Spatial regulation of cell motility and its fitness effect in a surface-attached bacterial community.

On a surface, microorganisms grow into a multi-cellular community. When a community becomes densely populated, cells migrate away to expand the community's territory. How microorganisms regulate surface motility to optimize expansion remains poorly understood. Here, we characterized surface motility of Proteus mirabilis. P. mirabilis is well known for its ability to expand its colony rapidly on a surface. Cursory visual inspection of an expanding colony suggests partial migration, i.e., one fraction of a population migrates while the other is sessile. Quantitative microscopic imaging shows that this migration pattern is determined by spatially inhomogeneous regulation of cell motility. Further analyses reveal that this spatial regulation is mediated by the Rcs system, which represses the expression of the motility regulator (FlhDC) in a nutrient-dependent manner. Alleviating this repression increases the colony expansion speed but results in a rapid drop in the number of viable cells, lowering population fitness. These findings collectively demonstrate how Rcs regulates cell motility dynamically to increase the fitness of an expanding bacterial population, illustrating a fundamental trade-off underlying bacterial colonization of a surface.

Stochastic activation of a family of TetR type transcriptional regulators controls phenotypic heterogeneity in Acinetobacter baumannii.

Phenotypic heterogeneity is an important mechanism for regulating bacterial virulence, where a single regulatory switch is typically activated to generate virulent and avirulent subpopulations. The opportunistic pathogen Acinetobacter baumannii can transition at high frequency between virulent opaque (VIR-O) and avirulent translucent subpopulations, distinguished by cells that form opaque or translucent colonies. We demonstrate that expression of 11 TetR-type transcriptional regulators (TTTRs) can drive cells from the VIR-O opaque subpopulation to cells that form translucent colonies. Remarkably, in a subpopulation of VIR-O cells, four of these TTTRs were stochastically activated in different combinations to drive cells to the translucent state. The resulting translucent subvariants exhibited unique phenotypic differences and the majority were avirulent. Due to their functional redundancy, a quadruple mutant with all four of these TTTRs inactivated was required to observe a loss of switching from the VIR-O state. Further, we demonstrate a small RNA, SrvS, acts as a "rheostat," where the levels of SrvS expression influences both the VIR-O to translucent switching frequency, and which TTTR is activated when VIR-O cells switch. In summary, this work has revealed a new paradigm for phenotypic switching in bacteria, where an unprecedented number of related transcriptional regulators are activated in different combinations to control virulence and generate unique translucent subvariants with distinct phenotypic properties.

Observing Bacterial Persistence at Single-Cell Resolution.

Within a bacterial population, there can be a subpopulation of cells with an antibiotic-tolerant persister phenotype characterized by long lag phase. Their long lag phase necessitates long (hours or days) periods of single-cell observation to capture high-quality quantitative information about persistence. We describe a method of single-cell imaging using glass bottom dishes and a nutrient agarose pad that allows for long-term single-cell microscopy observation in a stable environment. We apply this method to characterize the lag phase and persistence of individual Escherichia coli cells.

Pressure-Driven Mitochondrial Transfer Pipeline Generates Mammalian Cells of Desired Genetic Combinations and Fates.

Generating mammalian cells with desired mitochondrial DNA (mtDNA) sequences is enabling for studies of mitochondria, disease modeling, and potential regenerative therapies. MitoPunch, a high-throughput mitochondrial transfer device, produces cells with specific mtDNA-nuclear DNA (nDNA) combinations by transferring isolated mitochondria from mouse or human cells into primary or immortal mtDNA-deficient (ρ0) cells. Stable isolated mitochondrial recipient (SIMR) cells isolated in restrictive media permanently retain donor mtDNA and reacquire respiration. However, SIMR fibroblasts maintain a ρ0-like cell metabolome and transcriptome despite growth in restrictive media. We reprogrammed non-immortal SIMR fibroblasts into induced pluripotent stem cells (iPSCs) with subsequent differentiation into diverse functional cell types, including mesenchymal stem cells (MSCs), adipocytes, osteoblasts, and chondrocytes. Remarkably, after reprogramming and differentiation, SIMR fibroblasts molecularly and phenotypically resemble unmanipulated control fibroblasts carried through the same protocol. Thus, our MitoPunch "pipeline" enables the production of SIMR cells with unique mtDNA-nDNA combinations for additional studies and applications in multiple cell types.

Stable retention of chloramphenicol-resistant mtDNA to rescue metabolically impaired cells.

The permanent transfer of specific mtDNA sequences into mammalian cells could generate improved models of mtDNA disease and support future cell-based therapies. Previous studies documented multiple biochemical changes in recipient cells shortly after mtDNA transfer, but the long-term retention and function of transferred mtDNA remains unknown. Here, we evaluate mtDNA retention in new host cells using 'MitoPunch', a device that transfers isolated mitochondria into mouse and human cells. We show that newly introduced mtDNA is stably retained in mtDNA-deficient (ρ0) recipient cells following uridine-free selection, although exogenous mtDNA is lost from metabolically impaired, mtDNA-intact (ρ+) cells. We then introduced a second selective pressure by transferring chloramphenicol-resistant mitochondria into chloramphenicol-sensitive, metabolically impaired ρ+ mouse cybrid cells. Following double selection, recipient cells with mismatched nuclear (nDNA) and mitochondrial (mtDNA) genomes retained transferred mtDNA, which replaced the endogenous mutant mtDNA and improved cell respiration. However, recipient cells with matched mtDNA-nDNA failed to retain transferred mtDNA and sustained impaired respiration. Our results suggest that exogenous mtDNA retention in metabolically impaired ρ+ recipients depends on the degree of recipient mtDNA-nDNA co-evolution. Uncovering factors that stabilize exogenous mtDNA integration will improve our understanding of in vivo mitochondrial transfer and the interplay between mitochondrial and nuclear genomes.

Intracellular Photothermal Delivery for Suspension Cells Using Sharp Nanoscale Tips in Microwells.

Efficient intracellular delivery of biomolecules into cells that grow in suspension is of great interest for biomedical research, such as for applications in cancer immunotherapy. Although tremendous effort has been expended, it remains challenging for existing transfer platforms to deliver materials efficiently into suspension cells. Here, we demonstrate a high-efficiency photothermal delivery approach for suspension cells using sharp nanoscale metal-coated tips positioned at the edge of microwells, which provide controllable membrane disruption for each cell in an array. Self-aligned microfabrication generates a uniform microwell array with three-dimensional nanoscale metallic sharp tip structures. Suspension cells self-position by gravity within each microwell in direct contact with eight sharp tips, where laser-induced cavitation bubbles generate transient pores in the cell membrane to facilitate intracellular delivery of extracellular cargo. A range of cargo sizes were tested on this platform using Ramos suspension B cells with an efficiency of >84% for Calcein green (0.6 kDa) and >45% for FITC-dextran (2000 kDa), with retained viability of >96% and a throughput of >100 000 cells delivered per minute. The bacterial enzyme ß-lactamase (29 kDa) was delivered into Ramos B cells and retained its biological activity, whereas a green fluorescence protein expression plasmid was delivered into Ramos B cells with a transfection efficiency of >58%, and a viability of >89% achieved.

Complete genome sequence of Helicobacter pylori B128 7.13 and a single-step method for the generation of unmarked mutations.

BACKGROUND: Helicobacter pylori represents an interesting model of bacterial pathogenesis given that most infections are asymptomatic, while a minority of infections cause severe gastric disease. H pylori strain B128 7.13 is used extensively to understand H pylori pathophysiology. Due to extensive restriction-modification systems, the fact that only some H pylori strains are naturally transformable, the inability of common plasmid and transposon vectors to replicate in this bacterium, as well as the limited number of antibiotic cassettes that are functional in H pylori, there are relatively few genetic tools for the mutagenesis of this bacterium. MATERIALS AND METHODS: Here, we use PacBio and Illumina sequencing to reveal the complete genome sequence of H pylori B128 7.13. Furthermore, we describe a system to generate markerless and scarless mutations on the H pylori chromosome using the counter-selection marker, galactokinase from Escherichia coli. RESULTS: We show that this mutagenesis strategy can be used to generate in-frame insertions, gene deletions, and multiple independent mutations in B128 7.13. Using the closed genome as a reference, we also report the absence of second site chromosomal mutations and/or rearrangements in our mutagenized strains. We compare the genome sequence of H pylori B128 7.13 with a closely related strain, H pylori B8, and reveal one notable region of difference, which is a 1430 bp insertion encoding a H pylori-specific DUF874 family protein of unknown function. CONCLUSIONS: This article reports the closed genome of the important H pylori B128 7.13 strain and a mutagenesis method that can be adopted by researchers as an alternative strategy to generate isogenic mutants of H pylori in order to further our understanding of this bacterium.

CetZ tubulin-like proteins control archaeal cell shape.

Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homologue FtsZ establishes the cytokinetic ring that constricts during cell division. How such different roles of tubulin and FtsZ evolved is unknown. Studying Archaea may provide clues as these organisms share characteristics with Eukarya and Bacteria. Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ X-ray crystal structures showed the FtsZ/tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of CetZ proteins in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of the microbial rod shape is to facilitate swimming.

Role of inferior rectus botulinum toxin injection in vertical strabismus resulting from orbital pathology.

INTRODUCTION: We describe the uses and efficacy of botulinum toxin injection to the inferior rectus muscle in vertical strabismus resulting from orbital pathology. METHODS: A retrospective review of patients undergoing inferior rectus botulinum toxin injection between 1982 and 2006 for vertical strabismus due to orbital pathology. RESULTS: There were 13 patients identified; 6 with idiopathic orbital inflammatory syndrome (IOIS) including myositis, 3 with previous orbital wall fractures, 1 post-optic nerve sheath Schwannoma resection, 1 with lymphoma, 1 with metastasis, and 1 post-superior ophthalmic vein hemorrhage. A beneficial effect with inferior rectus botulinum toxin was obtained in 9/13 (69%) patients (mean follow-up 14 months). Resolution or improvement occurred in 4/6 (67%) patients with IOIS. Resolution was also obtained in the patient with previous optic nerve sheath Schwannoma resection, and the patient who sustained the superior ophthalmic vein hemorrhage. Improvement was noted in both the patient with lymphoma and in the patient with a metastasis. However, in patients with orbital wall fractures, only 1 of 3 patients obtained improvement. Benefit appeared independent of the size of vertical deviation, with 4/6 (67%) showing improvement with a pre-toxin angle 4-12 PD, and 5/7 (71%) improving with a pre-toxin angle 16-25 PD. Eleven patients received only 1 injection, one patient received 2, and another had an ongoing course of 8 injections. DISCUSSION: Vertical strabismus secondary to a range of orbital conditions, particularly inflammatory, often can be successfully managed by inferior rectus botulinum toxin injections.

The use of botulinum toxin to treat strabismus following retinal detachment surgery.

METHODS: Persistent symptomatic strabismus may be seen in up to 25% of patients post retinal detachment surgery. Surgical correction of ocular misalignment in this situation can be technically difficult to perform and the results are less predictable than in other forms of strabismus surgery. We report on our experience of the use of botulinum toxin injections as an alternative. RESULTS: A retrospective review was made of all patients who had received botulinum toxin injections in the management of their strabismus following retinal detachment surgery at our institution between November 1982 and January 2006. One hundred and forty patients fulfilled our criteria with a mean length of follow-up of 27 months. Thirty-three patients (23.6%) were symptom free following botulinum toxin injection alone and were discharged from the clinic, 50.5% of these after only 2 injections. Fusion was regained in 37.9%. The only factor associated with lack of restoration of fusion in the multivariate analysis was visual acuity in the injected eye. Complications were seen in 2.9% of patients. CONCLUSION: Botulinum toxin injections may represent a safe and acceptable alternative to surgery and may be particularly useful for those in whom surgery is contraindicated or not desired.

Beneficial effect of treatment for strabismus in patients with bilaterally poor vision, who cannot see their strabismus.

METHODS: Retrospective case note review of management of patients with bilaterally severely reduced vision presenting with symptomatic strabismus. RESULTS: There were 10 females and 7 males, mean age at treatment was 36 years, range 19 to 74 years. Thirteen patients had an exotropia ranging from 20 to 90(Δ) (mean 52(Δ)). Four had an esotropia ranging from 30 to 70(Δ) (mean 53(Δ)). Vision in each eye ranged from 6/24 Snellen to light perception, except for one patient with Usher's syndrome who had grossly restricted visual fields and vision of 6/12 and 6/36. The etiology was retinitis pigmentosa in 5 patients; 3 had cone dystrophy, 2 were high myopes. Other etiologies included CMV retinitis in both eyes secondary to HIV, bilateral optic atrophy post craniopharyngioma, and cortical visual impairment. All patients were unable to see well enough to assess their strabismus but were aware of its presence from family members and friends and wished improvement. Eleven patients underwent botulinum toxin injections; 2 of these then proceeded to surgery. A total of 8 patients had surgery. Post-operative angles ranged from 6(Δ) to 25(Δ) of esotropia. Five patients continue with maintenance toxin. Follow-up ranged from 3 months to 15 years. DISCUSSION/CONCLUSIONS: This is the first report of patients with bilaterally severely reduced vision requesting treatment for strabismus that they were unable to see for themselves. All patients were pleased they had undergone treatment for their strabismus despite being unable to see the outcome.

Clinical course and characteristics of acute presentation of fourth nerve paresis.

PURPOSE: Many cases of acute-onset cranial nerve paresis have benign etiologies such as microvascular occlusion. Most will resolve completely and neuroimaging is usually unnecessary. Few reports exist on acute fourth nerve paresis. METHODS: A retrospective review was conducted of all patients presenting with diplopia to the emergency department for 1 year caused by isolated fourth cranial nerve paresis from any cause including trauma. RESULTS: Thirty-two patients met the criteria, 26 (81%) males and 6 (19%) females, with an average age of 59.5 years (range: 14 to 80 years). Eighteen (56%) had a microvascular etiology with diabetes mellitus, hypertension, or both; 6 were already taking medication. Six (19%) had decompensating fourth nerve paresis (2 had hypertension and 1 had recent head trauma). Closed head trauma accounted for 2 patients, migraine and herpes zoster virus accounted for one each, and 4 remained unknown. Nineteen patients (59%) were prescribed prisms and 2 patients were given occlusion. Diplopia resolved without treatment in 23 patients (72%) within 2 weeks to 10 months, but 89% of patients with microvascular etiology resolved spontaneously. Three patients continued with prisms, one patient underwent surgery. CONCLUSION: The prognosis for complete and spontaneous resolution of microvascular fourth nerve paresis was excellent, with 89% completely resolved within 10 months.

The improving outcomes in intermittent exotropia study: outcomes at 2 years after diagnosis in an observational cohort.

BACKGROUND: The purpose of this study was to investigate current patterns of management and outcomes of intermittent distance exotropia [X(T)] in the UK. METHODS: This was an observational cohort study which recruited 460 children aged < 12 years with previously untreated X(T). Eligible subjects were enrolled from 26 UK hospital ophthalmology clinics between May 2005 and December 2006. Over a 2-year period of follow-up, clinical data were prospectively recorded at standard intervals from enrolment. Data collected included angle, near stereoacuity, visual acuity, control of X(T) measured with the Newcastle Control Score (NCS), and treatment. The main outcome measures were change in clinical outcomes (angle, stereoacuity, visual acuity and NCS) in treated and untreated X(T), 2 years from enrolment (or, where applicable, 6 months after surgery). Change over time was tested using the chi-square test for categorical, Wilcoxon test for non-parametric and paired-samples t-test for parametric data. RESULTS: At follow-up, data were available for 371 children (81% of the original cohort). Of these: 53% (195) had no treatment; 17% (63) had treatment for reduced visual acuity only (pure refractive error and amblyopia); 13% (50) had non surgical treatment for control (spectacle lenses, occlusion, prisms, exercises) and 17% (63) had surgery. Only 0.5% (2/371) children developed constant exotropia. The surgically treated group was the only group with clinically significant improvements in angle or NCS. However, 8% (5) of those treated surgically required second procedures for overcorrection within 6 months of the initial procedure and at 6-month follow-up 21% (13) were overcorrected. CONCLUSIONS: Many children in the UK with X(T) receive active monitoring only. Deterioration to constant exotropia, with or without treatment, is rare. Surgery appears effective in improving angle of X(T) and NCS, but rates of overcorrection are high.

Surgery for residual convergence excess esotropia.

The outcome of bilateral medial rectus posterior fixation sutures +/- central tenotomy was assessed as a secondary procedure for residual convergence excess esotropia in 11 patients. Ten had previously undergone bilateral medial rectus recessions. One had recess/resect surgery on the deviating eye. The average preoperative near angle was 30 prism diopters with a range of 16 to 45 prism diopters. Eight patients underwent bilateral medial rectus posterior fixation sutures with central tenotomy. Two had bilateral medial rectus posterior fixation sutures only, and one had bilateral medial rectus posterior fixation suture, a lateral rectus resection, and an inferior oblique disinsertion. The postoperative near angle ranged from 4-30 prism diopters, with mean of 12 prism diopters. Five patients demonstrated some stereopsis preoperatively, all needing bifocals. Postoperatively, nine patients demonstrated an improvement in stereopsis, none needing bifocals. Two showed smaller near angles and better control without bifocals. Final stereopsis ranged from 30 seconds of arc to 800 seconds of arc. We feel that bilateral medial rectus posterior fixation sutures with or without central tenotomy is a viable secondary procedure for residual convergence excess esotropia.

Role of inferior rectus botulinum toxin injection in iatrogenic vertical strabismus.

INTRODUCTION: We describe the uses and effectiveness of botulinum toxin to the inferior rectus muscle in iatrogenic vertical strabismus. METHODS: A retrospective review of our database to identify patients who had undergone inferior rectus botulinum toxin injection between 1982 and 2006 for iatrogenic vertical strabismus. RESULTS: There were 13 patients, 5 female, with an age range of 14 to 78 years. Eight left eyes were injected. The mean period of follow-up was 3.3 years. Etiologies included post-local anesthetic, post-plaque radiotherapy for malignant melanoma, adherence syndrome after inferior oblique surgery, and post-botulinum toxin to the levator muscle to produce a temporary therapeutic ptosis. Reversal on first injection occurred in 7 patients and occurred in a further 2 patients on second injection. Pre-injection binocularity was not demonstrable in 3 patients; 2 of these patients did have binocular vision demonstrable after botulinum toxin injection. Seven patients had one injection, 3 patients had two, 1 had three, 1 had five, and 1 had six. DISCUSSION: The data demonstrate the range of indications that can be successfully treated and managed with inferior rectus botulinum toxin injection in iatrogenic vertical strabismus.

Strabismus surgery in the over 60s--an update.

BACKGROUND: The proportion of individuals above retirement age is expanding and consequently there is increased demand for ophthalmic surgery, including strabismus. In 2001 we published a pilot study reviewing all strabismus operations performed on patients aged 60 and over between 1992 and 1999. Almost a decade later, we repeated the analysis, aiming to identify any longitudinal trends. METHODS: Using the hospital information system, we conducted a retrospective review of all elective strabismus surgery in patients aged 60 years and over, performed by one surgeon (JPL) between January 2000 and September 2008. All operations were under general anesthesia. RESULTS: We identified 237 strabismus operations (9.1% of total operations) that were performed in patients aged 60 or over. This is significantly higher compared with the proportion of elderly patients in the previous study (111 patients, 7% of total operations, P=0.015). Gender distribution was similar to our previous study (105 females, 51% vs 59 females, 53.1%, P=0.814). Ages ranged from 60 to 96 years (mean 69.5 ± 6.8). A decade ago, the more common diagnoses were fourth (22%) and sixth (10%) cranial nerve palsies, consecutive (14%) and secondary (9%) strabismus, whereas recently thyroid eye disease (18%), sixth nerve palsy (13%) and iatrogenic (11%) causes have overtaken the other categories (P=0.011). Adjustable sutures were undertaken in 144 patients (61%). CONCLUSION: Strabismus in elderly patients continues to be related to neurogenic etiologies but thyroid eye disease and iatrogenic causes are on the increase. This is the first 10-year longitudinal analysis to be presented, with the caveat that our data are obtained from a single practice.

The management of strabismus in patients with chronic progressive external ophthalmoplegia.

AIMS: To describe the clinical profiles and results of surgical and non-surgical interventions in a large cohort of patients with strabismus associated with chronic progressive external ophthalmoplegia (CPEO). METHODS: A retrospective case note review was conducted of all patients with a clinical diagnosis of CPEO referred to our institution's strabismus department in the United Kingdom between 1990 and 2008. RESULTS: The total number of patients in this series was 28 (12 male, 16 female). The main presenting symptoms were diplopia (14) or cosmetically objectionable strabismus (11). Three had symptoms unrelated to strabismus and were excluded from further analysis. The most common strabismus findings were large angle exotropias (mean 47 prism diopters), half of which had associated vertical deviations. Strabismus management consisted of prisms (3), occlusion (3), botulinum toxin (7), and surgery (8). Two patients were offered surgery but were deemed unfit for general anesthesia, and a further 2 declined surgery or defaulted follow-up. Three had no active treatment for strabismus. The most effective procedure for exotropia was maximal, bilateral lateral rectus recessions (8 mm or 17 mm from the limbus) with medial rectus resections (7 mm) using adjustable sutures. Patients who had less than maximal horizontal muscle surgery were inevitably undercorrected, even in the early postoperative period. Long-term stability of measurements was rarely achieved, with either progressive increase in the angle of exotropia, or with new onset vertical deviations. CONCLUSIONS: In selected patients with CPEO and exotropia, maximal bilateral surgery significantly improves ocular alignment and may relieve symptoms of diplopia, but due to the progressive nature of the disease, strabismus often recurs. Toxin can be invaluable in maintaining a satisfactory ocular alignment in patients with residual or progressive exotropias, who have undergone maximal horizontal rectus muscle surgery or multiple previous strabismus procedures.

Diagnostic use of botulinum toxin in patients with Duane syndrome.

INTRODUCTION: Duane syndrome is a difficult condition to treat. Patients and parents need to be informed that it will not resolve and it is not possible to create normal eye movements surgically. Botulinum toxin may be used to assess the likelihood of reducing the abnormal head posture and reducing the diplopia by increasing the field of binocular single vision. If results are favorable then surgery may be offered. This article is a retrospective review of patients with Duane syndrome treated with botulinum toxin using the toxin clinic database between 1980 and 2007. METHODS: Eighty-eight patients were identified, 48 females and 40 males. The average age at presentation was 29 years, range 5 to 68 years. The left eye was affected in 50 (57%) patients and 21 (24%) patients were affected bilaterally. The average angle was 28.6 +/- 18.4 Delta for the esotropic patients and 32.5 +/- 14.5 Delta for the exotropic patients. In 58 patients the medial rectus was injected, in 30 the lateral rectus. RESULTS: As a result of the outcome of botulinum toxin, 41 (46.5%) patients proceeded to surgery; 12 (14%) continued with maintenance toxin. Forty-seven (53%) demonstrated a long-term reduction in deviation. Transient complications were ptosis in 11 patients and induced vertical deviation in 10. CONCLUSION: This is the first study to explore the diagnostic role of botulinum toxin in Duane syndrome. It is a safe treatment that may also offer long-term benefits.

Alternate day exotropia.

INTRODUCTION: Cyclic strabismus is a rare disorder in which manifest strabismus and controlled latent deviation (heterophoria) alternates on a regular pattern which is usually over a 48-hr period. During the "straight" period the patient has a fully controlled heterophoria for all distances, with very good fusion and stereopsis. During the "squinting" period the patient has a constant manifest strabismus for all distances, with no demonstrable fusion and stereopsis. METHOD: We describe 2 cases of alternate day exotropia. One patient requires strabismus surgery to obtain constant binocularity. The second patient underwent surgery for an intermittent exotropia and subsequently developed an alternate day exotropia. The condition very gradually reduced over the years and the patient is now fully binocular. CONCLUSION: We report the first 2 cases of cyclic exotropia in non-Japanese female children presenting from about 1 to 3 years of age, without ocular or systemic pathology.