This is the largest retinal pathology series in multiple sclerosis to date—totalling nearly the full number of eyes that have previously been reported in the multiple sclerosis literature. Each of these cases was examined by several observers (blinded to the underlying diagnosis) in a uniform and consistent fashion to reduce bias. This is the only series to include subjects who were documented to suffer from relapsing remitting disease and to include an ‘other neurological disease’ control group. In addition, as almost all these cases were obtained prior to the advent of approved immunomodulatory treatment for multiple sclerosis, this series is likely to reflect the untreated natural pathology of the disease. Furthermore, in this series, immunohistochemistry was used, permitting more detailed evaluation of the abnormalities seen with haematoxylin and eosin. It is the first series to be assessed pathologically since the advent of OCT and was therefore analysed in the context of the growing interest in use of the inner retina to track disease in multiple sclerosis (Parisi et al.
; Trip et al.
; Fisher et al.
, Sepulcre et al.
; Henderson et al.
We have been able to confirm and reinforce results from previous smaller series. These include perivascular inflammation and fibrotic changes around vessels that did not have active inflammation. It also includes reduction in RGC density in the inner retina, demonstrating that inner retinal thinning in multiple sclerosis is not just a consequence of tissue atrophy but reflects neuronal and axonal loss. Given the size of our cohort, this analysis provides us with a more accurate point estimate of the real prevalence of these changes at end-of life in multiple sclerosis. Our careful clinical characterization of the cases allows us to document that these changes can be found throughout the course of disease and are not dependent on disease duration.
We have also documented a number of previously unreported pathological findings in this series, both in the retina and anterior chamber of multiple sclerosis eyes. For example, this series provides the first direct description of retinal neuronal loss beyond the RGC layer in multiple sclerosis. Despite the fact that the estimation of this injury was qualitative, it is prominent and frequent. It also comports with previously demonstrated but unexplained abnormalities in electroretinography performed in patients with multiple sclerosis (Coupland and Kirkham, 1982
; Forooghian et al.
Cell loss in the deeper layers of the retina could potentially be the consequence of a number of different pathologic mechanisms. The first possibility is that both the RGC and inner nuclear layer suffer injury as a consequence of a direct immune-mediated process. However, the inflammation we have observed is more focal than the neurodegeneration observed. Furthermore, immune cells were not routinely detected in the inner nuclear layer of the samples that demonstrated atrophy—though the possibility of perineuronal diffusion of immune cell products cannot be excluded. The second possibility is that both RGCs and horizontal/bipolar cells share a susceptibility to a neurodegenerative process in multiple sclerosis. Lastly the loss of horizontal and/or bipolar cells could result from retrograde transneuronal degeneration. It is striking that in all cases RGC loss was more pronounced than that seen in the inner nuclear layer. Furthermore, both (i) the absence of inner nuclear atrophy in ‘acute’ multiple sclerosis cases; and (ii) an observed correlation between the severity of inner nuclear layer injury and RGC loss would support this retrograde trans-synaptic model. Anterograde trans-synaptic degeneration leading to loss of neurons in the lateral geniculate nucleus has been previously described in multiple sclerosis (Evangelou et al.
), in glaucoma (Gupta et al.
) and after chemical injury to the optic nerve (Madigan et al.
). Furthermore, retrograde trans-synaptic degeneration has been demonstrated using OCT, showing retinal nerve fibre layer thinning in patients who had suffered injury to the posterior visual pathway (Jindahra et al.
). However, this would be the first histological demonstration of trans-synaptic degeneration in a retrograde pattern.
The distinctive architecture of the retina lends itself to investigating the cascade of nerve injury in multiple sclerosis including transneuronal degeneration. RGCs lie directly anterior to the inner nuclear cells that synapse with them. Furthermore, as a sensory organ the retina has primary afferent function—therefore the direction of loss can be defined. Recent pathological data from glaucoma suggest that in areas of retina corresponding to scotoma, there is decline in RGC density and associated loss of deeper retinal neurons (Lei et al.
). This may contribute to the permanence of scotomata and could even contribute to the extension of a scotoma at its edges. If the findings described here represent retrograde trans-synaptic degeneration in multiple sclerosis, they may provide insights regarding the mechanisms underlying ‘diffuse’ axonal loss in multiple sclerosis (distal from sites of inflammation, whether contemporaneous or previous). This may also provide us an excellent model for investigating retrograde trans-synaptic degeneration in neurological disease.
The frequent presence of retraction of the optic nerve head is significant for a number of reasons. First a deep cup, when identified on ophthalmoscopy, is considered a hallmark of glaucoma or vascular-mediated optic neuropathies. In contrast, toxic, nutritional and demyelinating optic neuropathies are associated with temporal pallor of the optic disc. This observation highlights that, while injury to nerve fibre layer may be relatively selective and the pattern of injury may be dependent on the mechanism, the disease-associated differences are not absolute. In essence this supports the contention that with longstanding optic neuropathy of any aetiology the ‘final common finding’ is the globally-pale disc. The histological presence of cupping is likely to result from the loss of nerve fibres, retraction and the presence of a gliomesodermal reaction in the optic nerve head. The fact that this is seen so frequently in longstanding multiple sclerosis argues that ongoing degenerative injury to the anterior visual pathway occurs in multiple sclerosis even in the absence of episodes of clinically evident acute optic neuritis and loss of nerve fibres and gliomesodermal reaction are secondary changes that compound the injury. The presence of gliomesodermal reaction implies that deformation of disc architecture can be the consequence of various mechanical processes (i.e. retraction of the optic nerve head by contraction of cytoskeletal elements versus axon loss of the arcuate bundles). Admittedly, we do not have data on whether any of the subjects studied had concomitant glaucoma, although reasonably this could only explain a modest proportion of the cupping seen as ~1–2% of patients in the UK over age 40 are thought to have open angle glaucoma (Kroese et al.
The findings reported here have important implications for OCT and other clinical retinal investigations in multiple sclerosis. OCT discerns retinal layers by using software detection algorithms based upon known pattern of backscatter off the healthy retina with incident low coherence infrared light (Blumenthal et al.
). It is unknown whether this backscatter pattern is modified by the pathological changes described here. The presence of perivascular inflammation and gliosis—especially given that the relevant vessels are situated in the inner retina—raises the possibility that retinal nerve fibre layer OCT thickness estimates may not be strictly measuring axons. Although this may undermine the reputed tissue specificity of OCT, newer, faster OCT methods (Fourier or spectral domain OCT) with improved image registration may permit us to identify the pathology described in this paper and modify our thickness estimating algorithms accordingly.
Descriptions of the pathologic features of multiple sclerosis in the retina have been based on a limited number of cases and predated a renewed interest in retinal injury in multiple sclerosis. Only Toussaint et al.
), who looked at the retinas of 15 patients with multiple sclerosis (and optic nerve in 17 additional patients), made any description of the clinical phenotype of disease. Unlike previous studies, the advent of agreed international criteria for subtype categorization of multiple sclerosis means that in this study it was possible to classify 80 of the 82 cases. However, some abnormalities described in this study confirmed those of earlier authors (ter Braak, 1933
; Rucker, 1944
; Gartner, 1953
; Fog, 1965
; Kerrison et al.
). A single case report described a patient with retinal periphlebitis identified at the time of diagnosis with detailed histopathology from the same location 14 years later at autopsy (Shaw et al.
). The retinal vascular changes observed in this patient closely resemble those seen in chronic plaques in the CNS in multiple sclerosis. In the present study, veins showing hyaline thickening of their walls but not inflammatory infiltrate were excluded in the assessment of retinal periphlebitis. Possibly, as in CNS plaques in multiple sclerosis, they were inflamed at some point in the disease. Furthermore, as a confirmation of the findings reported in this study, Gartner (1953
) described (in 10 patients) extensive gliosis as well as optic nerve atrophy, and reduction of RGC density. In Gartner’s sample gliosis was especially prominent in the optic nerve head, sometimes severe enough to lead to a scar deforming the architecture of the disc and optic nerve.
As with all autopsy-based pathological studies in multiple sclerosis, this study is limited by an inability to sample tissue other than at the end of life. The frequency with which perivascular inflammation was observed at this late stage in the disease raises various questions. Understanding how a tissue devoid of myelin is capable of maintaining a robust inflammatory response is important for understanding the operative mechanisms in progressive multiple sclerosis. Interestingly, in a recent study using a transgenic mouse model of multiple sclerosis, myelin-specific T cells also recognized neuronal autoantigen (Krishnamoorthy et al.
). Using the retina as a model may even help to reconcile our conceptions of multiple sclerosis as both a neuroinflammatory disease and a neurodegenerative disease.
Of particular note were the findings in the anterior uvea. Given the correlation between the uveal and retinal changes, we suspect that these changes are a response to a distressed retina (as has been postulated to be the operative mechanism in the rubeosis iridis found in association with diabetic retinopathy). Understanding the mechanisms of this process may provide important insights into the molecular response to tissue injury in grey matter in multiple sclerosis. We consider these changes to possibly be a correlate of inflammation seen in the leptomeninges with cortical injury in multiple sclerosis (Kutzelnigg et al.
). In addition, from our work it is unclear when in the course of disease these changes become clinically manifest, and whether they are detectable during life. Efforts at clinical confirmation will help us ascertain whether these findings could serve a diagnostic or prognostic purpose.
There are number of reports arguing for an association between multiple sclerosis and clinically identifiable uveitis. However, these clinical series have been controversial because of potential sampling bias, have estimated concomitant uveitis to be an infrequent finding (1–15%) and have typically localized the predominant inflammation to be in the intermediate uveal compartment (pars plana) (Graham et al.
; Biousse et al.
; Le Scanff et al.
). With regards to our pathological findings, we cannot be certain that either the iris neovascularization or the changes at the pupillary margin are secondary to inflammation. Still our observation that these iris changes occur along with cellular infiltrate in some cases suggests that they could be related phenomena.
Given the observation that perivascular inflammation was never uniformly identified in all blood vessels from a single eye and considering the sampling method, it is possible that the frequency with which these changes occur has been underestimated. It has been reported that perivenous sheathing most frequently occurs in vessels beyond the mid-periphery of the retina—a region which in the present study is likely to be under-sampled (Rucker, 1972
). However the results are in very close agreement with prior studies (Arnold et al.
; Birch et al.
; Schmidt et al.
A few significant additional limitations are worthy of mention. Although efforts were taken to insure the maintenance of blinding of the evaluating ophthalmic pathologist including (i) not disclosing the number or nature of control cases in the series; and (ii) having the evaluating pathologist review each pathological feature independently and in a specified order (optic disc and optic nerve last), we cannot rule out the possibility that the pathologist could have become partially unblinded by detecting pathology in a different location. Furthermore, our control group was primarily selected to account for the impact of (i) agonal changes; (ii) immune mediated injury; and (iii) treatment with glucocorticosteroids on retinal pathology (in order of importance). As a consequence, most of the control subjects suffered from peripheral nervous system demyelinating disorders. We therefore cannot rule out the possibility that similar pathological findings would be found in some other central nervous system disorders—especially those in which inflammation and involvement of the anterior visual pathway would be prominent (e.g. neurosarcoidosis). Lastly, although none of our patients had a concomitant significant ophthalmic condition documented in their available medical records, we cannot be certain that these records were exhaustive. Still, given the relatively low prevalence in the UK of the two most significant and common ophthalmic conditions, age related macular degeneration (3.5% > age 75; Evans et al.
) and glaucoma (1–2% > age 40; Kroese et al.
), it is unlikely that undiagnosed eye disease significantly confounds our results.
In conclusion, this is the largest retinal pathology series described in multiple sclerosis. It includes an embedded subset of control eyes essentially blinding the evaluating pathologist to the underlying diagnosis—demonstrating that the abnormalities seen are not an artefact of agonal processes or tissue preservation methods. This work confirms the common interpretation of OCT data that retinal atrophy in multiple sclerosis reflects loss of retinal ganglion cells and their axons. However, it also raises the question that other pathological processes involve the inner retina in multiple sclerosis and could confound interpretation of OCT measures. Gliosis and retinal inflammation (especially perivascular inflammation as inner retinal vessels lie in the retinal nerve fibre layer) could lead to increased thickness of retinal layers which is not due to expansion or swelling of neuronal/axonal elements. Furthermore, given OCT’s dependence on segmenting retinal layers based on presumed changes in tissue reflectivity at the interface of different retinal layers, OCT measures in multiple sclerosis should be interpreted with caution. We do not know how retinal inflammation or gliosis changes the backscatter of the low coherence infrared light source used in OCT. This series demonstrates that we cannot assume that retinal nerve fibre layer thickness measures in multiple sclerosis provide a pure estimate of axonal integrity in the anterior visual pathway. This does not diminish the capacity of OCT to serve as a meaningful measure of disease progression in multiple sclerosis, it only serves to provide greater context to prior observations and spur us forward in developing methods for distinguishing different pathological processes using OCT.
Ultimately, this work establishes that the retina is a site of important and underappreciated pathology in multiple sclerosis. We have demonstrated that retinal pathology in multiple sclerosis includes loss of RGCs and loss of neurons in the inner nuclear layer, further evidence that neuronal loss and atrophy in multiple sclerosis includes neuronal populations that are not myelinated. Finally we have documented a high rate of pathology in the anterior uvea, a phenomenon that may arise from either direct inflammation or as a consequence of the diffusion of factors from a distressed retina. Further investigation of these processes will provide valuable insights as we seek to uncover the mysteries of this destructive and capricious disease.