dopaminergic System of the Rodent Midbrain in the Dopaminergic System of the Rodent Midbrain Ji-Hyuk Park, MS, OT Therapeutic Sceicne Dept of Occupational Therapy Education University of Kansas Medical Center Background Background y Structure-function relationship in brain y Quantitative analysis for brain structure - 2D image analysis (systematic bias) - Design-based stereology (unbiased) y Quantitative research on DA system of rodent midbrain y Design-bases stereology provides standard method but biased 2D image analyses are still used in neuroscience Structure Structure-function relationship function relationship y Morphological and anatomical features related to function and behavior y Plasticity in these features due to - Therapeutic intervention - Neurological disorder y Morphological and anatomical changes - Therapeutic effects - Specific features of neurological disorder Quantitative Analysis Quantitative Analysis y Quantities analysis in neuroscience - Comparisons between groups - Correlation (structure-function) y Quantitative analyses - Brian imaging analysis - Light microscopic analysis : 2D image analysis : design-based stereology 2D image analysis 2D image analysis y Commonly used in neuroscience y Systematic bias (sampling/assumption) y Model-based stereology y Section analysis y Densitometry Design Design-based stereology based stereology y Theoretically unbiased - Systematic-random sampling - Probability theory (assumption free) y Precision (coefficient of error) y Disector method (total number) y Nucleator method (neuronal volume) y Cavalieri-point counting method (region volume)

Transcript of A Comparison of 2D and 3D Neuron Counting

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A Comparison of A Comparison of 2D Image Analysis and Design2D Image Analysis and Design--based Stereologybased Stereologyfor Morphological and Anatomical Changes for Morphological and Anatomical Changes in the Dopaminergic System of the Rodent Midbrainin the Dopaminergic System of the Rodent Midbrain

Ji-Hyuk Park, MS, OT

Therapeutic Sceicne

Dept of Occupational Therapy Education

University of Kansas Medical Center

BackgroundBackgroundStructure-function relationship in brainQuantitative analysis for brain structure- 2D image analysis (systematic bias)- Design-based stereology (unbiased)g gy ( )Quantitative research on DA system of rodent midbrainDesign-bases stereology provides standard method but biased 2D image analyses are still used in neuroscience

StructureStructure--function relationshipfunction relationship

Morphological and anatomical features related to function and behaviorPlasticity in these features due to- Therapeutic intervention p- Neurological disorderMorphological and anatomical changes - Therapeutic effects- Specific features of neurological disorder

Quantitative AnalysisQuantitative Analysis

Quantities analysis in neuroscience- Comparisons between groups- Correlation (structure-function)Quantitative analyses Q y- Brian imaging analysis - Light microscopic analysis : 2D image analysis: design-based stereology

2D image analysis2D image analysis

Commonly used in neuroscienceSystematic bias (sampling/assumption)Model-based stereologySection analysisDensitometry

DesignDesign--based stereology based stereology

Theoretically unbiased- Systematic-random sampling- Probability theory (assumption free)Precision (coefficient of error)( )Disector method (total number)Nucleator method (neuronal volume)Cavalieri-point counting method (region volume)

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DesignDesign--based Stereology (3D)based Stereology (3D)

Optical disector

TN = N X 1/SF X 1/AF X 1/TF

DesignDesign--based Stereologybased Stereology

Nucleator method

V = mean lm3 (4π/3)

DesignDesign--based Stereologybased Stereology

Cavalieri-point counting method

V = ΣP X a(p) X 1/SF X Section thickness

DesignDesign--based stereology versus 2D based stereology versus 2D image analysisimage analysis

Design-based stereology provides the standard of quantitative analysis2D image analysis commonly used without proofing reliability and accuracyp g y yMany studies using both analyses on DA system of rodent modelsDesign-based stereology challenging previous findings of 2D image analysis

DA system in Rodent modelsDA system in Rodent models

Chronic MPTP/probenecid mice in SNpc50% loss(3D) versus 30% loss(2D)Enriched environment rats in the SNpcSig difference(3D) versus No sig g ( ) gdifference (2D)Alcohol preferring (P) rats in the VTASig difference(3D) versus No sig difference (2D)

SignificanceSignificance

No comparison study between design-based stereology and 2D image analysis using same samples.Unknown accuracy of 2D image analysisy g yThis proposed study will help interpret the previous studies of 2D image analysis

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Purpose of StudyPurpose of Study

To compare 2D image analysis versus design-based stereology for investigation of morphological and anatomical changes in three different rodent models

Primary Research Question 1Primary Research Question 1

Does 2D image analysis detectmorphological and anatomical differences between two groups as design-based stereology does in rodent models?gy

Secondary Question 1Secondary Question 1

Does 2D image analysis detect differences as design-based stereology does in cell numbers?Does 2D image analysis detect differences Does 2D image analysis detect differences as design-based stereology does in cell size?Does 2D image analysis detect differences as design-based stereology does in region size?

Specific Aims 1Specific Aims 1

To compare neuron numbers between control and experimental group by 2D image analysis and design-based stereologygyTo compare neuron sizes between control and experimental groupTo compare region sizes between control and experimental group

Hypothesis 1Hypothesis 1

2D analysis will make different decisions from design-based stereology in detecting morphological and anatomical differences.

Primary Research Question 2Primary Research Question 2

Does 2D image analysis estimate morphological and anatomical differences between two groups as design-based stereology does in rodent models?gy

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Secondary Question 2Secondary Question 2

Does 2D image analysis estimate differences as design-based stereology does in cell numbers?Does 2D image analysis estimate g ydifferences as design-based stereology does in cell size?Does 2D image analysis estimate differences as design-based stereology does in region size?

Specific Aims 2Specific Aims 2

To compare differences of neuronal number between 2D image analysis and design-based stereologyTo compare differences of neuronal pvolume between 2D image analysis and design-based stereologyTo compare differences of region volume between 2D image analysis and design-based stereology

Hypothesis 2Hypothesis 2

Estimations of differences by 2D image analysis are different from estimations by design-based stereology in morphological anatomical features.

Animal SubjectsAnimal Subjects

Severe modelChronic MPTP/probenecid PD (MPD) mice for the SNpc and the VTAM d t d lModest modelAlcohol preferring (P) rats for the VTAEnriched environment (EE) rats for the SNpc

Chronic MPTP/Chronic MPTP/probenecidprobenecid PDPD20 C57BL/6 Mice (10-12 wk old, Male)Control (10) versus chronic MPD (10)10 dose of MPTP 0 ose o hydrocholoride (12.5 mg/kg in saline) with probenecid(250 mg/kg in dimethyl sufoxide)Administrated on a 5-week schedule with an interval of 3.5days

Enriched Environment (EE)Enriched Environment (EE)16 Sprague-Dawley rats (8 males, 8 females)8 rats EE versus 8 SE (random)Standard environment (SE)- Laboratory cage( 46mX 71mX 56m)cage(.46mX.71mX.56m)- food and water available ad libitumEnriched environment (EE)- Toys (alternated every three days)- Wire ladders, PVC pipes tunnel- At least two rats in each EE cage

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Alcohol Preferring (P)Alcohol Preferring (P)

16 adult wistarfemale rats8 P rats versus 8 NP ratsNo treatment

NAc

ApparatusApparatus

Stereology system- Software Stereologer- Nikon Eclipse 80i- Sony 3CCD camera

M t i d t- Motorized stage- 4X, 20X, 100X

NIH imageJ

Measurement MethodsMeasurement Methods DesignDesign--based stereologybased stereologyEstimate the total number of TH neurons(optical disector method)- Every 8th section containing the SNpc and the VTA - Outline the area (4X)Outline the area (4X)- Select counting frames (4X)- Count neurons (100X)Estimate mean neuronal volume(nucleator, number-weighted)- Measure the volume of each counted neurons(100X)

DesignDesign--based stereologybased stereology

Estimate total volume (cavalieri-point counting method)- Every 4th section containing the NAc- Outline the NAc (4X)( )- Select random grids within the NAc- Measure the thickness of each section (100X)

ModelModel--based stereologybased stereology

Estimate total neuronal number- Capture images of sections (20X)- Count all neurons in each section(ImageJ)( g J)- Abecrombie correction : Na/(D+t)Estimate mean neuronal diameter - Measure the area (A) of 10 neurons in each section(ImageJ) - d=

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ModelModel--based stereologybased stereology

Estimate total volume (cavalieri-area method)- Capture images of sections (4X)- Measure the area of the NAc (ImageJ)( g J)- Total volume = Total area X 4 X 60μm

Section analysis (2D)Section analysis (2D)

Estimate area neuronal density- Count all neurons in each section(ImageJ)- Measure the area in each section(imageJ)( g J)- Area density(D) = Total counted neurons/total area

Estimate optical density- Outline the area precisely - Measure optical density (ImageJ)

Section analysis (2D)Section analysis (2D)

Estimate neuronal diameter- Measure the largest diameter of 10 neurons in each section (ImageJ)

Total section area of the NAc- Measuring the area of the NAc (ImageJ)

Data AnalysisData Analysis

SPSS 17.0Student t-test (P<0.05)Control versus ExperimentalDesign based stereology versus 2D image Design-based stereology versus 2D image analyses

Control versus Experimental groupControl versus Experimental group

Three analyses to detect differences between control and experiment in each quantitative data

DesignDesign--based stereology versus 2D based stereology versus 2D image analysesimage analyses

Normalized in a percentage as respect to the mean of the control group

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Anticipated resultsAnticipated results

2D image analysis will detect only relatively large differences in morphological and anatomical features. Estimations of 2D image analysis will be g ydifferent from estimation of design-based stereology in morphological and anatomical differences between two groups.

ReferencesReferencesBaquet ZC, Williams D, Brody J, Smeyne RJ. A comparison of model-based (2D) and design-based (3D) stereological methods for estimating cell number in the substantia nigra pars compacta (SNpc) of the C57BL/6J mouse. Neuroscience. Jul 21 2009;161(4):1082-1090.

Bezard E, Dovero S, Belin D, et al. Enriched environment confers resistance to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and cocaine: involvement of dopamine transporter and trophic factors. J Neurosci. Dec 3 2003;23(35):10999-11007.

M PR P i i l d i f bi d l i d i f Mouton PR. Principles and practices of unbiased stereology : an introduction for bioscientists. Baltimore: Johns Hopkins University Press; 2002.

Schintu N, Frau L, Ibba M, Garau A, Carboni E, Carta AR. Progressive dopaminergicdegeneration in the chronic MPTPp mouse model of Parkinson's disease. NeurotoxRes. Aug 2009;16(2):127-139.

West MJ. New stereological methods for counting neurons. Neurobiology of aging. Jul-Aug 1993;14(4):275-285.

West MJ. Stereological methods for estimating the total number of neurons and synapses: issues of precision and bias.

Zhou FC, Zhang JK, Lumeng L, Li TK. Mesolimbic dopamine system in alcohol-preferring rats. Alcohol. Sep-Oct 1995;12(5):403-412.