Retina labeling

Fig. 2A. Radioactivity in SDS gels from 28 days post-crush and control retinas labeled as in Fig, 1, but after reassembly with carrier tubulin.

Elimination from the vitreous occurs by one of two pathways. This can be visualized by injecting fluorescent compounds and examining the concentration distribution in frozen sections obtained after a steady state has been established [230]. If the major route of elimination is by means of the re-tina/choroid, at steady state the lowest concentration would be in the vicinity of the retina. The contours observed in frozen sections of the rabbit eye obtained after intravitreal injection of fluorescein exhibit this pattern, with the highest concentration immediately behind the lens (Fig. 16A). Compounds not chiefly eliminated through the retina exit the vitreous by passive diffusion and enter the posterior aqueous, where they are eliminated by the natural production and outflow of aqueous humor. In such a situation, the contours would be perpendicular to the retina, with the highest concentration towards the rear of the vitreous cavity. This appears to be the case for fluorescently labeled dextran polymer, whose contours decrease in concentration toward the hyaloid membrane (Fig. 16B). 

The apparent retinal influx clearance,. Kin,retina expressed as mL/(min g retina), of the test substrate labeled with either [3 H] or [14C] from the circulating blood to the retina is determined by integration plot analysis. In brief, rats are anesthetized, followed by injection of the test substrate (e.g., an [3H]-labeled compound, about 10 /u.Ci/head) into the femoral vein. After collection of plasma samples, rats are decapitated and the retinas removed. The retinas are dissolved in 2 N NaOH and subsequently neutralized with 2 N HC1. The radioactivity of retinal cell lysates is measured by liquid scintillation spectrometry. As an index of the retinal distribution characteristics of the radiolabeled test substrate, the apparent retina-to-plasma concentration ratio (Vd) as a function of time is used. This ratio [Vd(Q] (mL/g retina) is defined as the amount of [3H] per gram retina divided by that per milliliter plasma, calculated over the time-period of the experiment. The Kjn,retina can be described by the following relationship ... 

In brief, the rats are anesthetized, followed by an injection of 0.2 mL of the test solution into the common carotid artery. The injection solution consists of a HEPES buffered Ringer s solution (containing 141 mM NaCl, 4 mM KC1, 2.8 mM CaCl2, and 10 mM HEPES, pH 7.4) which contains both the test substrate (e.g., a [3H]-labeled compound, about 10 /xCi) and a reference compound, which is highly extracted by the tissue (e.g., 0.1 /xCi [14C]n-butanol) in the presence or absence of transport inhibitors. If a [14C]-labeled compound is used as a test substrate, [3H]H20 can be selected as a reference compound. Rats are decapitated at 15 s after injection and the retina is removed. The retina is dissolved in 2 N NaOH and subsequently neutralized with 2 N HC1. The radioactivity is measured by liquid scintillation spectrometry. The RUI value, an index of the retinal distribution characteristics of the [3H] test substrate, is estimated using the following relationship ... 

Ambati et al. [44] measured the permeability of rabbit sclera to a series of fluorescein-labeled hydrophilic compounds and found that scleral permeability decreased with increasing molecular weight and molecular radius but was quite permeable to large molecules, such as IgG (145 kDa). Thus when targeting the retina by the topical or subconjunctival routes the sclera is not a rate-limiting barrier and the main barriers are beyond this tissue at the choroid, RPE, and ELM. 

Fig. 1. Representative fluorescent photomicrographs of retinal whole mounts showing the loss of Fluorogold (FG)-labeled RGCs in ischemic retina of rat. RGCs were retrogradely labeled with the fluorescent dye FG injected, under stereotaxic guidance, bilaterally into the superior colliculus of a rat 4 days after 50 min ischemia and sacrificed after additional 4 days. Obvious reduction of FG-labeled RGCs is evident in the retina undergone ischemia/reperfusion (panel B) as compared to the contralateral, nonischemic, retina (panel A). Photomicrographs were obtained from the peripheral area of the superior quadrant of the retina. Scale bar 50 fim.

When the oxonol backbone is stabilized by the addition of other, usually ring, structures, the new family is given a more descriptive name. In the case of the chromophores of biological vision, one choice would be to describe these molecules as retinienes to reflect their importance in the retina and their conjugated carbon backbone. However, the historical label retinenes has been used to describe a very similar family of monopolar materials with a conjugated carbon backbone that can be considered chromogens but not chromophores. In addition, the dipolar nature of the chromophores is not indicated. 

Color is essential to full enjoyment of the world. How we learn to classify and label colors is based largely on our culture. Because colors are not really out there but are constructed in our retinas and brains, it stands to reason that culture and psychology play a great role in our perceptions of color. People are drawn to color from an emotional standpoint. Color preferences are rooted in associations arising from our culture, how we were raised, our feelings about ourselves, and what we were taught. 

Fig. 4 A TH labeled (dopaminergic) amacrine cell of the rat retina. Note that the receptor layer is on the top and the faintly visible ganglion cell layer is on the bottom (from Mytileneou and Bodis-WoUner, Department of Neurology, The Mt. Sinai School of Medicine, around 1978 unpublished data)...

Retinal pigmental epithelia (RPE) are dopaminergic support cells in the neural retina. RPE cells on gelatin beads, also called Spheramine, produce levodopa (Watts et al., 2003), but there are no data yet on the consistency of dopamine production by these cells. An open label clinical trial of transplantation of Spheramine was conducted in six patients with PD (Bakay et al., 2004). Spheramine was transplanted into the striatum and show ed clinical effects over 24 months. Spheramine is now undergoing double bhnd placebo controlled trials in advanced PD. 

Development of culture systems in which distribution of various neurons could be studied under controlled conditions contributes to our understanding of these mechanisms. However, such experiments need reliable methods of labeling live neurons. Moreover, information regarding how the retinal mosaics respond to various pathological conditions, such as retinal degeneration (Sharma, 2001a, b Sharma et al., 2001), enhances our understanding of mosaic formation and compensatory mechanisms that may reside in the retina. 

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