Background. Different disease-modifying therapies (DMT) for multiple sclerosis (MS) have disparate effects on disability outcomes. Sweden has a leading position globally in initiating high-efficacy DMT instead of escalating DMT from 1(st)-line to high-efficacy DMT. With optical coherence tomography (OCT), retinal changes can be measured at a few micrometer level. OCT has been increasingly applied in diagnosing MS and monitoring disease course and therapeutic effect. Objective. We investigate the effects of 1(st)-line versus high-efficacy DMT for MS on retinal and brain atrophy and on functional outcomes during 6.8 years of escalating DMT. Materials and Methods. In this prospective longitudinal observational study, 18 MS patients were followed up for 6.8 years. Twelve of the patients were untreated at baseline. All patients underwent 1(st)-line DMT for median duration of 2.4 years and then switched to high-efficacy DMT for a median duration of 2.9 years. Findings from neurological examinations, MRI, and OCT measures were registered 2-4 times per year. Results. Ganglion cell-inner plexiform layer (GCIPL) thickness was significantly reduced during 1(st)-line DMT (73.75 mu m, p < 0.01) compared to baseline (76.38 mu m). During high-efficacy DMT, thickness reduction was slower (73.27 mu m, p < 0.05), and MRI contrast-loading lesions vanished (p < 0.01). However, brain parenchymal fraction (BPF) decreased during high-efficacy DMT compared to 1(st)-line DMT. Estimated models showed similar results. Conclusion. GCIPL decline was most profound during 1(st)-line DMT and diminished during high-efficacy DMT. MRI contrast lesions vanished during high-efficacy DMT. However, brain atrophy continued regardless of high-efficacy DMT.

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Purpose. To present real-life data of patients with macular edema (ME) secondary to central retinal vein occlusion (CRVO) treated with bevacizumab (BVZ); determine the possible influence of epiretinal membrane (ERM) on treatment efficacy; and compare treatment outcomes in a treat-and-extend regimen (TER) versus pro re nata (PRN). Methods. We carried out a retrospective analysis of 58 eyes (56 patients) with new-onset CRVO treated only with intravitreal bevacizumab according to TER or PRN. Outcome measures were best-corrected visual acuity (BCVA) and central retinal thickness (CRT) at baseline and 12 months after the first treatment, number of visits and injections, and presence of ERM confirmed by optical coherence tomography in the first 6 months. Results. At 12 months, the mean number of injections was 6.3 across all eyes, with significantly more injections given in TER (p < 0.001). Mean CRT improved from 627 mu m to 359 mu m (p < 0.001) in all eyes, with improvement noted in TER (p < 0.001), PRN (p < 0.001), ERM (p=0.003), and non-ERM (p < 0.001) subgroups. The mean BCVA gain was +13.6 letters, and the mean BCVA improved from 0.81 to 0.54 LogMAR (p < 0.001) in all eyes. BCVA improvement from baseline was significant in TER (p < 0.001) and non-ERM (p < 0.001) but not in PRN (p=0.08) or ERM (p=0.2) subgroups. Seven eyes, all receiving PRN treatment, developed neovascularization. Conclusions. Intravitreal bevacizumab according to either PRN or TER resolved edema and stabilized vision in the first 12 months, with TER yielding significant visual improvement and avoiding neovascular complications. ERM had no influence on bevacizumab efficacy in reducing ME in CRVO during 12 months of treatment.

Objective: We report that lumbar puncture (LP) with removal of cerebrospinal fluid (CSF) induced rebound intracranial hypertension with increased papilledema as monitored by optical coherence tomography (OCT). Background: Severe papilledema causes visual field loss and central vision damage if untreated. Fundoscopy is a key to diagnose papilledema, but is not sensitive enough to monitor therapeutic effects. Methods: OCT was applied to follow a 24-year-old woman with headache, visual dysfunction, severe bilateral papilledema, and elevated CSF opening pressure. She was first treated with serial LP, which led to symptom deterioration, increased CSF pressure, and increased the retinal nerve fiber layer (RNFL) thickness. She was then successfully treated with acetazolamide and furosemide. Results: OCT showed reduction of RNFL thickness directly after LP with CSF removal, accompanied with reduced CSF pressure. Increased RNFL thickness accompanied with worsened headache, visual dysfunction, and increased CSF pressure was observed on the next day after LP. Less than 24 h after start of medication, the symptoms had reversed and RNFL thickness was reduced. The patient was symptom-free 2 weeks after starting on medical treatment. Papilledema had vanished on fundoscopy 6 weeks after the therapy, and RNFL thickness was normalized at 3 months of follow-up. Conclusion: This case provides evidence that OCT is an objective and sensitive tool to monitor papilledema and its response to therapy, and thereby important to help in correct clinical decision-making.

Corneal neovascularization is a sight-threatening condition caused by angiogenesis in the normally avascular cornea. Neovascularization of the cornea is often associated with an inflammatory response, thus targeting VEGF-A alone yields only a limited efficacy. The NF-kappa B signaling pathway plays important roles in inflammation and angiogenesis. Here, we study consequences of the inhibition of NF-kappa B activation through selective blockade of the IKK complex I kappa B kinase beta (IKK2) using the compound IMD0354, focusing on the effects of inflammation and pathological angiogenesis in the cornea. In vitro, IMD0354 treatment diminished HUVEC migration and tube formation without an increase in cell death and arrested rat aortic ring sprouting. In HUVEC, the IMD0354 treatment caused a dose-dependent reduction in VEGF-A expression, suppressed TNF alpha-stimulated expression of chemokines CCL2 and CXCL5, and diminished actin filament fibers and cell filopodia formation. In developing zebrafish embryos, IMD0354 treatment reduced expression of Vegf-a and disrupted retinal angiogenesis. In inflammation-induced angiogenesis in the rat cornea, systemic selective IKK2 inhibition decreased inflammatory cell invasion, suppressed CCL2, CXCL5, Cxcr2, and TNF-alpha expression and exhibited anti-angiogenic effects such as reduced limbal vessel dilation, reduced VEGF-A expression and reduced angiogenic sprouting, without noticeable toxic effect. In summary, targeting NF-kappa B by selective IKK2 inhibition dampened the inflammatory and angiogenic responses in vivo by modulating the endothelial cell expression profile and motility, thus indicating an important role of NF-kappa B signaling in the development of pathologic corneal neovascularization.

Background: Nutritional visual defects are apparently uncommon nowadays in developed nations. Retinal change-related visual defects caused by hypovitaminoses may be underdiagnosed. Aim of the study: To investigate the retinal structural and functional changes in a patient with multivitamin deficiency before and during vitamin supplementation. Methods: A 51-year-old female had been on vegetarian diet as a child, and on restrict vegan diet during the last 2 years, developing severe bilateral deterioration of visual function and polyneuropathy. Blood test revealed low levels of vitamin A, B6 and D. The patient underwent examinations with optical coherence tomography (OCT), computerized visual field examination (VF), electroretinography (ERG), visual evoked potentials (VEP) and neurography before and after vitamin supplementation. Results: Visual acuity (VA) was 20/1000 and VF examination showed central scotoma in both eyes. Color vision was significantly affected. Full-field ERG showed normal rod and cone function, but a clearly reduced central peak was registered in multifocal ERG (mf-ERG), indicating impaired fovea function. VEP showed delayed latency and low amplitude of P100 in both eyes. Neurography showed sensory polyneuropathy. OCT showed significant thinning of macular ganglion cell plus inner plexiform layer (GCIPL) with rapid progression. Retinal nerve fiber layer (RNFL) was preserved and normal, which is in contrast to neuroinflammatory conditions. After 2.5 years of multivitamin supplementation, the visual functions were improved. GCIPL thickness was stable without further deterioration. Conclusions: Multivitamin deficiency results in progressive thinning of GCIPL with severe visual deterioration. In contrast to neuroinflammation, RNFL is preserved and normal. Stabilized GCIPL during vitamin supplementation was associated with improved visual function. OCT provides a sensitive and objective measure for differential diagnosis, monitoring retinal change and response to therapy.

Background: Optic neuritis (ON) is an inflammatory condition of the optic nerve. ON is associated with development of demyelinating diseases of the central nervous system (CNS). CNS lesions visualized by magnetic resonance imaging (MRI) and the finding of oligoclonal IgG bands (OB) in the cerebrospinal fluid (CSF) are used to stratify the risk of MS after a "first" episode of ON. However, the diagnosis of ON in absence of typical clinical manifestations can be challenging. Methods and Materials: Here we present three cases with changes in the optic nerve and ganglion cell layer in the retina over the disease course. (1) A 34-year-old female with a history of migraine and hypertension had suspect amaurosis fugax (transient vision loss) in the right eye. This patient developed MS four years later. Optical coherence tomography (OCT) showed dynamic changes of the thickness of peripapillary retinal nerve fiber layer (RNFL) and macular ganglion cell-inner plexiform layer (GCIPL) over time. (2) A 29-year-old male with spastic hemiparesis and lesions in the spinal cord and brainstem. Six years later he showed bilateral subclinical ON identified using OCT, visual evoked potentials (VEP) and MRI. The patient fulfilled diagnosis criteria of seronegative neuromyelitis optica (NMO). (3) A 23-year-old female with overweight and headache had bilateral optic disc swelling. With OCT and lumbar puncture, idiopathic intracranial hypertension (IIH) was excluded. Further investigation showed positive antibody for myelin oligodendrocyte glycoprotein (MOG). Conclusions: These three cases illustrate the importance of using OCT to facilitate quick, objective and accurate diagnosis of atypical or subclinical ON, and thus proper therapy.

Pathologic angiogenesis is involved in cancer and several blinding conditions such as wet age-related macular degeneration, proliferative retinopathies and corneal neovascularization.

In these dieseases, the angiogenic triggers are hypoxia and inflammation, and both involve the main angiogenic mediator, which is Vascular Endothelial Growth Factor (VEGF). Among available treatments, anti-VEGF often shows limited or temporary efficacy, while steroids are potentially responsible for many side-effects. This thesis presents a series of linked studies aimed at elucidating the early pathologic changes leading to inflammation and corneal neovascularization, and how various treatments affect this process. In this thesis, anti-inflammatory and anti-angiogenic treatments are applied in corneal neovascularization models, to identify VEGF-independent pathways and other novel factors as future therapy targets, as well as to investigate the endogenous modulation of angiogenesis.

A model of experimental neovascularization in the rat cornea was used as main model, where the neovascular response is triggered by a surgical suture placed into the cornea. Investigational treatments (anti-Vegf, dexamethasone, IMD0354, Gap27, or control substances) were then given topically, with the exception of IMD0354, which was given systemically. The effects in the cornea were studied in vivo with slit lamp photography to assess and quantify macroscopic vessel growth and using in vivo confocal microscopy (IVCM) to study cell infiltration and limbal vessel dilation and detect microscopic vessel sprouts; these examinations were performed longitudinally. Genomic analysis with RNA microarray, selected gene expression with q-RT-PCR, and selected protein expression in tissue (immunohistochemistry, immunofluorescence, Western blot) were performed at different time-points. Moreover, other experiments on cell cultures (HUVEC and HCEC), organ cultures (human corneas), ex vivo models (aortic rings) and in vivo studies (zebrafish vasculogenesis) were performed.

Dexamethasone suppressed limbal vasodilation and corneal neovascularization more than anti-Vegf, despite no difference in inflammatory cell infiltration into the cornea. Five-hundred eleven fewer genes were differentially expressed in dexamethasone-treated corneas relative to naïve corneas, compared to anti-Vegf. Among them, several major pro-angiogenic and pro-inflammatory factors and chemokines were suppressed only by dexamethasone and represent novel candidate factors to target in order to improve anti-VEGF treatment. On the other hand, selective inhibition of a single inflammatory pathway (NF-κB), despite showing similar early effects as dexamethasone in suppressing tissue inflammation, was not effective enough to suppress new vessel growth. The same factors suppressed by dexamethasone are also inhibited in endogenous modulation of angiogenesis. Surprisingly, dexamethasone activated several complement factors, which could possibly be beneficial in the anti-angiogenic response.

In a different therapeutic approach, promoting cell migration to accelerate epithelial wound closure similarly was not sufficient to avoid inflammation and angiogenesis in the cornea.

In conclusion, new and more effective treatments are needed for corneal inflammation and neovascularization with fewer side-effects. In this thesis, several novel factors and mechanisms related to inflammation are identified, factors that are not addressed by anti-Vegf therapy, and therefore represent interesting objects for further study, as they have the potential to be targets for adjuvant therapy. Specific anti-inflammatory treatment as well as therapeutic activation of endogenous regulatory pathways, and potentially complement modulation, might represent new strategies to improve anti-angiogenic therapy, but when used alone they do not seem to avoid corneal neovascularization.

Inflammatory angiogenesis is the pathogenic mechanism of various sight-threatening eye diseases, among them corneal neovascularization. Current treatment options include steroids which have undesirable side effects, or anti-VEGF which has only limited efficacy. In an inflammatory environment, however, angiogenesis can be stimulated by numerous factors not directly targeted by anti-VEGF therapy. The aim of this study was to induce corneal inflammation leading to angiogenesis, and investigate the early, differential effects of steroid and anti-VEGF therapy at the cellular, tissue, and gene expression levels. Fifty-two Wistar rats received a single intrastromal corneal suture to induce a controlled inflammatory angiogenic response. Rats were subsequently treated with dexamethasone, rat specific anti-VEGF, or goat IgG (control), topically 4 times daily for 7 days. In vivo confocal microscopy of the cornea was performed longitudinally from 5 h up to 7 d to investigate morphology at the cellular and tissue-level. In vivo photographic vessel analysis and immunohistochemistry were also performed. RT-PCR for VEGF-A, FGF-2, IL-6, TNF-alpha, CXCL2, CCL2, CCL3 and DLL4 was performed at 24 h, and for VEGF-A, IL-6, TNF-alpha, FGF-2, CXCL2, CCL2, and CCL3 at 7 days. Early infiltration of CD11b + myeloid cells into the cornea at 5 h post-suture was delayed by both treatments relative to controls; however neither treatment was able to suppress accumulation of myeloid cells at day 2 or 7. Limbal vessel dilation was inhibited at 5 h by both treatments, but only dexamethasone showed sustained effect until day 2. Early macrophage recruitment was also suppressed by dexamethasone (but not by anti-VEGF) until day 2. Dexamethasone furthermore suppressed corneal neovascularization at day 7 by over 90%, whereas suppression by anti-VEGF was 14%. Despite differential suppression of vessel dilation, macrophage recruitment, and vascular invasion, anti-VEGF and dexamethasone both down-regulated VEGF-A and IL-6 expression at 24 h with sustained effect to 7 d. They also both down regulated FGF-2 and TNF-alpha at 24 h and CCL2 at 7 d. In conclusion, anti-angiogenic treatments influence early, pre-angiogenic tissue activity such as limbal vessel dilation, inflammatory cell infiltration of the stroma, and macrophage recruitment. Importantly, the differential effects of steroids and anti-VEGF treatment in suppressing neovascular growth could not be attributed to differential inhibition of several major angiogenic and inflammatory factors in the early pre-sprouting phase, including IL-6, VEGF-A, FGF-2, TNF-alpha, CCL2, CCL3, CXCL2, or DLL4.

Angiogenesis as a pathological process in the eye can lead to blindness. In the cornea, suppression of angiogenesis by anti-VEGF treatment is only partially effective while steroids, although effective in treating inflammation and angiogenesis, have broad activity leading to undesirable side effects. In this study, genome-wide expression was investigated in a suture-induced corneal neovascularization model in rats, to investigate factors differentially targeted by dexamethasone and anti-Vegf. Topical treatment with either rat-specific anti-Vegf, dexamethasone, or normal goat IgG (sham) was given to sutured corneas for 48 hours, after which in vivo imaging, tissue processing for RNA microarray, and immunofluorescence were performed. Dexamethasone suppressed limbal vasodilation (P amp;lt; 0.01) and genes in PI3K-Akt, focal adhesion, and chemokine signaling pathways more effectively than anti-Vegf. The most differentially expressed genes were confirmed by immunofluorescence, qRTPCR and Western blot. Strong suppression of Reg3g and the inflammatory chemokines Ccl2 and Cxcl5 and activation of classical complement pathway factors C1r, C1s, C2, and C3 occurred with dexamethasone treatment, effects absent with anti-Vegf treatment. The genome-wide results obtained in this study provide numerous potential targets for specific blockade of inflammation and angiogenesis in the cornea not addressed by anti-Vegf treatment, as possible alternatives to broad-acting immunosuppressive therapy.

Background and PurposeThe connexin 43 (Cx43) mimetic peptide Gap27 was designed to transiently block the function of this gap junction. This study was undertaken to investigate the effect of Gap27 on corneal healing, inflammation and neovascularization. Experimental ApproachThe effect of Gap27 on wound healing, inflammation and vascularization was assessed in primary human corneal epithelial cells (HCEC) in vitro and whole human corneas ex vivo, and in an in vivo rat wound healing model. Key ResultsGap27 enhanced the wound closure of HCEC in vitro and accelerated wound closure and stratification of epithelium in human corneas ex vivo, but did not suppress the corneal release of inflammatory mediators IL-6 or TNF- in vivo. In human corneas ex vivo, F4/80 positive macrophages were observed around the wound site. In vivo, topical Gap27 treatment enhanced the speed and density of early granulocyte infiltration into rat corneas. After 7days, the expressions of TNF- and TGF1 were elevated and correlated with inflammatory cell accumulation in the tissue. Additionally, Gap27 did not suppress VEGF release in organotypic culture, nor did it suppress early or late VEGFA expression or neovascularization in vivo. Conclusions and ImplicationsGap27 can be effective in promoting the healing of superficial epithelial wounds, but in deep stromal wounds it has the potential to promote inflammatory cell migration and accumulation in the tissue and does not suppress the subsequent neovascularization response. These results support the proposal that Gap27 acts as a healing agent in the transient, early stages of corneal epithelial wounding.

Purpose: Treatment of corneal neovascularization can lead to vessel regression and recovery of corneal transparency. Here, we examined the response of the cornea to a repeated stimulus after initial vessel regression comparing the second wave of neovascularization with the first.

Methods: Corneal neovascularization was induced by surgical suture placement in the rat cornea for 7 days, followed by suture removal and a 30-day regression period. Corneas were then re-sutured and examined for an additional 4 days. Longitudinal slit-lamp imaging, in vivo confocal microscopy, and microarray analysis of global gene expression was conducted to assess the inflammatory and neovascularization response. Inhibitory effect of topical dexamethasone for repeat neovascularization was assessed.

Results: After initial robust neovascularization, 30 days of regression resulted in the recovery of corneal transparency; however, a population of barely functional persistent vessels remained at the microscopic level. Upon re-stimulation, inflammatory cell invasion, persistent vessel dilation, vascular invasion, and gene expression of Vegfa, Il1β, Il6, Ccl2, Ccl3, and Cxcl2 all doubled relative to initial neovascularization. Repeat neovascularization occurred twice as rapidly as initially, with activation of nitric oxide and reactive oxygen species, matrix metalloproteinase, and leukocyte extravasation signaling pathways, and suppression of anti-inflammatory LXR/RXR signaling. While inhibiting initial neovascularization, a similar treatment course of dexamethasone did not suppress repeat neovascularization.

Conclusions: Persistent vessels remaining after the initial resolution of neovascularization can rapidly reactivate to facilitate more aggressive inflammation and repeat neovascularization, highlighting the importance of achieving and confirming complete vessel regression after an initial episode of corneal neovascularization.

Inflammation in the normally immune-privileged cornea can initiate a pathologic angiogenic response causing vision-threatening corneal neovascularization. Inflammatory pathways, however, are numerous, complex and are activated in a time-dependent manner. Effective resolution of inflammation and associated angiogenesis in the cornea requires knowledge of these pathways and their time dependence, which has, to date, remained largely unexplored. Here, using a model of endogenous resolution of inflammation-induced corneal angiogenesis, we investigate the time dependence of inflammatory genes in effecting capillary regression and the return of corneal transparency. Endogenous capillary regression was characterized by a progressive thinning and remodeling of angiogenic capillaries and inflammatory cell retreat in vivo in the rat cornea. By whole-genome longitudinal microarray analysis, early suppression of VEGF ligand-receptor signaling and inflammatory pathways preceded an unexpected later-phase preferential activation of LXR/RXR, PPAR alpha/RXR alpha and STAT3 canonical pathways, with a concurrent attenuation of LPS/IL-1 inhibition of RXR function and Wnt/beta-catenin signaling pathways. Potent downstream inflammatory cytokines such as Cxcl5, IL-1 beta, IL-6 and Ccl2 were concomitantly downregulated during the remodeling phase. Upstream regulators of the inflammatory pathways included Socs3, Sparc and ApoE. A complex and coordinated time-dependent interplay between pro- and anti-inflammatory signaling pathways highlights a potential anti-inflammatory role of LXR/RXR, PPAR alpha/RXR alpha and STAT3 signaling pathways in resolving inflammatory corneal angiogenesis.

Inhibiting pathologic angiogenesis can halt disease progression, but such inhibition may offer only a temporary benefit, followed by tissue revascularization after treatment stoppage. This revascularization, however, occurs by largely unknown phenotypic changes in pathologic vessels. To investigate the dynamics of vessel reconfiguration during revascularization, we developed a model of reversible murine corneal angiogenesis permitting longitudinal examination of the same vasculature. Following 30 days of angiogenesis inhibition, two types of vascular structure were evident: partially regressed persistent vessels that were degenerate and barely functional, and fully regressed, non-functional empty basement membrane sleeves (ebms). While persistent vessels maintained a limited flow and retained collagen IV+ basement membrane, CD31+ endothelial cells (EC), and alpha-SMA+ pericytes, ebms were acellular and expressed only collagen IV. Upon terminating angiogenesis inhibition, transmission electron microscopy and live imaging revealed that revascularization ensued by a rapid reversal of EC degeneracy in persistent vessels, facilitating their phenotypic normalization, vasodilation, increased flow, and subsequent new angiogenic sprouting. Conversely, ebms were irreversibly sealed from the circulation by excess collagen IV deposition that inhibited EC migration and prevented their reuse. Fully and partially regressed vessels therefore have opposing roles during revascularization, where fully regressed vessels inhibit new sprouting while partially regressed persistent vessels rapidly reactivate and serve as the source of continued pathologic angiogenesis.

Therapeutics against pathologic new blood vessel growth, particularly those targeting vascular endothelial growth factor (VEGF) are of enormous clinical interest. In the eye, where anti-VEGF agents are in widespread clinical use for treating retinal and corneal blindness, only partial or transient efficacy and resistance to anti-VEGF agents are among the major drawbacks. Conversely, corticosteroids have long been used in ophthalmology for their potency in suppressing inflammation and angiogenesis, but their broad biological activity can give rise to side effects such as glaucoma and cataract. To aid in the search for more targeted and effective anti-angiogenic therapies in the eye, we present here a dataset comparing gene expression changes in dexamethasone versus anti-Vegfa treatment of inflammation leading to angiogenesis in the rat cornea. Global gene expression analysis with GeneChip Rat 230 2.0 microarrays was conducted and the metadata submitted to Expression Omnibus repository. Here, we present a high-quality validated dataset enabling genome-wide comparison of genes differentially targeted by dexamethasone and anti-Vegf treatments, to identify potential alternative therapeutic targets for evaluation.

Purpose Tuberculosis (TB) of the eye is a well-known extrapulmonary localization in high-incidence countries. Data on its relevance in developed countries are scanty. We aim to study the epidemiological and clinical pattern of ocular TB in a tertiary care institution of a western country. Methods From 2007 to 2010, consecutive patients with a diagnosis of isolated ocular TB or associated to extraocular TB were recruited. Patients with ophthalmological and clinical features of TB were treated with standard antitubercular therapy (ATT) and steroids in case of concomitant severe ocular inflammation. Results Seventeen cases of ocular and extraocular TB and 45 cases of isolated ocular TB were identified. The proportion of patients with ocular and extraocular TB in our local district was 8.1 %, with a proportion of 10.6 % for the isolated cases. In Cohort 1, only one patient was symptomatic for ocular impairment, and uveitis without inflammation was the most common presentation. On the contrary, in Cohort 2, all patients had visual impairment, mainly with bilateral involvement. 77.8 % of the patients showed an inflammatory pattern. ATT was administered for at least 9 months, in four cases with a short course of systemic corticosteroids. Eight cases in Cohort 2 showed recurrence after 1 year from diagnosis. Conclusions TB of the eye should not be forgotten, even in geographical areas not considered among endemic countries. Ocular evaluation is advisable in patients with pulmonary and extrapulmonary TB, as early detection may allow ATT to preserve visual acuity.