Effects of photobiomodulation therapy on the extracellular matrix of human dental pulp cell sheets.

Photobiomodulation therapy (PBMT) and the cell sheet (CS) technology improve processes relevant to tissue regeneration. The aim of this study was to investigate the effects of different PBMT parameters on the architecture (histology), protein composition (Western blotting and immunohistochemistry) and ultrastructure [scanning electron microscopy (SEM) and transmission electron microscopy (TEM)] of the extracellular matrix (ECM) synthesized by CSs composed by human dental pulp stem cells (hDPSCs). METHODS: Thawed cells were recharacterized by the expression profile of the surface molecules of mesenchymal stem cells (MSCs) using flow cytometry. Clonogenic medium supplemented with vitamin C (20 µg/ml) was used for obtaining the CSs. PBMT was performed with continuous-wave diode laser (660 nm, 20 mW, 0.028cm2, 0.71 W/cm2) in punctual and contact mode. The CSs were allocated in 3 experimental groups: Control: no further treatment; PBMT1 [4 s, 3 J/cm2 (lower energy density), 0.08 J/point] and PBMT2 [7 s, 5 J/cm2 (higher energy density), 0.14 J/point]. Statistical comparisons were performed (p ≤ .05). RESULTS: The cells presented the classical immunoprofile of MSCs. Type I and type III collagens and fibronectin were present in the ECM of the CSs. PBMT1 induced higher amount of fibronectin. The overall ultrastructure of the CSs in the PBMT1 was epithelial-like, whereas the PBMT2 leads to CSs with fusiform cells arranged in bundles. TEM identified a more mature ECM and signs of apoptosis and necrosis in the PBMT2 group. CONCLUSION: PBMT influence the composition and ultrastructure of the ECM of CSs of hDPSCs. Thus, PBMT, specifically when applied in the lower energy density, could be of importance in the determination of the mechanical quality of CSs, which may favor cell therapy by improving the CS transplantation approach.

[Switching the Treatment Regime from PRN (pro re nata, as needed) to T&E (Treat and Extend) for Intravitreal Therapy with Anti-VEGF Agents in Private Practice or Small Institutions]. / Umstellung des Therapieschemas pro re nata (PRN) auf Treat and Extend (T&E) in der intravitrealen Therapie mit Anti-VEGF-Medikamenten in der Praxis oder kleineren Institutionen.

In everyday practice, intravitreal therapy in an "as needed" regimen (pro re nata, PRN) is less predictable and requires more visits (monitoring and injections taken together) with poorer functional results than with the treat and extend (T&E) regimen. Current literature supports the benefit of a switch. However, practical advice is still missing. This article provides "best practice" recommendations for private practice or smaller institutions for the change from PRN to T&E. The requirements are organisational adjustments, staff training, definition of the scenario triggering the switch (A - functional or anatomical deterioration; B - general switch at a predefined date), counselling of the patients, defining benchmarks for the follow-up of the switch and preparing for higher capacity utilisation during the transition (shorter treatment intervals during the switching phase). Guidance is provided for each phase (a, preparation; b, transition; and c, follow-up).

Epigenetic regulation of melatonin receptors in neuropsychiatric disorders.

Melatonin, the primary indoleamine hormone of the mammalian pineal gland, is known to have a plethora of neuroregulatory, neuroprotective and other properties. Melatonergic signalling is mediated by its two GPCRs, MT1 and MT2 , which are widely expressed in the mammalian CNS. Melatonin levels and receptor expression often show a decrease during normal ageing, and this reduction may be accelerated in some disease states. Depleted melatonergic signalling has been associated with neuropsychiatric dysfunction and impairments in cognition, memory, neurogenesis and neurorestorative processes. The anticonvulsant and mood stabilizer, valproic acid (VPA), up-regulates melatonin MT1 and/or MT2 receptor expression in cultured cells and in the rat brain. VPA is known to affect gene expression through several mechanisms, including the modulation of intracellular kinase pathways and transcription factors, as well as the inhibition of histone deacetylase (HDAC) activity. Interestingly, other HDAC inhibitors, such as trichostatin A, which are structurally distinct from VPA, can also up-regulate melatonin receptor expression, unlike a VPA analogue, valpromide, which lacks HDAC inhibitory activity. Moreover, VPA increases histone H3 acetylation along the length of the MT1 gene promoter in rat C6 cells. These findings indicate that an epigenetic mechanism, linked to histone hyperacetylation/chromatin remodelling and associated changes in gene transcription, is involved in the up-regulation of melatonin receptors by VPA. Epigenetic induction of MT1 and/or MT2 receptor expression, in areas where these receptors are lost because of ageing, injury or disease, may be a promising therapeutic avenue for the management of CNS dysfunction and other disorders. LINKED ARTICLES: This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

Epigenetic induction of melatonin MT1 receptors by valproate: Neurotherapeutic implications.

We have reported that the anticonvulsant/mood stabilizer and histone deacetylase (HDAC) inhibitor valproate (VPA) induces expression of melatonin receptors both in vitro and in vivo, but the mechanisms involved were not known. Here we show that pharmacological inhibition of CREB, PKC, PI3K, or GSK3ß signaling pathways, which are known targets for VPA, do not prevent its upregulation of melatonin MT1 receptors in rat C6 glioma cells. M344, an HDAC inhibitor unrelated to VPA, mimics the effects of VPA on MT1 expression, whereas valpromide, a VPA derivative lacking HDAC inhibitory activity, does not. Furthermore, VPA, at a concentration which upregulates the MT1 receptor, induces histone H3 hyperacetylation along the length of the MT1 receptor promoter. These results show that an epigenetic mechanism involving histone acetylation underlies induction of MT1 receptor expression by VPA. Given the neuropsychiatric effects of melatonin coupled with evidence that VPA upregulates melatonin receptors in the rat brain, these findings suggest that the melatonergic system contributes to the psychotropic effects of VPA.

Altered melatonin MT1 receptor expression in the ventral midbrain following 6-hydroxydopamine lesions in the rat medial forebrain bundle.

The indoleamine hormone melatonin protects dopamine neurons in the rat nigrostriatal pathway following 6-hydroxydopamine lesioning, and an increase in striatal melatonin levels has been detected in this model of Parkinson's disease. Melatonin induces the expression of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, in the ventral midbrain, where G protein-coupled melatonin receptors are present. Based on the interaction between the melatonergic and dopaminergic systems, we hypothesized that 6-hydroxydopamine-induced degeneration of dopamine neurons would affect the expression of melatonin receptors in the nigrostriatal pathway. Following unilateral injection of 6-hydroxydopamine into the rat striatum or medial forebrain bundle, there was a significant increase in apomorphine-induced contralateral rotations in lesioned animals as compared to sham controls. A loss of tyrosine hydroxylase immunoreactivity and/or immunofluorescence in the striatum and substantia nigra was seen in animals lesioned in either the striatum or medial forebrain bundle, indicating degeneration of dopamine neurons. There were no significant differences in melatonin MT1 receptor protein expression in the striatum or substantia nigra, between intrastriatally lesioned animals and sham controls. In contrast, lesions in the medial forebrain bundle caused a significant increase in MT1 receptor mRNA expression (p<0.03) on the lesioned side of the ventral midbrain, as compared with the contralateral side. Given the presence of MT1 receptors on neurons in the ventral midbrain, these results suggest that a compensatory increase in MT1 transcription occurs to maintain expression of this receptor and neuroprotective melatonergic signaling in the injured brain.

Regional upregulation of hippocampal melatonin MT2 receptors by valproic acid: therapeutic implications for Alzheimer's disease.

We have reported that clinically relevant concentrations of valproic acid (VPA) upregulate the G protein-coupled melatonin MT1 receptor in rat C6 glioma cells, and both MT1 and MT2 receptors in the rat hippocampus. The melatonin MT2 receptor is relatively enriched in the hippocampus, where it is thought to be involved in modulating synaptic plasticity and cognitive function. Importantly, a significant decrease in MT2 expression has been observed in the hippocampus of Alzheimer's patients. Therefore, we examined whether the global upregulation of this receptor (and also the MT1) by VPA, observed in earlier RT-PCR and real time PCR studies, could be localized to more discrete hippocampal regions, which are involved in cognitive function. In situ hybridization of rat brain slices, following chronic VPA treatment (3mg/mL or 4mg/mL in drinking water), revealed a significant upregulation of the MT2 receptor mRNA in the CA1, CA2, CA3 and dentate gyrus (DG) regions of the rat hippocampus. In contrast, the MT1 receptor was not detected in the hippocampus by in situ hybridization. The significant induction of melatonin MT2 receptor expression by VPA in hippocampal regions involved in learning, memory and/or neural stem cell proliferation, suggests that a combinatorial therapeutic strategy involving VPA together with melatonin or other MT2 agonists, would be beneficial in neurodegenerative disorders such as Alzheimer's disease.

[Case report of osteo-odonto keratoprosthesis (Strampelli) and of Dacron keratoprosthesis (Pintucci)]. / Fallbeschreibung einer Osteo-odonto-Keratoprothese (Strampelli) und einer Dacron-fixierten-Keratoprothese (Pintucci).

BACKGROUND: In severe forms of ocular surface disorders keratoprostheses provide the ultimate possibility to restore vision. They are made of an optical cylinder integrated with a supporting biocompatible or biological haptic. CASE REPORT: We report on two patients with different types of keratoprostheses. An 88-year-old woman with ocular pemphigoid received in 1970 a bilateral osteo-odonto-keratoprosthesis (Strampelli). A 59-year-old man with refractory corneal ulcer after corneal grafting received in 2003 a keratoprosthesis with supporting Dacron tissue (Pintucci). RESULTS: The course 35 years after implantation of the osteo-odonto-keratoprosthesis was uneventful. Histologically there were no signs of loosening, rejection or infection. The autologous dentin, which was used for the fixation, was still present. The eye with the Dacron fixated prosthesis (Pintucci) had to be enucleated due to a loosening with endophthalmitis one and a half year after implantation. CONCLUSIONS: Keratoprostheses with autologous fixation often show good long-term results. On the other hand, prostheses with synthetic material are more often complicated by dislocation and inflammation.

Abuse of vasoconstrictive eyedrops mimicking an ocular pemphigoid.

PURPOSE: To describe conjunctival histopathologic alterations induced by excessive chronic astringent use. METHODS: Report of a case with clinical picture, epicutane test results, histologic workup of conjunctival biopsy using conventional staining, and immunohistochemical markers. RESULTS: A 45-year-old man using a phenylephrine preparation hourly for years presented with grotesque eye redness, fornix shortening, and scarring of puncta lacrimalia. Direct and indirect immunofluorescence were negative for ocular pemphigoid. Histology revealed signs of chronic inflammation and neovascularization in the conjunctiva. Symptoms resolved after cessation of therapy. CONCLUSIONS: Chronic abuse of decongestant eyedrops can produce a clinical picture resembling an ocular pemphigoid. Histology suggests that late onset immunoreaction and chronic vasoconstriction cause chronic inflammation and neovascularization, respectively.

Lentiviral-vector-mediated expression of murine IL-1 receptor antagonist or IL-10 reduces the severity of endotoxin-induced uveitis.

Uveitis is a sight threatening inflammatory disorder that remains a significant cause of visual loss. We investigated lentiviral gene delivery of interleukin 1 receptor antagonist (IL-1ra) or interleukin (IL)-10 to ameliorate murine endotoxin-induced uveitis (EIU). An human immunodeficiency virus-1-based vector containing the mIL-1ra or mIL-10 cDNA demonstrated high expression of biologically active cytokine. Following administration of Lenti.GFP into the anterior chamber, transgene expression was observed in corneal endothelial cells, trabecular meshwork and iris cells. To treat EIU, mice were injected with Lenti.IL-1ra, Lenti.IL-10 or a combination of these. EIU was induced 14 days after vector administration and mice were culled 12 h following disease induction. Lenti.IL-1ra or Lenti.IL-10-treated eyes showed significantly lower mean inflammatory cell counts in the anterior and posterior chambers compared with controls. The aqueous total protein content was also significantly lower in treated eyes, demonstrating better preservation of the blood-ocular barrier. Furthermore, the treated eyes showed less in vivo fluorescein leakage from inner retinal vessels compared with controls. The combination of both IL-1ra and IL-10 had no additive effect. Thus, lentiviral gene delivery of IL-1ra or IL-10 significantly reduces the severity of experimental uveitis, suggesting that lentiviral-mediated expression of immunomodulatory genes in the anterior chamber offers an opportunity to treat uveitis.

Glaucoma phenotype in a large Swiss pedigree with the myocilin Gly367Arg mutation.

AIMS: To characterize genotype, phenotype, and age-related penetrance in a Swiss pedigree with juvenile open-angle glaucoma (JOAG). METHODS: In a large Swiss family with history of glaucoma and 82 living members of four generations, we conducted molecular analysis and a detailed phenotype characterization in 52 family members. Mutation analysis was carried out using single-strand conformation polymorphism and DNA sequence analyses of the suspected candidate gene, myocilin (MYOC). RESULTS: We detected a Gly367Arg mutation in the MYOC gene of 13 family members. Nine of them (69.2%) had glaucoma: mean IOP 35.3 mm Hg, range 24-50 mm Hg; mean age at diagnosis 34.9 years, range 28-51 years. Two mutation carriers were glaucoma suspects, one (age 15) was unaffected, and one (age 16) not available for clinical examinations. Age-related glaucoma penetrance was 50% at 30 and 78% at 40. Untreated IOP resulted in rapid disease progression, whereas good IOP control, usually only by means of filtration surgery, could stabilize the disease. None of the wild-type members had glaucoma. CONCLUSIONS: This Swiss family is the largest reported Gly367Arg pedigree to date. The exact genotype and phenotype characterization allowed a reliable risk and prognosis assessment and targeted eye-care planning for the family. The study demonstrates the importance of genetic investigations in glaucoma families, carrying the potential of long-term socio-economic benefits.

[Retinal abnormalities in adult acute myeloid leukemia: semeiological features and prognostic correlations. Analysis of 178 cases]. / Anomalies rétiniennes dans la leucémie myéloïde aiguë de l'adulte: séméiologie et corrélations pronostiques. A propos de 178 cas.

INTRODUCTION: Adult patients with acute myeloid leukemia (AML) frequently present retinal abnormalities. We tried to find a relationship between fundus lesions and treatment responsiveness, prognosis, and several hematologic parameters. PATIENTS AND METHODS: We examined 178 adult patients with newly diagnosed AML. All patients were assigned to two groups regarding retinal parameters (1 or 2) and age (A or B). Group 1 included cases with retinal dysfunction classified as retinal abnormalities with impaired visual acuity; group 2 included cases with no or only minor retinal changes. Subgroup A included patients younger than 60 years (n=97), subgroup B patients older than 60 years (n=81). RESULTS: In this study, higher age and a lower Hb value were associated with retinal findings (group 1). Among the younger patients (subgroup A), 78% of those with complete remission had no retinal findings (group 2) compared to 18% of the nonresponders. In the elderly population (subgroup B), this ratio was 58% versus 19%. In the younger patients (subgroup A), the mean overall survival was 50 months if they had no retinal abnormalities (group 2) and 7 months in the case of retinal changes (group 1). In the older population (subgroup B), the ratio was 15 months versus 3 months, respectively. CONCLUSION: Retinal abnormalities in AML are generally associated with higher age, although they correlate with a shorter survival in both age groups. This association is stronger in younger patients.

The diagnostic yield of vitrectomy specimen analysis in chronic idiopathic endogenous uveitis.

PURPOSE: The low diagnostic yield of vitrectomy specimen analysis in chronic idiopathic uveitis (CIU) has been related to the complex nature of the underlying disease and to methodologic and tissue immanent factors in older studies. In an attempt to evaluate the impact of recently acquired analytic methods, the authors assessed the current diagnostic yield in CIU. METHODS: Retrospective analysis of consecutive vitrectomy specimens from patients with chronic endogenous uveitis (n = 56) in whom extensive systemic workup had not revealed a specific diagnosis (idiopathic) and medical treatment had not resulted in a satisfying clinical situation. Patients with acute postoperative endophthalmitis served a basis for methodologic comparison (Group 2; n = 21). RESULTS: In CIU, a specific diagnosis provided in 17.9% and a specific diagnosis excluded in 21.4%. In 60.7% the laboratory investigations were inconclusive. In postoperative endophthalmitis, microbiological culture established the infectious agent in 47.6%. In six of eight randomly selected cases, eubacterial PCR identified bacterial DNA confirming the culture results in three, remaining negative in two with a positive culture and being positive in three no growth specimens. A double negative result never occurred, suggesting a very high detection rate, when both tests were applied. CONCLUSIONS: The diagnostic yield of vitrectomy specimen analysis has not been improved by currently routinely applied methods in recent years in contrast to the significantly improved sensitivity of combined standardized culture and PCR analysis in endophthalmitis. Consequently, the low diagnostic yield in CIU has to be attributed to insufficient understanding of the underlying pathophysiologic mechanisms.

Vitrectomy for juvenile uveitis: prognostic factors for the long-term functional outcome.

PURPOSE: This study evaluated the long-term effect of pars plana vitrectomy (PPV) in children and adolescents with chronic uveitis on visual function, anatomical outcome, and the requirement of systemic treatment. Further, predictive preoperative factors associated with a beneficial visual outcome were assessed. METHODS: Retrospective review of 29 eyes of 23 consecutive paediatric and juvenile patients below 20 years of age with chronic uveitis who underwent a PPV for visually significant opacities in 25 eyes, vitreous haemorrhage in three eyes, and retinal detachment in one eye. The clinical diagnosis was chronic intermediate uveitis in 22 eyes and retinal vasculitis of different origin in seven eyes. RESULTS: LogMAR visual acuity improved from an average of 0.91 to 0.33 (P<0.001). Cystoid macular oedema (CME) was significantly reduced in eight of 10 eyes postoperatively (P=0.021). In the multiple regression analysis, a low preoperative logMAR visual acuity and the presence of a CME had a negative influence on the final logMAR visual acuity. Furthermore, the appearance of chronic uveitis relapses was significantly reduced from 15 eyes before to seven eyes after surgery (P=0.042). CONCLUSIONS: PPV has a beneficial effect on the course and the complications of chronic uveitis in paediatric and juvenile patients with respect to the anatomical and visual outcome. Preoperative logMAR visual acuity and clinically significant CME were the most accurate predictors for the functional outcome.

[Pars plana vitrectomy for idiopathic vitreous floaters]. / Pars-plana-Vitrektomie bei idiopathischen Glaskörpertrübungen.

BACKGROUND: The aim of this study was to evaluate the role of pars plana vitrectomy (PPV) in patients with persistent vitreous floaters (VF) in phakic (56.7 %) or pseudophakic (43.3 %) eyes. SUBJECTS AND METHODS: A retrospective study of 24 consecutive patients (30 eyes) who underwent a 2-port-PPV using indirect opthalmoscopy between 1992 and 2003 was carried out. Main outcome measures were postoperative visual acuity (PVA), incidence of postoperative complications and patient satisfaction, which has been assessed retrospectively using a detailed questionnaire. RESULTS: Symptoms resolved in all patients. PVA was significantly better (0.91 +/- 0.2 vs. 0.84 +/- 0.2 preoperative visual acuity) or equal in 25 patients (83.3 %). One pseudophakic patient (3.3 %) experienced a retinal detachment 48 months after surgery. In 5 of 17 phakic eyes (35 %) a cataract extraction had to be performed during the follow-up period. All patients were satisfied with their overall visual function. DISCUSSION: This study shows PPV to be a safe and effective primary treatment for visually disturbing VF. In spite of the small number of cases with a lower PVA (5 eyes/16.7 %), which in the most severe case corresponded to a reduction of VA from 1.0 to 0.6 due to a nuclear sclerosis of the lens, all patients were satisfied. As vitreoretinal complications may occur, a critical patient selection and a careful preoperative assessment of specific risks of vitrectomy are mandatory.

Effect of steroidal and non-steroidal drugs on the microglia activation pattern and the course of degeneration in the retinal degeneration slow mouse.

BACKGROUND: In hereditary retinal degeneration, microglia cells become activated, migrate through the outer nuclear layer (ONL) and accumulate in the subretinal space. Although this inflammatory process is not likely to be responsible for the onset of photoreceptor apoptosis, cytotoxic substances secreted by activated microglia could potentially accelerate and perpetuate the degenerative process. Anti-inflammatory drugs have been shown to modulate the microglia response in neurodegenerative disorders and potentially ameliorate the disease progression in various animal model systems. In this study we wanted to test the impact of the most commonly used anti-inflammatory drugs (acetylsalicylate and prednisolone) on the microglia activation pattern, the rate of caspase-3-dependent photoreceptor apoptosis and the course of the degeneration in the retinal degeneration slow (rds) mouse retina. METHODS: 169 pigmented rds mice and 30 CBA wild-type mice were used for this study. The treatment groups were injected daily with either acetylsalicylate (200 mg/kg) or prednisolone (2 mg/kg) i.p. from day 0 up to 3 months. Animals were sacrificed at days 10, 14, 16, 18, 20, 30, 40, 60 and 90. Cryoprotected frozen sections were immunostained with F4/80 and cleaved caspase-3 antibodies. The main outcome measures were the total microglia count in the subretinal space, the total cleaved caspase-3-positive cells in the ONL and the averaged number of photoreceptor rows in the midperipheral retina. RESULTS: Neither acetylsalicylate nor prednisolone reduced subretinal microglia accumulation in the rds mouse degeneration model. Moreover, they aggravated migration and accumulation in the early time course. The apoptotic cascade started earlier and was more pronounced in both treatment groups compared to the control group. The pace of retinal degeneration was not reduced in the treatment groups compared to the untreated control. In contrast, acetylsalicylate did significantly accelerate the photoreceptor cell degeneration in comparison to the prednisolone (p < 0.001) and to the control group (p < 0.001). CONCLUSIONS: Acetylsalicylate and prednisolone do not decrease the microglia response in the rds mouse and are not neuroprotective. More research is needed to clarify the molecular mechanisms which lead to photoreceptor cell death and to elucidate the complex role of microglia in inherited retinal degeneration.

[Capsular bag distention after implantation of foldable acrylic lenses]. / Hydrops des Kapselsacks nach Implantation faltbarer Acryllinsen.

BACKGROUND: Incomplete removal of the viscoelastic material during cataract surgery may lead to the early postoperative capsular block syndrome. In this retrospective case series, postoperative capsular bag distention after implantation of a foldable acrylic lens is reported and the sequelae are analyzed. PATIENTS AND METHOD: In a retrospective case series, 10 eyes of altogether 1674 eyes that underwent cataract surgery in the years 2001 and 2002 were identified with early postoperative capsular block syndrome. All 10 eyes had cataract removal with continuous curvilinear capsulorhexis, phacoemulsification and a foldable acrylic lens was placed in the capsular bag. In 4 of 10 eyes cataract extraction was combined with trabeculectomy. All patients had posterior Nd:YAG capsulotomy. Refraction was performed before and after capsulotomy. RESULTS: Postoperative examination showed capsular bag distention in all eyes. The mean postoperative spherical equivalent was -3.9 +/- 1.6 Dpt. As the target refraction was -1.2 +/- 1.0 Dpt there was an average of -2.7 +/- 1.4 Dpt of induced myopia. A mild increase in postoperative anterior chamber inflammation was noted only in 1 patient and shallowing of the anterior chamber was observed in another patient. Posterior Nd:YAG capsulotomy was performed 4.5 +/- 4.3 months after surgery. The mean refractive shift after Nd:YAG capsulotomy was 1.9 +/- 1.9 Dpt. CONCLUSIONS: Unexpected postoperative myopic correction was the main manifestation of capsular block syndrome in this series. Intraoperative use of miotics prohibiting optical control of complete viscoelastic removal may explain the high proportion of combined procedures. In the case of unexpected postoperative myopia the position of the intraocular lens within the bag should be checked and attention should be paid to possibly incompletely removed viscoelastic material.

[Filtering glaucoma surgery as outpatient procedure]. / Ambulante filtrierende Glaukomoperationen.

BACKGROUND: While cataract surgery is nowadays performed routinely as an outpatient procedure, performing filtering glaucoma surgery under these conditions remains questionable due to the more demanding perioperative management. PATIENTS AND METHODS: Outpatient filtering glaucoma surgery (trabeculectomy and combined phakoemulsification and trabeculectomy (phakotrab)) is performed at the Ophthalmology Department of Kantonsspital Winterthur when requested by the patient. This paper provides a retrospective review of all outpatient filtering glaucoma procedures performed in the last three and a half years. RESULTS: Forty-six filtering procedures (21 trabeculectomies and 25 phakotrabs) were performed in 45 eyes of 40 patients (50 - 84 years) as outpatient procedures. Mitomycin C was administered in 16/21 trabeculectomies and in 7/25 combined procedures. In the trabeculectomy group, intraocular pressure (IOP) was surgically lowered from 23.3 +/- 7.2 mm Hg (under 2.4 +/- 0.8 IOP-lowering medications) to 12.7 +/- 3.5 mm Hg (20/21 patients without medication). In the combined group, IOP was lowered from 20.8 +/- 6.3 (under 2.0 +/- 0.7 medications) to 13.7 +/- 2.7 mm Hg (only 5/25 patients still requiring IOP-lowering medications). In the latter group, the best corrected visual acuity was below 20/40 only in 2 eyes due to advanced glaucomatous optic atrophy. One patient developed relative intraocular hypotony (IOP 6 mm Hg), one patient required needling + 5-fluorouracil injection and one patient required surgical revision of the trabeculectomy after 14 months. DISCUSSION: Adequate patient selection and refined surgical technique (tight wound closure and releasable sutures or argon laser suturolysis) allow performing filtering glaucoma surgery as an outpatient procedure. Extended post-operative care during the first 2 months is the key for IOP-lowering success.

[Progress in somatic gene therapy of retinal degeneration in the animal model]. / Fortschritte in der somatischen Gentherapie von Netzhautdegenerationen am Tiermodell.

More than 60 genes responsible for human retinal dystrophies have already been identified. Most of them are either expressed in the photoreceptor or in the retinal pigment epithelium (RPE). Therefore these cells have become the target of new therapeutical strategies on a molecular level. The most promising approach at present is somatic gene therapy, which has been developed over the last years and the principle has now been established in animal models. For gene therapy of inherited retinal degeneration, as for gene therapy in general, gene transfer has to be proven to be not only efficient but also safe. This has recently been achieved using the adeno-associated virus (AAV) as a vector to express a therapeutic gene within the photoreceptor cell. It could be demonstrated in mouse and dog models of retinal degeneration that expression of the therapeutic transgene leads to anatomical and functional restitution of degenerating photoreceptors. A significant immune response to AAV has not been detected so far. In this paper the recent success of gene therapy of retinal degeneration in animal models is reviewed.

Kinetics of transgene expression in mouse retina following sub-retinal injection of recombinant adeno-associated virus.

Using confocal microscopy we have examined in detail the temporal and spatial pattern of green fluorescent protein expression following sub-retinal injection of recombinant adeno-associated virus (rAAV) in the mouse and have determined the effect of viral titre on the number and type of cells transduced. Our results suggest that some transgene expression occurs as early as three days after injection, and that transgene expression occurs beyond the area of retinal detachment. Vector titre appears to have a substantial effect on both transduction efficiency and the speed of onset of photoreceptor cell transduction. Our data suggests that we have not yet reached the limits of photoreceptor transduction efficiency using AAV vectors. An increase in titre could still lead to an improved transduction efficiency and faster onset of photoreceptor transduction. We failed to detect transfected cones even in areas where nearly 100% of the rods were transduced, but we found efficient and sustained RPE transduction.

Gene replacement therapy in the retinal degeneration slow (rds) mouse: the effect on retinal degeneration following partial transduction of the retina.

The retinal degeneration slow (rds or Prph2(Rd2/Rd2)) mouse, a model of recessive retinitis pigmentosa, lacks a functional gene encoding peripherin 2. This membrane glycoprotein is required for the formation of photoreceptor outer segment discs. The striking feature of the rds mouse is the complete failure to develop outer segments. We have previously examined the short-term effect of gene replacement therapy using an adeno-associated (AAV) vector and demonstrated induction of outer segments and improvement of photoreceptor function. Here we have extended our analysis and have demonstrated that the potential for ultrastructural improvement is dependent upon the age at which animals are treated, but the effect of a single injection on photoreceptor ultrastructure may be long-term. However, there was no significant effect on photoreceptor cell loss, irrespective of the date of administration, despite the improvements in morphology and function. Our investigation excluded procedure-related damage, vector toxicity and immune responses as major factors which might counteract the benefits of photoreceptor restoration, but suggested that transgene over-expression is of significance. These findings suggest that successful gene therapy in patients with photoreceptor defects may ultimately depend upon intervention in early stages of disease and upon accurate control of transgene expression.