Optical coherence tomography in patients with Wilson's disease

Abstract Background Morphological changes of retina in patients with Wilson's disease (WD) can be found by optical coherence tomography (OCT), and such changes had significant differences between neurological forms (NWD) and hepatic forms (HWD) of WD. The aim of this study was to evaluate the relationship between morphological parameters of retina and brain magnetic resonance imaging (MRI) lesions, course of disease, type of disease, and sexuality in WD. Methods A total of 46 WD patients and 40 health controls (HC) were recruited in this study. A total of 42 WD patients were divided into different groups according to clinical manifestations, course of disease, sexuality, and brain MRI lesions. We employed the Global Assessment Scale to assess neurological severity of WD patients. All WD patients and HC underwent retinal OCT to assess the thickness of inner limiting membrane (ILM) layer to retinal pigment epithelium layer and inner retina layer (ILM to inner plexiform layer, ILM–IPL). Results Compared to HWD, NWD had thinner superior parafovea zone (108.07 ± 6.89 vs. 114.40 ± 5.54 μm, p < .01), temporal parafovea zone (97.17 ± 6.65 vs. 103.60 ± 4.53 μm, p < .01), inferior parafovea zone (108.114 ± 7.65 vs. 114.93 ± 5.84 μm, p < .01), and nasal parafovea zone (105.53 ± 8.01 vs. 112.10 ± 5.44 μm, p < .01) in inner retina layer. Course of disease influenced the retina thickness. Male patients had thinner inner retina layer compared to female patients. Conclusion Our results demonstrated that WD had thinner inner retina layer compared to HC, and NWD had thinner inner retina layer compared to HWD. We speculated the thickness of inner retina layer may be a potential useful biomarker for NWD.

copper in posterior corneal layers. Sunflower cataracts (Rajappa et al., 2010), optic neuritis, optic disc pallor, and exotropia (Ala et al., 2007) are rare ophthalmological manifestations. Copper accumulates over the course of disease and causes different organs injury Dusek, Roos, et al., 2015; European Association for Study of Liver., 2012). Pathological injuries in brain are mainly located in basal ganglia, but copper may accumulate in other central nervous system (CNS) structures (Prashanth et al., 2005;Saatci et al., 1997).
Recent studies detected morphological changes of retina in WD patients by optical coherence tomography (OCT) Albrecht, Müller, Südmeyer, et al., 2012;Langwinska et al., 2017Langwinska et al., , 2016Svetel et al., 2021). Study found significant negative correlation between OCT parameters and UWDRS-part II in WD (Langwinska et al., 2017); patients with abnormal brain magnetic resonance imaging (MRI) had thinner macular and retinal nerve fiber layer (RNFL) in WD (Langwinska et al., 2016). These indicated OCT parameters of retina may be a potential biomarker in WD. Recently, several biomarkers of CNS injury in WD were found.
Studies indicated that serum neurofilament (sNFL) concentrations (Ziemssen et al., 2022), glial fibrillary acidic protein (Lin et al., 2021), microtubule-associated protein tau (Lekomtseva et al., 2019), and ubiquitin C-terminal hydrolase L1 (Shribman et al., 2021) may be a potential biomarker for CNS injury in WD. OCT is a fast, noninvasive, and dynamic technique and had been used in parkinsonian syndromes Albrecht, Müller, Südmeyer, et al., 2012) and multiple sclerosis (Saidha et al., 2015). The newest OCT devices can depict retinal changes at nearly the cellular level (Saidha et al., 2011). On the basis of previous studies, we want to investigate the relationship between OCT parameters of retina and brain MRI lesions, course of disease, type of disease, and sexuality in Chinese WD patients and, finally, relate OCT parameters of retina to brain MRI lesions, course of disease, type of disease, and sexuality in WD patients and identify potential ophthalmological biomarkers of WD.

MATERIALS AND METHODS
All patients provided informed consent. We collected 46 WD patients admitted to the First Affiliated Hospital of Guangdong Pharmaceutical The areas of measurement are marked in an image of the fundus. The inner retina thickness and full retina thickness were measured in scans centered on the fovea. S1, T1, I1, N1 mean superior, temporal, inferior, and nasal parafovea zones in 3 × 3 mm 2 centered on the fovea, S2, T2, I2, N2 mean superior, temporal, inferior, and nasal perifovea zone in 6 × 6 mm 2 centered on the fovea. (1) a contraindication to use 1% tropicamide, (2) patients who cannot undergo OCT examination due to severe involuntary movements (such as severe rest tremor), and (3) patients who were younger than 15year old. We also recruited 40 sexy-age match healthy controls (HCs).
Demographic and clinical characteristics of 42 WD patients are shown in Table 1.
According to clinical presentations, WD patients were divided into two groups, group of neurological (NWD) and group of hepatic (HWD).
We employed the Global Assessment Scale to assess the neurological severity of WD patients (Aggarwal et al., 2009

Statistical analysis
To avoid internal correlations, data were analyzed by using mean values of measurements taken from both eyes. Quantitative variables were described using mean and standard deviation (M ± SD). Group differences of demographic categorical variables were tested using the
In order to assess the relationship between OCT parameters and course of disease, all recruited patients were stratified into three groups: course of disease shorter than 5 years (<5-year group), 5−10 years (5-10-year group), and longer than 10 years (>10-year group).
There is no significant difference in the onset of age, age of diagnosis, course of disease, latency from diagnosis to treatment, and treatment duration between NWD and HWD.

OCT parameters in WD and HC
Compared to HC, WD had thinner superior parafovea zone, temporal parafovea zone, inferior parafovea zone, nasal parafovea zone, temporal perifovea zone, and nasal perifovea zone in inner retina layer (Table 2).

OCT parameters in NWD, HWD, and HC
Compared to HC, NWD had thinner parafovea zones, superior perifovea zone, and nasal perifovea zone in inner retina layer (Table 2).
Meanwhile, compared to HWD, NWD had thinner parafovea zones in inner retina layer (Table 2). There had no significant difference in OCT parameters between HWD and HC (Table 2).

OCT parameters in male and female
Previous studies showed that WD severity is associated with gender and the course of disease (Ferenci et al., 2019). In our study, we found that compared to female patients, male patients had thinner superior perifovea zone, inferior perifovea zone, and nasal perifovea zone in inner retina layer (Table 3).
layer (Table 4). It seems that the thickness of full retina layer is associated with the course of disease, and patients with longer course of disease are more likely to occur morphological change of retina.

OCT parameters in different brain injury groups
Previous study had detected macular and RNFL were thinner, especially in patients with brain MRI lesions (Langwinska et al., 2016); we using a slightly different approach divided patients into three groups based on different brain MRI lesions: group 1-present of basal ganglia injury but absent of brainstem injury (ABI group), group 2-present of basal ganglia injury and brainstem injury (PBI group), and group 3normal (no visual brain MRI lesions, MRI− group). In addition to brain stem and basal ganglia lesions, there are also other brain lesions in WD patients. A total of 13 patients had mild ventricular enlargement, and 10 patients had mild brain sulcus widening in our study. We only include the abnormal signals of basal ganglia and brainstem in our study.
In our study, there had no significant differences in OCT parameters between MRI− group and MRI+ group (ABI group + PBI group) (Table 5). PBI group had thinner nasal parafovea zone in inner retina layer compared to MRI− group (Table 6). However, there had no significant differences between ABI group and PBI group in OCT parameters (Table 6).

DISCUSSION
Our results indicated that WD had thinner parafovea zones and partial perifovea zones in inner retina layer compared to HC; NWD had thinner parafovea zones in inner retina layer compared to HWD.
There was no significant difference in OCT parameters between HWD and HC. Our study found morphological change of retina occurs at NWD, and thinning of inner retina layer in HWD may be associated with neurological system injury. Therefore, the thickness of inner retina layer may be a potential biomarker for monitoring disease progression.
OCT is a noninvasive high-resolution optical imaging technology; details of the principles of spectral-domain OCT had described in previous study (Nassif et al., 2004). In the last 20 years, the development of OCT had fundamentally changed retinal diagnosis (Puliafito et al., 1995(Puliafito et al., , 2010. Currently, due to a lack of direct access, human cerebral microvascular alterations are difficult to noninvasively monitor in vivo. Nevertheless, as the vasculature of retina and brain are similar in their anatomy and physiology (London et al., 2013), changes in the structure and function of brain and cerebral vessels may lead to changes in retina vessels and morphology. The embryological, structural, and functional continuity of retina with CNS makes the visual pathway a prime target for potential noninvasive investigations of neurodegeneration, such as neurodegeneration that occurs in course of Alzheimer's disease and multiple sclerosis (Greenberg et al., 2010). Note: S1,T1,I1,N1 (s1,t1,i1,n1) mean superior, temporal, inferior, and nasal parafovea in full retina layer (inner retina layer), S2,T2,I2,N2 (s2,t2,i2,n2) mean superior, temporal, inferior, and nasal perifovea in full retina layer (inner retina layer). M ± S = means ± standard deviation. Abbreviations: FRL, full retina layer; IRL, inner retina layer. Note: Compared to MRI−, # means p < .05. S1,T1,I1,N1 (s1,t1,i1,n1) mean superior, temporal, inferior, and nasal parafovea zones of full retina layer (inner retina layer) in 3 × 3 mm 2 centered on the fovea, S2,T2,I2,N2 (s2,t2,i2,n2) mean superior, temporal, inferior, and nasal perifovea zones of full retina layer (inner retina layer) in 6 × 6 mm 2 centered on the fovea. ABI group = present of basal ganglia injury, but absent of brainstem injury, PBI group = present of basal ganglia injury and brainstem injury, MRI− = no visual brain MRI injury. M ± S = Means ± standard deviation. Abbreviations: FRL, full retina layer; IRL, inner retina layer.

FRL (µm)M ± SD
Study showed that RNFL was thinner in WD compared to HC (Svetel et al., 2021). Our results indicated WD had thinner parafovea zones and partial perifovea zones in inner retina layer compared to HC. The reduction of RNFL thickness in WD reflects the degeneration of retinal ganglion cell axons and retinal ganglion cells themselves Albrecht, Müller, Südmeyer, et al., 2012). It is likely to account for the observed reduced thickness of inner retina layer in our study. Neuronal degeneration as a consequence of axonal damage due to copper deposition along the optic nerve and tract is a plausible explanation for the abovementioned observations. Langwinska et al. (2016) proved macular and RNFL were thinner, especially in patients with brain MRI lesions. In our study, we found there had no significant difference between MRI− group and MRI+ group (ABI group + PBI group). However, as we further divided the patients into three groups (ABI group, PBI group, and MRI− group) based on different brain MRI lesions, patients with more severe MRI lesions had thinner inner retina layer. We speculated thinning of inner retina layer is a slow process, aggravated with the aggravation of brain injury. Our results indicated that the thinning of inner retina layer in WD may be associated with neurologic injury. Svetel et al. (2021) found the course of disease did not influence the RNFL thickness. However, in our study, we found that compared to <5-year group, >10-year group had thinner superior parafovea zone and thinner nasal parafovea zone in full retina layer. We speculated the thinning of full retina layer in patients with a longer course of disease, which may be associated with retinal neurodegeneration caused by excess copper. In summary, with a prolonging course of disease, the full retina layer gradually became thinner, which may indicate the full retina layer as a useful index for the tracking course of disease in WD.
A study of a large cohort of WD patients confirmed there was a gender effect in index patients: Hepatic presentation was more common in females, and neurologic presentation was more common in males (Ferenci et al., 2019). Agnieszka et al. conducted a higher sNFL concentration (a marker of neurodegeneration) was observed in men (estrogens are thought to have a neuroprotective effect in neurodegenerative diseases) . In our study, compared to females, males had thinner partial perifovea zones in inner retina layer. It conducted our study was consistent with previous studies, that male was more prone to neurological damage.
The retina consists of axons and glia without myelin and may be a good structure for visualizing the degree of neurodegeneration (MacLaren et al., 1996). Direct oxidative stress and apoptosis are principal pathways of copper toxicity in WD, resulting in cell death in the affected tissues (Nagasaka et al., 2006). To a certain extent, the excess copper was neutralized by antioxidants, but an elevated level of reactive oxygen species beyond a certain limit might induce undesirable oxidative damage in the cells (Steinebach et al., 1994;Gaetke et al., 200). Therefore, the observation of significant differences of inner retina layer in NWD and HWD supported previous study that chronic copper-related degeneration affected unmyelinated fibers (Valenti, 2011).

CONCLUSION
Our results demonstrated that WD had thinner parafovea zones and partial perifovea zones in inner retina layer compared to HC, and NWD had thinner parafovea zones in inner retina layer compared to HWD.
This study confirmed the thickness of inner retina layer may be a potential useful biomarker for NWD, and OCT may be a useful tool for measuring the degree of neurodegeneration in WD patients and may play role in monitoring disease progression.

LIMITATIONS
Our study had several limitations. First, we only compared OCT parameters in ABI group and PBI group, but WD also existed in other brain MRI lesions. We will apply brain semiquantitative scale performed by Dusek Dusek, Roos, et al., 2015) or volumetric studies assessing brain atrophy performed by Smolinski et al. (2019) in future. Second, because of the small sample size of this study, we cannot conclude the specific significance of thinning of inner retina layer and full retina layer in WD. We will enlarge the sample and follow up to compare the changes of OCT parameters before and after treatment.

ACKNOWLEDGMENTS
We would like to thank all the subjects who participated in this study.

CONFLICT OF INTEREST STATEMENT
The authors declare that they have no conflict of interest.

DATA AVAILABILITY STATEMENT
The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.