Published: January 10th, 2019
A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.
Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.
The examination of the visual pathway, as a part of the central nervous system, has been proven to be an effective starting point in addressing not only ophthalmologic1,2,3,4,5, but also neurologic6,7,8,9,10,11,12,13,14,15,16 questions. In recent years, OCT and OKR have been identified as useful analytic, non-invasive tools to evaluate a large variety of retinopathies and retinal manifestations in various rodent models17,18,19,20,21,22,23,24,25. OCT allows for fast and high resolution in vivo visualization of the retinal morphology and structure in mice and rats, with results in good accordance with histological sections of the animals retinae26. OKR constitutes a fast and robust method to quantitatively assess visual function.
Many OCT devices allow simultaneous confocal scanning laser ophthalmoscopy (cSLO) imaging with different wavelengths, which provides diagnostic information about retinal pathologies, i.e., visualization of lipofuscin deposits or alterations of the retinal pigment epithelium27. Furthermore, in vivo imaging of fluorescence labelled cells in transgenic animals is possible28,29,30,31,32. However, the application of OCT technology in rodent models is still challenging, mainly because of the small eye size. Several commercially available devices require adaptations and often a different size of holder is required to image the animals of different species. Additionally, animals require anesthesia for measurement.
OKR devices can be used to assess the visual function in rodents. The animals are placed on a platform in the center of an actual or virtual cylinder displaying a moving grating, which the animals track with reflexive head and neck movements. This optokinetic response is reduced or eliminated in the case of the reduction or loss of visual function.
The aim of this protocol is to present a manual for the measurement of retinal thickness using a commercially available OCT device with a custom holder providing inhalant anesthesia. The protocol illustrates how to analyze volume scans using the software provided by the manufacturer. For visual testing, the aim is to provide instructions on how to use a commercially available system to assess the OKR.
All animal procedures were performed in compliance with the experimental guidelines approved by the regional authorities (State Agency for Nature, Environment and Consumer Protection; reference number 84-02.04.2014.A059) and conform to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and the European directive 2010/63/EU on the protection of animals used for scientific purposes.
1. Confocal Scanning Laser Ophthalmoscopy-optical Coherence Tomography
NOTE: The protocol for cSLO-OCT measurement is adaptable for all strains of laboratory mice and rats.
2. Optokinetic Response
NOTE: In the following, a detailed manual for OKR measurements of mice and rats is provided, which can be adapted to individual specific needs.
Using 3rd generation OCT imaging in myelin oligodendrocyte glycoprotein (MOG) peptide induced experimental autoimmune encephalomyelitis (EAE) mouse models, high-resolution morphological sections of the mouse retina were obtained. Using this technology, the protective capacities of different substances were demonstrated17. The thickness values of the inner retinal layers (IRL) obtained are in good accordance with the numbers of retinal ganglion cells (RGC) obtained by histological staining of retinal wholemounts (Figure 4).
OKR monitoring provides a functional readout of the neurodegeneration seen by OCT. In these experiments, visual function assessed as spatial frequency by OKR, and neuroaxonal damage assessed as IRL thinning by OCT, were in close correlation17. Various protocols can be employed to examine the visual acuity by changing the spatial or temporal frequency, contrast sensitivity, orientation or speed of the moving grid. In the EAE model, an improved spatial frequency of 0.05 cycles/degree (c/d) of animals treated with substance 1 was detected compared to untreated MOG-EAE mice (Figure 5).
Figure 1: Custom holder for OCT measurement. (A) OCT imaging of a C57BL/6J mouse using the custom holder33 and (B) rotational axis around the rodent eye. Rotation in the transverse plane (left) and in the axial plane (right) is demonstrated. This figure has been modified from Dietrich, M. et al.33. Please click here to view a larger version of this figure.
Figure 2: OCT post acquisition analysis. "1, 2, 3 mm" ETDRS grid on the 25 B-scan volume protocol (left). The thickness of retinal layers is provided for the different retinal sectors by the software (right). Please click here to view a larger version of this figure.
Figure 3: OKR measurement of mice and stimulus settings. (A) Top view through the camera analyzing a C57BL/6J mouse on the platform in the chamber. (B) User interface and settings of the OKR software. Please click here to view a larger version of this figure.
Figure 4: C57BL/6J mice with MOG EAE show an attenuated disease course when treated with substance 1 compared to untreated controls. (A) The degeneration of the inner retinal layers is reduced (B) and the clinical EAE score is attenuated during the EAE course when substance 1 was administered. Mice were scored daily, and OCT measurements were performed monthly over a period of 120 days. The graphs represent the mean and standard error of at least ten animals per group. (*p < 0.05, ***p < 0.001, area under the curve compared by ANOVA with Dunnett's post hoc test). (C) The IRL thickness change is in good accordance with RGC loss (***p < 0.001, by ANOVA with Dunnett's post hoc test compared to MOG untreated mice). Please click here to view a larger version of this figure.
Figure 5: OKR measurement of C57BL/6J mice with MOG-EAE. (A) OKR reveals an improved visual acuity of animals treated with substance 1 compared to untreated MOG EAE mice measured by spatial frequency threshold testing over a period of 120 days. The graphs represent the mean and standard error of at least six animals per group (**p < 0.01, ***p < 0.001, area under the curve compared by ANOVA with Dunnett's post hoc test). (B) Image of a C57BL/6J mouse in the testing chamber. Please click here to view a larger version of this figure.
This protocol provides an instruction for the thickness measurements and the examination of visual function in rodents. Visual readouts are increasingly used in translational research18,26,38,39,40 and are easily transferable to clinical trials. The significant advantage of OCT in comparison to histological investigations in animal experiments is that longitudinal analyses are possible allowing the investigation of dynamic pathological processes, largely reducing the variability and the number of animals needed per study. Furthermore, in vivo imaging with OCT is not subject to fixation, cutting or staining artifacts, which may affect the layer thickness in histological investigations.
However, the orthogonal orientation of the laser beam in all planes in relation to the retina is a critical step to ensure the quality and reproducibility of the thickness values. It requires some training of the investigator and is mandatory before the acquisition of OCT scans. Additionally, as the commercial devices are built for human applications, the quality of rodent OCT images is still inferior compared to B-scans of human patients. In the authors' experience, it may be difficult to distinguish the different inner retinal layers (retinal nerve fiber layer, ganglion cell layer and inner plexiform layer) during manual correction. We therefore recommend analyzing these layers as a compound readout (IRL).
The experimental setup provides an option for volatile anesthesia, e.g., inhalant isoflurane, which is, in our experience, safer and easier to control than injectable anesthesia, e.g., ketamine-xylazine41,42 and reduces the risk of premature awakening of rodents in case of longer acquisition times (e.g., when performing imaging of fluorescently labelled cells). In a preliminary study, volume scans were identified as the protocols with the highest validity and reliability. The inter-rater and test retest reliability was excellent when volume scans excluding the central part containing the optic disc were assessed with ICC (intra-class correlation coefficient) values above 0.85 for all assessments.
The measurement of the optokinetic response is based on the involuntary optokinetic reflex, which occurs in response to a continuously moving field. In rodents, in contrast to other species, the movement involves not only the eyes, but the whole head, which can easily be detected using the camera.
Distinguishing between "tracking" or normal behavioral movements of the animals requires some training of the investigator and it is important to be blinded for the experimental group. In addition, the animals need an adaption phase to accommodate to the experimental setting and during long-time measurement protocols, the animals have to be animated repeatedly to assure that "no tracking" is due to reaching the OKR threshold and not to decreasing attention. There is also a significant strain variability regarding the visual function of laboratory mice and rats43,44. The visual acuity of the rodent should therefore be evaluated before they are tested and some strains, such as SJL mice, may not even be suitable for OKR measurements, as they are homozygous for the allele Pde6brd1 (retinal degeneration 1).
In summary, the examination of retinal morphology and visual function in animal models allows for non-invasive, longitudinal investigations of structural and functional damage occurring in the context of EAE and may be helpful in other models involving the visual system, including but not limited to the models of retinopathies or optic nerve injury.
Unrelated to the work presented the authors declare the following financial disclosures:
Michael Dietrich received speaker honoraria from Novartis. Andrés Cruz-Herranz is a postdoctoral fellow of the National Multiple Sclerosis Society. Ari J. Green served on the scientific advisory board of MedImmune, Novartis, OCTIMS, Inception 5 Biosciences, and Bionure; is an associate editor of JAMA Neurology; was an editorial board member of Neurology; holds a patent for remyelination molecules and pathways; consulted for Inception 5 Sciences; received research support from Novartis Pharma OCTIMs, Inception Sciences SRA, NINDS, NIA, National MS Society, Sherak Foundation, and Hilton Foundation; holds stock or stock options in Inception 5; and served as an expert witness at Mylan v Teva Pharma. Hans-Peter Hartung has received fees for serving on steering committees from Biogen Idec, GeNeuro, Sanofi Genzyme, Merck, Novartis Pharmaceuticals, Octapharma, Opexa Therapeutics, Teva Pharmaceuticals, MedImmune, Bayer HealthCare, Forward Pharma, and Roche, fees for serving on advisory boards from Biogen Idec, Sanofi Genzyme, Merck, Novartis Pharmaceuticals, Octapharma, Opexa Therapeutics, Teva Pharmaceuticals, and Roche, and lecture fees from Biogen Idec, Sanofi Genzyme, Merck, Novartis Pharmaceuticals, Octapharma, Opexa Therapeutics, Teva Pharmaceuticals, MedImmune, and Roche. Philipp Albrecht received compensation for serving on Scientific Advisory Boards for Ipsen, Novartis, Biogen; he received speaker honoraria and travel support from Novartis, Teva, Biogen, Merz Pharmaceuticals, Ipsen, Allergan, Bayer Healthcare, Esai, UCB and Glaxo Smith Kline; he received research support from Novartis, Biogen, Teva, Merz Pharmaceuticals, Ipsen, and Roche. The other authors report no disclosures.
This work was supported by grants of the Dr. Robert Pfleger-Foundation and the Ilselore Luckow-Foundation, as well as Biogen and Novartis to PA. Figure 1B was reproduced from "Whole-body positional manipulators for ocular imaging of anaesthetized mice and rats: A do-it-yourself guide. Dietrich, M., Cruz-Herranz, A., Yiu, H., Aktas, O., Brandt, A. U., Hartung, HP., Green, A., Albrecht, P. BMJ Open Ophthalmology. 1 (1), e000008, 2017" with permission from BMJ Publishing Group Ltd.
|Heidelberg Spectralis HRA+OCT system
|Heidelberg Engineering, Germany
|ophthalmic imaging platform system
|Heidelberg Eye Explorer
|Heidelberg Engineering, Germany
|blue 25D non-contact lens
|Heidelberg Engineering, Germany
|lens for rodent mesurement
|CerebralMechanics Inc., Canada
|system for visual function analysis
|OptoMorty HD software
|CerebralMechanics Inc., Canada
|Inhalation Anesthetic Isoflurane
|Piramal Critical Care, Bethlehem, PA, USA
|Phenylephrin 2.5%-Tropicamide 0.5%
|University Hospital Düsseldorf, Germany
|Dr. Robert Winzer Pharma GmbH, Berlin, Germany
|ophthalmologic eye gel
|GraphPad Software Inc, San Diego, CA, USA
|statistical analysis software, Version 5.00
|IBM SPSS Statistics
|IBM Corporation, Armonk, New York, USA
|statistical analysis software, Version 20
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