A Rapid and Sensitive Automated Image‐Based Approach for In Vitro and In Vivo Characterization of Cell Morphology and Quantification of Cell Number and Neurite Architecture

Victor Tapias1, J. Timothy Greenamyre2

1 Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh, Pittsburgh, Pennsylvania, 2 Pittsburgh VA Healthcare System, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.33
DOI:  10.1002/0471142956.cy1233s68
Online Posting Date:  April, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Stereological methods for tissue cell counting, specifically for neuron quantification, decrease systematic error and sampling bias; however, they are tedious, labor intensive, and time consuming. Approaches for cell (neuron) quantification in vitro are not accurate, sensitive, or subsequently reproducible. Neuronal phenotype is related to alterations in cell morphology and neurite pattern. The techniques currently available for quantification of these features present several limitations. In this unit, we provide validated automated procedures for in vivo and in vitro quantification of cell number, morphological cell changes, and neurite morphometry in a fast, simple, and reliable manner. Our method counts up to 8 times as many neurons in less than 5% to 10% of the time required for stereological analysis (optical fractionator). In summary, this technology offers an unparalleled opportunity to examine features of cells at high resolution in a complex three‐dimensional environment. These techniques provide an exceptional in vivo and in vitro system for neurotoxicity studies, disease modeling, and drug discovery. Curr. Protoc. Cytom. 68:12.33.1‐12.33.22. © 2014 by John Wiley & Sons, Inc.

Keywords: neuroprotection; neurodegeneration; neurotoxicity; dendrites; 3‐D; reconstruction; neuron; neurites; morphology; quantification

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Immunofluorescence Quantification of Neurons and Astrocytes In Vivo
  • Basic Protocol 2: Immunofluorescence Quantification of Neurons In Vitro
  • Basic Protocol 3: Cell Morphology Assessment In Vivo
  • Basic Protocol 4: Assessment of Neurite Morphometry In Vivo and In Vitro
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Immunofluorescence Quantification of Neurons and Astrocytes In Vivo

  • 6‐ to 7‐month‐old male Lewis rats
  • Rotenone (see recipe)
  • 4% (w/v) paraformaldehyde in PBS (see appendix 2A for PBS)
  • 30% (w/v) sucrose
  • Cryoprotectant solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1% (v/v) Triton X‐100 in PBS (see appendix 2A for PBS)
  • Blocking solution: phosphate buffered saline (PBS; appendix 2A) containing 10% (v/v) normal (donkey) serum and 0.3% (v/v) Triton X‐100
  • Antibody diluent: 1% normal donkey serum and 0.3% (v/v) Triton X‐100 in PBS
  • Primary antibodies:
    • Mouse monoclonal antibody for microtubule associated protein 2 (MAP2; Millipore, cat. no. MAB378)
    • Sheep polyclonal antibody for tyrosine hydroxylase (TH; Millipore, cat. no. AB1542)
    • Rabbit polyclonal antibody for glial fibrillary acidic protein (GFAP; Millipore, cat. no. AB5804)
  • Secondary antibodies:
    • Cy3‐conjugated anti‐sheep antibody (Jackson ImmunoResearch, cat. no. 713–165–003)
    • Alexa Fluor 647–conjugated anti‐mouse antibody (Invitrogen, cat. no. A31571)
    • Alexa Fluor 488–conjugated anti‐rabbit antibody (Invitrogen, cat. no. A21206)
  • BisBenzimide H 33342 fluorochrome trihydrochloride dye (Sigma‐Aldrich, cat. no. B2261)
  • Mounting medium (e.g., Gelvatol)
  • Surgical instruments: scissors, forceps, #11 scalpel blade, spatula, razor blades
  • Glass petri dish
  • Freezing sliding microtome (unit 12.15)
  • 24‐well plates
  • 6‐well plates
  • Bench‐top agitator
  • Plus‐coated microscope slides (e.g., Fisher Scientific, cat. no. 12‐550‐15)
  • Glass coverslips (Fisher Scientific, cat. no. 12‐545‐82)
  • Automated upright fluorescence microscope (Nikon Eclipse 90i, cat. no. M319E04)
  • Cooled CCD camera (QImaging; Retiga EXi Fast 1394)
  • Linear Encoder System (Renishaw RGH22, cat. no. Y30D00)
  • Additional reagents and equipment for injection (Donovan and Brown, ) and euthanasia (Donovan and Brown, ) of rodents, cryosectioning (unit 12.15), and microscopy/data analysis ( protocol 1)

Basic Protocol 2: Immunofluorescence Quantification of Neurons In Vitro

  • 2‐ to 3‐month‐old day‐17 pregnant female Sprague‐Dawley rats
  • Neurobasal medium (see recipe)
  • 0.25% (1×) trypsin‐EDTA (Life Technologies, cat. no. 25200‐056)
  • 0.1 mg/ml poly‐D‐lysine hydrobromide (PDL; Sigma, cat. no. P7280)
  • Complete minimal essential medium (complete MEM; see recipe)
  • Neurobasal medium (see recipe)
  • Glial cell–derived neurotrophic factor (GDNF)
  • Rotenone (see recipe)
  • 4% (w/v) paraformaldehyde in PBS (see appendix 2A for PBS) supplemented with 0.02% (v/v) Triton X‐100 and 1 mM MgCl 2
  • 10% normal donkey serum in PBS (see appendix 2A for PBS)
  • Mounting medium (e.g., Gelvatol)
  • Surgical instruments (scissors and forceps)
  • Petri dishes
  • Circular glass coverslips
  • 24‐well tissue culture plates
  • Plus‐coated microscope slides (Fisher Scientific, cat. no. 12‐550‐15)
  • Automated upright fluorescence microscope (Nikon Eclipse 90i, cat. no. M319E04)
  • Cooled CCD camera (QImaging; Retiga EXi Fast 1394)
  • Linear Encoder System (Renishaw RGH22, cat. no. Y30D00)
  • Additional reagents and equipment for preparing primary ventral midbrain culture (Studer, ), counting cells with a hemacytometer ( appendix 3A), and determination of cell viability by trypan blue exclusion ( appendix 3B)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
PDF or HTML at Wiley Online Library


Literature Cited

Literature Cited
  Al‐Kofahi, K.A., Lasek, S., Szarowski, D.H., Pace, C.J., Nagy, G., Turner, J.N., and Roysam, B. 2002. Rapid automated three‐dimensional tracing of neurons from confocal image stacks. IEEE Trans. Inf. Technol. Biomed. 6:171‐187.
  Betarbet, R., Sherer, T.B., MacKenzie, G., Garcia‐Osuna, M., Panov, A.V., and Greenamyre, J.T. 2000. Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nat. Neurosci. 3:1301‐1306.
  Caiazzo, M., Dell'Anno, M.T., Dvoretskova, E., Lazarevic, D., Taverna, S., Leo, D., Sotnikova, T.D., Menegon, A., Roncaglia, P., Colciago, G., Russo, G., Carninci, P., Pezzoli, G., Gainetdinov, R.R., Gustincich, S., Dityatev, A., and Broccoli, V. 2011. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476:224‐227.
  Cannon, J.R., Tapias, V., Na, H.M., Honick, A.S., Drolet, R.E., and Greenamyre, J.T. 2009. A highly reproducible rotenone model of Parkinson's disease. Neurobiol. Dis. 34:279‐290.
  Cannon, J.R., Geghman, K.D., Tapias, V., Sew, T., Dail, M.K., Li, C., and Greenamyre, J.T 2013. Expression of human E46K‐mutated alpha‐synuclein in BAC‐transgenic rats replicates early‐stage Parkinson's disease features and enhances vulnerability to mitochondrial impairment. Exp. Neurol. 240:44‐56.
  Carlo, C.N., Stefanacci, L., Semendeferi, K., and Stevens, C.F. 2010. Comparative analyses of the neuron numbers and volumes of the amygdaloid complex in old and new world primates. J. Comp. Neurol. 518:1176‐1198.
  Cohen, A.R., Roysam, B., and Turner, J.N. 1994. Automated tracing and volume measurements of neurons from 3‐D confocal fluorescence microscopy data. J. Microsc. 173:103‐114.
  Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
  Donovan, J. and Brown, P. 2006b. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
  Gao, H.M., Zhou, H., Zhang, F., Wilson, B.C., Kam, W., and Hong, J.S. 2011. HMGB1 acts on microglia Mac1 to mediate chronic neuroinflammation that drives progressive neurodegeneration. J. Neurosci. 31:1081‐1092.
  Groszer, M., Erickson, R., Scripture‐Adams, D.D., Lesche, R., Trumpp, A., Zack, J.A., Kornblum, H.I., Liu, X., and Wu, H. 2001. Negative regulation of neural stem/progenitor cell proliferation by the Pten tumor suppressor gene in vivo. Science 294:2186‐2189.
  Gundersen, H.J. and Jensen, E.B. 1987. The efficiency of systematic sampling in stereology and its prediction. J. Microsc. 147:229‐263.
  Gundersen, H.J., Jensen, E.B., Kieu, K., and Nielsen, J. 1999. The efficiency of systematic sampling in stereology—reconsidered. J. Microsc. 193:199‐211.
  Kermer, P., Krajewska, M., Zapata, J.M., Takayama, S., Mai, J., Krajewski, S., and Reed, J.C. 2002. Bag1 is a regulator and marker of neuronal differentiation. Cell Death Differ. 9:405‐413.
  LaFerla, F.M., Troncoso, J.C., Strickland, D.K., Kawas, C.H., and Jay, G. 1997. Neuronal cell death in Alzheimer's disease correlates with apoE uptake and intracellular A‐beta stabilization. J. Clin. Invest. 100:310‐320.
  Longair, M.H., Baker, D.A., and Armstrong, J.D. 2011. Simple Neurite Tracer: open source software for reconstruction, visualization and analysis of neuronal processes. Bioinformatics 27:2453‐2454.
  Matheron, G. 1971. The theory of regionalized variables and its applications. École Nationale Supérieure des Mines de Paris. 211. Available at http://cg.ensmp.fr/bibliotheque/public/MATHERON_Ouvrage_00167.pdf.
  Mattson, M.P. 2000. Apoptosis in neurodegenerative disorders. Nat. Rev. Mol. Cell Biol. 1:120‐129.
  Morita, A., Yamashita, N., Sasaki, Y., Uchida, Y., Nakajima, O., Nakamura, F., Yagi, T., Taniguchi, M., Usui, H., Katoh‐Semba, R., Takei, K., and Goshima, Y. 2006. Regulation of dendritic branching and spine maturation by semaphorin3A‐Fyn signaling. J. Neurosci. 26:2971‐2980.
  Peng, H., Ruan, Z., Long, F., Simpson, J.H., and Myers, E.W. 2010. V3D enables real‐time 3D visualization and quantitative analysis of large‐scale biological image data sets. Nat. Biotechnol. 28:348‐353.
  Petrinovic, M.M., Hourez, R., Aloy, E.M., Dewarrat, G., Gall, D., Weinmann, O., Gaudias, J., Bachmann, L.C., Schiffmann, S.N., Vogt, K.E., and Schwab, M.E. 2013. Neuronal Nogo‐A negatively regulates dendritic morphology and synaptic transmission in the cerebellum. Proc. Natl. Acad. Sci. U.S.A. 110:1083‐1088.
  Salthun‐Lassalle, B., Hirsch, E.C., Wolfart, J., Ruberg, M., and Michel, P.P. 2004. Rescue of mesencephalic dopaminergic neurons in culture by low‐level stimulation of voltage‐gated sodium channels. J. Neurosci. 24:5922‐5930.
  Saxena, S. and Caroni, P. 2007. Mechanisms of axon degeneration: From development to disease. Prog. Neurobiol. 83:174‐191.
  Studer, L. 1997. Culture of substantia nigra neurons. Curr. Protoc. Neurosci. 00:3.3.1‐3.3.12.
  Tapias, V., Cannon, J.R., and Greenamyre, J.T. 2010. Melatonin treatment potentiates neurodegeneration in a rat rotenone Parkinson's disease model. J. Neurosci. Res. 88:420‐427.
  Tapias, V., Greenamyre, J.T., and Watkins, S.C. 2013. Automated imaging system for fast quantitation of neurons, cell morphology and neurite morphometry in vivo and in vitro. Neurobiol. Dis. 54:158‐168.
  Tapias, V., Cannon, J.R., and Greenamyre, J.T. 2014. Pomegranate juice exacerbates oxidative stress and nigrostriatal degeneration in Parkinson's disease. Neurobiol. Aging 35:1162‐1176.
  Toulorge, D., Guerreiro, S., Hild, A., Maskos, U., Hirsch, E.C., and Michel, P.P. 2011. Neuroprotection of midbrain dopamine neurons by nicotine is gated by cytoplasmic Ca2+. FASEB J. 25:2563‐2573.
  Welsbie, D.S., Yang, Z., Ge, Y., Mitchell, K.L., Zhou, X., Martin, S.E., Berlinicke, C.A., Hackler, L. Jr., Fuller, J., Fu, J., Cao, L.H., Han, B., Auld, D., Xue, T., Hirai, S., Germain, L., Simard‐Bisson, C., Blouin, R., Nguyen, J.V., Davis, C.H., Enke, R.A., Boye, S.L., Merbs, S.L., Marsh‐Armstrong, N., Hauswirth, W.W., DiAntonio, A., Nickells, R.W., Inglese, J., Hanes, J., Yau, K.W., Quigley, H.A., and Zack, D.J. 2013. Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death. Proc. Natl. Acad. Sci. U.S.A. 110:4045‐4050.
  West, M.J., Slomianka, L., and Gundersen, H.J. 1991. Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat. Rec. 231:482‐497.
  Zhang, Y., Zhou, X., Degterev, A., Lipinski, M., Adjeroh, D., Yuan, J., and Wong, S.T. 2007. A novel tracing algorithm for high throughput imaging screening of neuron‐based assays. J. Neurosci. Methods 160:149‐162.
PDF or HTML at Wiley Online Library