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A semiautomated targeted sampling method to assess optic nerve axonal loss in a rat model of glaucoma

Nature Protocols volume 5, pages 1642–1651 (2010)Cite this article

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Abstract

We have developed a fast, reliable and easily reproducible semiautomated quantitative damage grading scheme to assess axonal loss in the optic nerve after inducing ocular hypertension using a laser glaucoma model in adult rats. This targeted sampling method has been validated against complete axon counts, and compares favorably with a conventional, random sampling, semiquantitative method. In addition, we present a standardized method to quantify axons in a semiautomated way, using freely available ImageJ software, and describe in detail the method used to induce glaucoma. Our techniques can be easily implemented in any laboratory, thanks to the public availability of the software and the simplicity of the method. Depending on the number of animals used in a particular study, the whole process from experimental elevation of intraocular pressure to tissue processing and data analysis should take ∼40 d.

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Figure 1: IOP profile (mm Hg) of rats subjected to experimental glaucoma by trabecular meshwork photocoagulation.
Figure 2: Identification of zones with apparently homogenous damage in an optic nerve cross-section.
Figure 3: Correlation between axon quantification methods.

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Acknowledgements

K.R.M. holds a GlaxoSmithKline Clinician-Scientist Award. N.D.B. is supported by Fight for Sight (UK). We thank A. Hyatt for excellent technical assistance.

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Authors and Affiliations

  1. Centre for Brain Repair, Cambridge University, Cambridge, UK

    Nephtali Marina, Natalie D Bull & Keith R Martin

  2. UCL Neuroscience, Physiology and Pharmacology, University College London, London, UK

    Nephtali Marina

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  1. Nephtali Marina
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  2. Natalie D Bull
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Contributions

N.M. implemented and designed the protocol and performed experiments, analyzed data and wrote the paper; N.D.B. performed interobserver analysis and edited the paper; K.R.M designed and performed the experiments, analyzed data and provided critical discussion toward the writing of the paper.

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Correspondence to Keith R Martin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data

IOP calculations. An excel spreadsheet shows IOP values in a group of control rats that received intravitreal injections of a vehicle solution (0.1M PBS) 2 weeks before laser treatment. The following measures of IOP exposure were calculated for each eye: mean IOP, peak IOP, and positive integral IOP. (XLS 33 kb)

Supplementary Video 1

Experimental IOP elevation in anaesthetised rats. External translimbal treatment to the aqueous outflow area was delivered with a 532-nm laser. The laser beam is directed to the junction between the clear cornea and the sclera in order to treat the trabecular meshwork. Initial treatment is 40 to 60 spots of 50 µm size, 0.4-W power, and 0.6-second duration. (WMV 13435 kb)

Supplementary Fig. 1

Validation of automated axonal counts using Image J. The number of axons in the same micrograph taken from a glaucomatous optic nerve was evaluated with the automated “nucleus counter” plugin and with the manual “cell counter” plugin. The number of axons obtained by the ImageJ manual cell counter plugin was very similar to the counts obtained on the same image by the automated plugin nucleus counter (723 vs 755 axons, respectively). (PDF 8340 kb)

Supplementary Slideshow 1

Image capture and processing. A representative optic nerve cross section is presented to illustrate each step of the image capturing and digital processing in ImageJ. (PPT 8799 kb)

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Marina, N., Bull, N. & Martin, K. A semiautomated targeted sampling method to assess optic nerve axonal loss in a rat model of glaucoma. Nat Protoc 5, 1642–1651 (2010). https://doi.org/10.1038/nprot.2010.128

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