Acquiring Practical Population Estimates of Neurons Through Design-Based Stereology: Dissecting the Disector

ABSTRACT

A design-based stereological probe known as the optical disector is employed frequently to acquire unbiased neuronal population estimates from thick histologic sections. This methodology includes customizable parameters for systematic sampling through the X, Y, and Z axes of the region of interest (ROI). For the purpose of this study, cryosections of 40 μm nominal thickness were immunostained for tyrosine hydroxylase (TH) to detect dopaminergic neurons in the substantia nigra pars compacta (SNpc) of C57BL/6J mice. Stereological estimates of TH+ neuron populations were acquired using the Optical Fractionator Workflow module of the Stereo Investigator software system (MBF Bioscience). Because the histologic processing and immunostaining of thick sections may result in non-uniform distribution and density of TH+ neurons throughout the Z axis of the SNpc, various disector heights and placements were analyzed systematically to determine the degree to which disector height selection contributed substantially to the total population estimates. We concluded that the choice of disector height had a major influence on total population estimates of TH+ neurons in the SNpc. For studies that utilize the optical disector method to quantify neuronal population estimates, adequate preliminary sampling should be performed initially through the entire Z axis with extrapolation of the data in order to achieve accurate population estimates.

Daniel Zadory, Ellen Burton, Jeffrey Wolf. Experimental Pathology Laboratories, Inc.

OBJECTIVE

Evaluate the optical disector component of the design-based stereological probe, the optical fractionator, through various disector heights and placements to determine their impact on stereologically-derived TH+ neuron estimates in the SNpc of C57BL/6J mice.

RESULTS

CONCLUSIONS

Optimally, stereological estimates obtained using the optical fractionator approach

should be based on tissue sections in which the distribution of objects (e.g., neurons) is

uniform throughout the Z-depth. However, obtaining thick sections with entirely

uniform cell distributions can be technically challenging. The impact of disector height

and placement on total population estimates in thick histologic sections can be

established by sampling through the entire Z-depth. For the biphasic distribution pattern

that we typically encounter, the most consistent and most accurate results were obtained

when disectors were placed so as to encompass symmetrical portions of the z-depth

distribution curve. A recently released Stereo Investigator module called Resample

Disector allows for similar preliminary analysis of sampling parameters through the Z-

depth.

RESULTS

REFERENCES

• West M.J., Slomianka L., Gundersen H.J., (1991); Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat Rec; 231:482.

• Baquet Z.C., Williams D., Brody J., Smeyne R.J., (2009); 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; 161(4):1082-90. doi: 10.1016/j.neuroscience.2009.04.031.

• Torres E.M., Meldrum A., Kirik D., Dunnett S., (2006); An investigation of the problem of two-layered Immunohistochemical staining in paraformaldehyde fixed sections. Journal of Neuroscience Methods; 158, 66-74

• Carlo C.N., Stevens C.F., (2011); Analysis of differential shrinkage in frozen brain sections and its implications for the use of guard zones in stereology. Journal of Comparative Neurology; 519:2803-2810

• Gardella D., Hatton W.J., Rind H.B., Rosen G.D., von Bartheld C.S., (2003); Differential tissue shrinkage and compression in the z-axis: implications for optical disector counting in vibratome-, plastic- and cryosections. Journal of Neuroscience Methods; 124, 45-59

INTRODUCTION

The purpose of design-based stereology is to efficiently obtain precise and unbiased morphometric estimates of specific features in whole organs or anatomical structures by subsampling thru three-dimensional (3D) axes of the target region. Earlier two-dimensional (2D) model-based stereological methods relied on parameters ascertained through qualitative assumptions of volume, size, density, orientation, and shape that introduced varying degrees of bias. While both design-based and model-based stereological methods have strengths and weaknesses, it is imperative that these limitations are understood by researchers, so that reproducible, accurate, and unbiased estimates can be generated, and results from different laboratories can be reasonably compared (Baquet et al., 2009). A widely used design-based stereology probe, the optical fractionator, is employed frequently to quantify neurons in thick histologic sections as a measure of pharmacological efficacy or neurotoxicity. Through unbiased systematic uniform random sampling in a 3D space, this approach combines two elements of previously established stereological probes, the Optical Disector and Fractionator (West et al., 1991). Population estimates are derived through the following formula:

METHODS

• Specimen Collection and Processing Twenty male C57BL/6J mice of approximately 11-12 weeks old were anesthetized by intraperitoneal injection of sodium pentobarbital and transcardially perfused with physiological saline followed by 4% paraformaldehyde (PFA). The intact brains were removed and post-fixed in 4% PFA overnight at 4⁰ C, followed by separate immersion fixations of 10% and 30% sucrose solution at 4⁰ C for 24 hours and 48 hours, respectively, to initiate cryoprotection. Following cryoprotection, the brains were flash frozen for approximately 35 seconds in isopentane chilled to -40⁰ C.

METHODS

• Immunohistochemical Detection of TH+ Neurons in the SNpc Each brain was microtomed serially in the coronal plane (i.e., transversely along the rostral-caudal axis) at 40 µm intervals using a sliding microtome and systematically transferred into a 24-well plate containing 30% ethylene glycol solution and stored at -20⁰ C. A section interval of 1:3 was identified and briefly washed in 0.1 M phosphate buffered saline (PBS) followed by a 10 minute incubation in 3% H2O2 in PBS to quench endogenous peroxidase activity. Non-specific binding of antibodies to targets was blocked by immersion of the sections in a solution of 10% normal goat serum and 1% bovine serum albumin, made up in PBS for one hour at room temperature. Immunostaining was performed according to the standard avidin-biotin complex (ABC) method. Sections were then incubated in a solution containing the primary Rabbit Polyclonal Anti-Tyrosine Hydroxylase (EMD Millipore, Billerica, MA) antibody at a dilution of 1:4000 for 48 hours at 4⁰C, and then in the secondary antibody, biotinylated goat anti-rabbit IgG (Vector Laboratories, Inc.), at a dilution of 1:6000 and incubated for another 2 hours at room temperature. The brain sections were then incubated for 60 minutes in an ABC solution (Standard VectaStain Elite Kit, Vector Laboratories, Inc.) at room temperature. Visualization of the reaction was accomplished by immersion in 0.03% 3,3-diaminobenzidine (DAB, Sigma Fast Tablets, Sigma-Aldrich) for 1 minute at room temperature. Sections were then floated onto positively charged glass slides and air-dried for approximately 1 hour at room temperature. Following a succession of rinses with ethanol (100%, 95%, and 70%, respectively) and distilled water, sections were then counterstained FD Cresyl Violet Solution and cover slipped using a resinous mounting medium. • Design-Based Stereology The total numbers of TH+ neurons in the SNpc were estimated using the optical fractionator approach (West et. al, 1991) through the entire Z depth. Accordingly, neuron cell bodies were counted in a subsample of sections, section thicknesses, and section areas, and then the results were extrapolated to provide estimates of total number of TH+ neurons. Contours (virtual outlines) of the SNpc were drawn in Stereo Investigator (MBF, Williston, VT) for each transverse histologic brain section. The SNpc region is a dense region of neuron soma which regresses along the pars reticulata (SNr). Areas not included in the tracings were the SNr, pars laterials, ventral tegmental area, and immunoreactive fiber projections from the pars compacta. Sampling parameters for stereological cell counting were established in the Optical Fractionator Workflow module as follows: • Data Analysis Neuronal cell counts were exported to individual Excel spreadsheet files and neuronal populations through the Z-depth layers were assessed to determine the uniformity of distribution. As anticipated, neuronal cell densities of were consistently non-uniform throughout the Z-depth; however, the relative pattern of distribution was fairly symmetrical from the Z-depth midline to the top and bottom surfaces of the tissue sections. To further understand the quantitative impact this biphasic distribution has on total population estimates, we then superimposed disectors at various depths and calculated estimates of TH+ neuron numbers using the optical fractionator formula.

Nobj = 1/ssf × 1/asf × 1/tsf × ∑Q

Nobj = number of objects

ssf (section sampling fraction) = the number of SNpc sections evaluated / total

number of SNpc sections microtomed

asf (area sampling fraction) = counting frame size (microns) / counting frame

interval (microns)

tsf (thickness sampling fraction) = disector height (microns) / mean measured

section thickness (microns)

∑Q = the number of cells counted

Counting Frame Area (XY) (µm²) 36,00

Disector Height (Z) (µm) 1 19.0

Disector Volume (XYZ) (µm³) 68,400

Guard Zone Distance (µm) 0.0

Sampling Grid Area (XY) (µm²) 14,400

Section Evaluation Interval 3

Mean Estimates of TH+ Neurons Produced by the Optical Fractionator Probe (N = 20)

Total Markers Number

of Sections

Number of Sampling

Sites

Measured Defined

Mounted Thickness

Estimated Population by Mean Section

Thickness

C.E. Values (m=1)

680 (± 133.79)

13 (± 1.10)

385 (± 26.64)

15.93 (± 0.99)

6,859 (± 1,476)

0.05 (± 0.01)

0

20

40

60

80

100

120

0 -1 -2 -3 -4 -5 -6 -7 -8 -9 -10 -11 -12 -13 -14 -15 -16 -17 -18

No

. of

TH

+ N

eu

ron

s

Z Depth from top of Section (-1.00µm)

Mean Z-Depth Distribution of TH+ Neurons

7,795

14,229

2,680

7,628

9,178 8,953 8,729 8,716

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

Disector0-13

microns

Disector2-5

microns

Disector6-11

microns

Disector4-13

microns

Disector2-8

microns

Disector2-15

microns

Disector9-15

microns

Baquetet al.,2009

Est

imat

e N

o. o

f T

H+

Ne

uro

ns

Dissecting the Disector: Optical Fractionator Estimates of Total TH+ Neurons Relative to Disector

Placement

This distribution illustrates the non-uniform density of TH+ neurons as counted throughout the Z-depth. Note the symmetry of the biphasic pattern. Potential reasons for this biphasic distribution as described frequently in the literature include problems of antibody/stain penetration, differential tissue shrinkage, and differential tissue compression (Gardella et al., 2003), (Torres et al., 2006), (Carlo et al., 2011). To further understand the underlying cause of this non-uniform TH+ distribution along the Z-depth, we examined a representative tissue section from a unique perspective. A coronal 40 µm free-floating brain section containing the SNpc was immunostained as previously described, minus the cresyl violet counterstain. The immunostained section was then rotated perpendicularly and embedded on edge in optimal cutting temperature compound (O.C.T., Sakura Finetek). Cryosections were obtained transversely (i.e, sagittal plane) at 10 µm intervals through the region of the SNpc and counterstained in FD Cresyl Violet as previously described.

•(A) Stereo Investigator 20X Virtual Slice image of immunoreactivity through the sagittal plane (Z-Depth) of a 40 µm coronal section.

•(B) 100X image of the SNpc. The pattern of immunoreactivity correlates with the counted mean Z-depth distribution of TH+ neurons. Non-immunoreactive cells (cresyl violet) were observed infrequently in the middle portion of the z-depth.

•(C) Region distal to the SNpc. Immunoreactive neuronal processes are clearly visible throughout the Z-depth. Non-immunoreactive cells were distributed evenly throughout.

A

B

C

Disectors of varying heights were superimposed at different sampling depths within the extrapolated mean Z-depth distributions. To determine total population estimates relative to each disector placement, estimates of TH+ neuron numbers were calculated using the optical fractionator formula.

Disectors that encompassed symmetrical portions of the bisphasic curve produced numbers that were comparable to previous estimates of TH+ neurons in C57BL/6J mice: 8716 ±338 (range = 7546-9869, N = 10) (Baquet et al., 2009)

RESULTS