All procedures were approved by the University Health Network Animal Care Committee and performed in accordance with guidelines and regulations set by the Canadian Council on Animal Care.
1. Stereotactic injection
2. Brain sectioning and immunohistochemistry (IHC)
3. Confocal microscopy and image acquisition
4. Image analysis and quantitation
By applying the above methods to brain tissue collected 6 weeks after AAV injections, we demonstrated that stereotactic injection of AAV expressing mutant A53T α-syn (AAV-A53T) in the SNpc of rat brain results in a significant reduction in the density of dopaminergic neurons compared to injection of empty vector AAV (AAV-EV) as a control (Figure 5A,B). The mean number of TH-positive neurons/mm2 in the SNpc of rats injected with AAV-EV was 276.2 ± 34.7, and in the SNpc of rats injected with AAV-A53T was 41.2 ± 17 (P = 0.0003). Quantification of the number of dopaminergic neurons/mm2 in the SNpc is similar to previously published reports10, 11. For the methods described here, 4 sequential sections per animal were analyzed. Previous studies have shown significant differences with as little as 3 sections, but analysis can be further increased up to 12 sections to encompass the whole SNpc depending on the model and intervention being studied by the investigator.
Unbiased stereology was also performed as previously described12 on another set of brain sections from the same animals. Using this method, we also demonstrated that stereotactic injection of mutant A53T α-syn in the SNpc of rat brain results in a significant reduction in the estimated total number of TH-positive neurons in the SNpc, as compared to injection of EV-AAV (Figure 5C). Importantly, there was a strong correlation between the dopaminergic neuron density estimated using automated image analysis software and dopaminergic neuron number estimated using unbiased stereology (r = 0.8819, P=0.0007) (Figure 5D).
We also applied our methods using automated image analysis software to determine the number of TH-positive neurons/mm2 on the uninjected side of rats injected with AAV-A53T or AAV-EV. The mean number of TH-positive neurons/mm2 in the uninjected SNpc of rats injected with AAV-A53T was 123.2 ± 26.4, which was significantly greater than in the injected SNpc, which was 44.0 ± 15.8 (P = 0.0331) (Supplementary Figure 1A). The mean number of TH-positive neurons/mm2 in the uninjected SNpc of rats injected with AAV-EV (215.6 ± 35.5) was not significantly different from the injected SNpc (276.2 ± 34.7), confirming there was no degeneration due to injection with AAV-EV (Supplementary Figure 1B). We calculated these results as a percentage of injected/uninjected and found that animals injected with AAV-A53T had a 69% reduction compared to the AAV-EV animals (Supplementary Figure 1C).
Figure 1: Workflow of the method. Workflow demonstrating the steps required to inject AAVs, section and stain tissue, define a region of interest and optimize the software for counting of cells. Representative images of confocal tile scan, ROI definition, and quantitation of cells. Scale bar = 100 μm. Please click here to view a larger version of this figure.
Figure 2: Defining the region of interest. (A) Coronal brain section, including the SNpc immunostained for TH (green) from a rat injected with AAV-A53T α-syn. In rats with severe neurodegeneration (such as shown here), it can be difficult to identify the SNpc. (B) Temporarily increasing the absorption of the image can identify the structure and allow an accurate identification of the region of interest. Scale bar = 1 mm. Please click here to view a larger version of this figure.
Figure 3: Optimizing cell detection using the Cytonuclear method in HALO. Real-time tuning of the cytonuclear module allows the user to see changes in cell detection in real-time by altering Nuclear Contrast Threshold, Minimum Nuclear Intensity, Nuclear Segmentation Aggressiveness, and Nuclear Size. (A) Representative image with region of interest displayed. (B) Real-time tuning showing under-sampling in which the software does not detect all cells in the tuning window. (C) Over-sampling in which the software detects more cells than are evident in the tuning window. (D) Optimized tuning in which the correct number of cells are counted. Scale bar = 500 μm. Please click here to view a larger version of this figure.
Figure 4: HALO optimized settings within a defined region of interest. Representative images of completed analysis using optimized settings for cytonuclear detection in HALO. Scale bar = 500 μm. Please click here to view a larger version of this figure.
Figure 5: Expression of human mutant A53T α-syn in SNpc results in severe neurodegeneration at 6 weeks as quantified by HALO and unbiased stereology. (A) Representative images showing degeneration of TH-positive neurons in the SNpc 6 weeks after stereotactic injection of AAV-A53T α-syn at a titer of 3.4 x 1012 viral particles/mL. Immunofluorescent staining with anti-TH (green) and anti-α-syn (red) antibodies. Scale bar = 200 µm. (B) Quantification of the number of TH-positive neurons in the SNpc of rats injected with AAV-A53T α-syn or AAV-EV demonstrates that expression of mutant α-syn results in significant dopaminergic neuron loss. Unpaired t-test; n = 5 rats/group. Graph shows mean ± SEM, ***P < 0.001. (C) Representative images of colorimetric staining of dopaminergic neurons in the SNpc of AAV-A53T (left) or AAV-EV (right) injected rats used to perform unbiased stereology. Scale bar = 200 µm. (D) A significant correlation between HALO counting of TH-positive neurons/mm2 (y-axis) and unbiased stereology cell numbers (x-axis). Pearson correlation (r = 0.8819, P = 0.0007). Please click here to view a larger version of this figure.
Supplementary Figure 1: Significant unilateral neurodegeneration is observed in the SNpc of rats who received AAV-A53T injection. (A) Quantification of the number of TH-positive neurons in the injected or uninjected SNpc of rats that received a unilateral AAV-A53T stereotactic injection shows a significant decrease on the injected side. (B) Quantification of the number of TH-positive neurons in the injected or uninjected SNpc of rats that received unilateral AAV-EV stereotactic injection shows no significant changes. (C) Normalization to the uninjected contralateral side demonstrate a >50% decrease upon injection with AAV-A53T α-syn compared to AAV-EV. Unpaired t-test; n = 5 rats/group. Graphs show mean ± SEM, *P < 0.05, ***P < 0.001. Please click here to download this Supplementary Figure.
A-Syn Antibody | ThermoFisher Scientific | 32-8100 | |
ABC Elite | Vector Labs | PK-6102 | |
Alexa Fluor 488 secondary antibody | ThermoFisher Scientific | A-11008 | |
Alexa Fluor 555 secondary antibody | ThermoFisher Scientific | A-28180 | |
Alkaline phosphatase-conjugated anti-rabbit igG | Jackson Immuno | 111-055-144 | |
Biotinylated anti-mouse IgG | Vector Labs | BA-9200 | |
Bovine Serum Albumin | Sigma | A2153 | |
DAKO fluorescent mouting medium | Agilent | S3023 | |
HALO™ | Indica Labs | ||
Histo-Clear II | Diamed | HS202 | |
ImmPACT DAB Peroxidase substrate | Vector Labs | SK-4105 | |
LSM880 Confocal Microscope | Zeiss | ||
NeuN Antibody | Millipore | MAB377 | |
Normal Goat Serum | Vector Labs | S-1000-20 | |
OCT | Tissue-Tek | ||
Paraformaldehyde | BioShop | PAR070.1 | |
Sliding microtome | Leica | SM2010 R | |
Stereo Investigator | MBF Bioscience | ||
Sucrose | BioShop | SUC700 | |
TH Antibody | ThermoFisher Scientific | P21962 | |
VectaMount mounting medium | Vector Labs | H-5000 | |
Vector Blue Alkaline Phosphatase substrate | Vector Labs | SK-5300 | |
Zen Black Software | Zeiss | ||
Zen Blue Software | Zeiss |
Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson’s disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant α-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.
Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson’s disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant α-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.
Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson’s disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant α-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.