Binocular head-mounted chromatic pupillometry can detect structural and functional loss in glaucoma.

Aim: The aim of this study is to evaluate the utility of binocular chromatic pupillometry in detecting impaired pupillary light response (PLR) in patients with primary open-angle glaucoma (POAG) and to assess the feasibility of using binocular chromatic pupillometer in opportunistic POAG diagnosis in community-based or telemedicine-based services. Methods: In this prospective, cross-sectional study, 74 patients with POAG and 23 healthy controls were enrolled. All participants underwent comprehensive ophthalmologic examinations including optical coherence tomography (OCT) and standard automated perimetry (SAP). The PLR tests included sequential tests of full-field chromatic stimuli weighted by rods, intrinsically photosensitive retinal ganglion cells (ipRGCs), and cones (Experiment 1), as well as alternating chromatic light flash-induced relative afferent pupillary defect (RAPD) test (Experiment 2). In Experiment 1, the constricting amplitude, velocity, and time to maximum constriction/dilation were calculated in three cell type-weighted responses, and the post-illumination response of ipRGC-weighted response was evaluated. In Experiment 2, infrared pupillary asymmetry (IPA) amplitude and anisocoria duration induced by intermittent blue or red light flashes were calculated. Results: In Experiment 1, the PLR of POAG patients was significantly reduced in all conditions, reflecting the defect in photoreception through rods, cones, and ipRGCs. The variable with the highest area under the receiver operating characteristic curve (AUC) was time to max dilation under ipRGC-weighted stimulus, followed by the constriction amplitude under cone-weighted stimulus and the constriction amplitude response to ipRGC-weighted stimuli. The impaired PLR features were associated with greater visual field loss, thinner retinal nerve fiber layer (RNFL) thickness, and cupping of the optic disk. In Experiment 2, IPA and anisocoria duration induced by intermittent blue or red light flashes were significantly greater in participants with POAG than in controls. IPA and anisocoria duration had good diagnostic value, correlating with the inter-eye asymmetry of visual field loss. Conclusion: We demonstrate that binocular chromatic pupillometry could potentially serve as an objective clinical tool for opportunistic glaucoma diagnosis in community-based or telemedicine-based services. Binocular chromatic pupillometry allows an accurate, objective, and rapid assessment of retinal structural impairment and functional loss in glaucomatous eyes of different severity levels.

Evaluation of the Glaucomatous Macular Damage by Chromatic Pupillometry.

INTRODUCTION: This study aimed to examine the performance of binocular chromatic pupillometry for the objective and rapid detection of primary open-angle glaucoma (POAG), and to explore the association between pupillary light response (PLR) features and structural glaucomatous macular damage. METHODS: Forty-six patients (mean age 41.00 ± 13.03 years) with POAG and 23 healthy controls (mean age 42.00 ± 11.08 years) were enrolled. All participants underwent sequenced PLR tests of full-field, superior/inferior quadrant-field chromatic stimuli using a binocular head-mounted pupillometer. The constricting amplitude, velocity, and time to max constriction/dilation, and the post-illumination pupil response (PIPR) were analyzed. The inner retina thickness and volume measurements were determined by spectral domain optical coherence tomography. RESULTS: In the full-field stimulus experiment, time to pupil dilation was inversely correlated with perifoveal thickness (r = - 0.429, P < 0.001) and perifoveal volume (r = - 0.364, P < 0.001). Dilation time (AUC 0.833) showed good diagnostic performance, followed by the constriction amplitude (AUC 0.681) and PIPR (AUC 0.620). In the superior quadrant-field stimulus experiment, time of pupil dilation negatively correlated with inferior perifoveal thickness (r = - 0.451, P < 0.001) and inferior perifoveal volume (r = - 0.417, P < 0.001). The dilation time in response to the superior quadrant-field stimulus showed the best diagnostic performance (AUC 0.909). In the inferior quadrant-field stimulus experiment, time to pupil dilation (P < 0.001) correlated well with superior perifoveal thickness (r = - 0.299, P < 0.001) and superior perifoveal volume (r = - 0.304, P < 0.001). CONCLUSION: The use of chromatic pupillometry offers a patient-friendly and objective approach to detect POAG, while the impairment of PLR features may serve as a potential indicator of structural macular damage.

Use of a tissue clearing technique combined with retrograde trans-synaptic viral tracing to evaluate changes in mouse retinorecipient brain regions following optic nerve crush.

Successful establishment of reconnection between retinal ganglion cells and retinorecipient regions in the brain is critical to optic nerve regeneration. However, morphological assessments of retinorecipient regions are limited by the opacity of brain tissue. In this study, we used an innovative tissue cleaning technique combined with retrograde trans-synaptic viral tracing to observe changes in retinorecipient regions connected to retinal ganglion cells in mice after optic nerve injury. Specifically, we performed light-sheet imaging of whole brain tissue after a clearing process. We found that pseudorabies virus 724 (PRV724) mostly infected retinal ganglion cells, and that we could use it to retrogradely trace the retinorecipient regions in whole tissue-cleared brains. Unexpectedly, PRV724-traced neurons were more widely distributed compared with data from previous studies. We found that optic nerve injury could selectively modify projections from retinal ganglion cells in the hypothalamic paraventricular nucleus, intergeniculate leaflet, ventral lateral geniculate nucleus, central amygdala, basolateral amygdala, Edinger-Westphal nucleus, and oculomotor nucleus, but not the superior vestibular nucleus, red nucleus, locus coeruleus, gigantocellular reticular nucleus, or facial nerve nucleus. Our findings demonstrate that the tissue clearing technique, combined with retrograde trans-synaptic viral tracing, can be used to objectively and comprehensively evaluate changes in mouse retinorecipient regions that receive projections from retinal ganglion cells after optic nerve injury. Thus, our approach may be useful for future estimations of optic nerve injury and regeneration.

Reduced Dendritic Spines in the Visual Cortex Contralateral to the Optic Nerve Crush Eye in Adult Mice.

Purpose: To determine alteration of dendritic spines and associated changes in the primary visual cortex (V1 region) related to unilateral optic nerve crush (ONC) in adult mice. Methods: Adult unilateral ONC mice were established. Retinal nerve fiber layer (RNFL) thickness was measured by spectral-domain optical coherence tomography. Visual function was estimated by flash visual evoked potentials (FVEPs). Dendritic spines were observed in the V1 region contralateral to the ONC eye by two-photon imaging in vivo. The neurons, reactive astrocytes, oligodendrocytes, and activated microglia were assessed by NeuN, glial fibrillary acidic protein, CNPase, and CD68 in immunohistochemistry, respectively. Tropomyosin receptor kinase B (TrkB) and the markers in TrkB trafficking were estimated using western blotting and co-immunoprecipitation. Transmission electron microscopy and western blotting were used to evaluate autophagy. Results: The amplitude and latency of FVEPs were decreased and delayed at 3 days, 1 week, 2 weeks, and 4 weeks after ONC, and RNFL thickness was decreased at 2 and 4 weeks after ONC. Dendritic spines were reduced in the V1 region contralateral to the ONC eye at 2, 3, and 4 weeks after ONC, with an unchanged number of neurons. Reactive astrocyte staining was increased at 2 and 4 weeks after ONC, but oligodendrocyte and activated microglia staining remained unchanged. TrkB was reduced with changes in the major trafficking proteins, and enhanced autophagy was observed in the V1 region contralateral to the ONC eye. Conclusions: Dendritic spines were reduced in the V1 region contralateral to the ONC eye in adult mice. Reactive astrocytes and decreased TrkB may be associated with the reduced dendritic spines.

SP1-mediated upregulation of LINGO-1 promotes degeneration of retinal ganglion cells in optic nerve injury.

BACKGROUNDS: Insults to the axons in the optic nerve head are the primary cause of loss of retinal ganglion cells (RGCs) in traumatic, ischemic nerve injury or degenerative ocular diseases. The central nervous system-specific leucine-rich repeat protein, LINGO-1, negatively regulates axon regeneration and neuronal survival after injury. However, the upstream molecular mechanisms that regulate LINGO-1 signaling and contribute to LINGO-1-mediated death of RGCs are unclear. METHODS: The expression of SP1 was profiled in optic nerve crush (ONC)-injured RGCs. LINGO-1 level was examined after SP1 overexpression by qRT-PCR. Luciferase assay was used to examine the binding of SP1 to the promoter regions of LINGO-1. Primary RGCs from rat retina were isolated by immunopanning and RGCs apoptosis were determined by Tunnel. SP1 and LINGO-1 expression was investigated using immunohistochemistry and Western bolting. Neuroprotection was assessed by RGC counts, RNFL thickness, and VEP tests after inhibition of SP1 shRNA. RESULTS: We demonstrate that SP1 was upregulated in ONC-injured RGCs. SP1 was bound to the LINGO-1 promoter, which led to increased expression of LINGO-1. Treatment with recombinant Nogo-66 or LINGO-1 promoted apoptosis of RGCs cultured under serum-deprivation conditions, while silencing of SP1 promoted the survival of RGCs. SP1 and LINGO-1 colocalized and were upregulated in ONC-injured retinas. Silencing of SP1 in vivo reduced LINGO-1 expression and protected the structure of RGCs from ONC-induced injury, but there was no sign of recovery in VEP. CONCLUSIONS: Our findings imply that SP1 regulates LINGO-1 expression in RGCs in the injured retina and provide insight into mechanisms underlying LINGO-1-mediated RGC death in optic nerve injury.

Inhibition of the leucine-rich repeat protein lingo-1 enhances RGC survival in optic nerve injury.

Leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 1 (lingo-1) is selectively expressed on neurons and oligodendrocytes in the central nervous system and acts as a negative regulator in neural repair, implying a potential role in optic neuropathy. The aim of the present study was to determine whether adeno-associated virus serotype 2 (AAV2) vector-mediated transfer of lingo-1 short hairpin RNA could reduce nerve crush-induced axonal degeneration and enhance axonal regeneration following optic nerve (ON) injury in vivo. The expression of lingo-1 was knocked down in vivo using a green fluorescent protein (GFP)-tagged AAV2 encoding lingo-1 shRNA via intravitreal injection in adult Sprague-Dawley rats. Silencing effects of AAV2-lingo-1-shRNA were confirmed by detecting GFP labelling of RGCs, and by quantifying lingo-1 expression levels with reverse transcription-quantitative polymerase chain reaction and western blotting. Rats received an intravitreal injection of AAV2-lingo-1-shRNA or negative control shRNA. The ON crush (ONC) injury was performed 2 weeks after the intravitreal injection. RGC density, lesion volume of the injured ON and the visual electrophysiology [flash visual evoked potential (F-VEP)] at different time points post-injury were determined. Transduction with lingo-1-shRNA decreased lingo-1 expression levels and promoted RGC survival following ONC. Lingo-1-shRNA promoted ON tissue repair and functional recovery. The mechanism underlying the effect of AAV2-lingo-1-shRNA on RGCs may be the phosphorylation of protein kinase B (Akt) at Ser473 and activation of the Akt signaling pathway acting downstream of lingo-1. The results of the current study indicate that the inhibition of lingo-1 may enhance RGC survival and facilitate functional recovery following ON injury, representing a promising potential strategy for the repair of optic neuropathy.

Pterygial body epithelium domination of pterygial proliferation with TCF4 as a potential key factor.

AIM: To characterize the proliferative capacity of pterygial epithelium in different regions (head, neck and body) of pterygium and explore the function of transcription factor 4 (TCF4) in pterygium proliferation. METHODS: Thirty pterygium tissues and 10 normal conjunctival tissues were obtained from Zhongshan Ophthalmic Center (ZOC) and Guangdong Eye Bank, respectively. Proliferative capacity of head, neck and body in pterygial epithelium was measured using clonal analysis, fold growth analysis and expression profile of proliferative markers revealed by immunofluorescent staining and real-time PCR. The expression of TCF4 was highlighted by double immunofluorescent staining with other proliferation related markers such as proliferating cell nuclear antigen (PCNA) and ATP-binding cassette sub-family G member 2 (ABCG2). RESULTS: The proliferative potential of pterygial epithelium was higher than that of normal conjunctival epithelium. High expression levels of proliferative markers (P63α, PCNA and ABCG2) in pterygial body epithelium were observed in immunofluorescent staining and real-time PCR (P<0.05). Also, epithelial cells isolated from pterygial body demonstrated higher proliferative capacity in clonal analysis and fold growth analysis, than those isolated from the head and neck regions. The TCF4 expression in pterygial epithelium was similar to other proliferative markers (P63α, PCNA and ABCG2), as higher in pterygial body than head and neck. Moreover, TCF4 showed coexpression with other proliferation-related markers (PCNA and ABCG2) in the double immunofluorescent staining experiment. CONCLUSION: The proliferative capacity in pterygial body epithelium is prominent than the head and neck regions, and upregulated TCF4 may be associated with enhanced proliferation in the pterygium.

The inflammation influence on corneal surface after frontalis suspension surgery.

AIM: To study the influence of frontalis muscle flap suspension on ocular surface by analyzing the clinical features and inflammatory cytokines. METHODS: A prospective, observational case series. Thirty-one eyes of 25 patients with severe congenital blepharoptosis who underwent frontalis muscle flap suspension surgery with at least 6mo of follow-up were included in the study. The main outcome measures were margin reflex distance 1 (MRD1), degree of lagophthalmos, ocular surface disease index (OSDI), fluorescein staining (Fl), tear break-up time (BUT), Schirmer I test, and inflammatory cytokine assay. RESULTS: The degrees of lagophthalmos significantly increased after surgery. The OSDI scores significantly increased 1wk postoperatively and then decreased 4wk after operation. The Fl scores reflected corneal epithelial defects in sixteen patients at early stage postoperatively. The BUT and Schirmer I test values remained stable and did not show change compared to those before surgery. The inflammatory cytokines in conjunctival epithelial cells (including IL-1ß, IL-6, IL-8, TNF-α, and IL-17A) significantly increased 1wk after the surgery (P<0.001), then returned to the normal level at 24wk postoperatively. The levels of inflammatory cytokine IL-1ß, IL-6, IL-8, TNF-α, and IL-17A elevated significantly and were positively correlated with OSDI and Fl scores. CONCLUSION: Frontalis muscle flap suspension surgery results in lagophthalmos in early period of post-operation and relieved after months. The elevation of inflammatory cytokines level may participate in the occurrence of corneal epithelial defects at the early postoperative stage.

Tcf7l2 localization of putative stem/progenitor cells in mouse conjunctiva.

Conjunctival integrity and preservation is indispensable for vision. The self-renewing capacity of conjunctival cells controls conjunctival homeostasis and regeneration; however, the source of conjunctival self-renewal and the underlying mechanism is currently unclear. Here, we characterize the biochemical phenotype and proliferative potential of conjunctival epithelial cells in adult mouse by detecting proliferation-related signatures and conducting clonal analysis. Further, we show that transcription factor 7-like 2 (T-cell-specific transcription factor 4), a DNA binding protein expressed in multiple types of adult stem cells, is highly correlated with proliferative signatures in basal conjunctival epithelia. Clonal studies demonstrated that Transcription factor 7-like 2 (Tcf7l2) was coexpressed with p63α and proliferating cell nuclear antigen (PCNA) in propagative colonies. Furthermore, Tcf7l2 was actively transcribed concurrently with conjunctival epithelial proliferation in vitro. Collectively, we suggest that Tcf7l2 may be involved in maintenance of stem/progenitor cells properties of conjunctival epithelial stem/progenitor cells, and with the fornix as the optimal site to isolate highly proliferative conjunctival epithelial cells in adult mice.

Normative anthropometric analysis and aesthetic indication of the ocular region for young Chinese adults.

PURPOSE: The ocular region is of prime importance for the facial aesthetic outlook. Various anthropometric analyses for the periocular region have developed to ensure a pleasing postoperative appearance. However, little information exists for Chinese young adults. In this study, authors not only analyzed the periocular anthropometric characteristics, but, more importantly, searched out the most meaningful aesthetic indicators of the population. METHODS: The cross-sectional study was executed using two-dimensional photogrammetry acquired from 162 Chinese young adults (79 males, 83 females) between 20-30 years old. Anthropometric parameters including palpebral fissure length and height, intercanthal and outercanthal width, crease height, angle of endocanthion and exocanthion, axis of palpebral fissure, palpebral fissure index, canthal index, and angular index were acquired from standardized photographs. Then, 134 volunteers (20-30 years old) gave each photograph a score within 1-5 points to evaluate their ocular aesthetic attractiveness. The correlation between anthropometric parameters and aesthetic assessment was analyzed. RESULTS: A statistical difference between genders was found for palpebral fissure length and height, outercanthal width, angle of exocanthion, palpebral fissure index and canthal index (p < 0.05), with no statistical difference found for crease height between genders. Moreover, the palpebral fissure index, canthal index, crease height, and angle of exocanthion were significantly associated with aesthetic assessment. CONCLUSIONS: The normative anthropometric parameters are fundamental to interpret the morphology of eyes and to design plastic surgery for young Chinese adults. The parameters of palpebral fissure index, canthal index, crease height, and angle of exocanthion are strong indicators of aesthetic assessment.

Staged Surgery with Total Excision and Lamellar Reconstructive for Medium-sized Divided Nevus of the Eyelids.

BACKGROUND: To explore a prior treatment strategy for medium-sized (1.5-20 cm) divided nevus of the eyelids. METHODS: Six patients who suffered from divided nevus of eyelids were recruited to this prospective, case series study between July 2008 and January 2014 (4 male and 2 female patients). The patients' ages ranged from 14 to 29 years, with an average age of 24.5 years. All lesions were medium-sized (1.5-20 cm in diameter) and invaded eyelid margins and the posterior lamella of eyelids. Staged surgery involved total excision of lesions and then repair of the defects with advanced skin flaps and tarsoconjunctival flaps. Two staged surgeries were completed at intervals of at least 3 months. RESULTS: All of the patients were followed up at least 3 months after the second surgery. Malignant transformation and recurrence were not observed. All of the flaps survived well, and all of the donor sites were healed with inconspicuous scarring. The only complication was eyelash sacrifices, and 5 of 6 patients suffered from this complication. Excellent cosmetic results were gained in all patients, with the exception of 1 patient who thought his postoperative appearance was only good because of the impalpable disparity in color and thickness between the skin flaps and recipient sites. CONCLUSIONS: A staged surgery approach with the total excision of lesions and lamellar reconstructive procedures to repair the defect is a reasonable treatment strategy and can achieve satisfactory cosmetic results for medium-sized (1.5-20 cm in diameter) divided nevus of eyelid.

The influence of benign essential blepharospasm on dry eye disease and ocular inflammation.

PURPOSE: To study the influence of blepharospasm on dry eye disease by analyzing the clinical features, tear cytokine, and treatment response of patients with dry eye disease accompanied by benign essential blepharospasm. DESIGN: Prospective case series study. METHODS: Forty adults with a diagnosis of benign essential blepharospasm (BEB) and dry eye disease (DED) were consecutively recruited. Forty subjects with dry eye disease only and 40 healthy adults were recruited as eligible controls. A tear specimen was collected from all participants for cytokine analysis. The patients with benign essential blepharospasm were treated with botulinum neurotoxin type A. The main outcome measures were the following: (1) Ocular Surface Disease Index (OSDI) questionnaire; (2) clinical features, including tear break-up time (BUT), Schirmer І test, and fluorescein staining; (3) conjunctival impression cytology; and (4) multiplex cytokine immunobead assay. RESULTS: The symptoms of DED + BEB patients were significantly different from those of DED controls and healthy controls. Cytokine analysis in tear fluid also showed that tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-2, IL-17, and vascular endothelial growth factor levels were significantly increased in DED + BEB patients. In treatment, botulinum neurotoxin type A injection effectively relieved blepharospasm in all of the DED + BEB patients. Moreover, in this group of patients, OSDI decreased significantly after the botulinum neurotoxin type A injection, and BUT was increased as well. CONCLUSION: BEB may participate in the progress of inflammation in DED + BEB patients. Botulinum neurotoxin type A injections could effectively relieve the symptoms of DED + BEB patients and improve their ocular surface condition.