Fundus autofluorescence and retinal structure as determined by spectral domain optical coherence tomography, and retinal function in retinitis pigmentosa
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Autofluorescence
A simple and inexpensive apparatus and techniques are described for recording fluorographs of whole-body cryosections of mice injected with a fluorescent compound. The apparatus was constructed with commercial fluorescent tubes which emit UV light of 300-400nm and a single-lens reflex camera equipped with UV cut and Kodak Wratten filters.Autofluorescence of the whole-body section observed by using this technique was discussed and the effects of lapse of time after cryosectioning, fixation, and pH on the autofluorescence were also investigated. Autofluorescence of the skeletal muscle, myocardium, smooth muscle, brain, kidney cortex and pancreas was intensified with time after cryosectioning, but that of the Harderian gland was gradually faded. In other tissues, autofluorescence remained unchanged up to 2 weeks after cryosectioning. All fixatives used in this experiment reduced the autofluorescence of tissues and organs. Among the fixatives, 100% acetone had the least effect on autofluorescence. At pH4, autofluorescence of all tissues was considerably increased in comparison with pH7. With increasing pH, autofluorescence of most tissues was gradually reduced except for the bones and the Harderian gland.
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Abstract The manner in which new cells are added to the growing adult goldfish retina was examined using 3 H‐thymidine radioutography. Cell proliferation leading to the formation of neurons is restricted to the retinal margin at the ora terminalis . New retina is added in concentric rings, with slightly more growth dorsonasally. The rate of cell addition is variable, averaging 12,000 cells/ day. These new cells account for about 20% of the total increase in retinal area; the remaining 80% is due to hypertrophy, or expansin, of the retina. In contrast to all of the other retinal cells, the rods do not appear to participate in the retinal expansion. This hypothesized immobility of the rods would create a shearing strain between the retinal layers resulting in a shift in their position relative to the other cells. Were they to maintain synaptic contacts with the same horizontal and bipolar cells, the rod axons would have to be elongated peripherally or the post‐synaptic cell dendrites displaced centrally. Since neurons with this morphology have not been found in the goldfish retina, these observations suggest that the rods must be changing their synaptic connections as the retina grows.
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It is a well known fact that light emitted at a specific wavelength induces fluorescence in the human body. This kind of fluorescence is called autofluorescence. The application of autofluorescence diagnosis, on the other hand, is a more complicated system designed to detect faint autofluorescence inherent in tissues/cells. We have adopted this autofluorescence diagnosis method and developed a new autofluorescence endoscope imaging system called the SAFE‐1000. Normal mucosa emitting autofluorescence appears green on the monitor, while abnormal mucosa shows a dark image caused by the lack of autofluorescence.
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Abstract The pattern of retinal vasculative is described and the position at which cell proliferation at the ventral retinal margin is maximal was shown to be at the point of entry of the ventral blood vessels. To test whether there is a causal relation between retinal blood supply and retinal cell production, surgical inversion of the eye, transplantations and excisions of retina were done to change the pattern of retinal vasculature. The growth pattern of inverted eyes was normal with respect to the internal axes of the eyes. After excision of part of the retina or after fusion of retinal fragments to form compound eyes, the pattern of retinal cell proliferation was not correlated with the distribution of retinal blood vessels, but was correlated with the position(s) of the choroidal fissure(s).
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Autofluorescence can be a very disturbing factor in immunofluorescence microscopy. We present here a method to eliminate autofluorescence. The method is based on the fact that most autofluorescent compounds have broad-banded excitation and emission spectra, whereas specific fluorescent probes have narrow spectra. Two images are recorded and digitized, one at a wavelength exciting both the fluorescent probe and the autofluorescent molecules, and one at a wavelength exciting only the latter. Subtraction of the autofluorescence signal from the total fluorescence signal, using a self-developed computer program, results in an autofluorescence-free image. The procedure is demonstrated for elimination of elastin-derived autofluorescence in human lung alveoli and for elimination of lipofuscin-derived autofluorescence in human heart muscle. The autofluorescence signal is positively correlated with tissue section thickness (r = 0.93; p < 0.0001), and can be used to correct the specific fluorescence signals for section thickness.
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Immunofluorescence
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Spatial Pattern and Temporal Evolution of Retinal Oxygenation Response in Oxygen-Induced Retinopathy
To determine the spatial pattern and temporal evolution of the change in retinal partial oxygen pressure (DeltaPO(2)) associated with a murine oxygen-induced retinopathy (OIR) model of retinal neovascularization (NV).On P7, newborn C57BL/6 mice were exposed to 75% oxygen until postnatal day (P)12, followed by recovery in room air until P17 or P34. Control mice remained in room air until P17 or P34. At P17 and P34, functional magnetic resonance imaging (MRI) and a carbogen inhalation challenge was used to measure retinal DeltaPO(2). Retinal avascularity, distance from the optic nerve head to the vascular edge in the peripheral retina, and NV incidence and severity were measured in retinas stained with adenosine diphosphatase (ADPase).In P17 and P34 controls and in P34 OIR animals, retinas were fully vascularized without evidence of NV. In P17 OIR mice, there was a large central retinal capillary-free zone (22% +/- 3% of the entire retinal area, mean +/- SD) and 4 clockhours (range 1-7) of retinal NV at the border of the peripheral vascular and central acapillary retina in 100% (36/36) of the mice. In P17 OIR mice, retinal DeltaPO(2) over the vascularized far peripheral retina was not significantly (P > 0.05) different from the P17 control but was supernormal (P < 0.05) over the central capillary-free retina. However, no differences (P > 0.05) in retinal DeltaPO(2) were found between the P34 control and OIR groups.A reversible supernormal DeltaPO(2) was found only over the central acapillary retina during the appearance of retinal NV in a mouse OIR model. The present data show the applicability of carbogen-challenge functional MRI to the study of retinal DeltaPO(2) in vivo in eyes that are too small for the use of existing techniques.
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The oxygen requirements of different retinal layers are of interest in understanding the vulnerability of the retina to hypoxic damage in retinal diseases with an ischemic component. Here, we report the first measurements of retinal oxygen consumption in the visual streak of the rabbit retina, the region with the highest density of retinal neurons, and compare it with that in the less-specialized region of the retina underlying the vascularized portion of the rabbit retina. Oxygen-sensitive microelectrodes were used to measure oxygen tension as a function of retinal depth in anesthetized animals. Measurements were performed in the region of the retina containing overlying retinal vessels and in the center of the visual streak. Established mathematical analyses of the intraretinal oxygen distribution were used to quantify the rate of oxygen consumption in the inner and outer retina and the relative oxygen contributions from the choroidal and vitreal sides. Outer retinal oxygen consumption was higher in the visual streak than in the vascularized area (means ± SE, 284 ± 20 vs. 210 ± 23 nl O 2 ·min –1 ·cm –2 , P = 0.026, n = 10). However, inner retinal oxygen consumption in the visual streak was significantly lower than in the vascular area (57 ± 4.3 vs. 146 ± 12 nl O 2 ·min –1 ·cm –2 , P < 0.001). We conclude that despite the higher processing requirements of the inner retina in the visual streak, it has a significantly lower oxygen consumption rate than the inner retina underlying the retinal vasculature. This suggests that the oxygen uptake of the inner retina is regulated to a large degree by the available oxygen supply rather than the processing requirements of the inner retina alone.
Streak
Oxygen tension
Retinal Artery
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