Decreased visual function after patchy loss of retinal pigment epithelium induced by low-dose sodium iodate

PURPOSE: To correlate damage to the retinal pigment epithelium (RPE) with decreased visual function after the systemic administration of sodium iodate (NaIO(3)). METHODS: Damage was produced in mice by injection of 15, 25, or 35 mg/kg NaIO(3). Visual function was assessed with the cued water maze (WM) behavioral test and the optokinetic reflex (OKR) measurement at different times after injection. Autofluorescence in whole eye flatmounts was quantified, and hematoxylin and eosin staining of paraffin sections was performed to assess changes in the outer retina. RESULTS: After 15 mg/kg NaIO(3), cued WM test results were normal, whereas OKR measurements were significantly decreased at all times. Focal RPE loss began on day 21, but no significant damage to the outer nuclear layer was observed. After 25 mg/kg NaIO(3), the cued WM test was transitionally reduced and the OKR measurement again decreased at all times. Large areas of RPE loss occurred on day 14 with a reduced outer nuclear layer on the same day. With 35 mg/kg NaIO(3), the cued WM test was reduced beginning on day 14 with complete obliteration of the OKR beginning on day 3, large areas of RPE loss on the same day, and a reduced outer nuclear layer on day 7. CONCLUSIONS: Stable, patchy RPE loss was observed with a low concentration of NaIO(3). The OKR measurement showed changes in visual function earlier than the cued WM test and before histologic findings were observed.

Characteristics of rod regeneration in a novel zebrafish retinal degeneration model using N-methyl-N-nitrosourea (MNU)

Primary loss of photoreceptors caused by diseases such as retinitis pigmentosa is one of the main causes of blindness worldwide. To study such diseases, rodent models of N-methyl-N-nitrosourea (MNU)-induced retinal degeneration are widely used. As zebrafish (Danio rerio) are a popular model system for visual research that offers persistent retinal neurogenesis throughout the lifetime and retinal regeneration after severe damage, we have established a novel MNU-induced model in this species. Histology with staining for apoptosis (TUNEL), proliferation (PCNA), activated Müller glial cells (GFAP), rods (rhodopsin) and cones (zpr-1) were performed. A characteristic sequence of retinal changes was found. First, apoptosis of rod photoreceptors occurred 3 days after MNU treatment and resulted in a loss of rod cells. Consequently, proliferation started in the inner nuclear layer (INL) with a maximum at day 8, whereas in the outer nuclear layer (ONL) a maximum was observed at day 15. The proliferation in the ONL persisted to the end of the follow-up (3 months), interestingly, without ongoing rod cell death. We demonstrate that rod degeneration is a sufficient trigger for the induction of Müller glial cell activation, even if only a minimal number of rod cells undergo cell death. In conclusion, the use of MNU is a simple and feasible model for rod photoreceptor degeneration in the zebrafish that offers new insights into rod regeneration.

Optical imaging of postsynaptic odor representation in the glomerular layer of the mouse olfactory bulb.

Olfactory glomeruli are the loci where the first odor-representation map emerges. The glomerular layer comprises exquisite local synaptic circuits for the processing of olfactory coding patterns immediately after their emergence. To understand how an odor map is transferred from afferent terminals to postsynaptic dendrites, it is essential to directly monitor the odor-evoked glomerular postsynaptic activity patterns. Here we report the use of a transgenic mouse expressing a Ca(2+)-sensitive green fluorescence protein (GCaMP2) under a Kv3.1 potassium-channel promoter. Immunostaining revealed that GCaMP2 was specifically expressed in mitral and tufted cells and a subpopulation of juxtaglomerular cells but not in olfactory nerve terminals. Both in vitro and in vivo imaging combined with glutamate receptor pharmacology confirmed that odor maps reported by GCaMP2 were of a postsynaptic origin. These mice thus provided an unprecedented opportunity to analyze the spatial activity pattern reflecting purely postsynaptic olfactory codes. The odor-evoked GCaMP2 signal had both focal and diffuse spatial components. The focalized hot spots corresponded to individually activated glomeruli. In GCaMP2-reported postsynaptic odor maps, different odorants activated distinct but overlapping sets of glomeruli. Increasing odor concentration increased both individual glomerular response amplitude and the total number of activated glomeruli. Furthermore, the GCaMP2 response displayed a fast time course that enabled us to analyze the temporal dynamics of odor maps over consecutive sniff cycles. In summary, with cell-specific targeting of a genetically encoded Ca(2+) indicator, we have successfully isolated and characterized an intermediate level of odor representation between olfactory nerve input and principal mitral/tufted cell output.

Nerve Injury-Induced Neuropathic Pain Causes Disinhibition of the Anterior Cingulate Cortex

Neuropathic pain caused by peripheral nerve injury is a debilitating neurological condition of high clinical relevance. On the cellular level, the elevated pain sensitivity is induced by plasticity of neuronal function along the pain pathway. Changes in cortical areas involved in pain processing contribute to the development of neuropathic pain. Yet, it remains elusive which plasticity mechanisms occur in cortical circuits. We investigated the properties of neural networks in the anterior cingulate cortex (ACC), a brain region mediating affective responses to noxious stimuli. We performed multiple whole-cell recordings from neurons in layer 5 (L5) of the ACC of adult mice after chronic constriction injury of the sciatic nerve of the left hindpaw and observed a striking loss of connections between excitatory and inhibitory neurons in both directions. In contrast, no significant changes in synaptic efficacy in the remaining connected pairs were found. These changes were reflected on the network level by a decrease in the mEPSC and mIPSC frequency. Additionally, nerve injury resulted in a potentiation of the intrinsic excitability of pyramidal neurons, whereas the cellular properties of interneurons were unchanged. Our set of experimental parameters allowed constructing a neuronal network model of L5 in the ACC, revealing that the modification of inhibitory connectivity had the most profound effect on increased network activity. Thus, our combined experimental and modeling approach suggests that cortical disinhibition is a fundamental pathological modification associated with peripheral nerve damage. These changes at the cortical network level might therefore contribute to the neuropathic pain condition.

Methylnitrosourea (MNU)-induced retinal degeneration and regeneration in the zebrafish: histological and functional characteristics.

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial world. Animal models are of pivotal importance to study such diseases. In this regard the photoreceptor-specific toxin N-methyl-N-nitrosourea (MNU) has been widely used in rodents to pharmacologically induce retinal degeneration. Previously, we have established a MNU-induced retinal degeneration model in the zebrafish, another popular model system in visual research. A fascinating difference to mammals is the persistent neurogenesis in the adult zebrafish retina and its regeneration after damage. To quantify this observation we have employed visual acuity measurements in the adult zebrafish. Thereby, the optokinetic reflex was used to follow functional changes in non-anesthetized fish. This was supplemented with histology as well as immunohistochemical staining for apoptosis (TUNEL) and proliferation (PCNA) to correlate the developing morphological changes. In summary, apoptosis of photoreceptors occurs three days after MNU treatment, which is followed by a marked reduction of cells in the outer nuclear layer (ONL). Thereafter, proliferation of cells in the inner nuclear layer (INL) and ONL is observed. Herein, we reveal that not only a complete histological but also a functional regeneration occurs over a time course of 30 days. Now we illustrate the methods to quantify and follow up zebrafish retinal de- and regeneration using MNU in a video-format.

Quality control for retinal OCT in multiple sclerosis: validation of the OSCAR-IB criteria.

BACKGROUND Retinal optical coherence tomography (OCT) permits quantification of retinal layer atrophy relevant to assessment of neurodegeneration in multiple sclerosis (MS). Measurement artefacts may limit the use of OCT to MS research. OBJECTIVE An expert task force convened with the aim to provide guidance on the use of validated quality control (QC) criteria for the use of OCT in MS research and clinical trials. METHODS A prospective multi-centre (n = 13) study. Peripapillary ring scan QC rating of an OCT training set (n = 50) was followed by a test set (n = 50). Inter-rater agreement was calculated using kappa statistics. Results were discussed at a round table after the assessment had taken place. RESULTS The inter-rater QC agreement was substantial (kappa = 0.7). Disagreement was found highest for judging signal strength (kappa = 0.40). Future steps to resolve these issues were discussed. CONCLUSION Substantial agreement for QC assessment was achieved with aid of the OSCAR-IB criteria. The task force has developed a website for free online training and QC certification. The criteria may prove useful for future research and trials in MS using OCT as a secondary outcome measure in a multi-centre setting.

Investigation of retinal morphology alterations using spectral domain optical coherence tomography in a mouse model of retinal branch and central retinal vein occlusion.

Retinal vein occlusion is a leading cause of visual impairment. Experimental models of this condition based on laser photocoagulation of retinal veins have been described and extensively exploited in mammals and larger rodents such as the rat. However, few reports exist on the use of this paradigm in the mouse. The objective of this study was to investigate a model of branch and central retinal vein occlusion in the mouse and characterize in vivo longitudinal retinal morphology alterations using spectral domain optical coherence tomography. Retinal veins were experimentally occluded using laser photocoagulation after intravenous application of Rose Bengal, a photo-activator dye enhancing thrombus formation. Depending on the number of veins occluded, variable amounts of capillary dropout were seen on fluorescein angiography. Vascular endothelial growth factor levels were markedly elevated early and peaked at day one. Retinal thickness measurements with spectral domain optical coherence tomography showed significant swelling (p<0.001) compared to baseline, followed by gradual thinning plateauing two weeks after the experimental intervention (p<0.001). Histological findings at day seven correlated with spectral domain optical coherence tomography imaging. The inner layers were predominantly affected by degeneration with the outer nuclear layer and the photoreceptor outer segments largely preserved. The application of this retinal vein occlusion model in the mouse carries several advantages over its use in other larger species, such as access to a vast range of genetically modified animals. Retinal changes after experimental retinal vein occlusion in this mouse model can be non-invasively quantified by spectral domain optical coherence tomography, and may be used to monitor effects of potential therapeutic interventions.

Quantitative Analysis of Mouse Retinal Layers Using Automated Segmentation of Spectral Domain Optical Coherence Tomography Images.

PURPOSE Quantification of retinal layers using automated segmentation of optical coherence tomography (OCT) images allows for longitudinal studies of retinal and neurological disorders in mice. The purpose of this study was to compare the performance of automated retinal layer segmentation algorithms with data from manual segmentation in mice using the Spectralis OCT. METHODS Spectral domain OCT images from 55 mice from three different mouse strains were analyzed in total. The OCT scans from 22 C57Bl/6, 22 BALBc, and 11 C3A.Cg-Pde6b(+)Prph2(Rd2) /J mice were automatically segmented using three commercially available automated retinal segmentation algorithms and compared to manual segmentation. RESULTS Fully automated segmentation performed well in mice and showed coefficients of variation (CV) of below 5% for the total retinal volume. However, all three automated segmentation algorithms yielded much thicker total retinal thickness values compared to manual segmentation data (P < 0.0001) due to segmentation errors in the basement membrane. CONCLUSIONS Whereas the automated retinal segmentation algorithms performed well for the inner layers, the retinal pigmentation epithelium (RPE) was delineated within the sclera, leading to consistently thicker measurements of the photoreceptor layer and the total retina. TRANSLATIONAL RELEVANCE The introduction of spectral domain OCT allows for accurate imaging of the mouse retina. Exact quantification of retinal layer thicknesses in mice is important to study layers of interest under various pathological conditions.

The potential role of endometrial nerve fibers in the pathogenesis of pain during endometrial biopsy at office hysteroscopy

We aimed to evaluate whether nerve fibers are present in the endometrial layer of patients submitted to office hysteroscopy and their potential contribution to the pathogenesis of pain during that procedure. Through a prospective case-control study performed in tertiary centers for women's health, endometrium samples were collected during operative office hysteroscopy from 198 cycling women who previously underwent laparoscopy and/or magnetic resonance imaging investigation for infertility assessment. Samples were classified according to the degree of the pain patients experienced and scored from values ranging from 0 (absence of discomfort/pain) to 10 (intolerable pain) on a 10-cm visual analog scale (VAS). The presence of nerve fiber markers (S100, NSE, SP, VIP, NPY, NKA, NKB, NKR1, NKR2, and NKR3) in the endometrium was also evaluated by morphologic and immunohistochemical analyses. We found that S-100, NSE, NKR1, NK-A, NK-B, VIP, and NPY, were immunolocalized in samples of endometrium, in significantly (P < .01, for all) higher levels in samples collected from patients with VAS score > 5 (group A) than ≤ 5 (group B) and significantly (P < .0001 for all) positively correlated with VAS levels. A statistically significant (P = .018) higher prevalence of endometriosis and/or adenomyosis was depicted in patients of group A than group B. Data from the present study led us to conclude that nerve fibers are expressed at the level of the functional layer of the endometrium and may contribute to pain generation during office hysteroscopy, mainly in women affected by endometriosis and adenomyosis.

Fibroblast Growth Factor (FGF) Soluble Receptor 1 Acts as a Natural Inhibitor of FGF2 Neurotrophic Activity during Retinal Degeneration

Fibroblast growth factors (FGF) 1 and 2 and their tyrosine kinase receptor (FGFR) are present throughout the adult retina. FGFs are potential mitogens, but adult retinal cells are maintained in a nonproliferative state unless the retina is damaged. Our work aims to find a modulator of FGF signaling in normal and pathological retina. We identified and sequenced a truncated FGFR1 form from rat retina generated by the use of selective polyadenylation sites. This 70-kDa form of soluble extracellular FGFR1 (SR1) was distributed mainly localized in the inner nuclear layer of the retina, whereas the full-length FGFR1 form was detected in the retinal Muller glial cells. FGF2 and FGFR1 mRNA levels greatly increased in light-induced retinal degeneration. FGFR1 was detected in the radial fibers of activated retinal Muller glial cells. In contrast, SR1 mRNA synthesis followed a biphasic pattern of down- and up-regulation, and anti-SR1 staining was intense in retinal pigmented epithelial cells. The synthesis of SR1 and FGFR1 specifically and independently regulated in normal and degenerating retina suggests that changes in the proportion of various FGFR forms may control the bioavailability of FGFs and thus their potential as neurotrophic factors. This was demonstrated in vivo during retinal degeneration when recombinant SR1 inhibited the neurotrophic activity of exogenous FGF2 and increased damaging effects of light by inhibiting endogenous FGF. This study highlights the significance of the generation of SR1 in normal and pathological conditions.

Temporal distribution of the ganglion cell volleys in the normal rat optic nerve

We describe experiments on behaving rats with electrodes implanted on the cornea, in the optic chiasm, and on the visual cortex; in addition, two red light-emitting diodes (LED) are permanently attached to the skull over the left eye. Recordings timelocked to the LED flashes reveal both the local events at each electrode site and the orderly transfer of visual information from retina to cortex. The major finding is that every stimulus, regardless of its luminance, duration, or the state of retinal light adaptation, elicits an optic nerve volley with a latency of about 10 ms and a duration of about 300 ms. This phenomenon has not been reported previously, so far as we are aware. We conclude that the retina, which originates from the forebrain of the developing embryo, behaves like a typical brain structure: it translates, within a few hundred milliseconds, the chemical information in each pattern of bleached photoreceptors into a corresponding pattern of ganglion cell neuronal information that leaves via the optic nerve. The attributes of each rat ganglion cell appear to include whether the retinal neuropile calls on it to leave after a stimulus and, if so when, within a 300-ms poststimulus epoch. The resulting retinal analysis of the scene, on arrival at the cortical level, is presumed to participate importantly in the creation of visual perceptual experiences.

Sleep modifies retinal ganglion cell responses in the normal rat

Recordings were obtained from the visual system of rats as they cycled normally between waking (W), slow-wave sleep (SWS), and rapid eye movement (REM) sleep. Responses to flashes delivered by a light-emitting diode attached permanently to the skull were recorded through electrodes implanted on the cornea, in the chiasm, and on the cortex. The chiasm response reveals the temporal order in which the activated ganglion cell population exits the eyeball; as reported, this triphasic event is invariably short in latency (5–10 ms) and around 300 ms in duration, called the histogram. Here we describe the differences in the histograms recorded during W, SWS, and REM. SWS histograms are always larger than W histograms, and an REM histogram can resemble either. In other words, the optic nerve response to a given stimulus is labile; its configuration depends on whether the rat is asleep or awake. We link this physiological information with the anatomical fact that the brain dorsal raphe region, which is known to have a sleep regulatory role, sends fibers to the rat retina and receives fibers from it. At the cortical electrode, the visual cortical response amplitudes also vary, being largest during SWS. This well known phenomenon often is explained by changes taking place at the thalamic level. However, in the rat, the labile cortical response covaries with the labile optic nerve response, which suggests the cortical response enhancement during SWS is determined more by what happens in the retina than by what happens in the thalamus.

Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: Implications for glaucoma

Our group recently demonstrated that autoimmune T cells directed against central nervous system-associated myelin antigens protect neurons from secondary degeneration. We further showed that the synthetic peptide copolymer 1 (Cop-1), known to suppress experimental autoimmune encephalomyelitis, can be safely substituted for the natural myelin antigen in both passive and active immunization for neuroprotection of the injured optic nerve. Here we attempted to determine whether similar immunizations are protective from retinal ganglion cell loss resulting from a direct biochemical insult caused, for example, by glutamate (a major mediator of degeneration in acute and chronic optic nerve insults) and in a rat model of ocular hypertension. Passive immunization with T cells reactive to myelin basic protein or active immunization with myelin oligodendrocyte glycoprotein-derived peptide, although neuroprotective after optic nerve injury, was ineffective against glutamate toxicity in mice and rats. In contrast, the number of surviving retinal ganglion cells per square millimeter in glutamate-injected retinas was significantly larger in mice immunized 10 days previously with Cop-1 emulsified in complete Freund's adjuvant than in mice injected with PBS in the same adjuvant (2,133 ± 270 and 1,329 ± 121, respectively, mean ± SEM; P < 0.02). A similar pattern was observed when mice were immunized on the day of glutamate injection (1,777 ± 101 compared with 1,414 ± 36; P < 0.05), but not when they were immunized 48 h later. These findings suggest that protection from glutamate toxicity requires reinforcement of the immune system by antigens that are different from those associated with myelin. The use of Cop-1 apparently circumvents this antigen specificity barrier. In the rat ocular hypertension model, which simulates glaucoma, immunization with Cop-1 significantly reduced the retinal ganglion cell loss from 27.8% ± 6.8% to 4.3% ± 1.6%, without affecting the intraocular pressure. This study may point the way to a therapy for glaucoma, a neurodegenerative disease of the optic nerve often associated with increased intraocular pressure, as well as for acute and chronic degenerative disorders in which glutamate is a prominent participant.

Retinal engineering: engrafted neural cell lines locate in appropriate layers.

A major question in central nervous system development, including the neuroretina, is whether migrating cells express cues to find their way and settle at specific locations. We have transplanted quail neuroretinal cell lines QNR/D, a putative amacrine or ganglion cell, and QNR/K2, a putative Müller cell into chicken embryo eyes. Implanted QNR/D cells migrate only to the retinal ganglion and amacrine cell layers and project neurites in the plane of retina; in contrast, QNR/K2 cells migrate through the ganglion and amacrine layers, locate in the inner nuclear layer, and project processes across the retina. These data show that QNR/D and QNR/K2 cell lines represent distinct neural cell types, suggesting that migrating neural cells express distinct address cues. Furthermore, our results raise the possibility that immortalized cell lines can be used for replacement of specific cell types and for the transport of genes to given locations in neuroretina.

POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells.

The three members of the Brn-3 family of POU domain transcription factors are found in highly restricted sets of central nervous system neurons. Within the retina, these factors are present only within subsets of ganglion cells. We show here that in the developing mouse retina, Brn-3b protein is first observed in presumptive ganglion cell precursors as they begin to migrate from the zone of dividing neuroblasts to the future ganglion cell layer, and that targeted disruption of the Brn-3b gene leads in the homozygous state to a selective loss of 70% of retinal ganglion cells. In Brn-3b (-/-) mice other neurons within the retina and brain are minimally or not at all affected. These experiments indicate that Brn-3b plays an essential role in the development of specific ganglion cell types.