S-Adenosylmethionine Inhibits the Proliferation of Retinoblastoma Cell Y79, Induces Apoptosis and Cell Cycle Arrest of Y79 Cells by Inhibiting the Wnt2/ß-Catenin Pathway.

Retinoblastoma is one of the most common primary intraocular malignancies in young children. Traditional treatment methods such as chemotherapy often come with significant adverse effects, such as hearing loss, cognitive impairment, and vision loss. Therefore, there is an urgent need to explore a novel therapeutic drug that is both effective and safe. S-adenosylmethionine (SAM) is a natural compound known to exhibit anti-proliferative effects in various cancer cell lines. However, to date, no studies investigated the effects of SAM on retinoblastoma cells and its potential mechanisms of action. Therefore, this study aims to investigate the impact of SAM on retinoblastoma cells and explore its possible mechanisms of action, with the hope of providing new insights into the treatment of this disease. The optimal concentration of SAM was determined using the Cell Counting Kit-8 assay. The effect of SAM on retinoblastoma proliferation was assessed using the 5-ethynyl-2'-deoxyuridine cell proliferation assay. Y79 cells were subjected to hematoxylin and eosin stain and electron microscopy to observe any morphological changes induced by SAM. The stages of SAM's action on the retinoblastoma cell cycle and its apoptotic effects were measured using flow cytometry. The apoptotic effect of SAM on retinoblastoma was further confirmed using the TUNEL assay. Differential expression of related genes was detected through RT-PCR. In vivo subcutaneous tumor formation in nude mice and immunohistochemistry were employed to validate the effect of SAM on retinoblastoma-related phenotypes. Western blotting was conducted to investigate whether SAM modulated retinoblastoma-related phenotypes via the Wnt2/ß-catenin pathway. SAM arrested the cell cycle of retinoblastoma at the G1 phase, induced apoptosis of retinoblastoma cells through the Wnt2/ß-catenin pathway, and affected their morphology and even ultrastructure. In addition, in vitro and in vivo experiments demonstrated that SAM had an oncogenic effect on retinoblastoma. In this study, we verify in vitro and in vivo whether SAM inhibits the proliferation of retinoblastoma cell Y7, induces apoptosis and cell cycle arrest of Y79 cells by inhibiting the Wnt2/ß-catenin pathway, and affects the morphology and structure of retinoblastoma cell Y79.

Injectable Dendrimer Hydrogel Delivers Melphalan in Both Conjugated and Free Forms for Retinoblastoma.

We report the successful synthesis of an injectable dendrimer hydrogel (DH) carrying melphalan, a clinical drug for retinoblastoma treatment, in both conjugated and free forms. Polyamidoamine (PAMAM) dendrimer generation 5 (G5) is surface-modified with an acid-sensitive acetal-dibenzocyclooctyne linker and then undergoes azide-alkyne cycloaddition with melphalan-PEG-N3 conjugate to form G5-acetal-melphalan. During the DH gelation between G5-acetal-melphalan and PEG-diacrylate, free melphalan is added, resulting in a hydrogel (G5-acetal-melphalan-DH/melphalan) that carries the drug in both conjugated and free forms. Melphalan is slowly released from G5-acetal-melphalan-DH/melphalan, with the conjugated melphalan released more quickly at pH 5.3 due to acid-triggered acetal bond cleavage. The formulation's in vitro safety and efficacy were established on human corneal epithelia (HCE-2) and retinoblastoma cells (Y79). In an in vivo Y79 tumor xenograft model of retinoblastoma, intratumorally injected G5-melphalan-DH formulation prolonged tumor suppression. This injectable, multimodal, pH-responsive formulation shows promise for intravitreal injection to treat retinoblastoma.

[Liposome co-delivery of quercetin and doxorubicin in inhibiting retinoblastoma by regulating epithelial-mesenchymal transition process].

Regulating the process of epithelial-mesenchymal transition(EMT) is an essential strategy to inhibit tumor growth and metastasis. This study is based on the EMT process of retinoblastoma and constructs quercetin(QUE) and doxorubicin(DOX) co-loaded liposome(QD Lipo) to investigate the therapeutic effect and mechanisms of combined QUE and DOX treatment on retinoblastoma. Single-factor experiments were conducted to optimize the prescription process of QD Lipo. Eventually, spherical particles with a diameter of(108.87±1.93) nm, a PDI of 0.13±0.02, and a Zeta potential of(-34.83±1.92) mV were obtained. The encapsulation rates of QUE and DOX were 96.20%±4.40% and 91.17%±4.41%, respectively. Y79 human retinoblastoma cells were used as an in vitro cellular model, and confocal microscopy demonstrated that QD Lipo could enhance Y79 uptake efficiency. The CCK-8 assay confirmed that the optimal combination therapy effect of QUE and DOX occurred at a mass ratio of 1∶1 to 1∶2. Flow cytometry showed that QD Lipo enhanced the induction of apoptosis in Y79 cells. Western blot analysis revealed that QD Lipo significantly reduced the expression of EMT pathway-related proteins vimentin and α-SMA. Fluorescence assays detected a significant decrease in ROS levels in Y79 cells after treatment with QD. These results indicated that liposomal co-delivery of QUE and DOX can enhance drug delivery efficiency to retinoblastoma cells, inhibit the EMT process in retinoblastoma by downregulating ROS levels, and enhance the cytotoxicity of DOX against retinoblastoma.

A retrospective study on adjuvant chemotherapy in retinoblastoma: validation of the new recommendation against treatment for pT2a tumors based on the 8th AJCC classification.

BACKGROUND: Retinoblastoma (RB) is an intraocular malignant tumor detected in early childhood with variable global impact. Histopathological classification of the tumor in enucleated globes with RB is the key for the decision of adjuvant chemotherapy use. We aim to validate the use of adjuvant chemotherapy in cases with combined pre-laminar/intralaminar optic nerve (ON) invasion and focal choroidal invasion according to the American Joint Committee on Cancer (AJCC) 8th classification. METHODS: This is a retrospective study conducted at King Abdulaziz University Hospital (KAUH) and King Khalid Eye Specialist Hospital (KKESH) in Riyadh, Saudi Arabia of all RB cases who underwent enucleation over 22 years (2000 to 2021). The histopathological findings were reviewed to identify the enucleated globes classified as pT2a tumors, as an inclusion criterion. Basic demographic and clinical data were collected via chart review Simple descriptive and basic statistical analysis of the data was used where applicable. RESULTS: Thirty-one patients who had an enucleated globe with RB that fit into the above classification were included. Sixteen were males and 15 were females. The median age was 14 months (IQR = 14 months). Most of the patients (93.5%) had no family history of RB. The commonest presentation was leukocoria in 87.1% followed by squint in 32.3%. Fourteen patients (45.2%) were treated by enucleation alone while 17 patients (54.8%) received adjuvant chemotherapy. Out of these, 7 patients had unilateral RB and the remaining 10 patients had bilateral RB. None of our patients developed recurrence or metastatic disease irrespective of the indication for adjuvant chemotherapy use after a maximum period of follow up reaching 17.84 years and a median of 10.6 years (IQR = 5.92). CONCLUSIONS: In patients with 8th AJCC histopathological classification of pT2a, chemotherapy following enucleation might not be justified. The outcome in our untreated group of patients did not differ from the treated group with the absence of metastasis after a relatively long period of follow up with a median exceeding 10 years in both groups. Therefore, the risk and benefit of post enucleation adjuvant chemotherapy in the treatment of unilateral RB should be carefully decided and discussed with the primary caregivers taking into consideration the most recent evidence and recommendations in the literature.

Investigating druggable kinases for targeted therapy in retinoblastoma.

Retinoblastoma (RB) is a childhood retinal neoplasm and commonly treated with cytotoxic chemotherapeutic agents. However, these therapeutic approaches often lead to diverse adverse effects. A precise molecular therapy will alleviate these side effects and offer better treatment outcomes. Over the years, kinases have become potential drug targets in cancer therapy. Hence, we aimed to investigate genetic alterations of putative kinase drug targets in RB. Targeted exome sequencing was performed on 35 RB tumors with paired blood samples using a gene panel consisting of 29 FDA-approved kinase genes. Single nucleotide variants were analyzed for pathogenicity using an in-house pipeline and copy number variations (CNVs) were detected by a depth of coverage and CNVPanelizer. The correlation between genetic changes and clinicopathological features was assessed using GraphPad Prism. Three somatic mutations, two in ERBB4 and one in EGFR were identified. Two of these mutations (ERBB4 c.C3836A & EGFR c.A1196T) were not reported earlier. CNV analysis revealed recurrent gains of ALK, MAP2K2, SRC, STK11, and FGFR3 as well as frequent losses of ATM, PI3KCA and ERBB4. Notably, nonresponsive tumors had a higher incidence of amplifications in clinically actionable genes such as ALK. Moreover, ALK gain and ATM loss were strongly correlated with optic nerve head invasion. In conclusion, our study revealed genetic alterations of druggable kinases in RB, providing preliminary insights for the exploration of kinase-targeted therapy in RB.

Electroretinographic changes in the inner and outer retinal layers before and after intravenous chemotherapy for retinoblastoma.

PURPOSE: To study the inner and outer retinal functions using a full-field electroretinogram (ERG) before and after intravenous chemotherapy (IVC) in children with retinoblastoma (RB). METHODS: Of the 11 eyes, seven had RB and four were normal. All children were examined under anesthesia using a handheld ERG machine with a standard protocol - light-adapted single-flash ERG (fERG), photopic single-flash 3.0- and 30-Hz flickers, and photopic negative response (PhNR) amplitudes at 72 ms (P72). The amplitudes and peak times were compared before and after IVC. RESULTS: Post-chemotherapy tumor regressed in all seven eyes. Of the seven eyes, the fERG peak time (a-wave) was delayed in two eyes (29%), whereas the b-wave was delayed in six eyes (86%). The fERG amplitude height for a- and b-waves decreased in five eyes (71%) and six eyes (86%), respectively. In addition, photopic flicker 30-Hz b-wave peak time delayed in five eyes (71%), whereas the b-wave amplitude height decreased in six eyes (86%). Simultaneously, the P72 amplitude height decreased in six eyes (86%), whereas the P-ratio increased in all seven eyes (100%). In comparison, the ERG responses improved in three of the four contralateral normal eyes. Overall, the cone function improved in two eyes (29%), whereas cone bipolar cell and retinal ganglion cell (RGC) function improved in one eye (14%) each. CONCLUSION: Comparison of light-adapted ERG changes before and after IVC showed reduced amplitudes and delayed peak times for both a and b waveforms, as well as reduced PhNR amplitude attributable to bipolar and RGC injury.

PL3 CendR peptide shows specific uptake in cultured Y79 retinoblastoma cells with nucleolar accumulation.

Retinoblastoma is the most common pediatric intraocular malignant tumor affecting 1:15 000-1:20 000 live births. Even though the survival rate in developed countries is over 90 %, more efficient treatment options are needed for better vision salvage and reduction of the adverse effects. Therefore, we investigated fluorescein-labeled PL3 peptide targeting properties towards the Y79 retinoblastoma cell line in vitro. Through the application of cellular imaging and flow cytometry techniques, the PL3 peptide exhibited a rapid and specific internalization within Y79 cells, with subsequent translocation to the cell nuclei, showcasing notable accumulation in the nucleoli. This phenomenon was not present in other investigated cell lines and was not observable with similarly charged and length control peptide. However, the exact mechanism behind this Y79 cell line-specific nuclear and nucleolar targeting pattern remains elusive. In the future, this targeting process could facilitate specific treatment modalities of retinoblastoma with PL3 peptide-coupled drug delivery technologies.

Aqueous VEGF-A Levels as a Liquid Biopsy Biomarker of Retinoblastoma Vitreous Seed Response to Therapy.

Purpose: Regression of retinoblastoma vitreous seeds (VS) during intravitreal chemotherapy can be delayed, resulting in supernumerary injections. Similarly, VS relapse may not be clinically evident at first. A predictive biomarker of tumor regression and relapse could help guide real-time clinical decision making. Retinoblastoma is an oxygen-sensitive tumor; paradoxically, VS survive in the hypoxic vitreous. We hypothesized that VS elaborate pro-angiogenic cytokines. The purpose was to determine if pro-angiogenic cytokine signatures from aqueous humor could serve as a biomarker of VS response to treatment. Methods: Multiplex ELISA was performed on aqueous from rabbit eyes with human retinoblastoma VS xenografts to identify expressed proangiogenic cytokines and changes in aqueous cytokine levels during intravitreal treatment were determined. Confirmatory RNAscope in situ hybridization for VEGF-A was performed on human retinoblastoma tumor sections and VS xenografts from rabbits. For human eyes undergoing intravitreal chemotherapy, serial aqueous VEGF-A levels measured via VEGF-A-specific ELISA were compared to clinical response. Results: VEGF-A was highly expressed in human retinoblastoma VS in the xenograft model, and was the only proangiogenic cytokine that correlated with VS disease burden. In rabbits, aqueous VEGF-A levels decreased in response to therapy, consistent with quantitative VS reduction. In patients, aqueous VEGF-A levels associated with clinical changes in disease burden (regression, stability, or relapse), with changes in VEGF-A levels correlating with clinical response. Conclusions: Aqueous VEGF-A levels correlate with extent of retinoblastoma VS, suggesting that aqueous VEGF-A may serve as a predictive molecular biomarker of treatment response.

Safety of intravitreal chemotherapy in the management of retinoblastoma: A systematic review of the literature.

Intravitreal chemotherapy is used as a salvage therapy for retinoblastoma with persistent or recurrent vitreous seeding after primary treatment. To assess the safety of this technique, we conducted a systematic review of all studies reporting ocular toxicity data. Forty-eight trials involving 2751 eyes were included. The most common complications were cataract, retinal toxicity, and vitreous hemorrhage. However, severe and permanent adverse events were limited, while the risk of extraocular dissemination, a significant concern, was practically eliminated through preventive techniques. Globe salvage rates ranged from 29 % to 100 %. In conclusion, intravitreal chemotherapy seems to improve prognosis of eyes with advanced disease, with an acceptable safety profile. Nevertheless, most relevant studies are retrospective, and no randomized trials have been performed. Recognizing the challenges regarding the conduct of randomized studies for such a rare pediatric cancer, we believe that multicenter trials through international collaborations can significantly enhance the available information.

Utility of carboplatin therapeutic drug monitoring for the treatment of neonate and infant retinoblastoma patients in the United Kingdom.

BACKGROUND: Retinoblastoma is the most common intra-ocular malignancy in children and frequently presents in very young patients who commonly require intravenous carboplatin. Delivering this is challenging due to a lack of uniform dosing recommendations, rapid changes in physiological function and the risk of side-effects. METHODS: We conducted a retrospective review of neonates and infants in the UK with retinoblastoma, who have undergone carboplatin therapeutic drug monitoring (TDM). We report on the pharmacokinetic, treatment efficacy and toxicity data. RESULTS: In total, 29 patients (median age 5 weeks at treatment onset) underwent a total of 74 TDM guided cycles of chemotherapy, involving real time sampling and dose adjustment. An additional 13 patients underwent TDM sampling to modify doses between cycles. Without the adoption of TDM guided dosing, carboplatin exposures would have been ≥20% outside the target AUC in 38/78 (49%) of treatment cycles. Excellent responses and a reassuringly low incidence of toxicities were observed following dose adjustment, despite the young patient age and the implementation of dose increases in the majority of cases. CONCLUSIONS: Real time TDM is safe, effective and deliverable for neonates and infants receiving carboplatin for retinoblastoma and should be considered standard of care up to the age of 6 months.

Selective ophthalmic arterial injection using a balloon catheter for retinoblastoma: a seven-year clinical evaluation.

PURPOSE: To evaluate the effectiveness and safety of selective ophthalmic arterial injection (SOAI) for retinoblastoma utilizing a microballoon catheter system with an M chamber. STUDY DESIGN: Retrospective analysis. METHODS AND PATIENTS: This study was sanctioned by theNational Cancer Center Hospital' Independent Ethics Committee. The surgeon was a general interventional radiologist. After confirming that the distal internal carotid artery was not delineated by balloon occlusion and the ophthalmic artery was visualized using digital subtraction angiography, melphalan was manually administered. Notably, in cases presenting bilateral retinoblastoma, both eyes received treatment in a singular, low-dose procedure. Between July 2015 and December 2021, 125 patients with retinoblastoma (68 boys and 57 girls) underwent SOAI at our facility. The average age at initial treatment was 19.3 months. The study covered 250 procedures, with patients undergoing an average of 3.7 procedures. RESULTS: The success rate of the procedure was 99.2%, with a mean procedure duration of 18.3 min. Two distinct technical failures were recorded: one attributed to an internal carotid artery having a wide lumen and the other due to the ophthalmic artery remaining undetected on angiography post-balloon occlusion of the internal carotid artery. Adverse events were minimal but included bronchospasm post-procedure and severe orbital inflammation in 0.8% and 0.4% of cases, respectively. CONCLUSION: SOAI using the microballoon catheter with the M chamber is a feasible and safe procedure for the treatment of retinoblastoma. The success rate was 99.2%. This system can be recommended as intra-arterial chemotherapy for retinoblastoma.

Enhancement of vincristine sensitivity in retinoblastoma through Janus kinase inhibition by ruxolitinib.

Chemotherapy remains the main approach conserving vision during the treatment of retinoblastoma, the most prevalent eye cancer in children. Unfortunately, the development of chemoresistance stands as the primary reason for treatment failure. Within this study, we showed that prolonged exposure to vincristine led to heightened expression of JAK1 and JAK2 in retinoblastoma cells, while the other members of the JAK family exhibited no such changes. Employing a genetic intervention, we demonstrated the efficacy of depleting either JAK1 or JAK2 in countering vincristine-resistant retinoblastoma cells. In addition, the dual depletion of both JAK1 and JAK2 produced a more potent inhibitory outcome compared to the depletion of either gene alone. We further demonstrated that ruxolitinib, a small molecular inhibitor of JAK1/2, effectively reduced viability and colony formation in vincristine-resistant retinoblastoma cells. It also acts synergistically with vincristine in retinoblastoma cells regardless of inherent cellular and genetic heterogeneity. The effectiveness of ruxolitinib as standalone treatment against chemoresistant retinoblastoma, as well as its combination with vincristine, was validated in multiple retinoblastoma mouse models. Importantly, mice exhibited favorable tolerance to ruxolitinib administration. We confirmed that the underlying mechanism of ruxolitinib's action in chemoresistant retinoblastoma cells is the inhibition of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling. Our study reveals that the underlying mechanism driving ruxolitinib's impact on chemoresistant retinoblastoma cells is the inhibition of JAK/STAT signaling. This study reveals the contribution of JAK1/2 to the development of chemoresistance in retinoblastoma and underscores the effectiveness of targeting JAK1/2 as a strategy to sensitize retinoblastoma to chemotherapy.

Advancements in Nanosystems for Ocular Drug Delivery: A Focus on Pediatric Retinoblastoma.

The eye's complex anatomical structures present formidable barriers to effective drug delivery across a range of ocular diseases, from anterior to posterior segment pathologies. Emerging as a promising solution to these challenges, nanotechnology-based platforms-including but not limited to liposomes, dendrimers, and micelles-have shown the potential to revolutionize ophthalmic therapeutics. These nanocarriers enhance drug bioavailability, increase residence time in targeted ocular tissues, and offer precise, localized delivery, minimizing systemic side effects. Focusing on pediatric ophthalmology, particularly on retinoblastoma, this review delves into the recent advancements in functionalized nanosystems for drug delivery. Covering the literature from 2017 to 2023, it comprehensively examines these nanocarriers' potential impact on transforming the treatment landscape for retinoblastoma. The review highlights the critical role of these platforms in overcoming the unique pediatric eye barriers, thus enhancing treatment efficacy. It underscores the necessity for ongoing research to realize the full clinical potential of these innovative drug delivery systems in pediatric ophthalmology.

Intravitreal Topotecan for Vitreous Seeds in Retinoblastoma: A Long-term Review of 91 Eyes.

PURPOSE: To study the long-term efficacy of intravitreal topotecan (IVT) for vitreous seeds in eyes with retinoblastoma and risk factors for their recurrence. DESIGN: Retrospective, non-comparative, interventional study. PARTICIPANTS: Ninety-one eyes of 90 patients with retinoblastoma treated between January 2013 and April 2019. METHODS: Patients with recurrent or refractory vitreous seeds after completion of intravenous or intra-arterial chemotherapy were treated with IVT (30 µg/0.15 ml) by the safety-enhanced technique. The injection was repeated every 4 weeks until the regression of seeds. Patients with a minimum follow-up of 12 months were included in the analysis. MAIN OUTCOME MEASURES: Primary outcome measures were vitreous seed regression and eye salvage. Secondary outcomes were risk factors for vitreous seed recurrence after treatment with IVT, vision salvage, and complications of IVT. RESULTS: The median age of the patients was 18 months, with most having group D (n = 58 [64%]) and group E (n = 26 [29%]) retinoblastoma. Vitreous seeds were refractory in 46 eyes (51%) and recurrent in 45 eyes (49%). A total of 317 IVT injections were administered, with the median being 3 injections. The median number of IVT injections required was 2.5 injections for dust, 3 injections for sphere, and 5 injections for cloud morphologic features. Recurrence of vitreous seeds after IVT was seen in 17 eyes (19%) at a mean follow-up of 7.9 months. At a mean follow-up 34 months, vitreous seed regression was achieved in 88 eyes (97%) and eye salvage was achieved in 77 eyes (85%). Older age (P = 0.018) and recurrence of retinal tumor (15/17 eyes; P < 0.01) significantly increased the risk of vitreous seed recurrence. Cataract was the most common complication seen in 17 eyes (9%). CONCLUSIONS: Intravitreal topotecan at an every 3- to 4-week regimen is effective against both refractory and recurrent vitreous seeds. The vitreous seed morphologic features correspond to the number of injections required for regression. Increasing age and recurrence of retinal tumor increase the risk of vitreous seed recurrence after treatment with IVT. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Amniotic Membrane Augmentation for Enucleation After Chemotherapy in Retinoblastoma.

PURPOSE: To describe amniotic membrane augmentation for enucleation after chemotherapy in retinoblastoma. METHODS: This was a retrospective study of patients with retinoblastoma who underwent enucleation. The study also evaluated the utility of amniotic membrane grafting in enucleation after chemotherapy in eyes with retinoblastoma. RESULTS: In this study, 110 eyes of 107 patients were analyzed, and 49 patients had previous systemic chemotherapy, 13 eyes had previous intra-arterial chemotherapy, and 7 eyes had external beam radiation. Amniotic graft was used in 8 eyes (5 following IAC, 2 following systemic chemotherapy, and 1 after both). After IAC, 3 of 7 eyes without amniotic graft had implant exposure compared to 0 of 6 eyes with amniotic graft (P = .05). Pathological examination of the conjunctiva after intra-arterial chemotherapy showed goblet cell hypoplasia that hinders wound healing. CONCLUSIONS: Amniotic membrane augmentation improves wound integrity in patients with retinoblastoma, especially following intra-arterial chemotherapy. [J Pediatr Ophthalmol Strabismus. 2024;61(4):291-295.].

Assessment of Retinal Microvasculature and Choroidal Vascularity After Intra-arterial Chemotherapy for Retinoblastoma.

PURPOSE: To evaluate changes in retinal microvascular density and choroidal vascularity in patients with retinoblastoma (RB) after intra-arterial chemotherapy (IAC). DESIGN: Retrospective clinical cohort study. METHODS: This study included 12 unilateral RB eyes treated with IAC (RB tumor), 12 contralateral normal eyes (RB fellow), and 12 healthy controls. The macular retinal thickness and retinal microvascular structure, including the foveal avascular zone (FAZ) area, macular and peripapillary superficial vessel density (SVD), and deep vessel density (DVD), were measured by optical coherence tomography angiography (OCTA). The choroidal thickness (ChT) and choroidal vascularity, including total choroidal area (TCA), luminal area (LA), stromal area (SA), and choroidal vascularity index (CVI), were measured by spectral-domain optical coherence tomography (SD-OCT). A comparison among the 3 groups was conducted, and the correlations among the parameters were analyzed. RESULTS: Among the 3 cohorts, the foveal retinal thickness, SVD, DVD, ChT, TCA, LA, SA, and CVI were significantly lower in RB tumor compared to RB fellow and the control eyes (all P < .01). There were no significant differences in the parameters between the contralateral and control eyes. The correlation analyses indicated a significant negative correlation between the total melphalan dose and foveal and parafoveal DVD, ChT, and LA. CONCLUSIONS: The retinal microvascular density and choroidal vascularity were lower in unilateral RB treated with IAC, and seemed to be related to the total melphalan dose. There were no measurable changes in the contralateral eyes.