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  • 1.
    Barth, Henrik
    et al.
    Department of Ophthalmology, Lund University Hospital, Biomedicinskt centrum (BMC), Lund, Sweden.
    Crafoord, Sven
    Örebro University, School of Medical Sciences. Department of Ophthalmology, Örebro University Hospital, Örebro, Sweden.
    Andréasson, Sten
    Department of Ophthalmology, Lund University Hospital, Biomedicinskt centrum (BMC), Lund, Sweden.
    Ghosh, Fredrik
    Department of Ophthalmology, Lund University Hospital, Biomedicinskt centrum (BMC), Lund, Sweden.
    A cross-linked hyaluronic acid hydrogel (Healaflow(®)) as a novel vitreous substitute2016In: Graefe's Archives for Clinical and Experimental Ophthalmology, ISSN 0721-832X, E-ISSN 1435-702X, Vol. 254, no 4, p. 697-703Article in journal (Refereed)
    Abstract [en]

    Purpose: Vitrectomy requires the substitution of the natural vitreous, as well as tamponading of retinal breaks. Clinically available alternatives such as gas and silicone oil have side effects such as inflammation, secondary glaucoma, cataract, and a need for head posturing. In this study, a hydrogel of cross-linked sodium hyaluronic acid (Healaflow(®)) is evaluated for use as a novel vitreous substitute.

    Methods: A combined 25-20-gauge pars plana vitrectomy with posterior vitreous detachment was performed in the right eye of twelve pigmented rabbits, with subsequent injection of approximately 1 ml Healaflow(®). Clinical evaluation, measurement of intraocular pressure (IOP), and full-field ERG were performed postoperatively. The rabbits were sacrificed at different time-points between 42 and 105 days. After enucleation, the eyes were examined macroscopically, photographed, and prepared for histological examination with routine microscopy and immunohistochemistry.

    Results: Healaflow(®) was successfully used with standard surgical procedures and remained translucent but did lose most of its viscosity during the postoperative period. One rabbit was lost due to unrelated causes. In two eyes iatrogenic partial retinal detachments were seen, and in two eyes significant cataract developed due to intra-operative complications. ERG-recordings revealed no toxic effect on rod or cone function. Routine microscopy and immunohistochemistry demonstrated normal morphology with some Müller cell activation (up-regulation of glial acidic fibrillary protein, GFAP) compared to unoperated eyes and no significant DNA-fragmentation (TUNEL-assay).

    Conclusions: Healaflow® did not affect retinal morphology or function negatively during long-term use as a vitreous substitute, making it highly interesting in this setting. An estimated retention time of a few weeks suggests potential for use as a short-term tamponade. Future work will include an increased ratio of cross-linking to prolong the structural integrity of the gel.

  • 2.
    Barth, Henrik
    et al.
    Department of Ophthalmology, Lund University, BMC D10,, Lund, Sweden.
    Crafoord, Sven
    Örebro University, School of Medical Sciences. Department of Ophthalmology, Faculty of Medicine and Health, Örebro University Hospital, Orebro, Sweden.
    Arnér, Karin
    Department of Ophthalmology, Lund University, BMC D10, Lund, Sweden.
    Ghosh, Fredrik
    Department of Ophthalmology, Lund University, BMC D10, Lund, Sweden.
    Inflammatory responses after vitrectomy with vitreous substitutes in a rabbit model2019In: Graefe's Archives for Clinical and Experimental Ophthalmology, ISSN 0721-832X, E-ISSN 1435-702X, Vol. 257, no 4, p. 769-783Article in journal (Refereed)
    Abstract [en]

    PURPOSE: To investigate the inflammatory response of current and future potential vitreous substitutes in an experimental in vivo vitrectomy model.

    METHODS: Twenty-five gauge pars plana vitrectomy was performed in the right eye of 60 pigmented rabbits, with subsequent injection of 0.5-1.0 ml of Healaflow® (cross-linked hyaluronic acid, n = 12), Bio-Alcamid® (polyalkylimide, n = 8), silicone oil (n = 12), or balanced saline solution (BSS, n = 28). Postoperative clinical evaluation was performed; and the rabbits were sacrificed at 1 day, 1 week, or 1 month. The eyecups were then examined macroscopically; the retinas sectioned and stained with hematoxylin and eosin (Htx), and immunohistochemically labeled for glial fibrillary acidic protein (GFAP), CD45, galectin-3, CD68, and CD20. Unoperated left eyes from treated animals as well as eyes from untreated animals were used as controls.

    RESULTS: Vitrectomy without major complications was achieved in 46/60 eyes. The remaining 14 eyes were analyzed separately. One eye developed endophthalmitis after 1 week and was excluded. Eyes treated with Healaflow®, silicone oil, and BSS had a comparable appearance macroscopically and in Htx-stained sections, whereas Bio-Alcamid®-injected eyes exhibited increased macroscopic inflammation and severely affected retinas. GFAP upregulation was present in all treatment groups, most prominent in eyes treated with Bio-Alcamid® and silicone oil. Upregulation of CD45 and CD68 in the inner retina and vitreous space was most prominent with Bio-Alcamid® treatment, and these eyes together with their silicone oil-treated counterparts also displayed a stronger upregulation of CD20-labeled cells compared with remaining groups. General upregulation of galectin-3, mainly in the inner retina, was found in all groups. In eyes with perioperative complications, labeling of CD45, CD68, and especially GFAP was comparably high.

    CONCLUSIONS: We here describe differences in the postsurgery inflammatory profiles of existing and potential vitreous substitutes. Bio-Alcamid® and silicone oil display severe signs of gliosis and inflammation, whereas Healaflow® elicits minimal reactions comparable with BSS, highlighting its potential application as a vitreous substitute in a future clinical setting.

  • 3.
    Barth, Henrik
    et al.
    Department of Ophthalmology, Lund University, Lund, Sweden .
    Crafoord, Sven
    Örebro University, School of Medicine, Örebro University, Sweden. Department of Ophthalmology, Örebro University Hospital, Örebro, Sweden.
    O'Shea, Timothy M.
    Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology, Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, USA .
    Pritchard, Christopher D.
    Harvard-Massachusetts Institute of Technology, Division of Health Sciences and Technology, Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, USA .
    Langer, Robert
    Harvard-Massachusetts Institute of Technology, Division of Health Sciences and Technology, Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, USA .
    Ghosh, Fredrik
    Department of Ophthalmology, Lund University, Lund, Sweden .
    A new model for in vitro testing of vitreous substitute candidates2014In: Graefe's Archives for Clinical and Experimental Ophthalmology, ISSN 0721-832X, E-ISSN 1435-702X, Vol. 252, no 10, p. 1581-1592Article in journal (Refereed)
    Abstract [en]

    Purpose: To describe a new model for in vitro assessment of novel vitreous substitute candidates.

    Methods: The biological impact of three vitreous substitute candidates was explored in a retinal explant culture model; a polyalkylimide hydrogel (Bio-Alcamid (R)), a two component hydrogel of 20 wt.% poly (ethylene glycol) in phosphate buffered saline (PEG) and a cross-linked sodium hyaluronic acid hydrogel (Healaflow (R)). The gels where applied to explanted adult rat retinas and then kept in culture for 2, 5 and 10 days. Gel-exposed explants were compared with explants incubated under standard tissue culture conditions. Cryosections of the specimens were stained with hematoxylin and eosin, immunohistochemical markers (GFAP, Vimentin, Neurofilament 160, PKC, Rhodopsin) and TUNEL.

    Results: Explants kept under standard conditions as well as PEG-exposed explants displayed disruption of retinal layers with moderate pyknosis of all neurons. They also displayed moderate labeling of apoptotic cells. Bio-Alcamid (R)-exposed explants displayed severe thinning and disruption of retinal layers with massive cell death. Healaflow (R)-treated explants displayed normal retinal lamination with significantly better preservation of retinal neurons compared with control specimens, and almost no signs of apoptosis. Retinas exposed to Healaflow (R) and retinas kept under standard conditions showed variable labeling of GFAP with generally low expression and some areas of upregulation. PEG-exposed retinas showed increased GFAP labeling and Bio-Alcamid (R)-exposed retinas showed sparse labeling of GFAP.

    Conclusions: Research into novel vitreous substitutes has important implications for both medical and surgical vitreoretinal disease. The in vitro model presented here provides a method of biocompatibility testing prior to more costly and cumbersome in vivo experiments. The explant culture system imposes reactions within the retina including disruption of layers, cell death and gliosis, and the progression of these reactions can be used for comparison of vitreous substitute candidates. Bio-Alcamid (R) had strong adverse effects on the retina which is consistent with results of prior in vivo trials. PEG gel elicits reactions similar to the control retinas whereas Healaflow (R) shows protection from culture-induced trauma indicating favorable biocompatibility.

  • 4.
    Crafoord, Sven
    et al.
    Departments of Ophthalmology, Örebro University Hospital, Örebro, Sweden.
    Andreasson, Sten
    Departments of Ophthalmology, University of Lund, Lund, Sweden.
    Ghosh, Fredrik
    Departments of Ophthalmology, University of Lund, Lund, Sweden.
    Experimental vitreous tamponade using polyalkylimide hydrogel2011In: Graefe's Archives for Clinical and Experimental Ophthalmology, ISSN 0721-832X, E-ISSN 1435-702X, Vol. 249, no 8, p. 1167-74Article in journal (Refereed)
    Abstract [en]

    PURPOSE: To evaluate polyalkylimide as a possible vitreous tamponading agent.

    METHODS: A 20-gauge pars plana vitrectomy and posterior vitreous detachment were performed in the right eye of six pigmented rabbits. Approximately 1 ml of viscoelastic gel, polyalkylimide (Bio-Alcamid) was thereafter injected into the vitreous space. Full-field ERG and intraocular pressure (IOP, Tonopen) was measured pre-and postoperatively at regular intervals up to 28 days. At day 6 or 28, the rabbits were sacrificed and the eyes were examined macroscopically, photographed, and prepared for histological examination with routine microscopy.

    RESULTS: The viscoelastic hydrogel was successfully injected, and remained translucent with preserved gel properties throughout the postoperative period. The postoperative IOP was unchanged compared to preoperative values. Five of six eyes displayed retinal edema or pigmentary changes centrally while the periphery appeared intact. ERG recordings showed a radical decrease in rod- and cone-derived B-wave amplitudes. Histological examination confirmed varying degrees of edema combined with neuronal cell death within the retinal layers in the central part of the fundus, while the peripheral part appeared intact.

    CONCLUSION: Polyalkylimide displays favourable physical properties when used as a vitreous tamponade. However, the hydrogel causes functional and morphological retinal damage when in direct contact with the inner retina. Possible pathological mechanisms include osmotic imbalance and direct toxic effects, and modification of biochemical properties is warranted before clinical use will be possible.

  • 5.
    Makdoumi, Karim
    et al.
    Örebro University, School of Health and Medical Sciences.
    Bäckman, Anders
    Örebro University, School of Medical Sciences.
    Crafoord, Sven
    Örebro University, School of Medical Sciences.
    Response to: Bactericidal effect of photo-activated riboflavin using UVA2010In: Graefe's Archives for Clinical and Experimental Ophthalmology, ISSN 0721-832X, E-ISSN 1435-702X, Vol. 248, no 5, p. 757-758Article in journal (Refereed)
  • 6.
    Makdoumi, Karim
    et al.
    Örebro University, School of Health and Medical Sciences.
    Bäckman, Anders
    Mortensen, Jes
    Crafoord, Sven
    Evaluation of antibacterial efficacy of photo-activated riboflavin using ultraviolet light (UVA)2010In: Graefe's Archives for Clinical and Experimental Ophthalmology, ISSN 0721-832X, E-ISSN 1435-702X, Vol. 248, no 2, p. 207-212Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: To evaluate the antibacterial efficacy of photo-activated riboflavin using Ultraviolet A (UVA) on three bacterial strains commonly detected in keratitis. METHODS: Three bacterial strains (Staphylococcus epidermidis, Staphylococcus aureus and Pseudomonas aeruginosa) were cultured on blood/hematin-agar plates and dispersed in PBS. Dispersion was done of 10 microl of bacterial stock-solutions in 90 microl of RPMI, where different riboflavin molarities had been added, to achieve a bacterial concentration of 1-4 x 10 (4)/ml. Riboflavin end molarities before illumination were 0, 100, 200, 300 and 400 microM. Each solution had a negative control. The solutions were illuminated with UVA (365 nm) for 30 minutes (5.4 J/cm(2)) and then continued for a total time of 60 minutes (10.8 J/cm(2)). A count of CFU was conducted after incubation and results compared. RESULTS: In all tested strains, a slight decrease of bacteria was seen when exposed to UV for 30 minutes. A doubling of the UV dose showed a marked decrease of bacterial count in all bacteria tested. The combination of UV and riboflavin showed a more extensive reduction of CFU, confirming an interaction effect between UV and riboflavin. CONCLUSION: Riboflavin photo-activation using UVA (365 nm) can achieve an extensive eradication of bacteria, and the combination is more potent in reducing bacterial number than UV alone.

  • 7.
    Makdoumi, Karim
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Department of Ophthalmology, Örebro University Hospital, Region Örebro County, Örebro, Sweden.
    Bäckman, Anders
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Clinical Research Centre, Örebro University Hospital, Region Örebro County, Örebro, Sweden.
    Mortensen, Jes
    Dept. Ophthalmology, Örebro University Hospital, Region Örebro County, Örebro, Sweden.
    Magnuson, Anders
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Clinical Epidemiology and Biostatistic Unit, Örebro University Hospital, Örebro, Sweden.
    Crafoord, Sven
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Örebro University Hospital, Region Örebro County, Örebro, Sweden.
    Comparison of UVA- and UVA/riboflavin-induced growth inhibition of Acanthamoeba Castellanii2013In: Graefe's Archives for Clinical and Experimental Ophthalmology, ISSN 0721-832X, E-ISSN 1435-702X, Vol. 251, no 2, p. 509-514Article in journal (Refereed)
    Abstract [en]

    Purpose To investigate whether ultraviolet light (UVA) at 365 nm can inhibit/eliminate Acanthamoeba growth and if riboflavin would potentiate such an association.

    Method: Acanthamoeba castellanii in a fluid medium with a concentration of approximately 1.7 x 10(4) protozoa/ml were prepared with (0.01 %) and without riboflavin. Exposure of UVA (dose 5.475 J/cm(2)) took place twice, with each illumination period followed by culturing of 10 mu l in peptone yeast-extract glucose (PYG) medium for 7 days. Every suspension prepared had a non-exposed control solution. Determination of Acanthamoeba was conducted daily, by count in Burker chamber days 4 through 7 after exposure. Statistical analysis was done by repeated-measurement ANOVA and post-hoc analysis for unpaired samples.

    Results: The exposure of ultraviolet light resulted in an inhibited growth of Acanthamoeba compared to the non-exposed solutions, with a statistically significant reduction over time (p = 0.0003). The addition of riboflavin did not amplify the effect, and there were no tendencies for an interaction effect between UVA and riboflavin. The antiprotozoal effect of the UVA wavelength, utilized in CXL, is solely mediated by ultraviolet light, and riboflavin does not seem to amplify the antimicrobial efficacy.

  • 8.
    Makdoumi, Karim
    et al.
    Örebro University Hospital.
    Mortensen, Jes
    Orebro Univ Hosp, Orebro, Sweden..
    Crafoord, Sven
    Örebro University Hospital.
    Response to: evaluation of combined riboflavin and ultraviolet A as an alternative treatment for keratitis2013In: Graefe's Archives for Clinical and Experimental Ophthalmology, ISSN 0721-832X, E-ISSN 1435-702X, Vol. 251, no 3, p. 997-998Article in journal (Refereed)
  • 9.
    Makdoumi, Karim
    et al.
    Department of Ophthalmology, Örebro University Hospital, Örebro, Sweden.
    Mortensen, Jes
    Department of Ophthalmology, Örebro University Hospital, Örebro, Sweden; Department of Ophthalmology, Ryhov County Hospital, Jönköping, Sweden.
    Sorkhabi, Omid
    Department of Ophthalmology, Ryhov County Hospital, Jönköping, Sweden.
    Malmvall, Bo-Eric
    Department of Infectious Medicine, Futurum, the Academy of Health, Ryhov County Hospital, Jönköping, Sweden; Department of Infectious Medicine, Institution of Clinical and Experimental Medicine, University of Linköping, Linköping, Sweden.
    Crafoord, Sven
    Department of Ophthalmology, Örebro University Hospital, Örebro, Sweden.
    UVA-riboflavin photochemical therapy of bacterial keratitis: a pilot study2012In: Graefe's Archives for Clinical and Experimental Ophthalmology, ISSN 0721-832X, E-ISSN 1435-702X, Vol. 250, no 1, p. 95-102Article in journal (Refereed)
    Abstract [en]

    Background: The aim of this work as to investigate the photochemical interaction used in corneal crosslinking (CXL) as the primary therapy for bacterial keratitis.

    Methods: A prospective non-randomized study was conducted including 16 patients with a clinical diagnosis of bacterial keratitis. No patient had any prior antibiotic treatment for the current infection. Photography and microbial culturing of the infected cornea were performed. Riboflavin was topically administered for 20 min and ultraviolet light (UVA) exposure settings for treatment of keratoconus were used. After the procedure, clinical examinations were done at least once daily until signs of improvement had been established. The frequency of examinations was thereafter reduced. Antibiotic therapy was initiated if infectious progression was suspected. The trial was registered at ISCRTN.org (no: 21432643).

    Results: All eyes responded to the photochemical treatment with improvement in symptoms and signs of reduced inflammation. Epithelial healing was achieved in all cases. Antibiotic administration was necessary in two cases. One patient required a human amniotic membrane transplant.

    Conclusions: This trial illustrates that photosensitization of riboflavin using UVA at 365 nm has the potential to induce healing in patients with microbial keratitis. The results from the treatment of these 16 patients with corneal ulcers indicate that UVA-riboflavin photochemical therapy merits a controlled study in order to assess its efficacy and safety compared to antibiotics.

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