Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

Summary

Neuronal fiber length within a three-dimensional structure of a brain region is a reliable parameter to quantify specific neuronal structural integrity or degeneration. This article details a stereological quantification method to measure cholinergic fiber length within the nucleus basalis of Meynert in mice as an example.

Abstract

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a useful method to quantify neuronal profiles of various brain structures. Here we provide a software-based stereology protocol to estimate the total length of cholinergic fibers in the nucleus basalis of Meynert (NBM) of the basal forebrain. The method uses a space ball probe for length estimates. The cholinergic fibers are visualized by choline acetyltransferase (ChAT) immunostaining with the horseradish peroxidase-diaminobenzidine (HRP-DAB) detection system. The staining protocol is also valid for fiber and cell number estimation in various brain regions using stereology software. The stereology protocol can be used for estimation of any linear profiles such as cholinoceptive fibers, dopaminergic/catecholaminergic fibers, serotonergic fibers, astrocyte processes, or even vascular profiles.

Introduction

Quantitative estimates of length and/or density of nerve fibers in the brain are important parameters of neuropathological studies. The length of cholinergic, dopaminergic, and serotonergic axons in various brain regions are often correlated with the specific functions of the region. Because the distribution of these axons is generally heterogeneous, design-based stereology is used to avoid bias during sampling. The space ball probe of stereology has been designed to provide efficient and reliable measures of line-like structures such as neuronal fibers in a region of interest¹. The probe makes a virtual sphere that is imposed systematically in the tissue to measure line intersections with the surface of the probe. Because it is impossible to put sphere probes in the tissue for analysis, the commercially available software provides a virtual three-dimensional (3D) sphere, which is basically a series of concentric circles of various diameters that represent the surface of the sphere probe.

Selective cholinergic neurodegeneration is one of the consistent features of Alzheimer's disease (AD)^2,3,4. Dysfunctional cholinergic transmission is considered a causative factor for cognitive decline in AD. Cholinergic dysfunction is also evident in many other mental disorders such as Parkinson's, addiction, and schizophrenia. Different aspects of cholinergic neurodegeneration are studied in animal models (e.g., reduction in acetylcholine⁵, ChAT protein⁶, cholinergic fiber neurodegeneration in the vicinity of amyloid plaques⁶, and decrease in cholinergic fibers and synaptic varicosities^7,8). Fiber degeneration is believed to take place earlier than neuronal loss, because cholinergic neuronal loss is not always observed in studies. Most of the cholinergic neurons are in the basal forebrain and the brain stem, and their axons project to various brain regions such as the cortices and hippocampus. NBM is situated in the basal forebrain and found to be one of the commonly affected brain areas in AD.

The fractionator method of stereology is based on systematic random sampling of a tissue at multiple levels. Section sampling fraction (SSF) is the non-computer based systematic sampling of sections for the fractionator method of stereology. Area sampling fraction (ASF) is fractionation of an area of the region of interest in the section. Thickness sampling fraction (TSF) is the fractionation of the thickness of a section. The space ball probe allows us to quantify profiles of interest in a 3D sphere at fractionated locations. Here we use a space ball probe for estimation of the total length of cholinergic fibers in the NBM of mouse brain to illustrate the procedures. The current protocol provides details on tissue processing, sampling methods for stereology, immunohistochemical staining using the ChAT antibody, and unbiased stereology to estimate cholinergic fiber length and fiber density in the NBM of mouse brain.

Protocol

All procedures for using these animals have been approved by the Kansas City Veterans Affairs Medical Center Institutional Animal Care and Use Committee. Eighteen-month-old mice overexpressing Swedish mutant beta-amyloid precursor protein (APPswe) and their C57/BL6 WT littermates were used for the experiments. Details of breeding and genotyping is given in He et al.⁸.

1. Perfusion and tissue processing

1. Anesthetize mice using an intraperitoneal injection with ketamine (100 mg/kg) and xylazine (10 mg/kg). Pinch toes to confirm a lack of response before continuing⁹.
2. Transcardially perfuse with ~50 mL of ice cold 0.1M Dulbecco's phosphate buffered saline (DPBS) followed by 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB)¹⁰.
   CAUTION: PFA is toxic. Use personal protective equipment (PPE) when working with PFA.
3. Remove brains¹⁰ and immerse in 4% PFA in 0.1 M PB at 4 °C for postfixation for 24 h. Wash with DPBS 3x.
4. Change to 15% sucrose solution in 0.1 DPBS overnight and then 30% sucrose solution in 0.1 M DPBS for another 48 h at 4 °C.
5. Remove the tissue from the sucrose solution and freeze in a cryotome chamber preset to a temperature of -20 °C. Frozen samples can be stored in sealed tubes at -80 °C until sectioning.
6. Embed tissues in optimum cutting temperature embedding medium (see Table of Materials) and mount on a specimen disc. Cut serial 30 µm thick sections in a coronal plane using a cryotome and collect all the sections consequently in 24 well culture plates filled with cryoprotectant solution (30% glycerol, 30% ethylene glycol, 40% DPBS; Figure 1A–C). Store in a -20 °C freezer.
   NOTE: Thicker sections (e.g., 50 µm) are preferable when feasible. Make sure that the sections are kept in cutting order and all sections are completely in cryoprotectant. A 96 well plate can be used as an alternative to 24 well plates to collect sections.
7. Cover the wells with plate sealer to prevent evaporation and drying. Store plates at -20 °C until further use. Sections stored at -20 °C are stable for several months for ChAT immunohistochemistry.

2. Systematic section selection for IHC

NOTE: A pilot study should be done to know the total number of sections required to achieve an acceptable coefficient of error (CE). The CE value is an expression of the total amount of error in the sampling procedure. The lowest value represents the minimal error and a CE value lower than 0.1 is considered acceptable by the software used here (see Table of Materials)¹¹.

1. Identify the first and last sections for each brain by comparing morphological features with a standard mouse brain atlas such as Franklin and Paxinos. NBM begins at bregma -0.0mm and ends at -1.6 mm. Therefore, about 50 sections contain NBM. The total number of sections is one of the parameters required during stereology. The selection of every 8^th section (SSF = 1/8) gave a total of 6–7 sections for the analysis and yielded an acceptable CE for both volume estimation and fiber length estimation in our pilot study.
2. Randomly start with one of the first eight sections and then systemically sample every 8^th section until the last posterior section containing NBM (Figure 1C).

3. Immunohistochemistry

1. Transfer the cryopreserved sections to room temperature in cryoprotectant and then 0.1M phosphate buffer (PB) in 6 well plates.
2. Wash 3x in PB.
3. Incubate in 0.3% H₂O₂ in methanol for 15 min.
4. Wash 3x in Tris-buffered saline (TBS).
5. Incubate in 0.25% Triton X-100 in TBS for 30 min.
6. Block with 10% normal bovine serum (NBS) in 0.1% Triton X-100 in TBS for 30 min.
7. Incubate with a 1:1,000 dilution of goat anti-human ChAT primary antibodies (see Table of Materials) in TBS with 0.1% Triton X-100 and 1% NBS for 48 h at 4 °C.
8. Wash 3x in TBS at room temperature. Perform all further incubation and washing at room temperature.
9. Incubate with biotinylated bovine anti-goat secondary antibody (see Table of Materials) for 1 h.
10. Wash 3x in TBS.
11. Incubate with the avidin-biotin–peroxidase complex (see Table of Materials).
12. Wash 3x in TBS.
13. Develop using an enhanced DAB peroxidase substrate solution (see Table of Materials) according to the manufacturer's recommendations.
    CAUTION: DAB is a suspected carcinogen. It is toxic by contact and inhalation. Use PPE when working with DAB.
14. Wash the section a couple of times with distilled water and keep in Tris pH = 7.6.
15. Mount the sections on the gelatinized slides. All sections from one tissue can be put on the same slide. Air dry the sections, dehydrate 2x for 5 min each with 70%, 90%, 95%, and 100% ethanol, and then clear the sections with two 10 min xylene washes. Coverslip the sections using mounting medium (see Table of Materials).
    NOTE: The processing time of dehydration and clearing affects the thickness of the sections. Therefore, the same conditions should be used for all sections. In the current study, the mean value for the final thickness was 21.11 ± 0.45 µm.
16. Keep the sections in the fume hood to dry. The dry sections are ready for stereology.

4. Stereology

NOTE: See the Table of Materials for the microscope and software used. An immersion objective with a numerical aperture (NA) > 1.2 will be useful and should be used if required. Slides should be grouped according to genotype or treatment group and coded. The complete stereology for one study should be performed by the same person and the person performing the stereology should be blind to the identity of the individual slides or group examined^1,12,13.

1. Open a new study in the software. (File > New Study). A 'Study Initialization' dialog box will open. Fill out the study information, using Multi-level (Fraction Based) (Figure 2A).
2. Double click on 'Volume' under Parameters, which will open the Volume dialog box. Name the feature of interest and select 'Region Point Counting' probe. Click Next.
3. Double click on the next parameter, 'Length'.
   1. Provide a name of the feature (e.g., 'L') Select 'Sphere' probe and click Next.
4. Next, click on the 'Study Initialization' dialog box, which will open the 'Case Initialization' box. Fill in the case information. Groups must be coded before starting the study. The total number of sections is the number of sections starting from the first section containing the area of interest to the last section containing area of interest (see step 2.1). The section sampling interval is eight, because every 8^th section was selected for the IHC staining.
5. Clicking Next opens the 'Probe Initialization' dialog box, which will automatically set the lowest magnification for the region selection and volume estimation. Confirm the settings and check if the region of interest can be identified at the selected lower magnification. Double click on 'Volume' and fill 50,000 µm³ per point for the region volume fraction. Click Done.
6. Under Object (High) Magnification, set Length at 63x or 100x. Double click on 'Length' to open the Length-Sphere dialog box and set the diameter of the sphere to 10 µm. Click Done.
   NOTE: The guard zone should be determined based on the actual thickness of the sections. The operator must check the thickness of the sections at multiple sites to avoid any damage. If necessary, adjust the guard zone thickness based on section thickness.
7. Set appropriate values for frame area, frame height, guard zone, and frame spacing.
   NOTE: These values depend on the heterogeneity of the object profiles (fibers) in the region of study. A frame area of 400 µm², frame height of 10 µm, guard zone of 2 µm, and frame spacing of 300 µm gives acceptable CE values for fiber length estimation in NBM. A pilot study must be performed with these values before heading to the next case.
8. Follow the instructions provided by the software after each step. The instructions appear either as a dialog box and/or at the bottom of the screen.
9. Insert Section 1.
10. At low magnification (5x), define the region of interest by making an arbitrary boundary around the NBM. Click the Next button at the left corner of the video window. Follow the instructions and confirm all green points are in the NBM. The points can be included or excluded by clicking on the points.
    NOTE: There is no definite, set boundary around the NBM. The selection is mostly investigator-defined. The ChAT+ cholinergic neurons of the NBM can be observed in the internal capsule (ic) and globus pallidus (GP). In this protocol, the ic with ChAT+ cholinergic neurons and their projections (fibers) and whole GP is included in the NBM (Figure 1D).
11. Follow the instructions and change to 63x for fiber measurements at the current fraction. The 'Section Thickness' dialog box appears on the screen. Set the top and bottom surface of the section to measure the actual thickness of the section. The manual Z axis movement should be used. Click on Done.
    NOTE: If there is no fiber in the area, the step can be skipped to go to the next fraction location.
12. Move the Z axis slowly from top to bottom in the frame height of the section and mark all intersecting fibers on the surface of the virtual sphere probe (Figure 3). When done, click Next to go to the next location.
13. After completing all the fractions, the software will ask to insert the next section. Repeat steps 4.9–4.12 for all six or seven sections of the tissue.
    1. At the end, the software will generate a result for the case showing the CE values (Figure 4). If the CE is acceptable, proceed to the next case. File > New Case. If the CE is not acceptable (Figure 4A), the software provides recommendations to change some of the parameters. In many cases, decreasing the frame spacing decreases the CE values to an acceptable range (Figure 4B).
14. After completing all the cases, generate results for each case and group (File > Results).

5. Analysis and statistics

1. The software provides data for each sample and each group. The software itself calculates fractions such as SSF, ASF, and TSF, and provides the total reference volume (V_REF) and total length (L) of the fibers in the reference region (in this case, NBM) (Figure 4B). Export the data and save or copy to the statistical software of choice for between group analysis. Table 1 shows typical result data for statistical analysis. Analyze fiber density (Lv) by dividing the total fiber length with the reference volume.

Representative Results

Representative results are shown in Table 1 and Figure 5. Group C, which was decoded as APPswe group (APP), had significantly lower fiber length (Figure 5B) and fiber length density (Figure 5C) compared to their wild type (WILD) littermates. The results showed that there was no significant difference in the volume of the NBM between the two groups analyzed (Figure 5A).

Figure 1: Illustration of tissue processing and sampling used in the present study. Sample preparation and sampling. (A) The cerebellum and olfactory bulb are removed before mounting on the specimen disc. (B) Orientation of the brain on the specimen disc for section cutting. A shallow longitudinal incision (marked with a line) on the one hemisphere helps to identify the side of the brain in the sections. (C) Schematic diagram of a 24 well plate showing the systematic selection of every 8^th section from the 24 well plate (marked as 'X'). (D) Schematic diagram of coronal sections delimiting the borders of NBM in the systematically selected six sections. (E-G) Representative images of coronal sections immunostained for ChAT and location of NBM (outlined). (H) A high magnification image showing the NBM boundaries. LV = lateral ventricle; VL = ventrolateral thalamic nucleus; ic = internal capsule; GP = globus pallidus; CPu = caudate putamen (striatum); NBM = nucleus basalis of Meynert (represented as 'B' in Franklin and Paxinos mouse atlas). Scale bar = 1 mm (E-G), 200 µm (H). Please click here to view a larger version of this figure.

Figure 2: Screenshot images. (A) 'Study Initialization' (B) 'Case Initialization', and (C) 'Probe Initialization' dialog boxes. Please click here to view a larger version of this figure.

Figure 3: Sphere probe using an optical dissector. Representative screenshot images showing the four planes of a sphere and the marking of intersecting fibers. Please click here to view a larger version of this figure.

Figure 4: Representative results. (A) shows an unacceptable CE and (B) shows an acceptable CE for length estimation. The ASF, SSF, and TSF for each case is also presented in the results. Please click here to view a larger version of this figure.

Figure 5: Representative data and analysis. Graphical representation of volume (A), length (B), and length density (C) in the NBM of two groups. The data were analyzed within two different groups using the Student t-test. *p < 0.05. Please click here to view a larger version of this figure.

Group	Case #	Volume (µm^3)	Length (µm)	Lv (µm/µm^3)
B	1	926885302	16446282	0.018
B	2	856582400	19254528	0.022
B	3	1150520830	15980131	0.014
C	1	981056585	12108328	0.012
C	2	894169486	6905567	0.008
C	3	998618871	10359766	0.010
Statistics
Mean Group B		977996177.3	17226980.33	0.018
SD Group B		153490036.6	1771309.218	0.004
Mean Group C		957948314	9791220.333	0.010
SD Group C		55927744.89	2647567.494	0.002
TTEST B vs C		0.84	0.02	0.049

Table 1: Representative data and analysis. Volume and length values were directly copied from the results provided by the stereology software. Length density (Lv) was calculated by dividing the length values by the volume values of each case. p values were calculated using Student's t-test.

Discussion

Here we demonstrate a method to estimate the density of cholinergic fibers in the NBM using a space ball (sphere) probe. This probe estimates the total fiber length in the region of interest. The total length can be divided by the volume of the region to get the fiber density. To estimate the volume of the region, the Cavalieri point count method was used. The Cavalieri point count method is an unbiased and efficient estimator of a 3D reference volume for any region. The method calculates an estimate of the area on a cut surface of a section by counting points (representing area fractions) and then multiplying by the distance between two sections analysed¹¹. The method does not require labor intensive, accurate tracing of the perimeter of the region of interest. It is used in conjunction with optical fractionators to estimate the density of cells and fibers.

Stereological analysis requires a precise sampling method. The brain region of interest should be properly defined with staining. The NBM sits between the AP bregma -0.0 mm to -1.6 mm per the Franklin and Paxinos mouse atlas. For immunohistochemistry, sections should be systematically, randomly chosen, which means that the first section should be selected randomly and then other sections should be chosen systematically. For an adult mouse brain, the NBM consists of about 1,600 µm (anterior-posterior), which means about 53 coronal sections 30 µm thick. Then, if every 8^th section is selected, there will be 6−7 sections required to stain for stereological analysis. In our usual procedure, 6−7 sections are enough for estimating the total number of cholinergic fibers in the NBM. Analysis of the CE is suggested for verification at the beginning of the methodological optimization¹².

A proper staining methodology is the basic requirement for a study. ChAT staining for cholinergic fibers can be challenging, and many antibodies stain some of the cellular parts but not the distant axodendritic processes. Please refer to our previously described protocol for the relevant details regarding ChAT staining⁸.

The method essentially requires thick sections because histological processing causes shrinkage in the tissue. Ideally, sections of more than a 20 µm post-processing, final thickness (often thinner than the initial tissue section thickness) is required for space ball probes. Therefore, a section thickness of 50 µm is recommended. The shrinkage is generally uneven, and it can affect volumetric distortions within the tissue and therefore change in Lv value. For example, a multifold difference was observed in capillary length density when it was analyzed in vivo using multiphoton imaging^14,15. Considering this issue, it is more effective to report length per region instead of length per volume.

Although the given values in the protocol worked perfectly, a pilot study for an individual study is always advised. After completing a single tissue sample case, the software provides a CE value for the chosen sampling design. The CE value is used to estimate the precision of the estimate and can be calculated by several formulas. The software used here uses Gunderson's 1999 formula to analyze the CE and considers it acceptable if the values are less than 0.1^1,16. The sampling design scheme should be adjusted until the CE value becomes acceptable before the protocol is adapted for other samples. In general, an increased number of sampling (by reducing the frame interval) reduces the CE value. Practically no structure in the biological system is an ideal line profile, but ribbons or cylinders. Therefore, deciding the exact intersecting feature at a focusing point varies from person to person. Thus, the stereology of all samples of a particular study should be performed by same person. To avoid possible bias, the stereology operator should be blind to the sample identifier.

Reproducibility is a major concern in this method. One factor is the tissue shrinkage and deformities during sample processing which can be overcome to some extent by using in vivo confocal microscopy^13,14,15. The space ball requires high contrast staining and good imaging resolution to visualize structures. As fibers are not usually in linear form, the determination of intercepts remains mostly the operator's decision. Automated segmentation of histological features has been proposed to overcome this problem. However, this is not available yet¹³.

The advantage of using stereology is that it provides an unbiased scheme to analyse structures in a 3D tissue. The space ball probe provides isotropic fractions within tissue samples and therefore offers an unbiased approach to quantify fiber length. An alternative method to analyze fiber density is measuring the optical density of a histochemical staining. The staining intensity-based methods provide semiquantitative estimation of the staining density and may or may not be sensitive enough to differentiate the changes of cholinergic neurons and fibers in the NBM. Stereological methods using the space ball (sphere) probe uses the determination of fibers based on their visual characteristics and provides estimation of the real length of the fibers. The protocol can also be used to analyze other linear profiles in the brain, such as cholinoceptive fibers (using acetyl choline esterase histochemistry), dopaminergic or catecholaminergic fibers (tyrosine hydroxylase immunostaining), serotonergic fibers (serotonin immunostaining), vascular structures (CD31 immunostaining), or astrocyte processes (glial fibrillary acidic protein, GFAP, immunostaining)^{17,18,19,20,21}.

Materials

Name	Company	Catalog Number	Comments
ABC kit	Vector Laboratories	PK6100
Anti-ChAT Antibody	Millipore, MA, USA	AB144P
Bovine anti-goat IgG-B	Santacruz Biotechnology	SC-2347
Bovine Serum, Adult	Sigma-Aldrich, St. Louis, MO, USA	B9433
Cryostat	Lieca Microsystems, Buffalo Grove, IL, USA
Dulbecco's Phosphate Buffered Saline	Sigma-Aldrich, St. Louis, MO, USA	D5652
Ethylene Glycol	Sigma-Aldrich, St. Louis, MO, USA	324558
Glycerol	Sigma-Aldrich, St. Louis, MO, USA	G2025
Hydrogen Peroxide	Sigma-Aldrich, St. Louis, MO, USA	H1009
Immpact-DAB kit	Vector Laboratories	SK4105	Enhanced DAB peroxidase substrate solution
Ketamine	Westward Pharmaceuticals, NJ, USA	0143-9509-01
Microscope	Lieca Microsystems, Buffalo Grove, IL, USA	AF6000	Equipped with motorized stage and IMI-tech color digital camera
Optimum cutting temperature (O.C.T.) embedding medium	Electron Microscopy Sciences, PA, USA	62550-12
Paraformaldehyde	Sigma-Aldrich, St. Louis, MO, USA	P6148
Permount mounting medium	Electron Microscopy Sciences, PA, USA	17986-01
Stereologer Software	Stereology Resource Center, Inc. St. Petersburg, FL, USA	Stereologer2000	Installed on a Dell Desktop computer.
Triton X-100	Sigma-Aldrich, St. Louis, MO, USA	T8787
Trizma Base	Sigma-Aldrich, St. Louis, MO, USA	T1503	Tris base
Trizma hydrochloride	Sigma-Aldrich, St. Louis, MO, USA	T5941	Tris hydrochloride
Xylazine	Bayer, Leverkusen, Germany	Rompun
Xylenes, Histological grade	Sigma-Aldrich, St. Louis, MO	534056