Effects of optic nerve injury, glaucoma, and neuroprotection on the survival, structure, and function of ganglion cells in the mammalian retina.

Glaucoma is an optic neuropathy that originates with pressure-induced damage to the optic nerve. This results in the retrograde degeneration of ganglion cells in the retina, and a progressive loss of vision. Over the past several years, a number of studies have described the structural and functional changes that characterize ganglion cell degeneration in the glaucomatous eye, and following optic nerve injury. In addition, a variety of different strategies for providing neuroprotection to the injured retina have been proposed. Many of these are based on the use of brain-derived neurotrophic factor (BDNF), a particularly potent neuroprotectant in the mammalian eye and the basis of our research in this area. Of particular importance is the fact that BDNF not only promotes ganglion cell survival following damage to the optic nerve, but also helps to preserve the structural integrity of the surviving neurons, which in turn results in enhanced visual function. The studies presented here describe these attributes, and serve as the foundation for ongoing work that suggests a need to think beyond the eye in the development of future treatment strategies.

Experimental glaucoma in the primate induced by latex microspheres.

The injection of sterile latex microspheres into the anterior chamber of the eye is presented as a simple and cost effective method for inducing chronic elevation of intraocular pressure (IOP) and experimental glaucoma in primates. The microspheres produce elevated IOP primarily by restricting the outflow of aqueous humor through the trabecular meshwork located in the chamber angle. Different levels and durations of elevated IOP can be obtained by altering the frequency and number of microspheres injected. In comparison with other primate models of experimental glaucoma, the approach described here has the advantages of producing chronic elevations of IOP without the need for expensive ophthalmic equipment and personnel, surgical intervention or intraocular inflammation, and without compromising visibility of the optic disc, which is necessary for clinical assessment of the onset and progression of the disease.

Activation of NF-kappaB in airway epithelial cells is dependent on CFTR trafficking and Cl- channel function.

Polymorphonuclear leukocyte-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease and may be associated with CF transmembrane conductance regulator (CFTR) dysfunction as well as infection. Mutant DeltaF508 CFTR is mistrafficked, accumulates in the endoplasmic reticulum (ER), and may cause "cell stress" and activation of nuclear factor (NF)-kappaB. G551D mutants also lack Cl- channel function, but CFTR is trafficked normally. We compared the effects of CFTR mutations on the endogenous activation of an NF-kappaB reporter construct. In transfected Chinese hamster ovary cells, the mistrafficked DeltaF508 allele caused a sevenfold activation of NF-kappaB compared with wild-type CFTR or the G551D mutant (P < 0.001). NF-kappaB was also activated in 9/HTEo-/pCep-R cells and in 16HBE/pcftr antisense cell lines, which lack CFTR Cl- channel function but do not accumulate mutant protein in the ER. This endogenous activation of NF-kappaB was associated with elevated interleukin-8 expression. Impaired CFTR Cl- channel activity as well as cell stress due to accumulation of mistrafficked CFTR in the ER contributes to the endogenous activation of NF-kappaB in cells with the CFTR mutation.

BDNF enhances retinal ganglion cell survival in cats with optic nerve damage.

PURPOSE: To determine whether brain-derived neurotrophic factor (BDNF), a neuroprotectant in the small rat eye, might also serve as an effective neuroprotectant in larger vertebrate eyes. METHODS. A cat optic nerve crush model was combined with standard histologic staining and analysis techniques. Twenty-nine animals were studied, with the noninjected eye serving as the control eye. RESULTS: No treatment, or intravitreal injection of sterile water, resulted in an approximately 50% loss of ganglion cells 1 week after nerve crush. By contrast, the mean percentages of surviving ganglion cells measured in eyes receiving injections of 15, 30, 60, and 90 microg BDNF at the time of the nerve damage were 52%, 81%, 77%, and 70%, respectively. Similar values were obtained for ganglion cell density. Cell size measurements suggest a complex response among the different classes of cat ganglion cells; 30 microg BDNF treatment retained the highest number of large ganglion cells, whereas 90 microg minimized the loss of medium-sized neurons and retained normal proportions of large, medium, and small ganglion cells. CONCLUSIONS: The data show that BDNF is an effective neuroprotectant in primate-sized eyes after optic nerve injury. Although the amount required to achieve neuroprotection is much greater than that needed for the small rat eye (30 microg versus 0.5 microg), when differences in vitreal volume are considered, the effective dose is similar (0.01 microg BDNF/microl vitreal volume). High doses of BDNF induce inflammation and result in a decrease in total ganglion cell survival but appear necessary to save medium-sized neurons, which are affected most severely by nerve injury.

Suppression of arthritis and protection from bone destruction by treatment with TNP-470/AGM-1470 in a transgenic mouse model of rheumatoid arthritis.

OBJECTIVE: We assessed the clinical and histologic features of angiogenesis inhibition in a transgenic mouse model of arthritis that closely resembles rheumatoid arthritis (RA) in humans. METHODS: KRN/NOD mice, which spontaneously develop arthritis, were treated with TNP-470, an angiogenesis inhibitor. Disease was monitored by use of clinical indices and histologic examinations; circulating blood levels of vascular endothelial growth factor were determined by enzyme-linked immunosorbent assay. RESULTS: In the preventive protocol, with TNP-470 administration at a dosage of 60 mg/kg of body weight, the onset of arthritis was delayed and its clinical intensity was rather mild; 100% of placebo-treated transgenic mice developed arthritis that led to severe articular destruction. At a dosage of 90 mg/kg of TNP-470, the appearance of clinical signs was delayed for a longer period of time and disease was almost abolished. The therapeutic regimen alleviated clinical signs only when given during the very early stage of disease. Reductions in cartilage and bone destruction by TNP-470 treatment were observed histologically, a feature that was still evident at 30 and 80 days after injections were withdrawn. CONCLUSION: Our demonstration that in vivo administration of an angiogenesis inhibitor suppresses arthritis and protects from bone destruction provides new insight into the pathogenesis of the disease and opens new possibilities in the treatment of RA in humans.

Experimental glaucoma and cell size, density, and number in the primate lateral geniculate nucleus.

PURPOSE: To examine the effects that elevated intraocular pressure (IOP), a glaucoma risk factor, has on the size, density, and number of neurons in the primate lateral geniculate nucleus (LGN). METHODS: The monkey model of experimental glaucoma was combined with standard histologic staining and analysis techniques. Fourteen animals were examined. RESULTS: Mean IOPs higher than 40 mm Hg for 2.5, 4, 8, and 24 weeks resulted in reductions of 10% to 58% in the cross-sectional areas of LGN neurons receiving input from the glaucomatous eye. Reductions for animals with lower mean IOPs (37 and 28 mm Hg) for 16 and 27 weeks were 16% and 30%, respectively. Neurons receiving input from the normal eye also were reduced in size (4 -26%). No differential effect in cell size was seen for magnocellular versus parvocellular neurons. Elevation of IOP resulted in an increase in cell density in all layers of the LGN. The increase was approximately two times greater in parvocellular (59%) than magnocellular (31%) layers. When corrected for volumetric shrinkage of the LGN, the estimated loss of neurons was approximately four times greater in the magnocellular than parvocellular layers (38% versus 10%). CONCLUSIONS: Elevation of IOP affects the size, density, and number of neurons in the LGN, and the volume of the nucleus itself. Although higher mean pressures (more than 40 mm Hg) reduce the period during which these changes occur, comparable damage can be achieved by even moderate (28 -37 mm Hg) levels of elevated IOP. On the basis of cell loss, elevation of IOP appears to have a more profound degenerative effect on the magnocellular than on the parvocellular regions of the LGN.

Swelling and loss of photoreceptors in chronic human and experimental glaucomas.

OBJECTIVE: To determine whether outer retinal changes occur in chronic, presumed primary open-angle glaucoma (POAG). METHODS: The outer retinas from 128 human eyes with a diagnosis of chronic glaucoma (presumably POAG in most cases) and 90 control eyes were examined histologically by 3 masked observers for photoreceptor swelling and loss. Retinas from 9 rhesus monkeys with glaucoma induced experimentally by laser trabecular destruction were compared with 7 fellow (control) eyes. The mean pressure elevations in the eyes with laser trabecular destruction ranged from 26.6 to 53.6 mm Hg with durations varying from 7 to 33 weeks. RESULTS: Swelling of the red- and green-sensitive cones was observed in a statistically significantly greater proportion of human eyes with presumed POAG compared with the control eyes. Patchy loss of red/green cones and rods was also found in some of the glaucomatous retinas. In a subset of the human eyes with end-stage disease, cone swelling was a variable finding. Although no photoreceptor loss was found in the 9 monkey eyes with experimental glaucoma, 8 had swelling of their red/green cones that was remarkably similar to that seen in the human eyes. Swelling was not present in any of the control monkey eyes. CONCLUSIONS: The photoreceptors are affected by chronically elevated intraocular pressure. CLINICAL RELEVANCE: These findings may explain some of the abnormalities of color vision and the electrophysiological effects that have been observed in patients with POAG.

Angiogenesis: general mechanisms and implications for rheumatoid arthritis.

In rheumatoid arthritis, the vascular endothelium is among the key targets for circulating mediators of inflammation and controls the trafficking of cells and molecules from the bloodstream toward the synovial tissue. Local blood vessel proliferation allows the pannus to develop and grow, thereby promoting cartilage and bone destruction and joint remodeling. Angiogenesis, the production of new capillaries from preexisting blood vessels, is a key process in rheumatoid arthritis that involves multiple substances such as cytokines, chemokines, growth factors, cell adhesion molecules, proteinases, proteinase inhibitors, and matrix proteins. In animal models of arthritis, angiogenesis inhibitors have been found to improve clinical and radiological outcomes, opening up the possibility of therapeutic applications in humans. Before this possibility is realized, the steady accumulation of data on the mechanisms that regulate angiogenesis will have to continue until a clear picture of angiogenesis is formed.

Morphology of single ganglion cells in the glaucomatous primate retina.

PURPOSE: To examine the degenerative effects that prolonged elevation of intraocular pressure (IOP), a risk factor commonly associated with glaucoma, has on the morphology of single ganglion cells in the primate retina. METHODS: The monkey model of glaucoma was combined with intracellular staining techniques using an isolated retina preparation. Midget and parasol cells from normal and glaucomatous eyes were labeled intracellularly, and their axons, somas, and dendritic fields were compared using confocal microscopy. RESULTS: In midget and parasol cells, the earliest signs of pressure-induced degeneration involved structural abnormalities associated with the dendritic arbor. Reductions in axon thickness appeared later, with changes in soma size occurring concomitantly or slightly later. Chronic elevation of IOP resulted in a significant decrease in the mean soma sizes of midget and parasol cells, but only parasol cells showed a significant reduction in dendritic field size and axon diameter. Comparisons of eyes with different levels of optic nerve damage, based on cup- disc ratio, showed that the axons and dendritic fields of parasol cells were significantly smaller at lower cup-disc ratios than were those of midget cells, suggesting a possible differential effect. CONCLUSIONS: In glaucoma, retinal ganglion cells undergo a pattern of degeneration that originates with the dendritic arbor and ends with shrinkage of the cell soma. Although this pattern of degeneration implies early functional deficits and retinal ganglion cell atrophy that occurs earlier than previously thought, based on ganglion cell loss alone, it also suggests a window of opportunity for effective neuroprotection.

Dendritic field development of retinal ganglion cells in the cat following neonatal damage to visual cortex: evidence for cell class specific interactions.

A well-known feature of the mammalian retina is the inverse relation that exists in central and peripheral retina between the density of retinal ganglion cells and their dendritic field sizes. Functionally, this inverse relation is thought to represent a means by which retinal coverage is maintained, despite significant changes in ganglion cell density. While it is generally agreed that the dendritic fields of mature retinal ganglion cells reflect, in part, competitive interactions that occur during development, the issue of whether these interactions are cell class specific remains unclear. In order to examine this question, we used intracellular staining techniques and an in vitro, living retina preparation to compare the soma and dendritic field sizes of alpha and beta ganglion cells from normal retinae with those of cells located in matched areas of retinae in which the density of beta ganglion cells had been reduced selectively by neonatal removal of visual cortex areas 17, 18, and 19. Our intracellular data show that while an early, selective, reduction in beta cell density has little or no effect on the cell body and dendritic field sizes of mature alpha cells, it results in a 13% increase in the mean soma area and an 83% increase in the mean dendritic field area of surviving beta cells. This differential effect suggests that the soma and dendritic field sizes of alpha and beta ganglion cells in the mature cat retina result primarily from competitive interactions during development that are cell class specific.

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Synaptology of physiologically identified ganglion cells in the cat retina: a comparison of retinal X- and Y-cells.

It has long been known that a number of functionally different types of ganglion cells exist in the cat retina, and that each responds differently to visual stimulation. To determine whether the characteristic response properties of different retinal ganglion cell types might reflect differences in the number and distribution of their bipolar and amacrine cell inputs, we compared the percentages and distributions of the synaptic inputs from bipolar and amacrine cells to the entire dendritic arbors of physiologically characterized retinal X- and Y-cells. Sixty-two percent of the synaptic input to the Y-cell was from amacrine cell terminals, while the X-cells received approximately equal amounts of input from amacrine and bipolar cells. We found no significant difference in the distributions of bipolar or amacrine cell inputs to X- and Y-cells, or ON-center and OFF-center cells, either as a function of dendritic branch order or distance from the origin of the dendritic arbor. While, on the basis of these data, we cannot exclude the possibility that the difference in the proportion of bipolar and amacrine cell input contributes to the functional differences between X- and Y-cells, the magnitude of this difference, and the similarity in the distributions of the input from the two afferent cell types, suggest that mechanisms other than a simple predominance of input from amacrine or bipolar cells underlie the differences in their response properties. More likely, perhaps, is that the specific response features of X- and Y-cells originate in differences in the visual responses of the bipolar and amacrine cells that provide their input, or in the complex synaptic arrangements found among amacrine and bipolar cell terminals and the dendrites of specific types of retinal ganglion cells.

Synaptic inputs to physiologically identified retinal X-cells in the cat.

The cat retina contains a number of different classes of ganglion cells, each of which has a unique set of receptive field properties. The mechanisms that underlie the functional differences among classes, however, are not well understood. All of the afferent input to retinal ganglion cells are from bipolar and amacrine cell terminals in the inner plexiform layer, suggesting that the physiological differences among cat retinal ganglion cells might be due to differences in the proportion of input that they receive from these cell types. In this study, we have combined in vivo intracellular recording and labeling with subsequent ultrastructural analysis to determine directly the patterns of synaptic input to physiologically identified X-cells in the cat retina. Our primary aim in these analyses was to determine whether retinal X-cells receive a characteristic pattern of bipolar and amacrine cell input, and further, whether the functional properties of this cell type can be related to identifiable patterns of synaptic input in the inner plexiform layer. We reconstructed the entire dendritic arbor of an OFF-center X-cell and greater than 75% of the dendritic tree of an ON-center X-cell and found that 1) both ON- and OFF-center X-cells are contacted with approximately the same frequency by bipolar and amacrine cell terminals, 2) each of these input types is distributed widely over their dendritic fields, and 3) there is no significant difference in the pattern of distribution of bipolar and amacrine cell synapses onto the dendrites of either cell type. Comparisons of the inputs to the ON- and the OFF-center cell, however, did reveal differences in the complexity of the synaptic arrangements found in association with the two neurons; a number of complex synaptic arrangements, including serial amacrine cell synapses, were found exclusively in association with the dendrites of the OFF-center X-cell. Most models of retinal X-cell receptive fields, because their visual responses share a number of features with those of bipolar cells, have attributed X-cell receptive field properties to their bipolar cell inputs. The data presented here, the first obtained from analyzing the inputs to the entire dendritic arbors of retinal X-cells, demonstrate that these retinal ganglion cells receive nearly one-half of their input from amacrine cells. These results clearly indicate that further data concerning the functional consequences of amacrine cell input are needed to understand more fully visual processing in the X-cell pathway.

Synaptic connections between corticogeniculate axons and interneurons in the dorsal lateral geniculate nucleus of the cat.

Anatomical evidence is provided for direct synaptic connections by axons from visual cortex with interneurons in lamina A of the cat's dorsal lateral geniculate nucleus. Corticogeniculate axon terminals were labeled selectively with 3H-proline and identified by means of electron microscopic autoradiography. Interneurons in the lateral geniculate nucleus were stained with antibodies that had been raised against gamma aminobutyric acid (GABA). We found that corticogeniculate terminals synapsed with dendrites stained positively for GABA about three times as often as with unstained dendrites. Of the corticogeniculate terminals that contacted GABA-positive dendrites, 97% made synaptic connections with dendritic shafts. Only 3% synapsed with F profiles, the vesicle-filled dendritic appendages characteristic of lateral geniculate interneurons. These results suggest that the corticogeniculate pathway in the cat is directed primarily at interneurons and is organized synaptically to influence the integrated output of these cells, rather than the local interactions in which their dendritic specializations participate.

Morphology of single, physiologically identified retinogeniculate Y-cell axons in the cat following damage to visual cortex at birth.

It has been reported previously that neurons in the dorsal lateral geniculate nucleus (LGN) of cats with neonatal damage to visual cortex (KVC cats) have receptive fields that are abnormally large and that the receptive fields of these neurons sometimes do not appear to conform to the normal retinotopic order in the LGN. A primary aim of this study was to determine if these physiological abnormalities are related to inappropriate patterns of retinogeniculate connections. We therefore have analyzed the terminal arbors of retinogeniculate axons in adult cats that had received a lesion of visual cortex (areas 17, 18, and 19) on the day of birth. Single retinogeniculate axons were characterized physiologically and injected intracellularly with horseradish peroxidase. Consistent with earlier reports that neonatal removal of visual cortex results in a retrograde loss of retinal X-cells, all of the retinogeniculate axons that we recorded were from Y-cells. While the visual responses of these Y-cell axons were normal, the morphology of their terminal arbors in the LGN was abnormal. Retinal Y-cell axons in KVC cats have terminal fields in the A laminae of the LGN that are as large or larger than those of normal Y-cells. However, since the LGN in KVC cats is severely degenerated, single Y-cell arbors occupy a proportional volume of the LGN that is 12 times greater than normal. Thus an early lesion of visual cortex produces a severe mismatch between retinogeniculate axon arbor size and target size. Also, despite the normal size of retinogeniculate axon arbors in KVC cats, the number and density of terminal boutons are greatly decreased. Thus our morphological results suggest that the unusually large receptive fields of LGN cells in KVC cats and the relative lack of retinotopic precision in the LGN are due, at least in part, to anomalies in the relative size and distribution of retinogeniculate axon arbors that develop after neonatal removal of visual cortex.

Development of corticogeniculate synapses in the cat.

The development of corticogeniculate synapses was studied in 16 cats ranging in age from newborn to adult. Tritiated proline was injected into areas 17 and 18 of the visual cortex in order to label corticogeniculate terminals in lamina A of the dorsal lateral geniculate nucleus. The labeled terminals were then characterized ultrastructurally using electron microscopic autoradiography. Labeled synaptic profiles were found in newborn kittens, indicating that corticogeniculate connections are present in the cat at birth. Morphologically, however, many corticogeniculate endings in newborn and 1-week-old kittens are different from those in older animals in that they do not form well-defined terminal boutons, and their synaptic vesicles are often loosely packed. In kittens 2 weeks of age and older, corticogeniculate axons end as RSD terminals exclusively; i.e., they are relatively small in size and contain round, densely packed synaptic vesicles, and occasionally an electron-dense mitochondrion (Guillery: Z. Zellforsch. 99: 1-38, '69). However, not all RSD terminals in the LGN represent input from visual cortex. Injections of 3H-proline into the mesencephalic reticular formation also label RSD terminals selectively in the lateral geniculate nucleus. At all ages corticogeniculate axons make synaptic contacts with dendrites exclusively, and they are always presynaptic. This suggests that the essential pattern of corticogeniculate synapses is formed early and is not altered during subsequent development. Quantitatively, there is no significant change in the size of corticogeniculate terminals or their synaptic vesicles in kittens 2 weeks of age (the youngest measured) and older. In contrast, the synaptic contact lengths of these terminals decreases about 28% between 2 and 12 weeks. During this same period there is approximately a twofold increase in the density of corticogeniculate terminals in the neuropil of lamina A. Since the volume of neuropil in lamina A increases almost fourfold between 2 and 12 weeks, the doubling of corticogeniculate terminal density represents about an eightfold increase in terminal number. After 12 weeks there is little change in the length, density, or number of corticogeniculate synaptic contacts, which suggests that the morphological development of the corticogeniculate pathway is essentially complete by this age.

Genesis of interneurons in the dorsal lateral geniculate nucleus of the cat.

Most neurons in the A-laminae of the cat's dorsal lateral geniculate nucleus (LGN) are born between embryonic days 22 and 32. Whereas approximately 78% of these cells are destined to become geniculocortical relay cells, the remaining 22% of LGN neurons do not appear to establish connections with visual cortex, and therefore can be considered interneurons. In the present study we have combined the 3H-thymidine method for labeling dividing neurons with the retrograde horseradish peroxidase (HRP) method for identifying LGN relay cells in order to study specifically the genesis of interneurons in the cat's LGN. Developing LGN interneurons in 12 kittens were labeled with 3H-thymidine by injecting the radioactive label into the allantoic cavity of their pregnant mothers on different embryonic days. Approximately 8-22 weeks after birth LGN relay cells in the A-laminae were labeled retrogradely by injecting large volumes of HRP into visual cortex areas 17 and 18. LGN cells that could not be labeled retrogradely with HRP were considered to be interneurons. Our results show that interneurons are born on each of the embryonic days studied, E24-E30. This period represents approximately the middle two-thirds of the entire period of LGN neurogenesis. Although the birth rate for interneurons is not uniform, there is no indication from our data that interneurons and relay cells in the cat's LGN are born at different times during LGN neurogenesis.

The percentage of interneurons in the dorsal lateral geniculate nucleus of the cat and observations on several variables that affect the sensitivity of horseradish peroxidase as a retrograde marker.

Ten cats ranging in age from 4 weeks postnatal to adult received large bilateral injections of horseradish peroxidase (HRP) into cortical areas 17 and 18. In one cat additional unilateral injections of HRP were made into the lateral suprasylvian visual areas (PMLS). The purpose of these injections was to label relay cells in lamina A of the dorsal lateral geniculate nucleus (LGN), in order to distinguish them from neurons that could not be labeled retrogradely. Several factors thought to influence the effectiveness of HRP as a retrograde marker were varied in an effort to label as many relay cells as possible. These factors included the (1) rate and duration of HRP injections; (2) volume and concentration of HRP injected; (3) addition of L-alpha-lysophosphatidylcholine or dimethyl sulfoxide to the injected HRP; and (4) aldehyde and buffers used for fixation. In all experiments DAB (3,3'-diaminobenzidine tetrahydrochloride) was used as the chromogen, either alone or with the addition of cobalt chloride, nickel, and cobalt salts, or cobalt-glucose oxidase. In 1-micrometer plastic sections, the influence of each of the above factors and DAB methods was determined by measuring the percentage of unlabeled neurons and the cytoplasmic HRP grain density of cells that were labeled. Our results show that approximately 22% of the neurons in lamina A of the LGN remain unlabeled following injections of HRP into areas 17 and 18 alone or combined with injections into PMLS. The percentage of unlabeled cells is similar at each of the ages that we studied and is not affected significantly by any of the factors that were varied or DAB methods that were used. Cross-sectional area measurements show that unlabeled cells tend to be among the smallest neurons in lamina A. Regardless of age, the mean size of labeled neurons was about twice that of unlabeled cells. However, we found only a weak correlation between the size of a labeled cell and the cytoplasmic density of HRP grains. Thus it is unlikely that small cell body size alone can account for the unlabeled cells in lamina A, since small neurons can be as effective in transporting HRP retrogradely as large neurons. We therefore conclude that there is a distinct population of small neurons in lamina A of the LGN that do not project to cortex. Although we cannot rule out the possibility that these cells project subcortically, we believe that it is reasonable to regard them as interneurons.