COMPREHENSIVE NEUROAIDS CENTER (cnac)
Basic Science Core II: Animal Models, Behavioral Testing, Pathology, and Histology
This core assists researchers studying AIDS and brain disorders to utilize animal models for evaluating therapeutics and behavioral tests for improving treatments diagnosis and prevention. Moreover, this core provides services to researchers in analyzing human samples and animal tissues for improving diagnosis of disease and understanding the mechanisms involved in the pathogenesis of neuroAIDS.
We assist investigators in the development, analysis, and characterization of animal models with impaired nervous system function related to HIV-1 and AIDS, including phenotypic characterization of animals at the anatomical level and full-scale behavioral characterization of animals including memory, learning, and other cognitive functions, sensory and motor function, development and aging, neurological and neuromuscular assessment.
For additional information on animal models, please contact Jennifer Gordon, PhD.
For additional information on animal behavioral testing, please contact Mary Barbe, PhD.
We offer histological and immunohistochemical services for the evaluation of human autopsy and biopsy clinical samples, tissues harvested from experimental animal models, and cell cultures. More specifically, this core performs neuropathological evaluation of tissues from animal models and patients with neurological disorders including AIDS, PML, CNS neoplasia, and other neurodegenerative diseases, including peripheral neuropathies. Further, this core provides training in Laser Capture Microdissection (LCM) accessioning of tissue samples and nucleic acid extraction for downstream molecular biological applications, immunohistochemical labeling, photodocumentation, and quantitative analysis of neuronal density and expression of cellular and viral proteins using image analysis systems and methods. Through its microscope facilities, this core assists in training and use of the image capture and analysis techniques. In service hands-on training or seminars on core techniques are held from time to time. Please visit the CNAC website for upcoming sessions.
To schedule a neuropathology consultation, please email email@example.com. Consultations are available by appointment each week.
The Comprehensive NeuroAIDS Center (CNAC) Basic Science Core II: Animal Models, Behavioral Testing, Pathology, and Histology now has BIOQUANT LIFE SCIENCE Biomedical Research System software for quantification of findings from animal model and human biopsy and/or autopsy studies. Primary applications include developmental neuroscience; neuropathological changes in peripheral nerves brain and spinal cord; thresholded pixel counts of immunohistochemistry as well as immunofluorescence; thickness of layers, such as retinal layers or cortical gray or white matter; axon number and caliber; cellular mRNA (in situ hybridization) quantification; cell mapping and analysis; vascular cross section analysis, and more. Data can be collected either from scanned digital slides or from live imaging of slides on a microscope.
Mary Barbe, PhD, can be contacted for provide training in this software (firstname.lastname@example.org) and to further discuss how this software can be used to meet your research needs. The BIOQUANT software also has built-in procedural guides for ease of use, automated batch measurement as well as manual measurement capabilities, and pre-defined calculations for many quantification procedures.
This software can be used to quantify neuroinflammation using a semi-automated cell count method or by quantifying immunohistochemical and immunofluorescence labeling of activated macrophages, microglia, astrocytes, and more. These methods can be used to quantify neuroinflammatory and fibrotic responses in spinal cord, brain and peripheral nerves, such as shown in the figure at right in which epi-perineurium thickness was assayed as neurofibrosis, activated macrophages (labeled with CD68/ED-1) and activated T-cells (immunolabeled with TCR-αβ) increased over time, in a peripheral nerve neuritis model (Bove et al, J Neuroimmunol, 2009). A similar layer thickness method as shown in the above figure can be used to quantify the thickness of the corpus callosum in brain white matter injury model or loss of height of cortical gray matter.
The software can detect and separately quantify any color, as shown at left from Delcour et al, Behav Brain Res. 2012 15;232(1):233-44.Using a consistent "expression threshold," BIOQUANT rapidly quantifies the area of protein expression in specific anatomical regions. Comparisons between regions are done on the basis of area fraction of immunoreactive tissue. When using immunofluorescent markers, the expression level is quantified both by label intensity and area of expression. Similarly, cell counting can be performed automatically based on expression intensity.
Axon number and caliber can be quantified with this software. In semi-thin sections with myelin specific staining, BIOQUANT automatically detects myelinated axons. Detection is based on color, shape, and size. Shape filters automatically exclude obliquely cut axons. In the case of very small axons or partially myelinated axons, some manual editing may be necessary. A unique object recognition algorithm prevents the system from double-counting when scanning through adjacent overlapping fields of view. Calculated Indices for axon number and caliber include: nerve area, individual axon area, individual axon caliber, axon count, axon density (#/unit area), mean axon area and mean axon caliber.
With regard to cellular mRNA quantification, one can use the BIOQUANT Life Science software following S35 radiolabeling and silver grain emulsion dipping of slides to identify mRNA signals in tissue sections (such as shown at right and in Barbe et al, J Neuroscience Research, 1994). Using either standardized circular regions or free-drawn boundaries, BIOQUANT can analyze the degree of grain labeling in a cell population. Grains are automatically detected by color thresholding, and the software then calculates the grain density for each cell. Cells with a grain density more than five fold over the background grain density are considered significantly labeled. Calculated Indices include: Mean Grain Size, Estimated Background Grain Density, Estimated Background Grain Number, % Cellular Grain Area, Estimated Cellular Grain Number, Estimated Cellular Grain Density, and Cellular Grain Density Ratio.
Cell mapping and analysis is performed using automated tracing algorithms, in which BIOQUANT measures cross-sectional area in serially sectioned volumes of tissue. Based on cell-specific staining, BIOQUANT automatically counts cells within the volume of tissue. The contour and location of each cell is also recorded for use in the reconstruction, such as shown at right from Ruggieri et al, Neurourology and Urodynamics, 2011. Computed data include: Region Area / Volume, Cell Area, Cell Number, and Cellular Stain Intensity.
More information can be found at: http://lifescience.bioquant.com/. At this site, one can also find Sample Protocols (http://lifescience.bioquant.com/common-protocols). If your application is not listed, Dr. Mary Barbe can be consulted to aid development of a protocol for your application.
Jennifer Gordon, PhD
Mary F. Barbe, PhD
Basic Science Core II Co-Leader
Animal behavioral testing
Department of Anatomy and Cell Biology
Temple University School of Medicine
3500 N. Broad Street, 4th Fl. MERB