Electrophilic PPARγ ligands inhibit corneal fibroblast to myofibroblast differentiation in vitro: a potentially novel therapy for corneal scarring.

A critical component of corneal scarring is the TGFß-induced differentiation of corneal keratocytes into myofibroblasts. Inhibitors of this differentiation are potentially therapeutic for corneal scarring. In this study, we tested the relative effectiveness and mechanisms of action of two electrophilic peroxisome proliferator-activated receptor gamma (PPARγ) ligands: cyano-3,12-dioxolean-1,9-dien-28-oic acid-methyl ester (CDDO-Me) and 15-deoxy-Δ(-12,14)-prostaglandin J(2) (15d-PGJ(2)) for inhibiting TGFß-induced myofibroblast differentiation in vitro. TGFß was used to induce myofibroblast differentiation in cultured, primary human corneal fibroblasts. CDDO-Me and 15d-PGJ(2) were added to cultures to test their ability to inhibit this process. Myofibroblast differentiation was assessed by measuring the expression of myofibroblast-specific proteins (αSMA, collagen I, and fibronectin) and mRNA (αSMA and collagen III). The role of PPARγ in the inhibition of myofibroblast differentiation by these agents was tested in genetically and pharmacologically manipulated cells. Finally, we assayed the importance of electrophilicity in the actions of these agents on TGFß-induced αSMA expression via Western blotting and immunofluorescence. Both electrophilic PPARγ ligands (CDDO-Me and 15d-PGJ(2)) potently inhibited TGFß-induced myofibroblast differentiation, but PPARγ was only partially required for inhibition of myofibroblast differentiation by either agent. Electrophilic PPARγ ligands were able to inhibit myofibroblast differentiation more potently than non-electrophilic PPARγ ligands, suggesting an important role of electrophilicity in this process. CDDO-Me and 15d-PGJ(2) are strong inhibitors of TGFß-induced corneal fibroblast to myofibroblast differentiation in vitro, suggesting this class of agents as potential novel therapies for corneal scarring warranting further study in pre-clinical animal models.

Long-term neurochemical changes after visual cortical lesions in the adult cat.

Peripheral deafferentation alters cortical function and such alterations have been shown to affect the cortical expression of the calcium-binding proteins calbindin and parvalbumin and of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). To determine whether cortical deafferentation produces similar effects, we examined the long-term consequences of cortical lesions on the neurochemistry of interconnected cortical areas. We studied the reciprocal effects of localized damage to either visual cortical areas 17 and 18, or posteromedial lateral suprasylvian (PMLS) cortex in the adult cat. These areas are strongly interconnected and play an important role in the processing of visual information. Combined lesions of areas 17 and 18 caused a marked, topographically specific decrease in the proportion of neurons expressing calbindin in supragranular layers of PMLS cortex. Similarly, lesions of PMLS cortex caused topographically restricted decreases in calbindin expression within supragranular layers of areas 17 and 18, but not in other cortical areas with which PMLS is interconnected. To categorize the calbindin-positive neurons affected by such lesions, we carried out double-labeling experiments for the inhibitory neurotransmitter GABA. This investigation showed lesions of areas 17 and 18 to affect calbindin-positive excitatory and inhibitory neurons equally, but PMLS lesions had stronger effects on inhibitory, calbindin-positive neurons. This finding may represent differential damage to feed-forward vs. feed-back projections in the two types of lesions. Finally, the expression of parvalbumin and GABA was unchanged, even in zones of decreased calbindin immunoreactivity. Our results suggest that damage to adult visual cortical areas, whether striate or extrastriate, induces neurochemical changes in the supragranular corticocortical network to which these areas belong. That changes were restricted to calbindin expression suggests cell-specific and/or biochemical pathway-specific alterations in calcium homeostasis.

Perceptual deficits after lesions of inferotemporal cortex in macaques.

This study used a novel approach to examine a much studied question, the nature of visual deficits caused by lesions of the inferotemporal cortex (IT). Unlike many previous studies of IT lesions, we de-emphasized early, non-specific disruptions of testing caused by the lesions, and instead concentrated on permanent changes in thresholds. This approach produced unexpected results that suggest a re-evaluation of the traditional view of the role of the IT cortex in shape perception and such related visual abilities as perceptual invariances, visual grouping, the visibility of illusory contours and the performance of oddity discriminations. In addition, the measurement of stable, post-lesion hue discrimination thresholds gave us a different perspective on the severity of color vision deficits which result from lesions of the IT cortex. We found that shape distortion thresholds were not permanently elevated by IT lesions and, indeed, showed no greater transitory disruption than did other visual abilities. This result is inconsistent with the common view that IT is critical to shape discriminations. Two other visual abilities that would be expected to be disrupted by IT lesions - the visual grouping of misoriented line segments and shape invariances (failure of irrelevant stimulus changes to disrupt shape distortion thresholds) - were not affected by IT lesions. However, shape discriminations based on illusory contours and some oddity discriminations were severely and permanently affected. Our results also showed that IT lesions caused permanent, moderate to large impairments of color vision, but not color blindness. Bilateral damage to area TEO caused no disruption of performance on any of the abovediscriminations. Our results suggest that the IT cortex in macaques may be critical to the visibility of illusory contours and the performance of some oddity discriminations, that it plays some role in color perception, but that it is not essential for shape, grouping discriminations or perceptual shape invariances.

Deficits in complex visual perception following unilateral temporal lobectomy.

Although human temporal cortex is known to be important for short- and long-term memory, its role in visual perception is not well understood. In this study, we compared the performance of three patients with unilateral temporal lobectomies to that of normal controls on both "simple" and "complex" visual discriminations that did not involve explicit memory components. Two types of complex tasks were tested that involved discriminations secondary to texture segmentation. These were contrasted with simple discriminations using luminance-defined stimuli. Patients showed impaired thresholds only on tasks involving texture segmentation, performing as well as controls when the targets were defined by luminance rather than texture. The minimum stimulus presentation times for threshold performance were also measured for all tasks and found to be elevated in temporal lobectomy patients relative to controls. Although the magnitude of the deficits observed was substantial, loss was equivalent in ipsi- and contra-lesional regions of the visual field. Additional control experiments showed that the patients' perceptual deficits were not due, even in part, to disturbances of basic visual capacities such as acuity and contrast sensitivity. Our results indicate that temporal lobe damage disrupts complex, but not simple, visual discriminations throughout the visual field.

Retinal ganglion cells in the albino rat: revised morphological classification.

Rat retinal ganglion cells were traditionally classified on the basis of soma size and the morphology of their dendritic fields. However, in the past, techniques used to label ganglion cells (horseradish peroxidase, Golgi, or the neurofibrillar stain) did not always stain the axon and/or the entire dendritic field. In the present study, we have labelled retinal ganglion cells in the adult albino rat with the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI) or have intracellularly injected them with Neurobiotin. Such procedures enabled us to completely fill these neurons, and our findings prompted us to modify the existing retinal ganglion cell classification in the rat. First, cells were categorised into three groups on the basis of soma and dendritic field size: Group RG(A) cells have large somata and dendritic field diameters, Group RG(B) cells have small somata and dendritic field diameters, whereas Group RG(C) cells have small to medium-sized somata and medium-to-large dendritic field diameters. On the basis of dendritic field morphology and presence across the retina, each Group was then subdivided into subgroups. The significance of our results in terms of retinal ganglion cell function is discussed.

Excitatory convergence of Y and non-Y information channels on single neurons in the PMLS area, a motion area of the cat visual cortex.

We analysed the receptive field properties of neurons in the posteromedial lateral suprasylvian (PMLS) visual cortical area of anaesthetized cats in which there was selective conduction block of the largest (Y-type) fibres in one optic nerve. As in normal cats, in cats with selective block of one optic nerve the great majority of PMLS cells could be activated by photic stimulation through either eye. However, the responses evoked by stimulation via the eye with the selectively pressure-blocked optic nerve ('Y-blocked eye') were significantly weaker than those of the same cells evoked by the stimulation via the normal eye. Accordingly, eye dominance histograms were shifted markedly in favour of the normal eye. Furthermore, there was a significant shift towards lower preferred velocities when PMLS cells were photically stimulated via the Y-blocked eye. Finally, when stimulated via the Y-blocked eye, PMLS cells responded poorly or not at all to high stimulus velocities (> or = 100 degrees/s). On the other hand, a number of receptive field properties, such as the spatial organization of receptive fields, the size of the discharge fields, orientation tuning and direction selectivity indices, were not significantly affected by the removal of the Y input. We conclude that virtually all neurons in the PMLS area of the cat receive excitatory input from both Y and non-Y information channels, although the Y channel provides the dominant input and makes the principal contribution to the detection of high-velocity motion.

Neuronal cell death in the mammalian nervous system: the calmortin hypothesis.

1. This review is concerned with the calcium-dependent mechanisms involved in neuronal cell death. To this end, it provides definitions of the major types of cell death and then describes what is known of their occurrence during development and degeneration of the mammalian nervous system. 2. An analysis is presented of the different sources and compartments of calcium in neurons and of how these are related to the known calcium-dependent enzymes whose excess activation will lead to cell death. 3. The review uses the relatively large amount of pertinent information now available for other cell types, especially thymocytes, to reveal our limited knowledge of how calcium controls neuronal cell death. 4. In the final section, consideration is given to the identification of those factors that may mitigate against the calcium-dependent pathways leading to neuronal degeneration.

NADPH diaphorase expression in the rat retina after axotomy--a supportive role for nitric oxide.

The large majority of mammalian retinal ganglion cells degenerate following section of their axons in the optic nerve. It has been suggested that some axotomized retina ganglion cells die because of toxic agents produced within their immediate environment. Our hypothesis was that nitric oxide might be one of the toxic factors implicated in the death of adult retinal ganglion cells post-axotomy. In the first instance, we determined whether there were any changes in the retinal expression of NADPH diaphorase both 3 and 14 days following intraorbital section of the optic nerve in adult rats. Secondly, if nitric oxide was indeed implicated in the death of ganglion cells, then trophic factors which rescue these neurons might do so by decreasing the expression of nitric oxide synthase. Recently, we found that a collicular proteoglycan purified from the major target of retinal ganglion cells, the superior colliculus, rescued a greater proportion of adult ganglion cells from axotomy-induced death than most other known trophic factors. We thus injected this proteoglycan intraocularly after section of the optic nerve and examined its effect on the expression of NADPH diaphorase in the retina. Thirdly, an inhibitor of nitric oxide synthetase was repeatedly injected into the eye following the section of the optic nerve in order to determine if such a treatment might improve the survival of retinal ganglion cells. The present results indicate that section of the optic nerve does not alter the overall levels of NADPH diaphorase within the adult rat retina. Intraocular injections of the collicular proteoglycan actually increased the number of neurons expressing NADPH diaphorase, particularly in the ganglion cell layer. Finally, inhibition of nitric oxide synthetase following axotomy resulted in increased loss of retinal ganglion cells over a 2 week period when compared with controls. Our findings indicate that, rather than being toxic, small amounts of nitric oxide may be important for the survival of a proportion of injured retina ganglion cells.

Glial reactivity in the retina of adult rats.

The present study aimed to characterize the reaction of mammalian (rat) retinal macroglia (Müller cells and astrocytes) to disturbances of their environment in the form of intraorbital section of the optic nerve, intraocular insertion of a thin glass capillary (without damage to the retina) or a combination of both. Glial reactivity was assessed through the use of a battery of antibodies which recognise four different proteins--glial fibrillary protein (GFAP) and three other proteins designated respectively MA1, 4D6 and 4H11. Retinal astrocytes did not exhibit any changes in normally expressed GFAP or MA1. By contrast, the expression of GFAP and MA1 in Müller cells increased 14 days following section of the optic nerve and/or intravitreal insertions of a glass capillary. Three days postoperatively, the expression of GFAP, but not MA1, had already increased significantly in Müller cells. 4D6 and 4H11 proteins were not expressed in astrocytes. In Müller cells, the levels of these proteins increased significantly following combined optic nerve section and intraocular insertion of a glass capillary. Thus, a mechanical disturbance of the intraocular environment constitutes a more effective stimulus in increasing the expression of some Müllerian proteins than damage to the axons of retinal ganglion cells. Such changes have important implications for various ocular treatments that involve intraocular administration of drugs, as well as for the survival/regeneration potential of retinal ganglion cells undergoing Wallerian degeneration.

NADPH-diaphorase expression in neurons and glia of the normal adult rat retina.

In the rat retina, nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining has been described previously in a population of amacrine cells, most of which were located in the inner nuclear layer. In the present study, a number of parameters such as the nature of the fixative, the time of fixation and photointensification were optimised to obtain the strongest possible reaction for this enzyme. As a result, a very different staining pattern emerged: with short paraformaldehyde fixation, numerous neurons (identified as a combination of ganglion cells and amacrines) were labelled in the ganglion cell layer, NADPH-d-positive amacrine cells (described previously) were seen in the inner nuclear layer and Müller cells were labelled strongly, particularly in the inner retina. Glutaraldehyde fixation of the same duration resulted in the preferential staining of Müller cells while neurons appeared less reactive. Therefore, fixation conditions are a determining factor in the cellular localisation of NADPH-d in the rat retina. By taking fixation into account, future studies should gain more rigorous insights into the possible functions of this enzyme in the vertebrate retina.

Effect of collicular proteoglycan on the survival of adult rat retinal ganglion cells following axotomy.

Consistent with numerous previous studies, we have found that in adult rats 29% of cells retrogradely prelabelled by injections into retino-recipient nuclei are lost 1 week after intraorbital section of the optic nerve. This figure increases to 76% 2 weeks after axotomy. Intraocular injections of 150 ng of 480 kDa chondroitin sulphate proteoglycan purified from the superior colliculi of neonatal rats were performed every third day after axotomy. This procedure resulted in the loss of only 3 and 28% of the axotomized retinal ganglion cells 7 and 14 days respectively after optic nerve section. Intraocular injections of chondroitin sulphate type C, one of the sugar types present on the collicular proteoglycan, also resulted in a significant saving of axotomized ganglion cells (with the loss of only 48% 14 days after optic nerve lesion). These findings suggest that the collicular proteoglycan, and to a lesser extent its sugar moieties, substantially slows down the degeneration of adult retinal ganglion cells following axotomy.

Trophic effect of collicular proteoglycan on neonatal rat retinal ganglion cells in situ.

Naturally occurring neuronal death is widespread in the central nervous system of mammals. To date, the causes and mechanisms of such death are poorly understood. A major hypothesis is that developing neurons compete for limited amounts of trophic factor(s) released from their target centres as in the case of the peripheral nervous system and nerve growth factor. The present study aims to test this 'trophic hypothesis' in the mammalian central nervous system. In the rat, more than 50% of retinal ganglion cells die in the early post-natal period. Schulz and coworkers [57] purified a potential trophic agent from their major target, the superior colliculus, which was identified as a 480 kDa chondroitin sulfate proteoglycan. This proteoglycan or control solutions were injected into the eyes of rat pups during the post-natal part of the period of naturally occurring ganglion cell death. It was found that the collicular proteoglycan prevented the death of a significant number of the ganglion cells that would normally have been lost over a post-injection period of one or two days. The effect of the proteoglycan was dose- and time-dependent. These results support the notion that trophic interactions are a determining factor in the survival of retinal ganglion cells during the period of naturally occurring cell death. It is also the first time that a proteoglycan has been shown to possess neurotrophic properties in situ.

Effect of proteoglycan purified from rat superior colliculus on the survival of murine retinal ganglion cells.

Recently, Schulz and coworkers purified a chondroitin sulfate proteoglycan from the superior colliculus of the neonatal rat which promoted survival of neonatal rat retinal ganglion cells in vitro. The present work tests whether this factor supports the survival of axotomised retinal ganglion cells in vivo. To this effect, murine retinae 15 and 20 days after conception were explanted to the chorioallantoic membrane of live chicken embryos. The explants, which were left in the egg for 1, 2 or 7 days, differentiated and grew according to a normal timetable. Purified proteoglycan from neonatal rat superior colliculus was applied daily to one group of retinae while a control group received Ham's F-10 medium. Results indicated that application of proteoglycan resulted in the preferential survival of large cells in the ganglion cell layer, namely ganglion cells, for up to 7 days post-explantation. In addition, the proteoglycan had a significant short-term anti-traumatic effect on the ganglion cell layer of explants by causing a 72% decrease in the number of dead cells relative to controls 1 day post-explantation. It was concluded that the chondroitin sulfate proteoglycan purified from the superior colliculus of the neonatal rat promotes the survival of fetal and neonatal murine retinal ganglion cells in retinae explanted to the chorioallantoic membrane of the chick.

The origin and development of retinal astrocytes in the mouse.

Astrocytes, a class of glia which appear in the mammalian retina late in development, have been postulated either to originate in situ from Müller cells or extra-retinally from the optic stalk epithelium, only subsequently invading the eye. The site of origin and the developmental characteristics of retinal astrocytes were examined in the mouse, a species not previously studied for this purpose. Sections of normal eyes and stalks at different ages were examined. Cells positive for glial fibrillary acidic protein (GFAP) were first observed at post-conceptional day 17 at the optic disc end of the stalk. From this site, the GFAP-positive cells migrated into and across the retina at a rate of approximately 290 microns per day, reaching its edge by post-conceptional day 28. While migrating across the retina, the astrocytes progressively increased in size and morphological complexity, observations confirmed by measurement of their fractal dimension. Over the same period, a wave of differentiation swept along the stalk in the cranial direction. Further evidence that retinal astrocytes are born outside the retina emerged when foetal hemiretinae with or without optic stalks were explanted to the chorioallantoic membrane of the chick. When examined one to twelve days later, no explant cultured without the optic stalk contained GFAP-positive astrocytes, while explants with the stalk left attached contained relatively normal numbers of astrocytes. We observed, using fluorescence confocal microscopy, that retinal astrocytes in the mouse as in the rat, associate predominantly with blood vessels, not axonal bundles. It was of interest to determine whether this class of glia is essential to the normal cytoarchitectural development of the neural retina. Morphological analysis of the explants revealed no observable differences in cytoarchitecture or in the timing of developmental events between retinae maturing with or without astrocytes. It was therefore concluded that astrocytes may not be essential to the normal structural development of the murine retina.

Explantation of fetal murine retinae to the chorioallantoic membrane of the chicken embryo.

A technique is here described for the culture of mammalian retinal explants on the chorioallantoic membrane of the developing chicken embryo. As an integral part of the central nervous system, the mammalian retina is characterised by its highly organised laminar structure and developmental timetable. Study of its prenatal development is, however, difficult to undertake in utero. In an attempt to render the organ of vision more accessible experimentally, fetal mouse retinae were explanted across major species barriers to the live chorioallantoic membrane of the chick. From 26 experiments, 128 explants (70% of the total) were recovered and 27 possessed a cytomorphology apparently identical to that of age-matched controls. The surviving retinae were analysed using a specifically devised set of criteria and they had developed a normal laminar structure (ganglion cell, inner plexiform, inner nuclear, outer plexiform and outer nuclear layers) but increased numbers of pyknotic profiles were present and somal sizes in the ganglion cell layer were significantly smaller. Such patterns have been obtained in other studies, both in vivo and in vitro, in which retinae had no access to their major targets in the brain, the superior colliculus and lateral geniculate nucleus. Explantation to the chorioallantoic membrane is thus a viable alternative for experiments requiring tissue isolation from natural surroundings since the explants are accessible for manipulation and observation while interacting with the host chick embryo. Furthermore, the technique allows examination of retinal differentiation, offering the opportunity to answer a number of important questions regarding development in the central nervous system.

Grafting of fetal pancreata into neonatal rat brain.

Undigested fetal pancreatic tissue has been previously shown to have immunogenic properties, even after transplantation into the adult rat brain, a relatively immunoprivileged site. In the present study, iso-, allo-, and xenografts of fetal pancreas were placed into neonatal rat brain parenchyma and ventricles in order to determine the extent and quality of its survival in this environment. Adult recipients of the same tissue types were used as controls. Selective survival of insulin-staining beta cells was observed in neonates (n = 33) over the 6-week period of the experiment. Ducts and acini were gradually destroyed in allo- and xenografts, disappearing completely by the 42nd day, while there were no such changes in the isografts. The absence of an acute inflammatory reaction was noted, but there were varying degrees of lymphocytic infiltration, though small (20 +/- 4 lymphocytes per average graft area of 0.16 mm2) in all but one graft. This infiltrate was greatest in allografts, with a significant increase observed after 14 days, corresponding to the time when the ducts started to disappear. Other structures present included fibroblasts and blood vessels. The latter increased significantly with time after transplantation. Unlike isografts placed in the parenchyma of adult rats, allo- and xenografts were rejected from the earliest time observed, 7 days postoperatively. In summary, these data show that beta cells in rat fetal pancreas will survive when grafted across major allogeneic and xenogeneic barriers for up to 6 weeks, without utilization of any form of immunosuppression, provided the recipients are neonates.