Toward a Clinical Protocol for Assessing Rod, Cone, and Melanopsin Contributions to the Human Pupil Response

PURPOSE. To better understand the relative contributions of rod, cone, and melanopsin to the human pupillary light reflex (PLR) and to determine the optimal conditions for assessing the health of the rod, cone, and melanopsin pathways with a relatively brief clinical protocol. METHODS. PLR was measured with an eye tracker, and stimuli were controlled with a Ganzfeld system. In experiment 1, 2.5 log cd/m(2) red (640 +/- 10 nm) and blue (467 +/- 17 nm) stimuli of various durations were presented after dark adaptation. In experiments 2 and 3, 1-second red and blue stimuli were presented at different intensity levels in the dark (experiment 2) or on a 0.78 log cd/m(2) blue background (experiment 3). Based on the results of experiments 1 to 3, a clinical protocol was designed and tested on healthy control subjects and patients with retinitis pigmentosa and Leber`s congenital amaurosis. RESULTS. The duration for producing the optimal melanopsin-driven sustained pupil response after termination of an intense blue stimulus was 1 second. PLR rod-and melanopsin-driven components are best studied with low-and high-intensity flashes, respectively, presented in the dark (experiment 2). A blue background suppressed rod and melanopsin responses, making it easy to assess the cone contribution with a red flash (experiment 3). With the clinical protocol, robust melanopsin responses could be seen in patients with few or no contributions from the rods and cones. CONCLUSIONS. It is possible to assess the rod, cone, and melanopsin contributions to the PLR with blue flashes at two or three intensity levels in the dark and one red flash on a blue background. (Invest Ophthalmol Vis Sci. 2011; 52: 6624-6635) DOI: 10.1167/iovs.11-7586

Phosphene Thresholds Elicited by Transcorneal Electrical Stimulation in Healthy Subjects and Patients with Retinal Diseases

PURPOSE. To evaluate electrically evoked phosphene thresholds (EPTs) in healthy subjects and in patients with retinal disease and to assess repeatability and possible correlations with common ophthalmologic tests. METHODS. In all, 117 individuals participated: healthy subjects (n = 20) and patients with retinitis pigmentosa (RP, n = 30), Stargardt's disease (STG, n = 14), retinal artery occlusion (RAO, n = 20), nonarteritic anterior ischemic optic neuropathy (NAION, n = 16), and primary open-angle glaucoma (POAG, n = 17). EPTs were determined at 3, 6, 9, 20, 40, 60, and 80 Hz with 5+5-ms biphasic current pulses using DTL electrodes. Subjects were examined twice (test-retest range: 1-6 weeks). An empirical model was developed to describe the current-frequency relationship of EPTs. Visual acuity, visual field (kinetic + static), electrophysiology (RP, RAO, STG: Ganzfeld-electroretinography [ERG]/multifocal-ERG; POAG: pattern-ERG; NAION: VEP), slit-lamp biomicroscopy, fundus examination, and tonometry were assessed. RESULTS. EPTs varied between disease groups (20 Hz: healthy subjects: 0.062 +/- 0.038 mA; STG: 0.102 +/- 0.097 mA; POAG: 0.127 +/- 0.09 mA; NAION: 0.244 +/- 0.126 mA; RP: 0.371 +/- 0.223 mA; RAO: 0.988 +/- 1.142 mA). In all groups EPTs were lowest at 20 Hz. In patients with retinal diseases and across all frequencies EPTs were significantly higher than those in healthy subjects, except in STG at 20 Hz (P = 0.09) and 40 Hz (P = 0.17). Test-retest difference at 20 Hz was 0.006 mA in the healthy group and 0.003-0.04 mA in disease groups. CONCLUSIONS. Considering the fast, safe, and reliable practicability of EPT testing, this test might be used more often under clinical circumstances. Determination of EPTs could be potentially useful in elucidation of the progress of ophthalmologic diseases, either in addition to standard clinical assessment or under conditions in which these standard tests cannot be used meaningfully. (ClinicalTrials.gov number, NCT00804102.) (Invest Ophthalmol Vis Sci. 2012; 53: 7440-7448) DOI:10.1167/iovs.12-9612

Assessment of "non-recordable" electroretinograms by 9 Hz flicker stimulation under scotopic conditions

To refine methods of electroretinographical (ERG) recording for the analysis of low retinal potentials under scotopic conditions in advanced retinal degenerative diseases. Standard Ganzfeld ERG equipment (Diagnosys LLC, Cambridge, UK) was used in 27 healthy volunteers (mean age 28 +/- A SD 8.5 years) to define the stimulation protocol. The protocol was then applied in clinical routine and 992 recordings were obtained from patients (mean age 40.6 +/- A 18.3 years) over a period of 5 years. A blue stimulus with a flicker frequency of 9 Hz was specified under scotopic conditions to preferentially record rod-driven responses. A range of stimulus strengths (0.0000012-6.32 scot. cd s/mA(2) and 6-14 ms flash duration) was tested for maximal amplitudes and interference between rods and cones. Analysis of results was done by standard Fourier Transformation and assessment of signal-to-noise ratio. Optimized stimulus parameters were found to be a time-integrated luminance of 0.012 scot. cd s/mA(2) using a blue (470 nm) flash of 10 ms duration at a repetition frequency of 9 Hz. Characteristic stimulus strength versus amplitude curves and tests with stimuli of red or green wavelength suggest a predominant rod-system response. The 9 Hz response was found statistically distinguishable from noise in 38% of patients with otherwise non-recordable rod responses according to International Society for Clinical Electrophysiology of Vision standards. Thus, we believe this protocol can be used to record ERG potentials in patients with advanced retinal diseases and in the evaluation of potential treatments for these patients. The ease of implementation in clinical routine and of statistical evaluation providing an observer-independent evaluation may further facilitate its employment.

Haploinsufficiency of a Spliceosomal GTPase Encoded by EFTUD2 Causes Mandibulofacial Dysostosis with Microcephaly

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare sporadic syndrome comprising craniofacial malformations, microcephaly, developmental delay, and a recognizable dysmorphic appearance. Major sequelae, including choanal atresia, sensorineural hearing loss, and cleft palate, each occur in a significant proportion of affected individuals. We present detailed clinical findings in 12 unrelated individuals with MFDM; these 12 individuals compose the largest reported cohort to date. To define the etiology of MFDM, we employed whole-exome sequencing of four unrelated affected individuals and identified heterozygous mutations or deletions of EFTUD2 in all four. Validation studies of eight additional individuals with MFDM demonstrated causative EFTUD2 mutations in all affected individuals tested. A range of EPTUD2-mutation types, including null alleles and frameshifts, is seen in MFDM, consistent with haploinsufficiency; segregation is de novo in all cases assessed to date. U5-116kD, the protein encoded by EFTUD2, is a highly conserved spliceosomal GTPase with a central regulatory role in catalytic splicing and post-splicing-complex disassembly. MFDM is the fast multiple-malformation syndrome attributed to a defect of the major spliceosome. Our findings significantly extend the range of reported spliceosomal phenotypes in humans and pave the way for further investigation in related conditions such as Treacher Collins syndrome.

Transcorneal Electrical Stimulation Shows Neuroprotective Effects in Retinas of Light-Exposed Rats

PURPOSE. To examine the effects of transcorneal electrical stimulation (TES) on retinal degeneration of light-exposed rats. METHODS. Thirty-three Sprague Dawley albino rats were divided into three groups: STIM (n = 15) received 60 minutes of TES, whereas SHAM (n = 15) received identical sham stimulation 2 hours before exposure to bright light with 16,000 lux; healthy animals (n = 3) served as controls for histology. At baseline and weekly for 3 consecutive weeks, dark-and light-adapted electroretinography was used to assess retinal function. Analysis of the response versus luminance function retrieved the parameters Vmax (saturation amplitude) and k (luminance to reach 1/2Vmax). Retinal morphology was assessed by histology (hematoxylin-eosin [HE] staining; TUNEL assay) and immunohistochemistry (rhodopsin staining). RESULTS. Vmax was higher in the STIM group compared with SHAM 1 week after light damage (mean intra-individual difference between groups 116.06 mu V; P = 0.046). The b-wave implicit time for the rod response (0.01 cd.s/m(2)) was lower in the STIM group compared with the SHAM group 2 weeks after light damage (mean intra-individual difference between groups 5.78 ms; P = 0.023); no other significant differences were found. Histological analyses showed photoreceptor cell death (TUNEL and HE) in SHAM, most pronounced in the superior hemiretina. STIM showed complete outer nuclear layer thickness preservation, reduced photoreceptor cell death, and preserved outer segment length compared with SHAM (HE and rhodopsin). CONCLUSIONS. This sham-controlled study shows that TES can protect retinal cells against mild light-induced degeneration in Sprague Dawley rats. These findings could help to establish TES as a treatment in human forms of retinal degenerative disease. (Invest Ophthalmol Vis Sci. 2012;53:5552-5561) DOI: 10.1167/iovs.12-10037

Vitreous pharmacokinetics and electroretinographic findings after intravitreal injection of acyclovir in rabbits

OBJECTIVES: Acute retinal necrosis is a rapidly progressive and devastating viral retinitis caused by the herpesvirus family. Systemic acyclovir is the treatment of choice; however, the progression of retinal lesions ceases approximately 2 days after treatment initiation. An intravitreal injection of acyclovir may be used an adjuvant therapy during the first 2 days of treatment when systemically administered acyclovir has not reached therapeutic levels in the retina. The aims of this study were to determine the pharmacokinetic profile of acyclovir in the rabbit vitreous after intravitreal injection and the functional effects of acyclovir in the rabbit retina. METHODS: Acyclovir (Acyclovir; Bedford Laboratories, Bedford, OH, USA) 1 mg in 0.1 mL was injected into the right eye vitreous of 32 New Zealand white rabbits, and 0.1 mL sterile saline solution was injected into the left eye as a control. The animals were sacrificed after 2, 9, 14, or 28 days. The eyes were enucleated, and the vitreous was removed. The half-life of acyclovir was determined using high-performance liquid chromatography. Electroretinograms were recorded on days 2, 9, 14, and 28 in the eight animals that were sacrificed 28 days after injection according to a modified protocol of the International Society for Clinical Electrophysiology of Vision. RESULTS: Acyclovir rapidly decayed in the vitreous within the first two days after treatment and remained at low levels from day 9 onward. The eyes that were injected with acyclovir did not present any electroretinographic changes compared with the control eyes. CONCLUSIONS: The vitreous half-life of acyclovir is short, and the electrophysiological findings suggest that the intravitreal delivery of 1 mg acyclovir is safe and well tolerated by the rabbit retina.