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Neurostereology protocol for unbiased quantification of neuronal injury and neurodegeneration.

Golub VM, Brewer J, Wu X, Kuruba R, Short J, Manchi M, Swonke M, Younus I, Reddy DS - Front Aging Neurosci (2015)

Bottom Line: Regional tissue volume was determined by using the Cavalieri estimator, as well as cell density and cell number were determined by using the optical disector and optical fractionator.The protocol has greater predictive power for absolute counts as it is based on 3D features rather than 2D images.The total neuron counts were consistent with literature values from sophisticated systems, which are more expensive than our stereology system.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science Center, College of Medicine Bryan, TX, USA.

ABSTRACT
Neuronal injury and neurodegeneration are the hallmark pathologies in a variety of neurological conditions such as epilepsy, stroke, traumatic brain injury, Parkinson's disease and Alzheimer's disease. Quantification of absolute neuron and interneuron counts in various brain regions is essential to understand the impact of neurological insults or neurodegenerative disease progression in animal models. However, conventional qualitative scoring-based protocols are superficial and less reliable for use in studies of neuroprotection evaluations. Here, we describe an optimized stereology protocol for quantification of neuronal injury and neurodegeneration by unbiased counting of neurons and interneurons. Every 20th section in each series of 20 sections was processed for NeuN(+) total neuron and parvalbumin(+) interneuron immunostaining. The sections that contain the hippocampus were then delineated into five reliably predefined subregions. Each region was separately analyzed with a microscope driven by the stereology software. Regional tissue volume was determined by using the Cavalieri estimator, as well as cell density and cell number were determined by using the optical disector and optical fractionator. This protocol yielded an estimate of 1.5 million total neurons and 0.05 million PV(+) interneurons within the rat hippocampus. The protocol has greater predictive power for absolute counts as it is based on 3D features rather than 2D images. The total neuron counts were consistent with literature values from sophisticated systems, which are more expensive than our stereology system. This unbiased stereology protocol allows for sensitive, medium-throughput counting of total neurons in any brain region, and thus provides a quantitative tool for studies of neuronal injury and neurodegeneration in a variety of acute brain injury and chronic neurological models.

No MeSH data available.


Related in: MedlinePlus

A computer-driven stereology system. Microscope automation controller manages stage movements (XYZ) of Olympus BX53 microscope, fluorescence filter and encoder for stereology newCAST program. For epi-fluorescence, the fluorescence illumination, filter wheels and specific CCD camera system (ORCA) are shown.
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Figure 1: A computer-driven stereology system. Microscope automation controller manages stage movements (XYZ) of Olympus BX53 microscope, fluorescence filter and encoder for stereology newCAST program. For epi-fluorescence, the fluorescence illumination, filter wheels and specific CCD camera system (ORCA) are shown.

Mentions: The stereology system consists of an Olympus BX53 microscope (Olympus, Tokyo, Japan) fixed with a DP73 cooled digital color camera (Model: DP73-1-51, Olympus, Tokyo, Japan) or ORCA-R2 digital CCD camera (Hamamatsu, Hamamatsu City, Japan) for immunofluorescence images (Figure 1). A motorized stage (Model: H101ANNI, Prior Scientific, Rockland, MA, USA) controlled by universal microscope automation controller with encoder (Model: 500-H31XYZEF, ProScan III, Prior Scientific, Rockland, MA, USA) and Proscan III joystick (Model: P-PS3J100, Prior Scientific, Rockland, MA, USA) makes the protocol and hardware more user-friendly. The BX53 microscope is fixed with a 1.25× objective (PLAPON1.25×, numerical aperture (NA) = 0.04, working distance (WD) = 5.1 mm, Olympus), 10× objective (UPLSAPO10X2, NA = 0.4, WD = 3.1 mm, Olympus), 20× objective (UPLSAPO20XNA = 0.75, WD = 0.65 mm, Olympus) and 60× oil immersion objective (UPLSAPO60XO, NA = 1.35, WD = 0.15 mm, Olympus). Olympus immersion oil type-F (IMMOIL-F30CC, Olympus) was used with the 60× oil immersion objective. Appropriate cleaning supplies should be readily available for use such as lens cleaning solution, lens paper, and 99% ethanol for cleaning the slides. The stereology software used in this protocol is newCAST (Version: VIS4.6.1.630, Visiopharm, Denmark). A calibration slide with a standard Visiopharm calibration grid embedded on the glass sheet in the center is included with the newCAST VIS software. The calibration slide is to be used for calibrating the center of the view and size of the field-of-view under a series of magnifying lenses in the microscope. The calibration step has to be done before any stereology procedure.


Neurostereology protocol for unbiased quantification of neuronal injury and neurodegeneration.

Golub VM, Brewer J, Wu X, Kuruba R, Short J, Manchi M, Swonke M, Younus I, Reddy DS - Front Aging Neurosci (2015)

A computer-driven stereology system. Microscope automation controller manages stage movements (XYZ) of Olympus BX53 microscope, fluorescence filter and encoder for stereology newCAST program. For epi-fluorescence, the fluorescence illumination, filter wheels and specific CCD camera system (ORCA) are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4628120&req=5

Figure 1: A computer-driven stereology system. Microscope automation controller manages stage movements (XYZ) of Olympus BX53 microscope, fluorescence filter and encoder for stereology newCAST program. For epi-fluorescence, the fluorescence illumination, filter wheels and specific CCD camera system (ORCA) are shown.
Mentions: The stereology system consists of an Olympus BX53 microscope (Olympus, Tokyo, Japan) fixed with a DP73 cooled digital color camera (Model: DP73-1-51, Olympus, Tokyo, Japan) or ORCA-R2 digital CCD camera (Hamamatsu, Hamamatsu City, Japan) for immunofluorescence images (Figure 1). A motorized stage (Model: H101ANNI, Prior Scientific, Rockland, MA, USA) controlled by universal microscope automation controller with encoder (Model: 500-H31XYZEF, ProScan III, Prior Scientific, Rockland, MA, USA) and Proscan III joystick (Model: P-PS3J100, Prior Scientific, Rockland, MA, USA) makes the protocol and hardware more user-friendly. The BX53 microscope is fixed with a 1.25× objective (PLAPON1.25×, numerical aperture (NA) = 0.04, working distance (WD) = 5.1 mm, Olympus), 10× objective (UPLSAPO10X2, NA = 0.4, WD = 3.1 mm, Olympus), 20× objective (UPLSAPO20XNA = 0.75, WD = 0.65 mm, Olympus) and 60× oil immersion objective (UPLSAPO60XO, NA = 1.35, WD = 0.15 mm, Olympus). Olympus immersion oil type-F (IMMOIL-F30CC, Olympus) was used with the 60× oil immersion objective. Appropriate cleaning supplies should be readily available for use such as lens cleaning solution, lens paper, and 99% ethanol for cleaning the slides. The stereology software used in this protocol is newCAST (Version: VIS4.6.1.630, Visiopharm, Denmark). A calibration slide with a standard Visiopharm calibration grid embedded on the glass sheet in the center is included with the newCAST VIS software. The calibration slide is to be used for calibrating the center of the view and size of the field-of-view under a series of magnifying lenses in the microscope. The calibration step has to be done before any stereology procedure.

Bottom Line: Regional tissue volume was determined by using the Cavalieri estimator, as well as cell density and cell number were determined by using the optical disector and optical fractionator.The protocol has greater predictive power for absolute counts as it is based on 3D features rather than 2D images.The total neuron counts were consistent with literature values from sophisticated systems, which are more expensive than our stereology system.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science Center, College of Medicine Bryan, TX, USA.

ABSTRACT
Neuronal injury and neurodegeneration are the hallmark pathologies in a variety of neurological conditions such as epilepsy, stroke, traumatic brain injury, Parkinson's disease and Alzheimer's disease. Quantification of absolute neuron and interneuron counts in various brain regions is essential to understand the impact of neurological insults or neurodegenerative disease progression in animal models. However, conventional qualitative scoring-based protocols are superficial and less reliable for use in studies of neuroprotection evaluations. Here, we describe an optimized stereology protocol for quantification of neuronal injury and neurodegeneration by unbiased counting of neurons and interneurons. Every 20th section in each series of 20 sections was processed for NeuN(+) total neuron and parvalbumin(+) interneuron immunostaining. The sections that contain the hippocampus were then delineated into five reliably predefined subregions. Each region was separately analyzed with a microscope driven by the stereology software. Regional tissue volume was determined by using the Cavalieri estimator, as well as cell density and cell number were determined by using the optical disector and optical fractionator. This protocol yielded an estimate of 1.5 million total neurons and 0.05 million PV(+) interneurons within the rat hippocampus. The protocol has greater predictive power for absolute counts as it is based on 3D features rather than 2D images. The total neuron counts were consistent with literature values from sophisticated systems, which are more expensive than our stereology system. This unbiased stereology protocol allows for sensitive, medium-throughput counting of total neurons in any brain region, and thus provides a quantitative tool for studies of neuronal injury and neurodegeneration in a variety of acute brain injury and chronic neurological models.

No MeSH data available.


Related in: MedlinePlus