Allvar Gullstrand (1862-1930)--the gentleman with the lamp.

This is a biography of Allvar Gullstrand (1862-1930) on the occasion of the centennial of his 1911 Nobel Prize in physiology or medicine. We reviewed pertinent literature and we did archival studies at the Uppsala University Library and the Regional State Archives at Lund as well as the Nobel Archives at the Royal Swedish Academy of Sciences in Stockholm. Allvar Gullstrand was a brilliant scientist with an exceptional personality. He gave mathematical descriptions of the dioptric system of the human eye with unprecedented accuracy, and he invented and designed ophthalmological instruments of far-reaching importance. The two most valuable ones are the slit lamp and the reflexless ophthalmoscope. Both are in everyday use by any ophthalmologist in the world. Allvar Gullstrand is so far the only ophthalmologist who has been given a Nobel Prize for work in ophthalmology, and he deserved it well.

Early development of retinal subtypes in long-term cultures of human embryonic retina.

PURPOSE: To study early signs of neuronal and glial differentiation in the human embryonic retina. MATERIALS AND METHODS: 6.5-to 8-week-old human embryos were obtained from elective abortions. The neuroretinas were kept in culture as full-thickness sheets for 7-42 days. RESULTS: The control retinas consisted of a neuroblast cell layer and a thin marginal zone. Most explants displayed presence of retinal lamination, but also contained regions of disorganization. Vimentin labeling showed vertically arranged Müller cells in all explants. Recoverin-labeled photoreceptors appeared in explants kept 14 days and longer. By labeling with antibodies against PKC and parvalbumin, rod bipolar cells and amacrine cells could be seen in explants kept for 42 days in culture. CONCLUSIONS: We have shown that the embryonic full-thickness neuroretina can survive in a culture environment for at least 6 weeks, and can develop several types of the retina-specific neuronal and glial cells.

Compartmental localization of gamma-aminobutyric acid type B receptors in the cholinergic circuitry of the rabbit retina.

Although many effects of gamma-aminobutyric acid (GABA) on retinal function have been attributed to GABA(A) and GABA(C) receptors, specific retinal functions have also been shown to be mediated by GABA(B) receptors, including facilitation of light-evoked acetylcholine release from the rabbit retina (Neal and Cunningham [1995] J. Physiol. 482:363-372). To explain the results of a rich set of experiments, Neal and Cunningham proposed a model for this facilitation. In this model, GABA(B) receptor-mediated inhibition of glycinergic cells would reduce their own inhibition of cholinergic cells. In turn, muscarinic input from the latter to the glycinergic cells would complete a negative-feedback circuitry. In this study, we have used immunohistochemical techniques to test elements of this model. We report that glycinergic amacrine cells are GABA(B) receptor negative. In contrast, our data reveal the localization of GABA(B) receptors on cholinergic/GABAergic starburst amacrine cells. High-resolution localization of GABA(B) receptors on starburst amacrine cells shows that they are discretely localized to a limited population of its varicosities, the majority of likely synaptic-release sites being devoid of detectable levels of GABA(B) receptors. Finally, we identify a glycinergic cell that is a potential muscarinic receptor-bearing target of GABA(B)-modulated acetylcholine release. This target is the DAPI-3 cell. We propose, based on these data, a modification of the Neal and Cunningham model in which GABA(B) receptors are on starburst, not glycinergic amacrine cells.

Structural changes in the developing retina maintained in vitro.

The present study examined the emergence of structural remodeling in explanted neonatal rat retina. Immunohistochemical analysis demonstrated signs of glial and neuronal remodeling after 11 days in vitro and included the activation of Müller cells, the formation of ectopic neuropil areas and sprouting of photoreceptor terminals. We also observed that cholinergic and GABA-ergic amacrine cells displayed signs of disorganized laminations. These results demonstrate that retinal culturing initiates structural changes that show morphological similarities to glial and neuronal remodeling identified in retinitis pigmentosa retinas and experimentally detached retinas.

Nordic research in ophthalmology.

Nordic ophthalmologists and vision scientists are active in many fields of eye research. This is most evident at the biannual Nordic Congress of Ophthalmology, most recently held in Malmö in June 2004. The authors here review some of the research in vision and ophthalmology presented at this meeting or published recently by Nordic scientists. This paper does not represent a comprehensive review of all Nordic research in the field, but attempts to give an overview of some of the activities underway in eye research in this part of the world.

Cystatin C uptake in the eye.

BACKGROUND: As a secreted protein, cystatin C is assumed to play its role in the extracellular compartment, where it can inhibit virtually all cysteine proteases of families C1 (cathepsin B, L, S) and C13 (mammalian legumain-related proteases). Since many of its potential target enzymes in the eye reside in intracellular compartments, we sought evidence for a cellular uptake of the inhibitor in ocular tissues. METHODS: Fluorescence-labeled human cystatin C was injected intravitreally into normal rat eyes. Ocular tissues were subsequently examined using ELISA, fluorescence microscopy, and immunohistochemistry. Cystatin C uptake was additionally studied in an in vitro retina model. RESULTS: Cystatin C administered intravitreally in vivo is taken up into cells of the corneal endothelium and epithelium, the epithelial cells lining the ciliary processes, and into cells in the neuroretina (mostly ganglion cells) and the retinal pigment epithelium. The uptake is demonstrable also in vitro and was, in the neuroretina, found to be a high-affinity system, inhibited by cooling the specimens or by adding the microfilament polymerization inhibitor, cytochalasin D, to the medium. CONCLUSIONS: There is an active, temperature-dependent uptake system for cystatin C into several cell types in the cornea, ciliary body, and retina. The cell types that take up cystatin C are generally the same that contain endogenous cystatin C, suggesting that much or all cystatin C seen intracellularly in the normal eye may have been taken up from the surrounding extracellular space. The uptake indicates that the inhibitor may exert biological functions in intracellular compartments. It is also possible that this uptake system may regulate the extracellular levels of cystatin C in the eye.

Cystatin C in the anterior segment of rat and mouse eyes.

PURPOSE: Cystatin C is a mammalian cysteine protease inhibitor. This study describes the localization of cystatin C in the anterior segment of normal rat and mouse eyes. Cysteine proteases play an important role in protein degradation (e.g. of photoreceptor outer segments in the retinal pigment epithelium) and the balance between these proteases and their specific inhibitors is therefore of great interest. METHODS: Cells containing cystatin C were identified by immunohistochemistry and quantified by ELISA. Messenger RNA levels were analysed by quantitative real-time polymerase chain reaction. RESULTS: Cystatin C is present at biologically significant levels in the corneal epithelium, endothelium and stromal keratinocytes, lens epithelium, epithelial cells in the ciliary processes, aqueous humour and iris stromal cells. In the rat anterior segment, the highest cystatin C concentrations were found in the ciliary epithelium. CONCLUSIONS: Cystatin C is present in several cell types and is probably locally produced. The inhibitor is likely to be an important regulator of cysteine proteases in the retinal pigment epithelium, ciliary epithelium, aqueous humour, lens epithelium and in the corneal endothelium and epithelium.

Apoptotic cell death and microglial cell responses in cultured rat retina.

BACKGROUND: Optic nerve transection results in degeneration of axotomized retinal ganglion cells followed by the activation of resident microglial cells. METHODS: An organotypic culture of neonatal rat retina was used to examine the temporal aspect of retinal ganglion cell death and microglial cell recruitment. Retinas were fixed at various times after explantation and prepared for immunohistochemistry and lectin staining. RESULTS: Terminal deoxytransferase dUTP nick-end labeling (TUNEL) and immunohistochemistry for cleaved caspase-3 demonstrated a massive cascade of cell death in the ganglion cell layer (GCL) within hours after explantation. The rate of cell death in this layer was high and continued over a period of 48 h. In contrast, the rate of cell death was low in the outer nuclear layer (ONL) and apoptotic cells were evident after 6 days in vitro. Increases in the density of microglial cells in the GCL appeared to be recruited by proliferation within hours after explantation. In parallel, resident microglial cells also acquired an activated morphology as revealed by isolectin B(4) staining. Microglial cell activation in the GCL also included an upregulated expression for the lysosomal protein ED-1 and the cysteine protease inhibitor cystatin C. After 1 week of culture, immunolabeling for ED-1 demonstrated the presence of activated microglial cells also in the ONL. CONCLUSION: These data show rapid microglial cell recruitment and activation following the axotomy-induced cell death of differentiated ganglion cells. The processes of microglial cell activation and cell death are slower in the outer retina.

Standardized full-field electroretinography in rabbits.

We present a procedure for full-field ERG recording in rabbits, based on the human ERG standards published by the International Society for Clinical Electrophysiology of Vision (ISCEV). Following initial pilot experiments, six animals aged 3 months and 11 animals between 1 and 2 years were investigated. All animals displayed well detectable and reproducible separate cone and rod responses under appropriate stimulus conditions. The b-wave was smaller in young animals than in old, but there were no similar differences in the b-wave implicit times. The animals had to be lightly sedated, which was shown to have no adverse effects on the recordings. Standard deviations of normalized adult rabbit recordings were comparable to human recordings. The measurements were less precise in young animals. We suggest that our procedure is well suited for further scientific studies in this animal model.

Neuronal integration in an abutting-retinas culture system.

PURPOSE: Limited integration is consistently observed between subretinal transplants and host retinas. In the current study, an in vitro model system for studying connections forming between two abutting retinas was developed. METHODS: Neuroretinas were dissected from normal wild-type (WT) mice and green fluorescent protein (GFP) transgenic mice (obtained at postnatal days [P]0, P5, or P60), as well as from adult rd mice. Pieces from two different retinas (WT-WT, GFP-WT, GFP-rd) were placed side-by-side (contacting each other at the margins) or overlapping each other in organ cultures for 7 or 12 days. The abutting retinal pieces derived from animals of the same age (P5-P5; P60-P60) or of different ages (P0-P60; P5-P60). Retinal cells and fibers were visualized in wholemount preparations and in cross sections by immunocytochemistry using antibodies against neurofilament (NF+), neuronal nitric oxide synthase (NOS+), and protein kinase C (PKC+) and by GFP fluorescence (GFP+). RESULTS: In side-by-side pairs (WT-WT, GFP-WT), numerous horizontal cell fibers (NF+) and amacrine cell fibers (NOS+) crossed the interface between the two pieces, forming continuous plexiform layers. In overlapping pairs, NF+, NOS+, and PKC+ fibers displayed parallel plexiform layers, and no crossover of fibers was observed in any of the pair combinations examined (WT-WT, GFP-WT, GFP-rd). Some integration was seen only in small areas where the structure of both retinal pieces was disrupted at the interface. CONCLUSIONS: The results demonstrate the ability of neurites to extend between abutting retinas and to make appropriate target choices when they are placed side-by-side. However, this ability is limited when they overlap each other, similar to that observed in subretinal transplantation.

Cathepsin B in the rat eye.

BACKGROUND: Cathepsin B is a mammalian cysteine protease. The enzyme has been suggested to participate in the patophysiological processes of keratoconus as well as in the corneal response to infectious agents. This study describes the localization of cathepsin B in the rat eye. METHODS: Cathepsin B was identified in rat ocular tissues by Western blotting and immunohistochemistry. Cathepsin B mRNA levels were analyzed in the tissues by quantitative real-time cDNA amplification (QRT-PCR). RESULTS: Cathepsin B is present in the epithelium, in stromal cells and in the endothelium of the cornea. It is also present in the epithelium lining the ciliary processes, in occasional stromal cells in the iris, in the anterior subcapsular lens epithelium and in various cell types in the retina. At all locations cathepsin B is present in cytoplasmic granules, presumably lysosomes. QRT-PCR analysis detected cathepsin B mRNA in all these tissues in amounts correlating to the immunodetection results, suggesting that the enzyme detected is locally produced. CONCLUSIONS: Cathepsin B is present in several tissues and cell types throughout the rat eye. It is localized to cytoplasmic granules, presumably lysosomes. Our results suggest that it is probably also produced in the same cell types.

COX-2 inhibitors prolong trauma-induced elevations of iris hyaluronan.

PURPOSE: To investigate whether and how treatment with COX-2 inhibitors influences hyaluronan responses to a standardized trauma, argon laser induced iritis, in rabbits. METHODS: Two different COX-2 inhibitors were used, SC-236 and rofecoxib. The drugs were administered orally, 6 mg/kg/day and 1.5 mg/kg/day respectively. Iris and aqueous humor hyaluronan concentrations were measured with a radiometric assay at different time points after laser irradiation. RESULTS: The hyaluronan concentration in the iris increased 3-4-fold with a peak concentration of 129.1 microg/g wet weight 2 days after laser irradiation. It then decreased to normal values after 1 week. In eyes treated with either of the COX-2 inhibitors, iris hyaluronan concentrations did not decrease as rapidly and were significantly higher at day 4 and 7 when compared to drug untreated eyes. CONCLUSION: Treatment with COX-2 inhibitors prolongs trauma induced elevation of iris content of endogenous hyaluronan. This may be, at least partly, due to an inhibition of interstitial fluid pressure regulation.

Limitation of anatomical integration between subretinal transplants and the host retina.

PURPOSE: In previous studies of subretinal transplantation in rabbits, the host photoreceptor layer seemed to prevent the bridging of neuronal fibers between the graft and the host retina. The current study was undertaken to determine whether the same phenomenon occurs in transplants to the subretinal space of the vascularized retina of rats. Bridging of fibers was examined in transplants to animals of different genetic backgrounds (normal versus dystrophic rats), of different ages, and after different survival times. METHODS: Sprague-Dawley (SD) rat retinal tissue from embryonic day (E)18 was subretinally grafted to adult (60-day-old) normal SD rats, to RCS rats (32 and 73 days old), and to adult (60-day-old) transgenic P23H rats. After various survival times (28-183 days), transplanted retinas were processed for routine histology and immunocytochemistry. Antibodies against calbindin, neuronal nitric oxide synthase (NOS), and protein kinase C (PKC) were used to identify specific retinal cell types and their processes. RESULTS: The shape and position of the immunoreactive cell bodies indicated that the expected neuronal populations were labeled within the grafts and in the host retina. Labeled neuronal processes were also observed. In each case, NOS-, calbindin-, and PKC-immunolabeled fibers formed bridges between the graft and the host tissues. However, regardless of the extent of host photoreceptor cell loss, the age of the recipient, or the genetic background, bridging fibers were observed only in areas where the host photoreceptor layer was discontinuous or completely missing. CONCLUSIONS: The present study demonstrates that the host photoreceptor layer plays a role in limiting graft-host anatomical integration.

Effects of sympathetic denervation on the hyaluronan content of the anterior segment in the normal and traumatized rabbit eye.

PURPOSE: To determine whether there is any involvement of sympathetic nerves in the regulation of ocular hyaluronan production in the normal and traumatized rabbit iris. METHODS: Unilateral sympathetic denervation was performed by removing the right superior cervical ganglion. Hyaluronan concentrations in the iris and aqueous were measured with a radiometric assay at various time intervals after denervation. Peripheral iridectomy was also performed in both denervated and non-denervated eyes. RESULTS: Hyaluronan concentrations in the iris tissue after denervation were observed to have increased after 1 day, reaching a peak of 129.6 +/- 5.7 microg/g wet weight at day 3. Two weeks later, hyaluronan concentrations had fallen back to normal levels. Ocular trauma with peripheral iridectomy in denervated eyes caused an increase of hyaluronan content of up to 253.5 +/- 30.5 microg/g wet weight, which was not significantly different from hyaluronan concentrations observed after the same trauma in non-denervated eyes. CONCLUSION: Cervical sympathetic denervation results in a moderate increase of the hyaluronan content in the rabbit iris and does not appear to influence the hyaluronan response of the iris to trauma.