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.
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.
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 varicosities7,8). 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.
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
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.
3. Immunohistochemistry
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 examined1,12,13.
5. Analysis and statistics
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 8th 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 |
Statistik | ||||
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.
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 8th 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 imaging14,15. 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 0.11,16. 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 microscopy13,14,15. 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.
The authors have nothing to disclose.
This work was supported by grants to W.Z.S. from the Medical Research and Development Service, Department of Veterans Affairs (Merit Review 1I01 BX001067-01A2), the Alzheimer's Association (NPSPAD-11-202149), and resources from the Midwest Biomedical Research Foundation.
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 |