Patrick H. W. Chu

Porcine global flash multifocal electroretinogram: possible mechanisms for the glaucomatous changes in contrast response function.

PURPOSE The aim of this study was to obtain a better understanding of the cellular contributions to the porcine global flash mfERG by using a pharmacologic dissection method, together with the method using variation of stimulus contrast which has been used to demonstrate mfERG changes in human glaucoma. METHODS Global flash mfERGs with different stimulus-contrast settings (99%, 65%, 49% or 29%) were recorded from 14 eyes of ten 6-week-old Yorkshire pigs in control conditions and after suppression of inner retinal responses with inhalation of isoflurance (ISO), and injections of tetrodotoxin (TTX) and N-methyl-d-aspartic acid (NMDA). ON- and OFF-pathway responses were isolated by injection of 2-amino-4-phosphonobutyric acid (APB) and cis-2,3-piperidinedicarboylic acid (PDA). RESULTS The porcine global flash mfERG consisted of an early direct component (DC) and a late induced component (IC). ISO and TTX removed inner retinal contributions to the IC; NMDA application further abolished the oscillatory wavelets in the DC and removed the residual IC waveform. The inner retina contributed regular oscillation-like wavelets (W1, W2 and W3) to the DC and shaped the IC. After removing the inner retinal contributions, the porcine global flash mfERG waveform becomes comparable to that obtained with conventional mfERG stimulation. The remaining waveform (smoothed DC) was mainly contributed by the ON- and OFF-bipolar cells as revealed after APB or PDA injection. Photoreceptors contributed a small signal to the leading edge of N1. The characteristic of contrast response function of DC was demonstrated to be contributed by the inner retinal oscillation-like wavelets. CONCLUSION We believe that the DC of the porcine global flash mfERG is mainly composed of contributions from photoreceptors, and ON- and OFF-bipolar cells, where inner retinal activity partially shaped the DC with superimposed regular wavelets. However, the IC is dominated by inner retinal activity. The contrast response functions of DC consisted of both outer retinal response and oscillation-like wavelets of the inner retinal response. Both contain different characteristics during contrast modulation of the stimulus, where the changes of W2 of the inner retinal response seem independent of contrast modulation. The DC contrast response feature depends mainly on the relative contribution of inner retinal activities; the loss of inner retinal cells may alter the DC contrast response function, making it tend toward linearity.

Pharmacologically defined components of the normal porcine multifocal ERG

Multifocal electroretinograms (mfERG) from isoflurane anesthetized pigs were recorded and sequential application of TTX, NMDA, APB and PDA were used to identify contributions to the mfERG from inner retinal neurons, ON-pathway, OFF-pathway and photoreceptors. The cellular origins of the first-order kernel (K1) and the first slice of the second-order kernel (K2.1) porcine mfERG are contributed from both inner and outer retina. For the K1 waveform, the n1 involved responses of cone photoreceptors and OFF-bipolar cells. The leading edge of p1 is dominated by ON-bipolar cell depolarization. The rear edge of p1, n2 and p2 are dominated by ON-bipolar activities and shaped by the activities of OFF-bipolar cells and retinal cells with NMDAr and voltage-gated sodium channels other than ganglion cells. The p3 is mainly inner retinal activities. For the K2.1 waveform, the p1 and n1 are the summation of activities of ON-, OFF-bipolar cells and retinal cells rich in NMDAr and voltage-gated sodium channels other than ganglion cells. The p2 seems to be related to the ganglion cells. Better understanding of the cellular origins of the normal porcine mfERG will be useful for comparing and defining the functional changes that may occur in diseased retinas.

Multifocal electroretinogram in rhodopsin P347L transgenic pigs.

PURPOSE Neural ectopic rewiring in retinal degeneration such as retinitis pigmentosa (RP) may form functional synapses between cones and rod bipolar cells that cause atypical signal processing. In this study, the multifocal electroretinograms (mfERGs) of a large animal model of RP, the rhodopsin P347L transgenic (Tg) pig, were measured to examine the sources and nature of altered signal processing. METHODS mfERG responses from a 6-week-old Tg pig were recorded before and after sequential application of tetrodotoxin (TTX), N-methyl-D-aspartate (NMDA), 2-amino-4-phosphonobutyric acid (APB), and cis-2,3-piperidinedicarboylic acid (PDA), to identify contributions to the retinal signal from inner retinal neurons, the ON-pathway, the OFF-pathway, and photoreceptors. The mfERG response contributions from different retinal components of in the Tg eyes were estimated and compared with control data from eyes of age-matched wild-type (WT) pigs. RESULTS There was a prominent difference in the estimates of the inner retinal response and ON-bipolar cell pathway contribution between the Tg and WT mfERG responses. In particular, the early components of the inner retinal contribution were obviously altered in the Tg mfERG. The inner retinal components at approximately 24 and 40 ms appeared to be inverted. Differences in the estimates of OFF-bipolar cell pathway contributions were minimal. There was no change in cone cell responses in the Tg mfERG. CONCLUSIONS In Tg retinas, ectopic synapses formed between cones and rod bipolar cells probably altered signal processing of the ON-bipolar cell pathway. In response to the altered visual signal input from the outer retina, signal processing in inner retinal neurons was also modified.

Effect of Lycium Barbarum (Wolfberry) Polysaccharides on Preserving Retinal Function after Partial Optic Nerve Transection

Lycium Barbarum Polysaccharides (LBP) are the active components of Wolfberry (a traditional Chinese medicine) which has long been used for improving visual function. This study aims to investigate localized changes of retinal function in a partial optic nerve transection (PONT) model, and effects of LBP on visual function. The multifocal electroretinograms (mfERG) were obtained from 30 eyes of 30 Sprague-Dawley rats. The rats were divided into 6 groups (five treatment groups and one control group). Starting from the first day of the experiment, the rats in the (PONT+LBP) group and the (LBP) group were dosed with LBP; rats in the (PONT+PBS (phosphate buffered saline)) group and the (PBS) group were dosed with PBS via nasogastric tube every day until euthanized. The dorsal part of the optic nerve was transected in the (PONT), (PONT+LBP) and (PONT+PBS) groups at the end of week 1 (day 7 after LBP or PBS feeding began). The mfERG was measured at three time points: week 2, week 3 and week 5. Significant reduction of P1 and PhNR amplitudes of the mfERG were observed in all retinal regions a week after PONT. Feeding with LBP prior to PONT preserved retinal function. All mfERG responses returned to the normal range in the superior retina, which corresponds to the transected dorsal region of the optic nerve, while most of the inferior retinal responses were significantly increased at week 4 after PONT. The ventral part of the retina had secondary degeneration which was not only limited to the ganglion cell layer, but is a widespread effect affecting the outer retina. LBP altered the functional reduction caused by PONT by regulating the signal from the outer retina.

A novel missense mutation in the NYX gene associated with high myopia

Myopia is a complex eye disorder. The X‐linked form of complete congenital stationary night blindness (CSNB1A) is usually associated with moderate to high myopia, and is caused by mutations in the NYX gene. We explored if NYX mutations could be associated with high myopia, but not CSNB1A.

Effect of inner retinal dysfunction on slow double-stimulation multifocal electroretinogram.

Purpose This study investigated the retinal adaptive mechanism in inner retinal dysfunction using the slow double-stimulation multifocal electroretinogram (mfERG) paradigm. Methods Slow double-stimulation mfERG responses were recorded from 15 eyes of 15 4-month-old Mongolian gerbils in control conditions and after suppression of inner retinal responses with injections of tetrodotoxin (TTX) and N-methyl-d-aspartic acid (NMDA). The stimulation consisted of five video frames: the two initial frames with multifocal flashes were triggered by two independent m-sequences, followed by three dark video frames. The results were compared with findings in humans: 7 subjects with glaucoma and 31 age-matched normal subjects were measured using the same mfERG protocol. Results The stimulation generates two responses (M1 and M2) from the two independent multifocal frames. The M1:M2 ratio showed a significant reduction after administration of TTX+NMDA in the animal study. This matched with the human glaucoma findings. Glaucoma subjects generally have a reduced M1:M2 ratio; this ratio showed a sensitivity of 86%, with a specificity of 84% for differentiating normal eyes from glaucomatous eyes. Conclusion This stimulation paradigm provides a method of measuring temporal visual characteristics. The M1:M2 ratio acts as an indirect functional indicator of retinal adaptation, which may be abnormal in the diseased retina. Further development of this method may help to describe the functional variation in the diseased retina and to predict the occurrence of a range of retinopathies.

Forward and backward adaptive effects in global flash multifocal electroretinogram stimulation

Purpose:  The present study investigated retinal adaptive responses in concert with the modulation of forward and backward adaptation induced by periodic global flashes using the global flash multifocal electroretinogram (mfERG). Methods:  Six normal subjects were recruited for global flash mfERG measurements, which consisted of 103 scaled hexagonal elements followed by a global flash frame. In experiments I and II, with constant luminance maintained in both local and global flash frames, the number of dark frames was independently varied and these frames were either inserted prior to or following the global flash frame to investigate the forward or backward adaptive effect of the global flash on the mfERG. In experiment III, the number of dark frames was fixed but the luminance of the global flash frame was varied with constant luminance of the focal flash. This was used to demonstrate that the adaptive effect related not to time but to variation of luminance. Results:  All the central, para-central and peripheral direct component amplitudes were found to be significantly influenced by variation of the number of dark frames (p < 0.01). Reducing the forward adaptive effect of the global flash enhanced the direct component response and it became steady after five dark frames were inserted following the global flash. Reducing the backward adaptive effect of the global flash also enhanced the direct component response but it started reducing after four dark frames were inserted prior to the global flash frame. These changes were different with luminance modulation of the global flash intensity with fixed dark frames, while the direct component amplitude grew approximately linearly with decreasing mean luminance of the global flash stimulation. Conclusion:  The retina plays a major role in visual adaptation. Both forward and backward adaptive effects of the global flash on the direct component have been illustrated in this study. The results show that the forward and backward adaptive phenomena in the global flash mfERG are different and demonstrate that backward adaptation is found at the retinal level.Purpose:  The present study investigated retinal adaptive responses in concert with the modulation of forward and backward adaptation induced by periodic global flashes using the global flash multifocal electroretinogram (mfERG).

Temporal interactive response is resistant to cloudy ocular media in the slow double-stimulation multifocal electroretinogram

Aim To examine the influence of cloudy media on the slow double-stimulation multifocal electroretinogram (mfERG). Methods Slow double-stimulation mfERG responses were measured from 26 subjects with normal ocular health under normal and light scattering conditions (induced using acrylic sheets) (Experiment 1) and another nine cataract patients before and after cataract surgery (Experiment 2). The amplitudes and implicit times of the first (M1) and second (M2) stimulation were compared under normal and light scattering conditions in Experiment 1 and they were compared under precataract and postcataract surgery in Experiment 2. Results Compared with control conditions (normal and postcataract surgery), the M1 amplitude in the central region was significantly reduced in light scattering conditions (acrylic sheets and precataract surgery); the M2 amplitude and both M1 and M2 implicit times of all regions examined were moderately affected in precataract surgery. The M1:M2 amplitude ratio and implicit time ratio were virtually unaffected in cloudy media for either central or mid-peripheral regions. Conclusion Cloudy media affects the mfERG amplitude and implicit time in the slow double-stimulation, but does not affect the response ratio (ie, M1:M2 amplitude ratio and implicit time ratio) between the two stimulations. This suggests that the ratio analysis can be applied in patients with mild to moderately cloudy ocular media to evaluate the functional integrity of the retina.

Human Electroretinal Responses to Grating Patterns and Defocus Changes by Global Flash Multifocal Electroretinogram

The electrical response of the retina was examined as a function of retinal region, using stimuli of various spatial frequencies in the first experiment. In the second experiment, the regional response of the retina to defocus at high and low spatial frequencies was investigated. Twenty three subjects were recruited for global flash multifocal electroretinogram (mfERG) in experiment 1. Black and white gratings (printed on plastic transparent sheets) of four spatial frequencies (SF), 0.24, 1.2, 2.4 and 4.8 cycle per degree were presented in front of the mfERG stimulation. The amplitudes and implicit times of the direct (DC) and induced (IC) components of mfERG responses were pooled into six concentric rings for analysis. There was low amplitude DC at low SF, which increased with increasing SF, and which decreased with increasing eccentricity. The IC was high in amplitude at all SF and reduced in amplitude with increasing eccentricity. Our findings suggested that outer and inner retina had different characteristics in processing spatial details. In experiment 2, Twenty-three young adults were recruited for mfERG measurement. The retinal electrical responses for low (0.24cpd) and high (4.8cpd) SF under fully corrected conditions of short-term negative defocus (-2D) and short term positive defocus (+2D) conditions were measured. There was a sign-dependent response to defocus in the DC response, mainly in peripheral regions. The sign dependent response at low SF was more obvious than that at high SF, and was located more peripherally. The IC response showed no clear trends for either defocus condition. The human retina seems to have a decoding system for optical defocus, which was tuned for low spatial frequency, and was located in the retinal near periphery.

Secondary Degeneration After Partial Optic Nerve Injury and Possible Neuroprotective Effects of Lycium Barbarum (Wolfberry)

Secondary degeneration occurs commonly in a range of neurodegenerative diseases, including glaucoma. Partial optic nerve transection (PONT) model was established in the last decade and was good for studying secondary degeneration in retinas and optic nerves. The results from the published papers about PONT showed that the mechanisms—apoptosis, necrosis, autophagy, oxidative stress, calcium overload, mitochondria, activation of c-jun, water channel change, and glial cells (microglia, astrocytes and oligodendrocytes)—were involved in secondary degeneration after PONT. In addition to the cell bodies and the axons of retinal ganglion cells (RGCs), other cells in the layers outside the ganglion cell layer were also affected according to the measurement of multifocal electroretinogram (mfERG) by our group. Lycium barbarum (L. barbarum) is a traditional medicine in the oriental world and has long been used as a functional food and for medicinal purposes. The data from our group and others showed that the polysaccharides extracted from L. barbarum (LBP) were neuroprotective in different animal models, including the PONT model. Our results showed that LBP could inhibit secondary degeneration of the cell bodies of RGCs rather than primary degeneration as well as preserve the function of retinas measured by mfERG. These effects are related with the antioxidant function of LBP, inhibition of c-jun N-terminal kinase (JNK) pathway in the retinas after PONT. Other possible mechanisms involved in LBP’s neuroprotective effects for secondary degeneration are immunomodulatory effects, preservation of synapses, and modulation of autophagy.