Eye (2005) 19, 491–498 & 2005 Nature Publishing Group All rights reserved 0950-222X/05 $30.00 www.nature.com/eye 1 2 1 Histological S Frenkel , JE Morgan and EZ Blumenthal REVIEW measurement of retinal nerve fibre layer thickness Abstract Introduction Purpose Accurate assessment of the retinal The last decade has seen a dramatic increase in nerve fibre layer (RNFL) is central to the the number of articles addressing the clinical diagnosis and follow-up of glaucoma. The estimation of retinal nerve fibre layer (RNFL) in vivo measurement of RNFL thickness by a thickness in glaucoma. The measurement of this variety of digital imaging technologies is parameter may play a central role in the early becoming an important measure for early detection of glaucomatous damage given the detection, as well as for follow-up, of changes that can be seen in the RNFL prior to glaucomatous damage. However, when the onset of visual field loss.1–5 In parallel, the drawing clinical inference concerning the implementation of RNFL evaluation both by state of the RNFL, it is important to have clinical assessment as well as with imaging valid reference data on RNFL thickness in devices in routine clinical practice of glaucoma both healthy and diseased eyes. In this patients is on the rise. By contrast, we know review, we summarize the knowledge relatively little about histological estimates of currently available about RNFL thickness RNFL thickness in normal and diseased human in human and primate eyes. eyes. Methods A review of the literature on While each of the commercially available histological analysis of RNFL thickness imaging devices (confocal scanning laser in the context of glaucomatous damage. ophthalmoscopy, scanning laser polarimetry, 1 Conclusions Six studies have so far analysed optical coherence tomography2) has had its own Department of RNFL thickness. Despite the diverse study set of normative RNFL thickness values Ophthalmology, Hebrew University - Hadassah methodology taken, a consistent feature of all collected, these values have not been validated Medical Center, Jerusalem the data is that the superior and inferior histologically. While clinical studies have shown Israel quadrants of the peripapillary retina are that these devices hold considerable clinical thicker than the nasal and temporal quadrants; promise in the detection of disease, the extent to 2University of Wales College that the RNFL thickness rapidly diminishes which they provide a true estimate of RNFL of Medicine, Heath Park with increasing distance from the disc margin; thickness is not certain. The paucity of Cardiff, UK and that apparently at different locations the histological data may reflect a view that Correspondence: ratio of axons to supportive tissue varies accurate measurement of the RNFL is secondary EZ Blumenthal significantly. We conclude that limited data are to the ability of any given device to diagnose Department of available to describe the normal variation in disease. In clinical terms, this approach may Ophthalmology, RNFL thickness in the normal human have some merit. However, the lack of Hadassah University eye. Further studies may help better clinical–histological correlations limits Hospital PO Box 12000 characterize the RNFL thickness in health and comparison between devices and can hinder the Jerusalem 91120, Israel disease and to facilitate the correlation with recognition of imaging artefacts that might arise Tel: þ 972-2-677-6580 clinical methods for nerve fibre layer with some RNFL measurement techniques. Given Fax: þ 972-2-675-8926 assessment. the potential importance of RNFL thickness, the E-mail: eblumenthal@ Eye (2005) 19, 491–498. doi:10.1038/sj.eye.6701569 paucity of high-quality data on retinal nerve md.huji.ac.il Published online 27 August 2004 fibre thickness is a drawback. In part it reflects Received: 16 January 2004 technical difficulties in the histological analysis Accepted: 12 March 2004 Keywords: retinal nerve fibre layer; retina; of the RNFL. In this review, some of these issues Published online: 27 August glaucoma; histology; optic nerve; ganglion cell are highlighted and discussed. 2004 RNFL thickness measurement S Frenkel et al 492 Approaches for quantifying glaucomatous damage in glaucoma diagnosis and follow-up, far more prevalent histologically than analysis of the RNFL.2,20.A few studies have reported the approximate cup-to-disc ratio of the optic Before focusing on the RNFL thickness as a means for disc head in enucleated eyes as a means of differentiating quantifying glaucomatous damage histologically, we normal from glaucomatous eyes.13,21 Nevertheless, we would like to acknowledge that different approaches are not aware of a study providing an in-depth exist for quantifying ganglion cells and their axons evaluation of the morphometry of the optic disc head in histologically in the enucleated eye. histological sections of enucleated eyes. This is perhaps owing to the topographical changes, most notably Ganglion cell body counts swelling, seen at the level of the optic disc, occurring shortly after death. Death of ganglion cells is the common final pathway event in glaucoma pathophysiology. Ganglion cell dropout can therefore provide an accurate assessment RNFL thickness of the damage inflicted by the disease process. Two different approaches utilizing ganglion cell counts, come As compared to optic nerve axonal counts that can to mind: first, total counts or density maps of ganglion accurately sum up the total number of ganglion cells cell bodies. Second, staining diseased or dying cells in the in the eye, but without providing information on focal ganglion cell layer, such as apoptotic cells stained using dropout, RNFL thickness can provide data on focal the TUNEL method.6,7 Visualizing dying ganglion cells RNFL loss, and hence on focal ganglion cell loss. This can provide powerful insight to disease presence and can be achieved by peripapillary measurements of the rate of loss at the moment of tissue harvesting. While an RNFL thickness, owing to the retinotopic organization of 22,23 appealing approach, total retinal ganglion cell counts the nerve bundles entering the optic disc. The have been performed on very few human eyes, most of availability of RNFL thickness measurements obtained them normal.8–12 This histological approach, while via several imaging devices in living human eyes, and potentially powerful, is extremely labour intensive. the rationale in quantifying RNFL dropout via thickness measurements, makes this histological approach relevant to clinical diagnosis and follow-up, perhaps more Optic nerve axonal counts relevant than the other three approaches discussed This is a common histological approach that has been above. used as an endpoint for evaluating glaucomatous damage in animal models,13 as well as in healthy14–16 and glaucomatous17 human eyes. Axons in the optic nerve can RNFL axonal counts be relatively easily stained and visualized owing to the A fifth approach for quantifying glaucomatous damage myelin sheath surrounding each individual axon; such a involves actual counts of RNFL axons. Focal axonal sheath is lacking from the intraocular (RNFL) portion of counts may hold promise in becoming, perhaps, a gold the ganglion cell axon. Gross topographic representation standard for evaluating treatment modalities in animal of the retina in the optic nerve cross-section might enable, models. Axonal counts circumvent the assumption of though to a very limited extent, identification of focal a correlation between RNFL thickness and actual 18 damage. This technique is labour intensive even when axonal contents, thus eliminating the contribution of the computerized imaging analysis tools have automated nonaxonal components of the RNFL. Axonal counts also portions of the task. There appears to be an approximately bypass the confounder effect of calibre differences found two-fold interindividual variability in the total nerve fibre between axons, and the possibility of a preferential loss 19 counts of normal optic nerves, somewhat limiting the of certain calibre axons. Lastly, axonal counts are not as ability of this approach to detect damage in the optic affected by post-mortem and fixation (shrinkage or nerve when analysed at a single point in time. In contrast, expansion) artefacts. 14 Balazsi et al. found far less variability in total axonal The intraocular portion of the ganglion cells’ axon is counts of normal human optic nerves. not covered by myelin, therefore the calibre of some of them spans less than 1 mm in diameter, bordering the resolution limits of light microscopy. Thus, identifying Optic nerve head morphometryFa histological and counting individual axons requires the use of evaluation electron microscopy.24 It seems that the high expense and Clinically, photographic and imaging analyses of the labour involved in electron microscopy as well as the optic nerve head topography are standard approaches need to summate enormous quantities of topographically Eye RNFL thickness measurement S Frenkel et al 493 aligned data are some of the reasons why this approach Tissue preparation considerations has not taken the lead despite its potential advantages. Nearly every research group used its own method for fixing the specimens, including: choice of fixative chemical, embedding material (paraffin, epoxy resin, RNFL thickness measurements: methodological issues historesin, plastic), duration of fixation (48–96 h), method of sectioning (glass knife, microtome, frozen sections), Tissue considerations thickness of sections (1, 2, 60 mm), and method of viewing Accurate assessment and quantification of retinal the slides (light, differential interference contrast, or structure is difficult. The principal difficulty is the period electron microscopy). These differences probably led to of time elapsing from death to fixation during which the significant differences in the amount of tissue shrinkage retinal nerve fibre layer swells. In contrast with human occurring during sample preparation.14 Ogden26 tissue, such changes are not seen in animal models when addressed the issue of tissue shrinkage in his method of the eye is perfused and fixed at the precise time of death, fixation and estimated it to be about 10–15%.