Subretinal hyperreflective material in regions of atrophy and fibrosis in eyes with neovascular age-related macular degeneration.

BACKGROUND: Subretinal hyperreflective material (SHRM) is a significant biomarker for poor visual outcomes in neovascular age-related macular degeneration (nAMD); however, its relationship with fibrosis and atrophy is not well understood. This study aims to evaluate the relationship between SHRM, atrophy, and fibrosis in eyes receiving antivascular endothelial growth factor therapy for nAMD. METHODS: Post-hoc analysis of the 65 patients enrolled in the SEVEN-UP study, a multicenter cross-sectional study of patients originally enrolled in the ANCHOR and MARINA trials of ranibizumab. Color fundus photographs (CFP) were reviewed and manually segmented to define regions of atrophy and fibrosis. SHRM borders on OCT volume scans were manually delineated, and thickness measurements were computed and compared in corresponding regions of atrophy and fibrosis on the CFPs. RESULTS: Of the 65 subjects, 51 eyes showed atrophy and/or fibrosis on CFP and were included in the final analysis. Both atrophy and fibrosis regions exhibited SHRM on OCT. The mean SHRM thickness on OCT was significantly greater in CFP-fibrosis regions (44.19 ± 46.95 µm) compared with CFP-atrophy regions (14.28 ± 13.35 µm; p < 0.001). Additionally, the average maximum height of SHRM in fibrotic regions (268.04 ± 130.05 µm) was significantly thicker than in atrophic regions (121.95 ± 51.17 µm; p < 0.001). CONCLUSIONS: Although atrophy and fibrosis are thought to be different end-stage outcomes in eyes with nAMD, they both demonstrate SHRM on OCT; the main distinction being thickness. Given these similarities, these regions of nAMD-associated atrophy may be better-termed "atrosis" to distinguish these lesions from typical atrophy in the absence of neovascular disease.

Role of the superior salivatory nucleus in parasympathetic control of choroidal blood flow and in maintenance of retinal health.

The vasodilatory pterygopalatine ganglion (PPG) innervation of the choroid is under the control of preganglionic input from the superior salivatory nucleus (SSN), the parasympathetic portion of the facial motor nucleus. We sought to confirm that choroidal SSN drives a choroid-wide vasodilation and determine if such control is important for retinal health. To the former end, we found, using transscleral laser Doppler flowmetry, that electrical activation of choroidal SSN significantly increased choroidal blood flow (ChBF), at a variety of choroidal sites that included more posterior as well as more anterior ones. We further found that the increases in ChBF were significantly reduced by inhibition of neuronal nitric oxide synthase (nNOS), thus implicating nitrergic PPG terminals in the SSN-elicited ChBF increases. To evaluate the role of parasympathetic control of ChBF in maintaining retinal health, some rats received unilateral lesions of SSN, and were evaluated functionally and histologically. In eyes ipsilateral to choroidal SSN destruction, we found that the flash-evoked scotopic electroretinogram a-wave and b-wave peak amplitudes were both significantly reduced by 10 weeks post lesion. Choroidal baroregulation was evaluated in some of these rats, and found to be impaired in the low systemic arterial blood pressure (ABP) range where vasodilation normally serves to maintain stable ChBF. In retina ipsilateral to SSN destruction, the abundance of Müller cell processes immunolabeled for glial fibrillary acidic protein (GFAP) and GFAP message were significantly upregulated. Our studies indicate that the SSN-PPG circuit mediates parasympathetic vasodilation of choroid, which appears to contribute to ChBF baroregulation during low ABP. Our results further indicate that impairment in this adaptive mechanism results in retinal dysfunction and pathology within months of the ChBF disturbance, indicating its importance for retinal health.

Effect of the Decrease in Luminance Noise Range on Color Discrimination of Dichromats and Trichromats.

Color vision assessment can be done using pseudoisochromatic stimuli, which has a luminance noise to eliminate brightness differences between the target and background of the stimulus. It is not clear the influence of the luminance noise on color discrimination. We investigated the effect of change in the luminance noise limits on color discrimination. Eighteen trichromats and ten congenital dichromats (eight protans, two deutans) had their color vision evaluated by the Cambridge Colour Test, and were genetically tested for diagnostic confirmation. The stimuli were composed of a mosaic of circles in a 5° circular field. A subset of the circles differed in chromaticity from the remaining field, forming a letter C. Color discrimination was estimated in stimulus conditions differing in luminance noise range: (i) 6-20 cd/m2; (ii) 8-18 cd/m2; (iii) 10-16 cd/m2; and (iv) 12-14 cd/m2. Six equidistant luminance values were used within the luminance noise limits with the mean stimulus luminance maintained constant under all conditions. A four-alternative, forced-choice method was applied to feed a staircase procedure to estimate color discrimination thresholds along eight chromatic axes. An ellipse model was adjusted to the eight color discrimination thresholds. The parameters of performance were threshold vector lengths and the ellipse area. Results were compared using the Kruskal-Wallis test with a significance level of 5%. The linear function model was applied to analyze the dependence of the discrimination parameters on the noise luminance limits. The first derivative of linear function was used as an indicator of the rate of change in color discrimination as a function of luminance noise changes. The rate of change of the ellipse area as a function of the luminance range in dichromats was higher than in trichromats (p < 0.05). Significant difference was also found for individual thresholds in half of the axes we tested. Luminance noise had a greater effect on color discrimination ability of dichromats than the trichromats, especially when the chromaticities were close to their protan and deutan color confusion lines.

Defective Choroidal Blood Flow Baroregulation and Retinal Dysfunction and Pathology Following Sympathetic Denervation of Choroid.

Purpose: We sought to determine if sympathetic denervation of choroid impairs choroidal blood flow (ChBF) regulation and harms retina. Methods: Rats received bilateral superior cervical ganglionectomy (SCGx), which depleted choroid of sympathetic but not parasympathetic innervation. The flash-evoked scotopic ERG and visual acuity were measured 2 to 3 months after SCGx, and vasoconstrictive ChBF baroregulation during high systemic arterial blood pressure (ABP) induced by LNAME was assessed by laser Doppler flowmetry (LDF). Eyes were harvested for histologic evaluation. Results: ChBF increased in parallel with ABP in SCGx rats over an ABP range of 90% to 140% of baseline ABP, while in sham rats ChBF remained stable and uncorrelated with ABP. ERG a- and b-wave latencies and amplitudes, and visual acuity were significantly reduced after SCGx. In SCGx retina, Müller cell GFAP immunolabeling was upregulated 2.5-fold, and Iba1+ microglia were increased 3-fold. Dopaminergic amacrine cell fibers in inner plexiform layer were reduced in SCGx rats, and photoreceptors were slightly depleted. Functional deficits and pathology were correlated with impairments in sympathetic regulation of ChBF. Conclusions: These studies indicate that sympathetic denervation of choroid impairs ChBF baroregulation during elevated ABP, leading to choroidal overperfusion. This defect in ChBF regulation is associated with impaired retinal function and retinal pathology. As sympathetic ChBF baroregulatory defects have been observed in young individuals with complement factor H (CFH) polymorphisms associated with risk for AMD, our results suggest these defects may harm retina, perhaps contributing to AMD pathogenesis.

Neural control of choroidal blood flow.

The choroid is richly innervated by parasympathetic, sympathetic and trigeminal sensory nerve fibers that regulate choroidal blood flow in birds and mammals, and presumably other vertebrate classes as well. The parasympathetic innervation has been shown to vasodilate and increase choroidal blood flow, the sympathetic input has been shown to vasoconstrict and decrease choroidal blood flow, and the sensory input has been shown to both convey pain and thermal information centrally and act locally to vasodilate and increase choroidal blood flow. As the choroid lies behind the retina and cannot respond readily to retinal metabolic signals, its innervation is important for adjustments in flow required by either retinal activity, by fluctuations in the systemic blood pressure driving choroidal perfusion, and possibly by retinal temperature. The former two appear to be mediated by the sympathetic and parasympathetic nervous systems, via central circuits responsive to retinal activity and systemic blood pressure, but adjustments for ocular perfusion pressure also appear to be influenced by local autoregulatory myogenic mechanisms. Adaptive choroidal responses to temperature may be mediated by trigeminal sensory fibers. Impairments in the neural control of choroidal blood flow occur with aging, and various ocular or systemic diseases such as glaucoma, age-related macular degeneration (AMD), hypertension, and diabetes, and may contribute to retinal pathology and dysfunction in these conditions, or in the case of AMD be a precondition. The present manuscript reviews findings in birds and mammals that contribute to the above-summarized understanding of the roles of the autonomic and sensory innervation of the choroid in controlling choroidal blood flow, and in the importance of such regulation for maintaining retinal health.

Influence of Spatial and Chromatic Noise on Luminance Discrimination.

Pseudoisochromatic figures are designed to base discrimination of a chromatic target from a background solely on the chromatic differences. This is accomplished by the introduction of luminance and spatial noise thereby eliminating these two dimensions as cues. The inverse rationale could also be applied to luminance discrimination, if spatial and chromatic noise are used to mask those cues. In this current study estimate of luminance contrast thresholds were conducted using a novel stimulus, based on the use of chromatic and spatial noise to mask the use of these cues in a luminance discrimination task. This was accomplished by presenting stimuli composed of a mosaic of circles colored randomly. A Landolt-C target differed from the background only by the luminance. The luminance contrast thresholds were estimated for different chromatic noise saturation conditions and compared to luminance contrast thresholds estimated using the same target in a non-mosaic stimulus. Moreover, the influence of the chromatic content in the noise on the luminance contrast threshold was also investigated. Luminance contrast threshold was dependent on the chromaticity noise strength. It was 10-fold higher than thresholds estimated from non-mosaic stimulus, but they were independent of colour space location in which the noise was modulated. The present study introduces a new method to investigate luminance vision intended for both basic science and clinical applications.

Stimulation of Baroresponsive Parts of the Nucleus of the Solitary Tract Produces Nitric Oxide-mediated Choroidal Vasodilation in Rat Eye.

Preganglionic parasympathetic neurons of the ventromedial part of the superior salivatory nucleus (SSN) mediate vasodilation of orbital and choroidal blood vessels, via their projection to the nitrergic pterygopalatine ganglion (PPG) neurons that innervate these vessels. We recently showed that the baroresponsive part of the nucleus of the solitary tract (NTS) innervates choroidal control parasympathetic preganglionic neurons of SSN in rats. As this projection provides a means by which blood pressure (BP) signals may modulate choroidal blood flow (ChBF), we investigated if activation of baroresponsive NTS evokes ChBF increases in rat eye, using Laser Doppler Flowmetry (LDF) to measure ChBF transclerally. We found that electrical activation of ipsilateral baroresponsive NTS and its efferent fiber pathway to choroidal SSN increased mean ChBF by about 40-80% above baseline, depending on current level. The ChBF responses obtained with stimulation of baroresponsive NTS were driven by increases in both choroidal blood volume (ChBVol; i.e., vasodilation) and choroidal blood velocity (ChBVel; possibly due to orbital vessel dilation). Stimulation of baroresponsive NTS, by contrast, yielded no significant mean increases in systemic arterial blood pressure (ABP). We further found that the increases in ChBF with NTS stimulation were significantly reduced by administration of the neuronal nitric oxide (NO) synthase inhibitor Nω-propyl-l-arginine (NPA), thus implicating nitrergic PPG terminals in the NTS-elicited ChBF increases. Our results show that the NTS neurons projecting to choroidal SSN do mediate increase in ChBF, and thus suggest a role of baroresponsive NTS in the BP-dependent regulation of ChBF.

Disinhibition of neurons of the nucleus of solitary tract that project to the superior salivatory nucleus causes choroidal vasodilation: Implications for mechanisms underlying choroidal baroregulation.

Preganglionic neurons in the superior salivatory nucleus (SSN) that mediate parasympathetic vasodilation of choroidal blood vessels receive a major excitatory input from the baroresponsive part of the nucleus of the solitary tract (NTS). This input appears likely to mediate choroidal vasodilation during systemic hypotension, which prevents decreases in choroidal blood flow (ChBF) due to reduced perfusion pressure. It is uncertain, however, how low blood pressure signals to NTS from the aortic depressor nerve (ADN), which fires at a low rate during systemic hypotension, could yield increased firing in the NTS output to SSN. The simplest hypothesis is that SSN-projecting NTS neurons are under the inhibitory control of ADN-receptive GABAergic NTS neurons. As part of evaluating this hypothesis, we assessed if SSN-projecting NTS neurons, in fact, receive prominent inhibitory input and if blocking GABAergic modulation of them increases ChBF. We found that SSN-projecting NTS neuronal perikarya identified by retrograde labeling are densely coated with GABAergic terminals, but lightly coated with excitatory terminals. We also found that, infusion of the GABA-A receptor antagonist GABAzine into NTS increased ChBF. Our results are consistent with the possibility that low blood pressure signals from the ADN produce vasodilation in choroid by causing diminished activity in ADN-receptive NTS neurons that tonically suppress SSN-projecting NTS neurons.

Color Discrimination Is Affected by Modulation of Luminance Noise in Pseudoisochromatic Stimuli.

Pseudoisochromatic stimuli have been widely used to evaluate color discrimination and to identify color vision deficits. Luminance noise is one of the stimulus parameters used to ensure that subject's response is due to their ability to discriminate target stimulus from the background based solely on the hue between the colors that compose such stimuli. We studied the influence of contrast modulation of the stimulus luminance noise on threshold and reaction time color discrimination. We evaluated color discrimination thresholds using the Cambridge Color Test (CCT) at six different stimulus mean luminances. Each mean luminance condition was tested using two protocols: constant absolute difference between maximum and minimum luminance of the luminance noise (constant delta protocol, CDP), and constant contrast modulation of the luminance noise (constant contrast protocol, CCP). MacAdam ellipses were fitted to the color discrimination thresholds in the CIE 1976 color space to quantify the color discrimination ellipses at threshold level. The same CDP and CCP protocols were applied in the experiment measuring RTs at three levels of stimulus mean luminance. The color threshold measurements show that for the CDP, ellipse areas decreased as a function of the mean luminance and they were significantly larger at the two lowest mean luminances, 10 cd/m(2) and 13 cd/m(2), compared to the highest one, 25 cd/m(2). For the CCP, the ellipses areas also decreased as a function of the mean luminance, but there was no significant difference between ellipses areas estimated at six stimulus mean luminances. The exponent of the decrease of ellipse areas as a function of stimulus mean luminance was steeper in the CDP than CCP. Further, reaction time increased linearly with the reciprocal of the length of the chromatic vectors varying along the four chromatic half-axes. It decreased as a function of stimulus mean luminance in the CDP but not in the CCP. The findings indicated that visual performance using pseudoisochromatic stimuli was dependent on the Weber's contrast of the luminance noise. Low Weber's contrast in the luminance noise is suggested to have a reduced effect on chromatic information and, hence, facilitate desegregation of the hue-defined target from the background.

The identification and neurochemical characterization of central neurons that target parasympathetic preganglionic neurons involved in the regulation of choroidal blood flow in the rat eye using pseudorabies virus, immunolabeling and conventional pathway tracing methods.

The choroidal blood vessels of the eye provide the main vascular support to the outer retina. These blood vessels are under parasympathetic vasodilatory control via input from the pterygopalatine ganglion (PPG), which in turn receives its preganglionic input from the superior salivatory nucleus (SSN) of the hindbrain. The present study characterized the central neurons projecting to the SSN neurons innervating choroidal PPG neurons, using pathway tracing and immunolabeling. In the initial set of studies, minute injections of the Bartha strain of the retrograde transneuronal tracer pseudorabies virus (PRV) were made into choroid in rats in which the superior cervical ganglia had been excised (to prevent labeling of sympathetic circuitry). Diverse neuronal populations beyond the choroidal part of ipsilateral SSN showed transneuronal labeling, which notably included the parvocellular part of the paraventricular nucleus of the hypothalamus (PVN), the periaqueductal gray, the raphe magnus (RaM), the B3 region of the pons, A5, the nucleus of the solitary tract (NTS), the rostral ventrolateral medulla (RVLM), and the intermediate reticular nucleus of the medulla. The PRV+ neurons were located in the parts of these cell groups that are responsive to systemic blood pressure signals and involved in systemic blood pressure regulation by the sympathetic nervous system. In a second set of studies using PRV labeling, conventional pathway tracing, and immunolabeling, we found that PVN neurons projecting to SSN tended to be oxytocinergic and glutamatergic, RaM neurons projecting to SSN were serotonergic, and NTS neurons projecting to SSN were glutamatergic. Our results suggest that blood pressure and volume signals that drive sympathetic constriction of the systemic vasculature may also drive parasympathetic vasodilation of the choroidal vasculature, and may thereby contribute to choroidal baroregulation during low blood pressure.

Low number of luminance levels in the luminance noise increases color discrimination thresholds estimated with pseudoisochromatic stimuli.

In pseudoisochromatic stimuli the presence of spatial and luminance noise forces the subject to discriminate the target from the background solely on the basis of chromaticity difference. Color-blind subjects may show difficulty to identify the target due to the elimination of borders and brightness clues caused by the luminance and spatial noise. Few studies have fully described the features of pseudoisochromatic stimuli. Fewer investigators have focused their studies in the effects of specific pseudoisochromatic parameters on color discrimination. We used the Cambridge Color Test (CCT) to investigate the influence on color discrimination thresholds due to the number of luminance levels present in the luminance noise. The CCT default has six luminance steps; however, in our investigation a total of eight different conditions were tested from 2 to 16 luminance steps. It was found that the CCT provided very robust values for color discrimination thresholds, which were degraded only for very small number of luminance steps. When the number of steps was increased, the color discrimination thresholds improved from 2 to 6 luminance steps and gradually reached a plateau for 10 or more luminance steps. The area of color discrimination ellipses as a function of luminance steps matches the relative proportion of ineffective contrasts between mosaic patches as a function of luminance steps, assuming that contrast becomes ineffective for values 18.6% or less. The lower number of color and luminance interactions in these conditions could explain the measured increase of color discrimination thresholds. The primary conclusion from this investigation was that results from pseudoisochromatic tests should have their parameters described in more detail. This type of description would allow a better understanding of the results provided, interpretations, and therefore cross study comparison of results obtained from different laboratories.

Platelets, glycoprotein Ib-IX, and von Willebrand factor are required for FeCl(3)-induced occlusive thrombus formation in the inferior vena cava of mice.

Venous thromboembolism is a leading cause of death from cardiovascular disease. Despite the importance of the glycoprotein (GP) Ib-IX/von Willebrand factor (vWF) axis in arterial thrombosis, its requirement in venous, not venule thrombosis in response to endothelial injury (not stenosis or stasis) is uncharacterized. GPIbα-vWF participation in FeCl(3)-induced thrombus formation was evaluated in the inferior vena cava (IVC). Stable, occlusive thrombus formation in response to FeCl(3)-induced injury of the IVC was studied. FeCl(3) (20% FeCl(3), 10 minutes)-induced occlusive thrombosis required platelets as confirmed by a lack of occlusion in thrombocytopenic mice, and stable occlusion in control animals. No IVC occlusion was observed using GPIbα-deficient animals, a model of the human Bernard-Soulier syndrome (BSS). Transgenic IL-4 R/GPIbα mice (lack murine GPIbα, but express the extracellular domain of the human interleukin (IL-4 receptor fused to the transmembrane and cytoplasmic domains of human GPIbα) were studied to determine if the absence of IVC occlusion in the BSS mouse was caused by GPIbα extracellular domain deficiency rather than platelet BSS phenotype associated abnormalities. As with GPIbα knock-out mice, no occlusion was observed in the IVC of IL-4 R/GPIbα mice. The IVC of vWF-deficient mice also failed to occlude in response to FeCl(3) treatment. The chimeric protein GPIbα(2V)-Fc prevented occlusion, demonstrating that GPIbα-vWF A1 domain interaction is required for FeCl(3)-induced stable thrombus formation in the IVC. Therefore, FeCl(3)-induced stable, occlusive thrombus formation in the IVC is platelet, and apparently GPIbα-vWF interaction dependent, despite the large diameter and low venous flow rate in the IVC.

Age-related impairment in choroidal blood flow compensation for arterial blood pressure fluctuation in pigeons.

PURPOSE: Choroidal vessels compensate for changes in systemic blood pressure (BP) so that choroidal blood flow (ChBF) remains stable over a BP range of approximately 40 mm Hg above and below basal. Because of the presumed importance of ChBF regulation for maintenance of retinal health, we investigated if ChBF compensation for BP fluctuation in pigeons fails with age. METHODS: Transcleral laser Doppler flowmetry was used to measure ChBF during spontaneous BP fluctuation in anesthetized pigeons ranging in age from 0.5 to 17 years (pigeons can live approximately 20 years in captivity). RESULTS: ChBF in <8-year-old pigeons remained near 100% of basal ChBF at BPs ranging 40 mm Hg above and below basal BP (95 mm Hg). Baroregulation failed below approximately 50 mm Hg BP. In ≥8-year-old pigeons, ChBF compensation was absent at >90 mm Hg BP, with ChBF linearly following BP. Over the 60 to 90 mm Hg range, ChBF in ≥8-year-old pigeons was maintained at 60-70% of young basal ChBF. Below approximately 55 mm Hg, baroregulation again followed BP linearly. CONCLUSIONS: Age-related ChBF baroregulatory impairment occurs in pigeons, with ChBF linear with above-basal BP, and ChBF failing to adequately maintain ChBF during below-basal BP. Defective autonomic sympathetic and parasympathetic neurogenic control, or defective myogenic control, may cause these baroregulatory defects. In either case, overperfusion during high BP may cause oxidative injury to the outer retina, whereas underperfusion during low BP may result in deficient nutrient supply and waste removal, with both abnormalities contributing to age-related retinal pathology and vision loss.

Immunohistochemical localization of AMPA-type glutamate receptor subunits in the nucleus of the Edinger-Westphal in embryonic chick.

The Edinger-Westphal nucleus (EW) in birds is responsible for the control of pupil constriction, accommodation, and choroidal blood flow. The activation of EW neurons is mediated by the neurotransmitter glutamate, in large part through AMPA-type glutamate receptors (GluRs), whose behavior varies according to the subunit composition. We investigated the developmental expression of the GluR subunits in EW of the chick (Gallus gallus) using immunohistochemistry on tissue from embryonic days 10 through 20 (E10-E20). Of the three antibodies used, one recognized the GluR1 subunit, another the GluR4 subunit, and the third recognized a sequence common to GluR2 and GluR3 subunits. No immunolabeling of EW neurons for any GluR subunits was observed prior to E12, although immunolabeling was seen in somatic oculomotor prior to E12. At E12, immunoreactivity for each of the three antibodies was in only approximately 2% of EW neurons. By E14, the abundance of GluR1+ perikarya in EW had increased to 13%, and for GluR2/3 had increased to 48%. The perikaryal abundance of the immunoreactivity for GluR1 and GluR2/3 declined to 3% and 23%, respectively, by E16. At E14, 33% of EW neurons immunolabeled for GluR4, and their frequency increased to 43% by E16, and remained at that approximate percentage through hatching. The increased expression of GluR1 and GluR4 in EW at E14 coincides with the reported onset of the expression of the calcium-binding protein parvalbumin, and the calcium currents associated with AMPA receptors formed by these two subunits may play a role in the occurrence of parvalbumin expression.

Projections from the hypothalamic paraventricular nucleus and the nucleus of the solitary tract to prechoroidal neurons in the superior salivatory nucleus: Pathways controlling rodent choroidal blood flow.

Using intrachoroidal injection of the transneuronal retrograde tracer pseudorabies virus (PRV) in rats, we previously localized preganglionic neurons in the superior salivatory nucleus (SSN) that regulate choroidal blood flow (ChBF) via projections to the pterygopalatine ganglion (PPG). In the present study, we used higher-order transneuronal retrograde labeling following intrachoroidal PRV injection to identify central neuronal cell groups involved in parasympathetic regulation of ChBF via input to the SSN. These prominently included the hypothalamic paraventricular nucleus (PVN) and the nucleus of the solitary tract (NTS), both of which are responsive to systemic BP and are involved in systemic sympathetic vasoconstriction. Conventional pathway tracing methods were then used to determine if the PVN and/or NTS project directly to the choroidal subdivision of the SSN. Following retrograde tracer injection into SSN (biotinylated dextran amine 3K or Fluorogold), labeled perikarya were found in PVN and NTS. Injection of the anterograde tracer, biotinylated dextran amine 10K (BDA10K), into PVN or NTS resulted in densely packed BDA10K+terminals in prechoroidal SSN (as defined by its enrichment in nitric oxide synthase-containing perikarya). Double-label studies showed these inputs ended directly on prechoroidal nitric oxide synthase-containing neurons of SSN. Our study thus establishes that PVN and NTS project directly to the part of SSN involved in parasympathetic vasodilatory control of the choroid via the PPG. These results suggest that control of ChBF may be linked to systemic blood pressure and central control of the systemic vasculature.

Choroidal blood flow compensation in rats for arterial blood pressure decreases is neuronal nitric oxide-dependent but compensation for arterial blood pressure increases is not.

Choroidal blood flow (ChBF) compensates for changes in arterial blood pressure (ABP) and thereby remains relatively stable within a +/-40 mmHg range of basal ABP in rabbits, humans and pigeons. In the present study, we investigated if ChBF can compensate for increases and decreases in ABP in rats. ChBF was continuously monitored using laser Doppler flowmetry in anesthetized rats, and ABP measured via the femoral artery. At multiple intervals over a 2-4 h period during which ABP varied freely, ChBF and ABP were sampled and the results compiled across rats. We found that ChBF remained near baseline over an ABP range from 40 mmHg above basal ABP (90-100 mmHg) to 40 mmHg below basal ABP, but largely followed ABP linearly below 60 mmHg. Choroidal vascular resistance increased linearly as BP increased above 100 mmHg, and decreased linearly as BP declined from basal to 60 mmHg, but resistance declined no further below 60 mmHg. Inhibition of nitric oxide (NO) formation by either a selective inhibitor of neuronal nitric oxide synthase (NOS) (N(omega)-propyl-L-arginine) or a nonselective inhibitor of both neuronal NOS and endothelial NOS (N(omega)-nitro-l-arginine methyl ester) did not affect compensation above 100 mmHg ABP, but did cause ChBF to linearly follow declines in BP below 90 mmHg. In NOS-inhibited rats, vascular resistance increased linearly with BP above 100 mmHg, but remained at baseline below 90 mmHg. These findings reveal that ChBF in rats, as in rabbits, humans and pigeons, compensates for rises and/or declines in arterial blood pressure so as to remain relatively stable within a physiological range of ABPs. The ChBF compensation for low ABP in rats is dependent on choroidal vasodilation caused by neuronal NO formation but not the compensation for elevated BP, implicating parasympathetic nervous system vasodilation in the ChBF compensation to low ABP.

Essential and synergistic roles of RP1 and RP1L1 in rod photoreceptor axoneme and retinitis pigmentosa.

Retinitis pigmentosa 1 (RP1) is a common inherited retinopathy with variable onset and severity. The RP1 gene encodes a photoreceptor-specific, microtubule-associated ciliary protein containing the doublecortin (DCX) domain. Here we show that another photoreceptor-specific Rp1-like protein (Rp1L1) in mice is also localized to the axoneme of outer segments (OSs) and connecting cilia in rod photoreceptors, overlapping with Rp1. Rp1L1-/- mice display scattered OS disorganization, reduced electroretinogram amplitudes, and progressive photoreceptor degeneration, less severe and slower than in Rp1-/- mice. In single rods of Rp1L1-/-, photosensitivity is reduced, similar to that of Rp1-/-. While individual heterozygotes are normal, double heterozygotes of Rp1 and Rp1L1 exhibit abnormal OS morphology and reduced single rod photosensitivity and dark currents. The electroretinogram amplitudes of double heterozygotes are more reduced than those of individual heterozygotes combined. In support, Rp1L1 interacts with Rp1 in transfected cells and in retina pull-down experiments. Interestingly, phototransduction kinetics are normal in single rods and whole retinas of individual or double Rp1 and Rp1L1 mutant mice. Together, Rp1 and Rp1L1 play essential and synergistic roles in affecting photosensitivity and OS morphogenesis of rod photoreceptors. Our findings suggest that mutations in RP1L1 could underlie retinopathy or modify RP1 disease expression in humans.

Sustained upregulation of glial fibrillary acidic protein in Müller cells in pigeon retina following disruption of the parasympathetic control of choroidal blood flow.

Choroidal blood flow in pigeon eyes is light driven and controlled by a parasympathetic input from ciliary ganglion (CG) neurons that receive input from the medial subdivision of the ipsilateral nucleus of Edinger-Westphal (EWM). EWM lesions diminish basal ChBF and irreversibly prevent ipsilateral light-evoked increases in ChBF, presumably rendering the retina mildly ischemic. To characterize the location, severity, and time course of the retinal abnormality caused by an EWM lesion, we quantitatively analyzed the cellular and regional extent of Müller cell glial fibrillary acidic protein (GFAP) immunolabeling up to nearly a year after an EWM lesion. We found that unilateral EWM lesions greatly increased Müller cell GFAP throughout the entire retinal depth and topographic extent of the affected eye, up to nearly a year post lesion. By contrast, destruction of the pupilloconstrictive pretectum or of the pupilloconstrictive part of lateral EW (EWL) did not appreciably increase Müller cell GFAP. Thus, the large increase in Müller cell GFAP following an EW lesion is attributable to an ongoing defect in choroidal vasodilatory function rather than to chronic pupil dilation. The Müller cell GFAP increase was greater ipsilateral than contralateral to the EWM destruction for the retinal territory deep to the heavily CG-innervated superior and temporal choroid, but not for the retinal territory deep to the poorly CG-innervated inferior and nasal choroid. The GFAP increase was light-dependent, since it did not occur in EW-lesioned birds housed in dim illumination. Our results show that the chronic vascular insufficiency caused by the loss of the EWM-mediated parasympathetic control of choroidal blood flow leads to a significant and sustained increase in retinal Müller cell GFAP. This increase could be a sign of a disturbance in retinal homeostasis that eventually leads to retinal injury and impaired visual function.

Choroidal blood flow in pigeons compensates for decreases in arterial blood pressure.

While it had once been thought that choroidal blood flow (ChBF) does not compensate for changes in perfusion pressure, recent studies have shown that ChBF in rabbits and humans does compensate for changes in arterial blood pressure (ABP) and thereby remains relatively stable within a physiological range of ABPs. In this study, we sought to determine if ChBF in birds can compensate for decreases in ABP, either spontaneously occuring or caused by blood withdrawal. ChBF was continuously monitored using laser Doppler flowmetry in anesthetized pigeons, and at the same time ABP was measured via the brachial artery. In studies of spontaneous fluctuation in ABP, ChBF and ABP were analyzed at regular intervals over a 2-3 hr period, while for blood withdrawal studies, blood was transiently withdrawn via the brachial artery. In both paradigms, ChBF remained near baseline over an ABP range from basal (about 90 mmHg) to about 55 mmHg, followed ABP nearly linearly below 50 mmHg, and showed no compensation below 40 mmHg. The blood withdrawal studies further showed that the compensation was more rapid with small acute declines in ABP than with larger declines. These findings reveal that ChBF in pigeons, as in rabbits and humans, compensates for declines in ABP so as to remain relatively stable within a physiological range of ABPs. Given the phylogenetic distance between humans and rabbits on one hand and birds on the other, these results suggest that choroidal compensation for ABP declines may be a common ocular mechanism among warm-blooded vertebrates.

Effect of polyamine depletion on cone photoreceptors of the developing rabbit retina.

PURPOSE: To measure the concentrations of polyamines, determine their cellular and subcellular localization, and analyze effects of their depletion in developing rabbit retina. METHODS: Isolated retinas at different developmental stages were analyzed for polyamine content by high-performance liquid chromatography (HPLC). An antibody against polyamines was used to localize endogenous stores in both freshly harvested retinas and neonatal retinal explants. To determine the effects of polyamine depletion on immature retina, neonatal explants were cultured in the presence or absence of alpha-difluoromethylornithine (DFMO), an inhibitor of the polyamine synthetic enzyme ornithine decarboxylase (ODC). Similar studies were also performed on dissociated cell cultures. Tissue was assessed using standard histologic stains as well as cell-specific markers (peanut agglutinin for cone photoreceptors and calbindin for horizontal cells). RESULTS: Retinal polyamine content was highest at birth, remained relatively high during the first postnatal week, and then steadily decreased to adult levels. At all ages analyzed, spermine concentration was higher than putrescine or spermidine; however, the differential was greatest in the adult. Polyamine immunoreactivity was localized to distal processes of both rods and cones during development. Strong immunoreactivity was maintained in adult cone inner and outer segments; comparatively weak staining was observed in the adult rods. Heavy staining of ganglion cells was present throughout development but was localized in the cytoplasm in immature cells and in the nucleus in the adult. Amacrine cells stained only in the adult. Polyamine depletion caused a disruption of immature cones, evident in the loss of their somata in the outer nuclear layer, in their processes in the outer plexiform layer in retinal explants, and in their decreased association with horizontal cells in dissociated cell culture. CONCLUSIONS: The relatively high concentrations of polyamines in neonatal retina and their discrete localization in developing photoreceptor outer segments and ganglion cells suggests an important role for these compounds in development. The disruption of cone-specific markers in polyamine-depleted retinas indicates a specific reliance on polyamines for expression of normal cone morphology or morphologic development. These developmental effects may involve polyamine-sensitive ion channels, which are known to exist in retina, or direct interactions with specialized cytoskeletal elements within outer segments.