AAV-mediated transduction and targeting of retinal bipolar cells with improved mGluR6 promoters in rodents and primates.

Adeno-associated virus (AAV) vectors have been a powerful gene delivery vehicle to the retina for basic research and gene therapy. For many of these applications, achieving cell type-specific targeting and high transduction efficiency is desired. Recently, there has been increasing interest in AAV-mediated gene targeting to specific retinal bipolar cell types. A 200-bp enhancer in combination with a basal SV40 promoter has been commonly used to target transgenes into ON-type bipolar cells. In the current study, we searched for additional cis-regulatory elements in the mGluR6 gene for improving AAV-mediated transduction efficiency into retinal bipolar cells. Our results showed that the combination of the endogenous mGluR6 promoter with additional enhancers in the introns of the mGluR6 gene markedly enhanced AAV transduction efficiency as well as made the targeting more selective for rod bipolar cells in mice. Furthermore, the AAV vectors with the improved promoter could target to ON bipolar cells with robust transduction efficiency in the parafovea and the far peripheral retina of marmoset monkeys. The improved mGluR6 promoter constructs could provide a valuable tool for genetic manipulation in rod bipolar cells in mice and facilitate clinical applications for ON bipolar cell-based gene therapies.

Endogenous rhythms in axial length and choroidal thickness in chicks: implications for ocular growth regulation.

PURPOSE: To determine whether the diurnal rhythms in axial length and choroidal thickness in the chick eye are endogenous circadian rhythms. METHODS: Six chickens, 14 days of age, were put into darkness for 4 days. Beginning on the 3rd day, ocular dimensions were measured using high-frequency A-scan ultrasonography, in darkness, at 6-hour intervals over 48 hours. Five age-matched chickens reared in a normal light/dark (L/D) cycle and measured at 6-hour intervals for 5 days were controls. RESULTS: The rhythms in axial length and choroidal thickness persist in constant darkness. The phases of these rhythms are approximately in antiphase to one another, similar to those of eyes in a L/D cycle; however, the peak of the rhythm in axial length occurs slightly earlier relative to that of eyes in L/D (12 PM versus 3 PM; P: < 0.05, one-tailed t-test). By the 3rd day in darkness, the rate of growth is significantly higher than that in L/D (117 versus 72 microm/24 hours; P: < 0.01), and the choroid becomes significantly thinner (159 versus 210 microm; P: < 0.0001). CONCLUSIONS: The rhythms in axial length and choroid thickness are circadian rhythms, driven by an endogenous oscillator. The phase of the rhythm in axial length in constant darkness is slightly phase-advanced relative to eyes in L/D and thus is similar to eyes that are deprived of form vision. These findings suggest that in the absence of visual input, the eyes revert to a "default" growth state and that the similarities between the effects of constant darkness and of form deprivation suggest that deprivation may represent a type of "constant" condition.

Diurnal illumination patterns affect the development of the chick eye.

Exposure to continuous illumination disrupts normal ocular development in young chicks, causing severe corneal flattening, shallow anterior chambers and progressive hyperopia ('constant light (CL) effects'). We have studied the minimum requirements of a diurnal light cycle to prevent CL effects. (1) Seven groups of 10 chicks were reared under a 0 (constant light, CL), or 1, 2, 3,4, 6, or 12/12 h (normal) light-dark cycles. It was found that CL effects were prevented if the dark period was 4 h or longer. Below 4 h, the effects were dose-dependent and inversely correlated with the amplitude of the Fourier component of illumination at 1 cycle per day (CPD). (2) Three groups of 20 chicks were exposed to 4 h of darkness distributed differently over 24 h to vary the amplitude of the Fourier component at 1 CPD. It was found that complete suppression of the CL effects required that the 4 h of darkness were given in one block and at the same time each day. Our results show that normal ocular development in the chick requires a minimum of 4 h darkness per day, provided at the same time of the day without interruption, and suggest that the light-dark cycle interacts with a linear or weakly nonlinear oscillating system.

Form deprivation myopia in mature common marmosets (Callithrix jacchus).

PURPOSE: Experimental manipulations of visual experience are known to affect the growth of the eye and the development of refractive state in a variety of species including human and nonhuman primates. For example, it is well established that visual form deprivation causes elongation of the eye and myopia. The effects of such manipulations have generally been examined in neonatal or juvenile animals. Whether adolescent common marmosets (a new world primate) are susceptible to form deprivation myopia was studied. METHODS: Five adolescent marmosets were used in this study. Monocular form deprivation was induced by lid closure for 12 to 20 weeks, starting between 299 and 315 days of age. The effects of deprivation were assessed with keratometry, A-scan ultrasonography, and cycloplegic refractions. Both eyes (treated and fellow control) were measured before lid-closure, at the end of the deprivation period, and several times over the following 8 to 12 weeks. RESULTS: Adolescent marmosets are susceptible to visual form deprivation myopia. The experimental eyes showed significant axial elongation and myopia relative to the fellow control eyes. These changes were smaller, however, than those observed in younger eyes deprived for comparable periods. Like juvenile animals, the adolescent marmosets did not show recovery from myopia over the period monitored. CONCLUSIONS: The period for susceptibility to form deprivation myopia in the marmoset monkey extends beyond the early developmental period when ocular growth is rapid and emmetropization normally takes place. Visual form deprivation in adolescent marmosets with adult-sized eyes results in increased ocular growth and myopia. These data suggest that visual factors may influence the growth and refractive development of the human eye after puberty and may be involved in late-onset myopia.

Decreased proteoglycan synthesis associated with form deprivation myopia in mature primate eyes.

PURPOSE: The rate of proteoglycan synthesis was measured in the scleras of adolescent marmosets that had undergone monocular form deprivation to characterize the scleral extracellular matrix changes associated with the development of myopia in a mature primate. METHODS: Form deprivation myopia was induced in adolescent marmosets by unilateral lid suture for an average of 108 days. After the lids were reopened, the axial lengths and refractions were measured at intervals for up to 39 weeks. At the end of the study period, sclera were isolated and immediately radiolabeled with 35SO4 in organ culture. Proteoglycan synthesis rates were determined by measurement of 35SO4 incorporation into cetylpyridinium chloride-precipitable glycosaminoglycans after digestion of the scleral samples with proteinase K. Collagen content was determined by measurement of total hydroxyproline in scleral digests. Newly synthesized proteoglycans were separated on a Sepharose CL-4B molecular sieve column and identified by their core proteins by Western blot analyses. RESULTS: Lid suture resulted in myopia due to a significant increase in vitreous chamber depth. After Sepharose CL-4B chromatography, newly synthesized scleral proteoglycans isolated from normal, form-deprived, and contralateral control eyes, resolved into one major peak that eluted in the position of decorin, a small chondroitin-dermatan sulfate proteoglycan. After digestion of the major peak with chondroitinase ABC, an approximately 45-kDa core protein was detected by Western blot analyses, confirming the presence of decorin. Form deprivation resulted in a significant reduction in the rate of proteoglycan synthesis in the posterior sclera (-43.55%, P < or = 0.001). Proteoglycan synthesis was also significantly reduced in the posterior sclera of form-deprived eyes relative to total collagen content (-36.19%, P < or = 0.01) and was negatively correlated with the rate of vitreous chamber elongation in the deprived eye (r2 = 0.779, P < or = 0.05). CONCLUSIONS: Significant extracellular matrix remodeling occurs in the posterior sclera of the adolescent primate eye during vitreous chamber elongation and myopia development. The negative correlation between vitreous chamber elongation rates and the synthesis rates of decorin in form-deprived eyes suggests that proteoglycan synthesis within the posterior sclera plays a role in the regulation of ocular size and refraction in the adolescent marmoset.

Choroidal thickness changes during altered eye growth and refractive state in a primate.

PURPOSE: In the chick, compensation for experimentally induced defocus involves changes in the thickness of the choroid. The choroid thickens in response to imposed myopic defocus and thins in response to imposed hyperopic defocus. This study was undertaken to determine whether similar choroidal changes occur in the primate eye with induced refractive errors. METHODS: Thirty-three common marmosets were used. Eyes in 26 monkeys served as untreated control eyes, and eyes in 7 received 3 weeks of monocular lid suture to induce changes in eye growth and refractive state. Refractive errors were measured using refractometry and retinoscopy, and axial ocular dimensions, including choroidal thickness, were measured using high-frequency A-scan ultrasonography. Eyes were measured before the lids were sutured and at frequent intervals after lid opening. RESULTS: In the marmoset, choroidal thickness ranges from 88 to 150 microm and increases significantly during the first year of life. Monocular lid suture initially results in short, hyperopic eyes that then become elongated and myopic. In these animals the choroids of both the experimental and the fellow control eyes also increase in thickness with age but additionally show interocular differences that vary significantly with the relative changes in vitreous chamber depth and refraction. In eyes that are shorter and more hyperopic than control eyes the choroids are thicker, and in eyes that are longer and more myopic than control eyes the choroids are thinner. CONCLUSIONS: In marmosets, the thickness of the choroid increases during postnatal eye growth. Superimposed on this developmental increase in choroidal thickness there are changes in thickness that are correlated with the induced changes in eye size. These changes are small (<50 microm) in comparison with those observed in the chick, contributing to less than a diopter change in refractive error.

[Current role of thrombendarterectomy in chronic peripheral obstructive arteriopathy]. / Ruolo attuale della tromboendoarteriectomia (TEA) nelle arteriopatie ostruttive croniche periferiche.

BACKGROUND: The authors report the present indications for thromboendarterectomy (TEA) and the advantages it offers for the surgical treatment of steno-obstructive lesions of aortoiliac and femoro-popliteal regions. They illustrate this method and the necessary instruments and describe the technique in detail, for the good outcome of revascularization. METHODS: Then personal surgical experience in 46 patients during the period from January 1985 to December 1995, is reported. Epidemiological data (sex, average age, risk-factors, concomitant diseases), different types of surgical operations (TEA "open air", TEA "semiclosed", TEA in association with bypass), clinical and instrumental preoperating study, invasive or not, medical therapy during and after operation, immediate and remote check-up are then reported. RESULTS AND CONCLUSIONS: The results in terms of cumulative pervious vessel and salvage of the arm, compared with different stages of the disease and a 5 years- follow-up are reported. The effectiveness of TEA is confirmed in the symptomatic improvement and salvage of the arm, and the lower costs, when the indications and the accuracy of surgical technique are respected.

Factors controlling the dendritic arborization of retinal ganglion cells.

The effects of changing retinal ganglion cell (RGC) density and availability of presynaptic sites on the development of RGC dendritic arbor in the developing chick retina were contrasted. Visual form deprivation was used to induce ocular enlargement and expanded retinal area resulting in a 20-30% decrease in RGC density. In these retinas, RGC dendritic arbors increased in a compensatory manner to maintain the inner nuclear layer to RGC convergence ratio in a way that is consistent with simple stretching; RGC dendritic arbors become larger with increased branch lengths, but without change in the total number of branches. In the second manipulation, partial optic nerve section was used to produce areas of RGC depletion of approximately 60% in the central retina. This reduction in density is comparable to the density of locations in the normal peripheral retina. In RGC depleted retinas, dendritic arbor areas of RGCs in the central retina grow to match the size of normal peripheral arbors. In contrast to the expanded case, two measures of intrinsic arbor structure are changed in RGC-depleted retinas; the branch density of RGC dendrites is greater, and the relative areas of the two arbors of bistratified cells are altered. We discuss the potential roles of retinal growth, local RGC density, and availability of presynaptic terminals in the developmental control of RGC dendritic arbor.

Functional architecture of area 17 in normal and monocularly deprived marmosets (Callithrix jacchus).

The organization of the primary visual cortex (VI) of the common marmoset (Callithrix jacchus) was studied both physiologically and by means of transneuronal labelling of geniculocortical afferents. We addressed the question whether monocular deprivation (MD) could stabilize segregation into ocular dominance (OD) columns, which are not seen in normal adult marmosets but are present in juvenile animals (Spatz, 1979, 1989). Properties of neurons in normal marmosets closely resembled those of other New-World and Old-World monkeys and orderly tangential progressions of preferred orientation were observed. However, in contrast to species that display well-defined OD columns, neurons of layer 4 in V1 of normal adult marmosets received balanced inputs from the two eyes. Early MD (even though followed by prolonged binocular experience into adulthood) resulted in a reduction of cell size in laminae of the lateral geniculate nucleus with input from the deprived eye and a dramatic overall shift in ocular dominance towards the nondeprived eye in the cortex. However, isolated clusters of cells dominated by the deprived eye were found in both layers 4 and 6. Injection of lectin-conjugated horseradish peroxidase (WGA-HRP) into the deprived eye revealed elongated patches of terminal label, about 350 microns wide, in flat-mounted sections through layer 4. Afferent segregation was sharper and more regular in the region of V1 representing parafoveal visual space than in that representing the fovea. Our findings support the notion that all Old-World and New-World monkeys possess the capacity for segregation of geniculocortical afferents into OD columns.

The mechanism of lenticular accommodation in chicks.

In the chick eye, accommodation for near objects is brought about by changes in the focal length of the lens and by changes in the corneal radius of curvature. Several different mechanisms of lenticular accommodation have been proposed for the avian eye. These include a role for the ciliary muscle, a role for the iris muscle, and a role for changes in intraocular pressure. We have studied accommodation in the chick eye using electrical stimulation of the Edinger-Westphal nucleus, electric-field stimulation of enucleated eyes, in vitro measurement of changes in back vertex distance of the lens, and histology. We present evidence showing that, in the chick eye, lenticular accommodation is induced primarily by a contraction of the muscle fibers at the peripheral edge of the iris. During accommodation, the peripheral muscle fibers of the iris contract to apply a force through the ciliary processes to the anterior equatorial surface of the lens. This increases the focal power of the lens. When accommodation is relaxed, the lens is returned to its unaccommodated state by the elasticity of the pectinate ligament and the ciliary body. Contractions of the posterior ciliary muscle and changes in intraocular pressure, forces that have previously been proposed to play major roles in lenticular accommodation, are shown to be of secondary importance only.

Constant light produces severe corneal flattening and hyperopia in chickens.

In this study we report on the effects of constant light (CL) on the refractive development and ocular morphology of White Leghorn chicks (Cornell K-strain). Refractive state and corneal curvature were measured by IR photoretinoscopy and IR keratometry respectively. The axial lengths of the ocular components were measured by A-scan ultrasonography. We find that constant light produces significant hyperopia compared to controls in as few as 10 days (7.4 vs 4.0 D). This is apparently the result of flatter than normal corneal curvature (radius of curvature: 3.22 vs 3.08 mm) as vitreous chamber depth is significantly deeper in CL eyes than controls at that age (5.6 vs 5.1 mm). In contrast to other reports, if CL rearing is continued for longer periods the hyperopia progresses, even though vitreous chamber depth continues to increase. After 11 weeks of CL severe hyperopia was observed (18.2 vs 2.8 D). Long term CL is also found to produce shallow anterior chambers, corneal thickening, lenticular thinning and cataracts, and damage to the retina, pigment epithelium, and choroid.

Differences in eye growth and the response to visual deprivation in different strains of chicken.

Several laboratories studying visual deprivation myopia in the domestic chick report varying degrees of axial elongation and myopia induced by similar visual deprivation techniques. In this study we tested the hypothesis that in different strains of chick the eyes respond differently to visual deprivation. We compared under identical conditions two strains of White Leghorn chick commonly used in ocular development research--the Cornell-K strain (K) and Washington H & N Strain (H/N). The normal development of the eye was found to vary significantly between these strains of White Leghorn chicks. The K strain normally develops flatter corneas, thicker lenses, and larger eyes than the H/N strain. The response to visual deprivation also varies significantly between strains. For example, we find that 2 weeks of visual deprivation in the K strain results in less elongation of the vitreous chamber and flattening of the cornea yielding lower levels of induced myopia compared to the H/N strain. Our results show that while visual experience clearly affects normal ocular development in both strains of chick, the nature of the effect depends upon not only the type and duration of the experience but the genetics of the subject population as well.

The mechanism of corneal accommodation in chicks.

Corneal accommodation can account for up to 9 D of accommodation in a freely behaving chick. We have explored the possibility that changes in corneal curvature are due to changes in intraocular pressure (IOP) during accommodation. In an in vitro preparation we demonstrate that increasing the pressure will tend to flatten the cornea. We have used electrical stimulation of the Edinger-Westphal (EW) nucleus to further test the pressure hypothesis in vivo by recording IOP changes in the eye during EW-stimulated accommodation and by artificially modulating the IOP to assess the effects on corneal curvature. During EW stimulation there is an increase in IOP on the order of 1-3 mmHg which tends to flatten the curvature of the cornea, thus eliminating changes in IOP as a possible mechanism of corneal accommodation. Slit-lamp observations of accommodative changes at the corneo-scleral margin and electrical stimulation of dissected eyes in vitro indicate that corneal accommodation is mediated by a contraction of the ciliary muscles, which exerts a pull on the inner lamella of the cornea, flattening the peripheral cornea and increasing the curvature of the central cornea. Histological examination of the ciliary region of the eye confirms the appropriate positioning of the ciliary muscles. We conclude that corneal accommodation in the chick eye is accomplished by a ciliary muscle-mediated mechanism.

Visual optics and retinal cone topography in the common marmoset (Callithrix jacchus).

The common marmoset (Callithrix jacchus) is a small, diurnal, New World monkey amenable to vision research. In this paper we describe the visual optics and cone photoreceptor topography of the normal adult marmoset. Paraxial optical ray-tracing shows that the marmoset eye is well represented as a scaled-down version of the human eye. The density of foveal and perifoveal cone photoreceptors in the marmoset is as high, and in peripheral retina higher, than those reported in humans and macaques. The foveal acuity predicted by the Nyquist limits set by the cone mosaic (30 c/deg) is in agreement with behavioral measures of visual acuity. Foveal depth of focus is remarkably small (< 0.2 D) for an eye of this size (axial length about 11 mm). Estimates of the amplitude of accommodation using infrared photorefraction indicate that the marmoset is capable of more than 20 D of accommodation.

Ocular development and visual deprivation myopia in the common marmoset (Callithrix jacchus).

The normal postnatal ocular development of the common marmoset (Callithrix jacchus) and the effects of visual deprivation on eye growth and refractive state are described. The marmoset normally undergoes a developmental process of emmetropization from high hyperopia at birth. This emmetropization is easily disrupted by visual deprivation produced by lid-suture. Myopia and axial elongation of the vitreous chamber are induced by visual deprivations of 12, 5, and 3 weeks duration. The development of axial myopia after 3 weeks of visual deprivation differs from longer duration deprivations in that the experimental eyes are initially shorter than normal and hyperopic at the end of the visual deprivation period, but subsequently become longer than normal and myopic. Visual deprivation myopia in the marmoset persists even after the deprivation is discontinued and a visual signal is restored. In all experimental groups, the development of the eye in response to the cessation of visual deprivation shows no slowing of vitreous chamber enlargement; the axial enlargement relative to the control eye is either maintained or increases and produces significantly greater myopia. These results suggest that the visual control of postnatal eye growth in the marmoset may be unidirectional in its response to visual experience and able only to increase the growth rate of the vitreous chamber, possibly after an initial delay.

Synchrotron X-ray diffraction and histochemical studies of normal and myopic chick eyes.

Synchrotron X-ray diffraction patterns were obtained from the cornea and posterior sclera of control and myopic chicks. No significant differences was found in the interfibrillar or in the intermolecular spacing of the collagen fibrils from the corneas of control and myopic chicks. The intermolecular spacing of myopic sclera was shown to be significantly (p < 0.01) higher than in controls. Sclera and cornea from normal and myopic chicks were stained for proteoglycans using the 'critical electrolyte' method of Scott and Orford (1981). In the sclera, two morphologically distinct types of proteoglycans could be distinguished; one small and usually elongated (approximately 20 nm), the other larger and irregularly shaped. The small proteoglycans were seen binding preferentially to the 'd' and 'e' bands of the collagen fibrils. Small proteoglycans were also present within the fibrils, these were usually approximately 10 nm in diameter although sizes up to 30 nm were observed. Longitudinal sections of fibrils revealed that these intrafibrillar proteoglycans were chiefly orientated parallel to the axis of the collagen fibrils, and preferentially located along the gap region of the fibrils. No difference was observed in the binding sites of the proteoglycans between normal and myopic sclera. The larger proteoglycans were also seen aggregated into clumps, which were usually associated with spaces between collagen bundles. The differences between control and myopic sclera appear to be quantitative rather than qualitative suggesting that the scleral expansion in myopia is an enhanced form of normal scleral growth.

[Compression of the sigmoid of gynecological origin]. / Compressione del sigma di natura ginecologica.

The authors report a case of extrinsic stenosis of the sigmoid colon due to inflammation of the uterus and adjoining parts. Attention is drawn to the possibility that the radiological images of this condition may be similar to neoplastic diseases.

Neonatal eye growth and emmetropisation--a literature review.

The refractive development of the neonatal eye has been the subject of much study and debate. In this paper the hypothetical mechanisms of emmetropisation and their relationship to the development of refractive errors will be reviewed. The evidence supporting visual feedback control of eye growth will be described, and the role of ocular accommodation will be discussed.

The regulation of eye growth and refractive state: an experimental study of emmetropization.

During growth the vertebrate eye achieves a close match between the power of its optics and its axial length with the result that images are focused on the retina without accommodative effort (emmetropia). The possibility that vision is required for the regulation of eye growth was studied experimentally in chicks made myopic or hyperopic by different visual manipulations. After discontinuing these visual manipulations, the eyes returned quickly to emmetropia mainly by adjusting the growth of their vitreous chambers; growth stopped in eyes recovering from myopia and continued in eyes recovering from hyperopia. Because both hyperopic and myopic eyes were already larger than normal controls, the difference in growth indicates that refractive error, rather than eye size per se, guides the eye toward emmetropia. Evidence is also presented for nonvisual shape-related control of eye growth, but this is slow-acting and cannot explain the emmetropization from induced refractive errors. Both the visually guided and shape-related mechanisms work even in eyes with the optic nerve cut, indicating that the two mechanisms are local to the eye. Although the optic-nerve-sectioned eye can sense the sign of a refractive error and initially adjust growth accordingly, it eventually overshoots emmetropia and reverses the sign of the initial refractive error. Whether this is due to loss of feedback from the central nervous system or retinal ganglion cells is unclear.

Developing eyes that lack accommodation grow to compensate for imposed defocus.

The eyes of growing chicks adjust to correct for myopia (eye relatively long for the focal length of its optics) or hyperopia (eye relatively short for the focal length of its optics). Eyes made functionally hyperopic with negative spectacle lenses become myopic and long, whereas eyes made functionally myopic with positive spectacle lenses become hyperopic and short. We report here that these compensatory growth adjustments occur not only in normal eyes but also in eyes unable to accommodate (focus) because of lesions to the Edinger-Westphal nuclei. Thus, at least in chicks, accommodation is not necessary for growth that reduces refractive errors during development, and may not be necessary for the normal control of eye growth.