2009-12-06

Background
A Comparison of 2D Image Analysis and Design-based Stereology Designfor Morphological and Anatomical Changes 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

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

StructureStructure-function relationship
Morphological and anatomical features related to function and behavior Plasticity in these features due to - Therapeutic intervention p - Neurological disorder Morphological and anatomical changes - Therapeutic effects - Specific features of neurological disorder

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

2D image analysis
Commonly used in neuroscience Systematic bias (sampling/assumption) Model-based stereology Section analysis Densitometry

DesignDesign-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)

2009-12-06

DesignDesign-based Stereology (3D)

Optical disector

DesignDesign-based Stereology
Nucleator method

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

V = mean lm3 (4/3)

DesignDesign-based Stereology
Cavalieri-point counting method

DesignDesign-based stereology versus 2D image analysis

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

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

DA system in Rodent models

Chronic MPTP/probenecid mice in SNpc 50% loss(3D) versus 30% loss(2D) Enriched environment rats in the SNpc Sig difference(3D) versus No sig g ( ) g difference (2D) Alcohol preferring (P) rats in the VTA Sig difference(3D) versus No sig difference (2D)

Significance
No comparison study between designbased stereology and 2D image analysis using same samples. Unknown accuracy of 2D image analysis y g y This proposed study will help interpret the previous studies of 2D image analysis

2009-12-06

Purpose 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 1

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

Secondary Question 1
Does 2D image analysis detect differences as design-based stereology does in cell numbers? 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 1
To compare neuron numbers between control and experimental group by 2D image analysis and design-based stereology gy To compare neuron sizes between control and experimental group To compare region sizes between control and experimental group

Hypothesis 1
2D analysis will make different decisions from design-based stereology in detecting morphological and anatomical differences.

Primary Research Question 2

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

2009-12-06

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

Specific Aims 2
To compare differences of neuronal number between 2D image analysis and design-based stereology To compare differences of neuronal p volume between 2D image analysis and design-based stereology To compare differences of region volume between 2D image analysis and designbased stereology

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

Animal Subjects
Severe model Chronic MPTP/probenecid PD (MPD) mice for the SNpc and the VTA Modest M d t model d l Alcohol preferring (P) rats for the VTA Enriched environment (EE) rats for the SNpc

Chronic MPTP/probenecid PD MPTP/probenecid

20 C57BL/6 Mice (1012 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 5week schedule with an interval of 3.5days

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 libitum Enriched environment (EE) - Toys (alternated every three days) - Wire ladders, PVC pipes tunnel - At least two rats in each EE cage

2009-12-06

Alcohol Preferring (P)

16 adult wistar female rats 8 P rats versus 8 NP rats No treatment
NAc

Apparatus
Stereology system - Software Stereologer - Nikon Eclipse 80i - Sony 3CCD camera - M t i d stage Motorized t - 4X, 20X, 100X NIH imageJ

Measurement Methods

DesignDesign-based stereology
Estimate the total number of TH neurons (optical disector method) - Every 8th section containing the SNpc and the VTA - 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 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 stereology
Estimate total neuronal number - Capture images of sections (20X) - Count all neurons in each section ( g J) (ImageJ) - Abecrombie correction : Na/(D+t) Estimate mean neuronal diameter - Measure the area (A) of 10 neurons in each section(ImageJ) - d=

2009-12-06

ModelModel-based 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 60m

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)

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

Control versus Experimental group

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

DesignDesign-based stereology versus 2D image analyses

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

2009-12-06

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

References
Baquet 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 1methyl-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. Mouton PR. P i i l and practices of unbiased stereology : an introduction for M PR Principles d i f bi d l i d i f bioscientists. Baltimore: Johns Hopkins University Press; 2002. Schintu N, Frau L, Ibba M, Garau A, Carboni E, Carta AR. Progressive dopaminergic degeneration in the chronic MPTPp mouse model of Parkinson's disease. Neurotox Res. Aug 2009;16(2):127-139. West MJ. New stereological methods for counting neurons. Neurobiology of aging. JulAug 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 alcoholpreferring rats. Alcohol. Sep-Oct 1995;12(5):403-412.