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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

Illustration of morphological identification of principal neurons and interneurons in the brain. (A) NeuN(+) granule cells in the hippocampus dentate gyrus (DG) region. (B) NeuN(+) pyramidal cells in the hippocampus CA1 region. (C) PV(+) interneurons in the hippocampus DG region. (D) Cartoon illustration of link between principle neuron and interneurons in the brain.
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Figure 5: Illustration of morphological identification of principal neurons and interneurons in the brain. (A) NeuN(+) granule cells in the hippocampus dentate gyrus (DG) region. (B) NeuN(+) pyramidal cells in the hippocampus CA1 region. (C) PV(+) interneurons in the hippocampus DG region. (D) Cartoon illustration of link between principle neuron and interneurons in the brain.

Mentions: We optimized a stereology protocol to suit targeted neuron counts within the rat hippocampus (Table 2; Figure 3). Before starting any slide, a quality check was done in every section based on our standard expectations for general brain architecture and immunostaining pattern. If there was any deviation from that expectation, such as staining issues, the concerns were resolved by re-staining the sample in a new set of sections. After successful verification of the sections and software parameters, the principle and interneuron cell counts were performed using various in-built features of the program (Figure 4). Principle cells and interneurons were identified based on their typical morphology and their anatomical context (Figure 5). Our optimized protocol allowed an unbiased quantification of total neurons in the hippocampus subfields in approximately 3 h, with the possibility of the completion of up to three animals in a single day. The protocol was utilized for quick quantification of NeuN(+) total neurons and PV(+) interneurons in the hippocampus subfields CA1, CA2, CA3, DG and DH (Figure 6).


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)

Illustration of morphological identification of principal neurons and interneurons in the brain. (A) NeuN(+) granule cells in the hippocampus dentate gyrus (DG) region. (B) NeuN(+) pyramidal cells in the hippocampus CA1 region. (C) PV(+) interneurons in the hippocampus DG region. (D) Cartoon illustration of link between principle neuron and interneurons in the brain.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Illustration of morphological identification of principal neurons and interneurons in the brain. (A) NeuN(+) granule cells in the hippocampus dentate gyrus (DG) region. (B) NeuN(+) pyramidal cells in the hippocampus CA1 region. (C) PV(+) interneurons in the hippocampus DG region. (D) Cartoon illustration of link between principle neuron and interneurons in the brain.
Mentions: We optimized a stereology protocol to suit targeted neuron counts within the rat hippocampus (Table 2; Figure 3). Before starting any slide, a quality check was done in every section based on our standard expectations for general brain architecture and immunostaining pattern. If there was any deviation from that expectation, such as staining issues, the concerns were resolved by re-staining the sample in a new set of sections. After successful verification of the sections and software parameters, the principle and interneuron cell counts were performed using various in-built features of the program (Figure 4). Principle cells and interneurons were identified based on their typical morphology and their anatomical context (Figure 5). Our optimized protocol allowed an unbiased quantification of total neurons in the hippocampus subfields in approximately 3 h, with the possibility of the completion of up to three animals in a single day. The protocol was utilized for quick quantification of NeuN(+) total neurons and PV(+) interneurons in the hippocampus subfields CA1, CA2, CA3, DG and DH (Figure 6).

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