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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.