SURFACE STRUCTURE OF ISOLATED NEURONS : Detachment of Nerve Terminals during Axon Regeneration.

Freehand, isolated neuronal perikarya from the hypoglossal nucleus of the rabbit have been examined with light-and electron-microscopy (transmission and scanning). The surface of the cell bodies was largely covered with spherical particles which were 0.5-2 micro in diameter. Transmission electron microscopy proved that the spherical particles were synaptic nerve terminals. Crush of the hypoglossal nerve which leads to chromatolysis and swelling of the neuronal cell bodies results in a conspicuous reduction in the number of terminals attached to the surface of hypoglossal neurons. This effect was observed both for isolated neurons and in tissue sections. The effect is considered in relation to earlier reported variations in the adherence of neuropil to isolated neuronal perikarya. The functional importance of nerve ending detachment in connection with nerve injury is discussed.

Nej1p, a cell type-specific regulator of nonhomologous end joining in yeast.

Mutant yeast strains lacking the silencing proteins Sir2p, Sir3p, or Sir4p have a defect in a DNA double-strand break (DSB) repair pathway, called nonhomologous end joining (NHEJ). Mutations in sir genes also lead to the simultaneous expression of a and alpha mating type information, thus generating a nonmating haploid cell type with many properties shared with a/alpha diploids. We addressed whether cell type or Sir proteins per se regulate NHEJ by investigating the role of a novel haploid-specific gene in NHEJ. This gene, NEJ1, was required for efficient NHEJ, and transcription of NEJ1 was completely repressed in a/alpha diploid and sir haploid strains. The NEJ1 promoter contained a consensus binding site for the a1/alpha2 repressor, explaining the cell type-specific expression. Expression of Nej1p from a constitutive promoter in a/alpha diploid and sir mutant strains completely rescued the defect in NHEJ, thus showing that Sir proteins per se were dispensable for NHEJ. Nej1p and Lif1(P), the yeast XRCC4 homolog, interacted in two independent assays, and Nej1p localized to the nucleus, suggesting that Nej1p may have a direct role in NHEJ.

Kluyveromyces lactis Sir2p regulates cation sensitivity and maintains a specialized chromatin structure at the cryptic alpha-locus.

In Saccharomyces cerevisiae, transcriptional silencing of the cryptic mating type loci requires the formation of a heterochromatin-like structure, which is dependent on silent information regulator (Sir) proteins and DNA sequences, called silencers. To learn more about silencing, we characterized the mating type loci from the yeast Kluyveromyces lactis. The K. lactis MAT, HMRa, and HMLalpha loci shared flanking DNA sequences on both sides of the loci presumably acting as recombinational targets during mating type switching. HMRa contained two genes, the a1 gene similar to the Saccharomyces a1 gene and the a2 gene similar to mating type genes from other yeasts. K. lactis HMLalpha contained three genes, the alpha1 and alpha2 genes, which were similar to their Saccharomyces counterparts, and a novel third gene, alpha3. A dam-methylase assay showed Sir-dependent, but transcription-independent changes of the chromatin structure of the HMLalpha locus. The HMLalpha3 gene did not appear to be part of the silent domain because alpha3p was expressed from both MATalpha3 and HMLalpha3 and sir mutations failed to change the chromatin structure of the HMLalpha3 gene. Furthermore, a 102-bp silencer element was isolated from the HMLalpha flanking DNA. HMLalpha was also flanked by an autonomously replicating sequence (ARS) activity, but the ARS activity did not appear to be required for silencer function. K. lactis sir2 strains grown in the presence of ethidium bromide (EtBr) accumulated the drug, which interfered with the essential mitochondrial genome. Mutations that bypassed the requirement for the mitochondrial genome also bypassed the EtBr sensitivity of sir2 strains. Sir2p localized to the nucleus, indicating that the role of Sir2p to hinder EtBr accumulation was an indirect regulatory effect. Sir2p was also required for growth in the presence of high concentrations of Ni(2+) and Cu(2+).

Impairment of contrast sensitivity function (CSF) as a measure of disability glare.

A method for quantitative measurements of disability glare in clinical practice is presented. Glare is induced by a circular fluorescent tube which surrounds a sinusoidal grating displayed on a monitor. The threshold contrast that is needed for detection of the grating is measured with and without presence of the glare light. This is repeated for several different spatial frequencies. The discrepancy between the contrast sensitivity function (CSF) obtained with and without glare light was used to calculate the glare score (n) as a measure of disability glare. This was done for normals and cataract patients. The results show that an increasing glare score is related to an increase in turbidity of the optic media, while visual acuity had a weak correlation to the glare score. We also studied the relation glare score versus luminance and found that normals had a glare score that was almost independent of luminance level, while the cataract patients had a marked decrease in glare sensitivity when the luminance decreased. Most patients had a glare score that corresponded to their glare problems. These findings indicate a potential for using this psychophysical disability glare test method in industry, transport, and clinical ophthalmology. Some sources of methodological error inherent in the test method are evaluated and discussed.

Contrast sensitivity in the presence of a glare light. Theoretical concepts and preliminary clinical studies.

A method is presented for quantitative measurements of the glare effect of light scattered in the ocular media. The contrast sensitivity function is measured with a television display system. A bright light source is introduced into the field of vision, and the resultant decrease in contrast sensitivity is mea-ured. It is further used to calculate a scattering factor which is a direct measure of the intraocular light scattering. The scattering factor shows a marked increase in patients with early cataracts even if their visual acuity is not affected.

The 'light scattering factor'. Importance of stimulus geometry, contrast definition, and adaptation.

PURPOSE: Paulsson and Sjöstrand have suggested that the light scattering factor (LSF) can be estimated by using the equation: LSF = L/E (M2/M1-1). Here L is the space average luminance of the target, E is the illuminance of the glare source, and M2 and M1 are modulation contrast thresholds in the presence and absence of the glare source. To compensate for change of adaptation. Abrahamsson and Sjöstrand later modified the above equation by introducing a correction factor (CF): LSF = L/E ((CF) (M2/M1-1). The purpose of this study is to analyze the validity of the above equations. METHODS: The importance of stimulus geometry, contrast definition, background luminance, and glare illumination is studied through theoretical analysis and comparison with earlier studies. Stimulus geometry and contrast definition are studied through optical modeling. Adaptation is modeled according to the laws of Weber and DeVries-Rose. RESULTS: The choice of contrast definition may corrupt the result by a factor of 2. At background luminance levels above approximately 10 cd/m2, the Paulsson-Sjöstrand equation agrees well with theory. At lower background levels, the Abrahamsson-Sjöstrand equation is used with correction factors derived from adaptation measurements. Using this equation and earlier published data from glare testing performed at 2 cd/m2, the results are found to be in fair agreement with the light scattering theory. CONCLUSIONS: Glare testing using the Paulsson-Sjöstrand equation is found to be valid as long as the measurements are performed at high luminance levels (above 10 cd/m2), with targets of low spatiotemporal frequencies (e.g., 2 cpd and 1 Hz) and with the use of a properly chosen definition of contrast. At lower luminance levels, the Abrahamsson-Sjöstrand equation may be used with well-derived correction factors.

Proteolysis in human lens epithelium determined by a cell-permeable substrate.

PURPOSE: To develop a system for continuous evaluation of proteolytic activity in human lens epithelium and to characterize factors of importance for the regulation of proteolytic activity in lens epithelial cells. METHODS: Human lens epithelial cells were obtained during cataract surgery. Capsule epithelium specimens consisted of the central parts of the anterior capsule and the underlying lens epithelium. The sample, with the cell-permeable substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, was placed in a chamber, which was placed in a thermostat-controlled aluminum block. Fluorescence changes were continuously measured by the fiber optics of the luminometer, which was placed 5 mm above the buffer surface. RESULTS: After administration of substrate to the medium overlying the cells, the substrate was degraded at a relatively slow rate. Approximately 10 picomoles of amino-4-methylcoumarin were formed per minute. A significant increase of proteolytic activity could be observed after application of 1 microM ionomycin or 2 microM thapsigargin. No leakage of lactate dehydrogenase from the cells was observed during these procedures. Basal proteolytic activity was totally inhibited by the proteasome inhibitor lactacystin. Lactacystin also attenuated the response to ionomycin and thapsigargin. CONCLUSIONS: Human lens epithelium responds to increased Ca levels from external or internal stores with an increased proteolytic activity that may be mediated by calpain, by the proteasome, or by both. This calcium-dependent change in proteolytic activity may be of importance in the development of cataract.

Caspase and proteasome activity during staurosporin-induced apoptosis in lens epithelial cells.

PURPOSE: To determine what caspases are activated during staurosporin-induced apoptosis in cultured bovine lens epithelial cells (BLECs), to study the time course of caspase activation in relation to morphologic changes, and to investigate the effect of caspase and/or proteasome inhibition on apoptosis. METHODS: BLECs were incubated with staurosporin at different concentrations or for different times. Phosphatidylserine (PS) externalization was detected by annexin-V labeling, nuclear morphology was studied by staining with Hoechst 33342 stain (Hoechst, Frankfurt, Germany), and the percentage of apoptotic cells was determined by the TdT-dUTP terminal nick-end labeling (TUNEL) assay. The activity of caspase-1, -2, -3, -4, -8, and -9 as well as the chymotrypsin-like activity of the proteasome was measured by the use of fluorogenic peptide substrates. Inhibition of the proteasome was performed by incubation with 10 microM lactacystin, and caspases were inhibited by 1 microM Z-DEVD-FMK or 20 microM Z-VAD-FMK. RESULTS: Staurosporin treatment caused a dose- and time-dependent increase in the number of apoptotic cells and in caspase-3 activity. Activation of caspase-2, -4, -8, and -9 was also seen. Caspase activity was increased after 3 hours' incubation with 1 microM staurosporin, which is also the time when most cells became annexin-V-positive. Nuclear changes indicative of apoptosis, viewed with both Hoechst and TUNEL staining, appeared after 4 to 6 hours of staurosporin incubation. Incubation of BLECs with lactacystin caused reduction of proteasome activity and increased apoptosis, evidenced in both the TUNEL assay and caspase-3 activation. Preincubation of lens epithelial cells with caspase inhibitors caused complete inhibition of lactacystin- or staurosporin-induced caspase-3 activation (Z-DEVD-FMK/Z-VAD-FMK) and also of caspase-2, -4, -8, and -9 (Z-VAD-FMK), but the reduction in TUNEL-positive cells was only partial. PS translocation and DNA fragmentation after staurosporin treatment occurred despite complete caspase blockade. CONCLUSIONS: Staurosporin-induced apoptosis in BLECs involves activation of several caspases. Inhibition of the proteasome causes caspase-3 activation and apoptosis. Both staurosporin- and lactacystin-induced apoptosis can be executed in a caspase-independent manner. The present data are useful for understanding of proteolytic mechanisms during apoptosis in lens epithelial cells, which may be an important event in normal lens development as well as in some types of cataract.

Slow axonal transport or proteins; blockade by interruption of contact between cell body and axon.

The influence of ligation and colchicine treatment on the axonal transport of slowly migrating [3H]leucine-labelled proteins was studied in the vagus nerve of the rabbit. Two days after [3H]leucine labelling of the dorsal motor nucleus of the vagus nerve, ligation or local application of 60 mM colchicine immediately blocked the further progression of slowly migrating proteins distal to the site of treatment. Application of 50-100 mug colchicine to the nerve cell bodies 2 days after labelling blocked the transport of slowly migrating proteins within the next 24 h. It is suggested that contact between nerve cell body and the axon is necessary for the maintenance of the slow transport of proteins in these nerves.

Changes in synaptic function induced by blockage of axonal transport in the rabbit optic pathway.

This study was undertaken to elucidate the physiological significance of material involved in the rapid axonal transport. The effects of colchicine-induced inhibition of axonal transport in the retinal ganglion cells on the electrophysiological properties of the retrobulbar visual pathways were investigated in Albino rabbits. An impaired signal transmission to the contralateral visual cortex, superior colliculus and lateral geniculate body following flash light stimulation as well as direct optic nerve stimulation appeared 4--6 days after an intravitreous injection of 10--25 microgram colchicine. It was concluded that inhibition of the fast axonal transport within the retinal ganglion cells interferes with transsynaptic signal transmission from optic nerve terminals in the subcortical nuclei. This indicates a functional relationship between material supplied via the rapid phase of axonal transport and an unimpaired transsynaptic signal transmission, previously not revealed in the central nervous system of mammals.

Graded inhibition of retrograde axonal transport by compression of rabbit vagus nerve.

Effects of experimental compression at different pressures on retrograde axonal transport were studied in rabbit vagus nerve. Proteins in the sensory neurones were radiolabelled by injection of [3H]leucine into the nodose ganglion. Sixteen hours after labelling, a small compression chamber and/or ligatures were applied around the cervical part of the vagus nerve for 8 h. Compression of the vagus nerve at 20, 30 and 200 mm Hg pressure induced a graded inhibition of both retrograde and anterograde transport of the radiolabelled proteins. Neither retrograde nor anterograde transport was affected by the presence of the non-inflated chamber. The results indicate that compression at pressures similar to those found in human carpal tunnel syndrome can block retrograde axonal transport. The consequences of inhibition of retrograde and anterograde axonal transport for the metabolism in the nerve cell bodies are discussed.

Action of chloroquine on in vivo RNA and protein biosynthesis in the retina and the optic pathway of the rabbit.

The in vivo action of chloroquine on RNA and protein metabolism in the optic pathway of the albino or pigmented rabbit was examined. To study the acute effects, chloroquine in a dose of 500 mug was injected into the vitreous body of one eye. The following day the animals were injected into both eyes with [3H]uridine or [3H]leucine. At various time intervals following the isotope injections the retinal synthesis and the axonal transport of labelled RNA or protein was studied. The results showed no significant difference between the drug-treated and the control side with respect to synthesis and axonal transport of RNA or protein. Nor was any selective effect noticed on the synthesis of different RNA fractions from the retina. In long-term experiments chloroqune (100 mg/kg body weight) was administered via their drinking water 3 days a week for a period of 6 or 8 months. Following an intraocular injection of [3H]leucine no significant change in rapid axonal transport could be found in those chronically treated rabbits.

Anisometropic and strabismic amblyopia in the age group 2 years and above: a prospective study of the results of treatment.

Forty-four children aged 2-9 years with strabismic and anisometropic amblyopia were prospectively followed up during amblyopia treatment. The efficacy of optimised treatment in terms of number of cured children, time to achieve cure, and rate of initial improvement of visual acuity was evaluated in relation to age at start of treatment, type and initial degree of amblyopia, and adherence to treatment regimen. Compliance with treatment was the most critical factor predicting a successful outcome. Among the compliant children 35 out of 36 were cured (visual acuity difference between amblyopic and non-amblyopic eyes not more than one line) within five months regardless of age, treatment regimen, and type or initial degree of amblyopia as compared with none in the group with low compliance. Most of these compliant children were cured within three months, with shorter treatment times on average for the younger children. The initial improvement of visual acuity was also faster at 2 years than at 4 years of age. Anisometropes with moderate amblyopia at the start of treatment were over-represented in the group with low compliance. We conclude that early diagnosis of strabismus in combination with general population screening at the age of 4 to detect amblyopia caused by anisometropia or microstrabismus seems to be efficacious for the cure of most cases. The major factor in treatment failure was found to be inadequate adherence to the treatment regimen.

A clinical study of radiation cataract formation in adult life following gamma irradiation of the lens in early childhood.

AIMS: To analyse long term effects on the lens of radium irradiation during infancy. METHODS: An infant cohort (n = 20, median age 6 months) treated for skin haemangioma with one or two radium-226 needles located at or within the orbital rim was examined 30 to 45 years after gamma radiation. Detailed information about the treatment procedure was available for all cases. Subcapsular opacities were graded semiquantitatively according to a scale based on extent and density of the opacities. RESULTS: A high prevalence of light to moderate posterior, subcapsular, and cortical cataract formation was found in the lenses on the treated side irradiated with a mean dose ranging from approximately 1 to 8 Gy. The cataract formation increased as a function of dose. The presence of subcapsular punctate opacities and vacuoles in the lenses on the untreated side receiving irradiation of an estimated dose varying around 0.1 Gy indicates a higher sensitivity than expected. CONCLUSION: The growing lens during infancy is sensitive to radium irradiation at doses lower than those previously stated. The eye lens seems suitable for studies of effects of low dose radiation since damaged cells are retained in the lens for a lifetime.

Resolution, separation of retinal ganglion cells, and cortical magnification in humans.

We present direct comparisons of resolution thresholds and quantitative estimates of retinal ganglion cell separation in humans with reported functional magnetic resonance imaging estimates of the human linear cortical magnification factor. Measurements of resolution thresholds (MAR), retinal ganglion cell (GC) densities, and linear cortical magnification factor (M) values were taken from the literature. Our objective was to analyse the apparent overrepresentation of human central vision in the visual cortex and to determine whether the cause of this is an effect of the uneven distribution of GC in the retina and/or that central GC have more devoted cortical area per cell. The reserved amount of cortical distance per retinal unit, i.e. the product of M on the one hand and effective GC separation, MAR, and GC receptive field separation on the other, indicates an overrepresentation of the fovea and immediately surrounding retina in the human striate cortex due to an increase in devoted cortical distance per central GC or resolution unit. This cannot be explained by lateral displacement of foveal ganglion cells nor by peripheral scaling, but rather by an additional magnification in the retino-cortical pathway.

Natural history of infantile anisometropia.

AIMS/BACKGROUND: In a previous study longitudinal changes of anisometropia were investigated. It was shown that anisometropia arises and vanishes during the emmetropisation process and that the associated risk for amblyopia is low. The aim of this study was to follow acuity and refraction longitudinally in children with marked anisometropia at 1 year of age. METHODS: Refractive errors and visual acuity were estimated every sixth month for a selected group of 20 children with marked anisometropia > or = 3.0 D (spherical equivalent) at 1 year of age from approximately 3 to 10 years of age. RESULTS: The children could be classified into three groups. In six subjects the anisometropia increased (mean 1.4 D) and they all developed amblyopia. The remaining children could be classified into two groups of equal size. One group developed no amblyopia and the anisometropia decreased with a mean of 3.0 D. The seven remaining children developed amblyopia and/or strabismus; the mean anisometropia decrease was 1.2 D. CONCLUSION: Anisometropia at 1 year of age that is larger or equal to 3.0 D will in 90% of the cases still be there at 10 years of age. There is a substantial risk of this group developing amblyopia (60%).

Course of exfoliation and simplex glaucoma after primary trabeculectomy.

AIM: To study the course of exfoliation and simplex glaucoma with respect to intraocular pressure (IOP) regulation and visual field survival after primary trabeculectomy. METHODS: Postoperative IOP regulation and complications were analysed prospectively in 95 patients. Mean follow up was 46 months. Visual field survival was studied by high pass resolution perimetry (HRP) in a subsample of 28 patients. RESULTS: Medical treatment was reinstated in 42% of exfoliation and in 36% of simplex glaucoma. In these patients, mean medicine free survival time, last untreated IOP, and mean IOP at the end of follow up were similar for both glaucoma types. Among patients with controlled postoperative IOP without added medication, mean IOP at the end of follow up was significantly lower in exfoliation glaucoma. Visual field deterioration and the pattern of complications were similar for both glaucoma types. CONCLUSION: The effect of trabeculectomy on IOP regulation was good in both types of glaucoma, and somewhat better in exfoliation glaucoma. The magnitude of IOP lowering could not separate patients with continued visual field deterioration from those in whom visual fields remained stable. Visual field preservation was similar for both glaucoma types.

Contrast sensitivity and acuity relationship in strabismic and anisometropic amblyopia.

The contrast sensitivity function (CSF) and visual acuity were determined in children and adults with unilateral amblyopia due to strabismus or anisometropia with central fixation. The preschool children were examined repeatedly during occlusion treatment. All amblyopes had CSF deficits. The CSF was characterised by its peak value (the maximal sensitivity, Smax, and the spatial frequency at which Smax occurs, Frmax) calculated by a single peak least-square regression method. The two amblyopic groups showed discrepancies in relationship of both Smax and Frmax versus visual acuity both initially and during treatment. The strabismic cases had a more marked visual acuity deficit in relation to the contrast sensitivity losses, whereas these parameters are affected similarly in anisometropic amblyopes. The relationship between recovery of visual acuity and CSF during the initial month of occlusion treatment was of prognostic significance for the outcome of visual acuity improvement.

Refraction changes in children developing convergent or divergent strabismus.

Strabismus and amblyopia were studied in a cohort of children born in 1979 or 1980 in the area of Västerås, Sweden. Forty percent of the children had participated in a voluntary eye examination at 1 year of age. All children diagnosed as strabismic and/or amblyopic between 1979 and 1988 at any of the three eye clinics in the area were included in this study. Strabismic cases were mostly detected by the parents while microstrabismus and straight eye amblyopia were found at the general 4 years of age screening at children's health centres. In 57 cases with (n = 31) and without amblyopia (n = 41) it was possible to obtain several refraction values between 1 and 6 years of age. In this study we concentrated on manifest esotropia and exotropia. The aim of the study was to describe changes of refraction before and after onset of strabismus and to establish risk indicators that identified populations at risk of developing strabismus. We found that patients with esotropia show a more pronounced hypermetropia than exotropic cases at the time of detection of strabismus. This difference becomes more definite over time, since hypermetropia increased in the deviating eye in the esotropic cases while refractive errors remained stationary in most of the exotropic eyes. It was also apparent that anisometropia frequently developed after onset of strabismus in esotropic cases in contrast to exotropic cases. An increasing refractive error in the deviating esotropic eye could be combined with an emmetropisation of the fixating eye.

Changes in visual acuity from 4 to 12 years of age in children operated for bilateral congenital cataracts.

AIMS: To investigate the long term effects of age at surgery on the development of visual acuity (VA) by measuring VA from preschool age to puberty. Furthermore, to report the VA levels at 12 years of age in a geographically based cohort of operated congenital bilateral cataracts. METHODS: All children born in four western counties of Sweden between January 1980 and December 1993 who were diagnosed with congenital cataracts were included in a longitudinal prospective study. The monocular VA of the better eye in 38 subjects was analysed at 4, 7, 10, and 12 years of age, with 20 total and 18 partial cataracts. The mean follow up time was 9.3 years after surgery. RESULTS: The final value of VA was 0.4 or above for approximately 50% of the subjects at 12 years of age. Visual acuity improved to a considerable extent after school age, especially in children who underwent surgery between the ages of 7 weeks and 1 year. Results for partial cataracts were favourable compared to those for total cataracts, reaching a mean of approximately 0.5 at age 12. The mean VA in the group of total congenital cataracts operated on before 7 weeks of age achieved higher values of VA at 4 years of age compared to children with total cataracts operated on between 7 weeks and 1 year of age. However, no statistically significant difference in VA results among these groups could be proved. CONCLUSION: Visual acuity improves to a considerable extent after school age in children with delayed visual development caused by congenital cataracts. Surgery within 7 weeks results in a more rapid development of VA, initially.