Quality improvement strategies for organizational change: a multiphase observational study to increase insight into nonparticipating organizations.

BACKGROUND: The scope of implementation research is often restricted to the analysis of organizations that participate voluntarily in implementation interventions. The recruitment of participants for a quality improvement collaborative increases awareness of the specific innovation. The objective of this multiphase observational study was to identify differences between organizations that participated in a large-scale implementation project aiming to improve perioperative care, functional recovery, and length of hospital stay after gynecologic surgery and organizations that did not participate. A secondary objective was to explore how perioperative practice changed among nonparticipants. METHODS: Of the seven gynecology departments of nonparticipating Dutch hospitals, five agreed to participate in a retrospective analysis. Baseline data of participating hospitals' (N = 19) characteristics, time to functional recovery, and length of hospital stay were compared. Outcome measures for the subsequent pre-post awareness study in the five nonparticipating hospitals were: (1) overall adherence to predefined evidence-based perioperative elements; and (2) change in functional recovery and length of hospital stay. Multivariable regression models, adjusted for baseline characteristics, were used for analysis. RESULTS: In retrospect, nonparticipating and participating hospitals did not differ in baseline characteristics, functional recovery, and length of hospital stay. In three of the five nonparticipating hospitals, adherence to the selected evidence-based perioperative elements increased significantly after awareness of the trial (overall mean difference 9.7%, 95% CI 6.9 to 12.5%, p <  0.001). Linear regression models revealed no statistically significant or clinically relevant differences in time to functional recovery (mean difference - 0.2 days, 95% CI -0.7 to 0.2, p = 0.319) or length of hospital stay (mean difference - 0.4 days, 95% CI -1.3 to 0.5, p = 0.419) in the nonparticipating hospitals. None of these hospitals managed to reduce time to functional recovery or length of hospital stay significantly. CONCLUSIONS: No differences in perioperative outcomes between the nonparticipating and participating hospitals were identified at baseline. Despite the statistically significant improvement in overall evidence-based perioperative care, the awareness raised by recruitment activities alone was not enough to reduce time to functional recovery and length of hospital stay in nonparticipating hospitals. Insight into the trends of nonparticipants is valuable to existing implementation effectiveness research.

Interdepartmental Spread of Innovations: A Multicentre Study of the Enhanced Recovery After Surgery Programme.

BACKGROUND: Spread of evidence-based innovations beyond pioneering settings is essential to improve quality of care. This study aimed to evaluate the influence of a national project to implement 'Enhanced Recovery After Surgery' (ERAS) among colorectal teams on the spread of this innovation to gynaecological procedures. METHODS: A retrospective observational multicentre study was performed of a consecutive sample of patients who underwent major elective gynaecological surgery in 2012-2013. Ten Dutch hospitals (294 patients) had participated in a colorectal breakthrough project implementing ERAS on a nationwide basis and were assigned to the intervention group. Thirteen hospitals (390 patients) that had not participated in this project acted as controls. Outcome measures were time to functional recovery and total length of postoperative hospital stay. Multilevel models adjusted for clustering and baseline demographics were used for analysis. The uptake of ten selected perioperative care elements was evaluated for each hospital. RESULTS: The estimated mean difference (95% confidence interval) between the intervention and control hospitals was -0.3 (-0.9 to 0.3) days in the time to recovery and 0.2 (-0.8 to 1.3) days in the total length of hospital stay. The mean (± standard deviation) absolute rate of implemented perioperative care elements per hospital was 28.9 ± 14.9% in the control, versus 29.3 ± 11.1% in the intervention group (p = 0.934). CONCLUSION: Initial implementation effects seem to be restricted to the participating teams and do not automatically spread to other surgical teams in the same hospital.

Enhanced recovery pathways in abdominal gynecologic surgery: a systematic review and meta-analysis.

INTRODUCTION: Enhanced recovery pathways have been widely accepted and implemented for different types of surgery. Their overall effect in abdominal gynecologic surgery is still underdetermined. A systematic review and meta-analysis were performed to provide an overview of current evidence and to examine their effect on postoperative outcomes in women undergoing open gynecologic surgery. MATERIAL AND METHODS: Searches were conducted using Embase, Medline, CINAHL, and the Cochrane Library up to 27 June 2014. Reference lists were screened to identify additional studies. Studies were included if at least four individual items of an enhanced recovery pathway were described. Outcomes included length of hospital stay, complication rates, readmissions, and mortality. Quantitative analysis was limited to comparative studies. Effect sizes were presented as relative risks or as mean differences (MD) with 95% confidence intervals (CI). RESULTS: Thirty-one records, involving 16 observational studies, were included. Diversity in reported elements within studies was observed. Preoperative education, early oral intake, and early mobilization were included in all pathways. Five studies, with a high risk of bias, were eligible for quantitative analysis. Enhanced recovery pathways reduced primary (MD -1.57 days, 95% CI CI -2.94 to -0.20) and total (MD -3.05 days, 95% CI -4.87 to -1.23) length of hospital stay compared with traditional perioperative care, without an increase in complications, mortality or readmission rates. CONCLUSION: The available evidence based on a broad range of non-randomized studies at high risk of bias suggests that enhanced recovery pathways may reduce length of postoperative hospital stay in abdominal gynecologic surgery.

Sustainability of professionals' adherence to clinical practice guidelines in medical care: a systematic review.

OBJECTIVES: To evaluate (1) the state of the art in sustainability research and (2) the outcomes of professionals' adherence to guideline recommendations in medical practice. DESIGN: Systematic review. DATA SOURCES: Searches were conducted until August 2015 in MEDLINE, CINAHL, EMBASE, Cochrane Central Register of Controlled Trials (CENTRAL) and the Guidelines International Network (GIN) library. A snowball strategy, in which reference sections of other reviews and of included papers were searched, was used to identify additional papers. ELIGIBILITY CRITERIA: Studies needed to be focused on sustainability and on professionals' adherence to clinical practice guidelines in medical care. Studies had to include at least 2 measurements: 1 before (PRE) or immediately after implementation (EARLY POST) and 1 measurement longer than 1 year after active implementation (LATE POST). RESULTS: The search retrieved 4219 items, of which 14 studies met the inclusion criteria, involving 18 sustainability evaluations. The mean timeframe between the end of active implementation and the sustainability evaluation was 2.6 years (minimum 1.5-maximum 7.0). The studies were heterogeneous with respect to their methodology. Sustainability was considered to be successful if performance in terms of professionals' adherence was fully maintained in the late postimplementation phase. Long-term sustainability of professionals' adherence was reported in 7 out of 18 evaluations, adherence was not sustained in 6 evaluations, 4 evaluations showed mixed sustainability results and in 1 evaluation it was unclear whether the professional adherence was sustained. CONCLUSIONS: (2) Professionals' adherence to a clinical practice guideline in medical care decreased after more than 1 year after implementation in about half of the cases. (1) Owing to the limited number of studies, the absence of a uniform definition, the high risk of bias, and the mixed results of studies, no firm conclusion about the sustainability of professionals' adherence to guidelines in medical practice can be drawn.

Diffusion of Enhanced Recovery principles in gynecologic oncology surgery: is active implementation still necessary?

OBJECTIVE: Spontaneous diffusion of the evidence-based Enhanced Recovery After Surgery (ERAS) program from an early adopter department (colorectal surgery) to other closely related departments (gynecologic surgery) within the same hospital could be expected. Given this diffusion hypothesis, this quality improvement study examines the value of active implementation of ERAS in addition to spontaneous diffusion. METHODS: A nonrandomized, pre-post intervention study was conducted at a tertiary referral hospital. Prospective data of consecutive patients who underwent abdominal surgery between March, 2010 and March, 2011 for gynecologic malignancies were collected and compared with those of a historical cohort of patients treated before the structured implementation of ERAS by an expert team. Outcomes were length of hospital stay, length of functional recovery, and compliance to protocol care elements. RESULTS: Seventy-seven patients treated after structured implementation of ERAS were compared with 38 patients included in the historical cohort. Most women had surgery for ovarian or endometrial cancer (48% and 37% respectively). Postoperative care mostly lacked ERAS elements and needed to be actively implemented. With structured implementation, a reduced time to functional recovery (median 3 versus 6 days, p<0.001) and a shorter length of hospital stay (5 versus 7 days, p<0.001) were achieved. CONCLUSIONS: After several years of practicing ERAS in colorectal surgery, spontaneous spread of ERAS principles to gynecologic oncology surgery occurred partially. The results of this study underscore the need for a structured and supported pro-active process to implement the ERAS program in a complete and successful way.

Long-term efficacy of ciliary muscle gene transfer of three sFlt-1 variants in a rat model of laser-induced choroidal neovascularization.

Inhibition of vascular endothelial growth factor (VEGF) has become the standard of care for patients presenting with wet age-related macular degeneration. However, monthly intravitreal injections are required for optimal efficacy. We have previously shown that electroporation enabled ciliary muscle gene transfer results in sustained protein secretion into the vitreous for up to 9 months. Here, we evaluated the long-term efficacy of ciliary muscle gene transfer of three soluble VEGF receptor-1 (sFlt-1) variants in a rat model of laser-induced choroidal neovascularization (CNV). All three sFlt-1 variants significantly diminished vascular leakage and neovascularization as measured by fluorescein angiography (FA) and flatmount choroid at 3 weeks. FA and infracyanine angiography demonstrated that inhibition of CNV was maintained for up to 6 months after gene transfer of the two shortest sFlt-1 variants. Throughout, clinical efficacy was correlated with sustained VEGF neutralization in the ocular media. Interestingly, treatment with sFlt-1 induced a 50% downregulation of VEGF messenger RNA levels in the retinal pigment epithelium and the choroid. We demonstrate for the first time that non-viral gene transfer can achieve a long-term reduction of VEGF levels and efficacy in the treatment of CNV.

Non-viral gene therapy for GDNF production in RCS rat: the crucial role of the plasmid dose.

Glial cell line-derived neurotrophic factor (GDNF) is one of the candidate molecules among neurotrophic factors proposed for a potential treatment of retinitis pigmentosa (RP). It must be administered repeatedly or through sustained releasing systems to exert prolonged neuroprotective effects. In the dystrophic Royal College of Surgeon's (RCS) rat model of RP, we found that endogenous GDNF levels dropped during retinal degeneration time course, opening a therapeutic window for GDNF supplementation. We showed that after a single electrotransfer of 30 µg of GDNF-encoding plasmid in the rat ciliary muscle, GDNF was produced for at least 7 months. Morphometric, electroretinographic and optokinetic analyses highlighted that this continuous release of GDNF delayed photoreceptors (PRs) as well as retinal functions loss until at least 70 days of age in RCS rats. Unexpectedly, increasing the GDNF secretion level accelerated PR degeneration and the loss of electrophysiological responses. This is the first report: (i) demonstrating the efficacy of GDNF delivery through non-viral gene therapy in RP; (ii) establishing the efficacy of intravitreal administration of GDNF in RP associated with a mutation in the retinal pigment epithelium; and (iii) warning against potential toxic effects of GDNF within the eye/retina.

The outer limiting membrane (OLM) revisited: clinical implications.

PURPOSE: The outer limiting membrane (OLM) is considered to play a role in maintaining the structure of the retina through mechanical strength. However, the observation of junction proteins located at the OLM and its barrier permeability properties may suggest that the OLM may be part of the retinal barrier. MATERIAL AND METHODS: Normal and diabetic rat, monkey, and human retinas were used to analyze junction proteins at the OLM. Proteome analyses were performed using immunohistochemistry on sections and flat-mounted retinas and western blotting on protein extracts obtained from laser microdissection of the photoreceptor layers. Semi-thin and ultrastructure analyses were also reported. RESULTS: In the rat retina, in the subapical region zonula occludens-1 (ZO-1), junction adhesion molecule (JAM), an atypical protein kinase C, is present and the OLM shows dense labeling of occludin, JAM, and ZO-1. The presence of occludin has been confirmed using western blot analysis of the microdissected OLM region. In diabetic rats, occludin expression is decreased and glial cells junctions are dissociated. In the monkey retina, occludin, JAM, and ZO-1 are also found in the OLM. Junction proteins have a specific distribution around cone photoreceptors and Müller glia. Ultrastructural analyses suggest that structures like tight junctions may exist between retinal glial Müller cells and photoreceptors. CONCLUSIONS: In the OLM, heterotypic junctions contain proteins from both adherent and tight junctions. Their structure suggests that tight junctions may exist in the OLM. Occludin is present in the OLM of the rat and monkey retina and it is decreased in diabetes. The OLM should be considered as part of the retinal barrier that can be disrupted in pathological conditions contributing to fluid accumulation in the macula.

Toxoplasma gondii: flat-mounting of retina as a new tool for the observation of ocular infection in mice.

Ocular toxoplasmosis is the principal cause of posterior uveitis and a leading cause of blindness. Animal models are required to improve our understanding of the pathogenesis of this disease. The method currently used for the detection of retinal cysts in animals involves the observation, under a microscope, of all the sections from infected eyes. However, this method is time-consuming and lacks sensitivity. We have developed a rapid, sensitive method for observing retinal cysts in mice infected with Toxoplasma gondii. This method involves combining the flat-mounting of retina - a compromise between macroscopic observation and global analysis of this tissue - and the use of an avirulent recombinant strain of T. gondii expressing the Escherichia coli beta-galactosidase gene, visually detectable at the submacroscopic level. Single cyst unilateral infection was found in six out of 17 mice killed within 28 days of infection, whereas a bilateral infection was found in only one mouse. There was no correlation between brain cysts number and ocular infection.

Influence of age on retinochoroidal healing processes after argon photocoagulation in C57bl/6j mice.

PURPOSE: To analyze the influence of age on retinochoroidal wound healing processes and on glial growth factor and cytokine mRNA expression profiles observed after argon laser photocoagulation. METHODS: A cellular and morphometric study was performed that used 44 C57Bl/6J mice: 4-week-old mice (group I, n=8), 6-week-old mice (group II, n=8), 10-12-week-old mice (group III, n=14), and 1-year-old mice (group IV, n=14). All mice in these groups underwent a standard argon laser photocoagulation (50 microm, 400 mW, 0.05 s). Two separated lesions were created in each retina using a slit lamp delivery system. At 1, 3, 7, 14, 60 days, and 4 months after photocoagulation, mice from each of the four groups were sacrificed by carbon dioxide inhalation. Groups III and IV were also studied at 6, 7, and 8 months after photocoagulation. At each time point the enucleated eyes were either mounted in Tissue Tek (OCT), snap frozen and processed for immunohistochemistry or either flat mounted (left eyes of groups III and IV). To determine, by RT-PCR, the time course of glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and monocyte chemotactic protein-1 (MCP-1) gene expression, we delivered ten laser burns (50 microm, 400 mW, 0.05 s) to each retina in 10-12-week-old mice (group III', n=10) and 1-year-old mice (group IV', n=10). Animals from Groups III' and IV' had the same age than those from Groups III and IV, but they received ten laser impacts in each eye and served for the molecular analysis. Mice from Groups III and IV received only two laser impacts per eye and served for the cellular and morphologic study. Retinal and choroidal tissues from these treated mice were collected at 16 h, and 1, 2, 3, and 7 days after photocoagulation. Two mice of each group did not receive photocoagulation and were used as controls. RESULTS: In the cellular and morphologic study, the resultant retinal pigment epithelium interruption expanse was significantly different between the four groups. It was more concise and smaller in the oldest group IV (112.1 microm+/-11.4 versus 219.1 microm+/-12.2 in group III) p<0.0001 between groups III and IV. By contrast, while choroidal neovascularization (CNV) was mild and not readily identifiable in group I, at all time points studied, CNV was more prominent in the (1-year-old mice) Group IV than in the other groups. For instance, up to 14 days after photocoagulation, CNV reaction was statistically larger in group IV than in group III ((p=0.0049 between groups III and IV on slide sections and p<0.0001 between the same groups on flat mounts). Moreover, four months after photocoagulation, the CNV area (on slide sections) was 1,282 microm(2)+/-90 for group III and 2,999 microm(2)+/-115 for group IV (p<0.0001 between groups III and IV). Accordingly, GFAP, VEGF, and MCP-1 mRNA expression profiles, determined by RT-PCR at 16 h, 1, 2, 3, and 7 days postphotocoagulation, were modified with aging. In 1-year-old mice (group IV), GFAP mRNA expression was already significantly higher than in the younger (10-12 week) group III before photocoagulation. After laser burns, GFAP mRNA expression peaked at 16-24 h and on day 7, decreasing thereafter. VEGF mRNA expression was markedly increased after photocoagulation in old mice eyes, reaching 2.7 times its basal level at day 3, while it was only slightly increased in young mice (1.3 times its level in untreated young mice 3 days postphotocoagulation). At all time points after photocoagulation, MCP-1 mRNA expression was elevated in old mice, reaching high levels of expression at 16 h and day 3 respectively. CONCLUSIONS: Our results were based on the study of four different age groups and included not only data from morphological observations but also from a molecular analysis of the various alterations of cytokine signaling and expression. One-year-old mice demonstrated more extensive CNV formation and a slower pace of regression after laser photocoagulation than younger mice. These were accompanied by differences in growth factors and cytokine expression profiles indicate that aging is a factor that aggravates CNV. The above results may provide some insight into possible therapeutic strategies in the future.

PlGF-1 and VEGFR-1 pathway regulation of the external epithelial hemato-ocular barrier. A model for retinal edema.

VEGF is considered as an important factor in the pathogenesis of macular edema. VEGF induces the rupture of the blood retinal barrier and may also influence the retinal pigment epithelial (RPE) outer retinal barrier. The aim of this work was to analyze the influence of the VEGF receptor pathways in the modulation of the RPE barrier breakdown in vitro and in vivo. The ARPE19 human junctions in culture are modulated by VEGF through VEGFR-1 but not through VEGFR-2. PlGF-1, that is a pure agonist of VEGFR-1, is produced in ARPE-19 cells under hypoxic conditions and mimics VEGF effects on the external retinal barrier as measured by TER and inulin flux. In vivo, the intravitreous injection of PlGF-1 induces a rupture of the external retinal barrier together with a retinal edema. This effect is reversible within 4 days. VEGF-E, that is a pure agonist of VEGFR-2, does not induce any acute effect on the RPE barrier. These results demonstrate that PlGF-1 can reproduce alterations of the RPE barrier occurring during diabetic retinopathy.

[Distribution of transferrin and transferrin receptor of the type 1 in the process of formation of the rat eye retina in early postnatal ontogenesis].

By the method of indirect immunohistochemistry, distribution of transferrin and of transferrin receptor of the type (TFR1) was studied in the formed rat eye retina at the period of early postnatal ontogenesis (from birth to opening of eyelids). It has been established that the character of distribution of these proteins and intensity of specific staining change dependent on the retina formation stage. Retina of the newborn rat is characterized by diffuse transferrin distribution in nuclear retina layers (in the neuroblast layer--NBL) and in the ganglionic cell layer (GCL) as well as in the eye pigment epithelium (PE); relative immunoreactivity to transferrin is not high. At the 5th postnatal day, immunoreactivity to transferrin is maximal and is revealed both in nuclear and in plexiform layers of retina and in the eye PE, the greatest signal being characteristic of NBL. At the 10th postnatal day the transferrin signal intensity in retina decreases, specific staining is revealed in GCL, PE, and in the area of formed outer segments of photoreceptors. At the 15th postnatal day, transferrin is revealed in GCL, in outer and inner photoreceptor segments and in the eye PE. TFR1 is present in all retinal layers at all stages of the retina formation; the relative immunoreactivity to TFR1 sharply rises beginning from the 10th postnatal day; correlation between distribution of transferrin and TFR1 is detected in the entire retina of newborn rats as well as in the external retina area at subsequent stages of its development. A possible role of transferrin at various stages of formation of retinal is discussed.

Influence of triamcinolone intravitreal injection on retinochoroidal healing processes.

To analyze the effects of triamcinolone intravitreal injection on the wound healing processes after argon laser retinal photocoagulation, wild type C57BL/6J mice, 8-12 weeks old underwent a standard argon laser photocoagulation protocol. After pentobarbital anesthesia and pupil dilatation, argon laser lesions were induced (50microm, 400mW, 0.05s). Two photocoagulation impacts created two disc diameters from the optic nerve in both eyes. The photocoagulated mice were divided into four groups: Group I (n=12), photocoagulation controls, did not receive any intravitreous injection. Group II (n=12), received an intravitreous injection of 1microl of balanced salt solution (BSS). Group III (n=12), received an intravitreous injection of 1microl containing 15microg of triamcinolone acetonide (TAAC) in BSS. Two mice from each of these three groups were sacrificed at 1, 3, 7, 14 days and 2 and 4 months after photocoagulation. Group IV (n=10) received 1.5, 3, 7.5, 15, or 30microg of TAAC and were all sacrificed on day 14. The enucleated eyes were subjected to systematic analysis of the cellular remodeling processes taking place within the laser lesion and its vicinity. To this purpose, specific antibodies against GFAP, von Willebrand factor, F4/80 and KI67 were used for the detection of astrocytes, activated Müller cells, vascular endothelial cells, infiltrating inflammatory cells and actively proliferating cells. TUNEL reaction was also carried out along with nuclear DAPI staining. Temporal and spatial observations of the created photocoagulation lesions demonstrate that 24h following the argon laser beam, a localized and well-delineated affection of the RPE cells and choroid is observed in mice in Groups I and II. The inner retinal layers in these mice eyes are preserved while TUNEL positive (apoptotic) cells are observed at the retinal outer nuclear layer level. At this stage, intense staining with GFAP is associated with activated retinal astrocytes and Müller cells throughout the laser path. From day 3 after photocoagulation, dilated new choroidal capillaries are detected on the edges of the laser lesion. These processes are accompanied by infiltration of inflammatory cells and the presence of proliferating cells within the lesion site. Mice in Group III treated with 15microg/mul of triamcinolone showed a decreased number of infiltrating inflammatory cells and proliferating cells, which was not statistically significant compared to uninjected laser treated controls. The development of new choroidal capillaries on the edges of the laser lesion was also inhibited during the first 2 months after photocoagulation. However, on month 4 the growth of new vessels was observed in these mice treated with TAAC. Mice of Group IV did not show any development of new capillaries even with small doses. After argon laser photocoagulation of the mouse eye, intravitreal injection of triamcinolone markedly influenced the retina and choroid remodeling and healing processes. Triamcinolone is a powerful inhibitor of the formation of neovessels in this model. However, this inhibition is transient. These observations should provide a practical insight for the mode of TAAC use in patients with wet AMD.

Placental growth factor-1 and epithelial haemato-retinal barrier breakdown: potential implication in the pathogenesis of diabetic retinopathy.

AIMS/HYPOTHESIS: Disruption of the retinal pigment epithelial (RPE) barrier contributes to sub-retinal fluid and retinal oedema as observed in diabetic retinopathy. High placental growth factor (PLGF) vitreous levels have been found in diabetic patients. This work aimed to elucidate the influence of PLGF-1 on a human RPE cell line (ARPE-19) barrier in vitro and on normal rat eyes in vivo. METHODS: ARPE-19 permeability was measured using transepithelial resistance and inulin flux under stimulation of PLGF-1, vascular endothelial growth factor (VEGF)-E and VEGF 165. Using RT-PCR, we evaluated the effect of hypoxic conditions or insulin on transepithelial resistance and on PLGF-1 and VEGF receptors. The involvement of mitogen-activated protein kinase (MEK, also known as MAPK)/extracellular signal-regulated kinase (ERK, also known as EPHB2) signalling pathways under PLGF-1 stimulation was evaluated by western blot analysis and specific inhibitors. The effect of PLGF-1 on the external haemato-retinal barrier was evaluated after intravitreous injection of PLGF-1 in the rat eye; evaluation was by semi-thin analysis and zonula occludens-1 immunolocalisation on flat-mounted RPE. RESULTS: In vitro, PLGF-1 induced a reversible decrease of transepithelial resistance and enhanced tritiated inulin flux. These effects were specifically abolished by an antisense oligonucleotide directed at VEGF receptor 1. Exposure of ARPE-19 cells to hypoxic conditions or to insulin induced an upregulation of PLGF-1 expression along with increased transcellular permeability. The PLGF-1-induced RPE cell permeability involved the MEK signalling pathway. Injection of PLGF-1 in the rat eye vitreous induced an opening of the RPE tight junctions with subsequent sub-retinal fluid accumulation, retinal oedema and cytoplasm translocation of junction proteins. CONCLUSIONS/INTERPRETATION: Our results indicate that PLGF-1 may be a potential regulation target for the control of diabetic retinal and macular oedema.

The role of PKCzeta in NMDA-induced retinal ganglion cell death: prevention by aspirin.

Intravitreal NMDA injection has been shown to induce the excitotoxic loss of retinal cells. The retinal ganglion cell apoptosis induced by NMDA is thought to play an important role in retinal ischemia injury and NMDA-injected rat has been used as a model of neuronal loss in diseases such as glaucoma. In this experimental model, we studied the early effects of NMDA leading to the degeneration of retinal ganglion cells. PKCzeta regulates the NF-kappaB pathway in cellular responses to various stresses and we have shown that aspirin inhibits purified human PKCzeta. We therefore investigated the molecular mechanism by which retinal cells limit ocular injury following NMDA treatment. We found that the NMDA-induced apoptosis of ganglion cells was mediated, at least partly, by PKCzeta. This enzyme was activated early in the cellular response to NMDA. Prolonged activation was followed by PKCzeta cleavage, and nuclear translocation of the C-terminal region of this protein-a critical event for the survival of retinal cells. We also found that pretreatment with aspirin or the coinjection of NMDA with a specific PKCzeta inhibitor counteracted the effects of NMDA. These findings provide new insight into the role played by PKCzeta in neuronal loss in glaucoma.

Krypton laser photocoagulation induces retinal vascular remodeling rather than choroidal neovascularization.

The purpose of this study is to analyze the retina and choroid response following krypton laser photocoagulation. Ninety-two C57BL6/Sev129 and 32 C57BL/6J, 5-6-week-old mice received one single krypton (630 nm) laser lesion: 50 microm, 0.05 s, 400 mW. On the following day, every day thereafter for 1 week and every 2-3 days for the following 3 weeks, serial sections throughout the lesion were systematically collected and studied. Immunohistology using specific markers or antibodies for glial fibrillary acidic protein (GFAP) (astrocytes, glia and Muller's cells), von Willebrand (vW) (vascular endothelial cells), TUNEL (cells undergoing caspase dependent apoptosis), PCNA (proliferating cell nuclear antigen) p36, CD4 and F4/80 (infiltrating inflammatory and T cells), DAPI (cell nuclei) and routine histology were carried out. Laser confocal microscopy was also performed on flat mounts. Temporal and spatial observations of the created photocoagulation lesions demonstrate that, after a few hours, activated glial cells within the retinal path of the laser beam express GFAP. After 48 h, GFAP-positive staining was also detected within the choroid lesion center. "Movement" of this GFAP-positive expression towards the lasered choroid was preceded by a well-demarcated and localized apoptosis of the retina outer nuclear layer cells within the laser beam path. Later, death of retinal outer nuclear cells and layer thinning at this site was followed by evagination of the inner nuclear retinal layer. Funneling of the entire inner nuclear and the thinned outer nuclear layers into the choroid lesion center was accompanied by "dragging" of the retinal capillaries. Thus, from days 10 to 14 after krypton laser photocoagulation onward, well-formed blood capillaries (of retinal origin) were observed within the lesion. Only a few of the vW-positive capillary endothelial cells stained also for PCNA p36. In the choroid, dilatation of the vascular bed occurred at the vicinity of the photocoagulation site and around it. Confocal microscopy demonstrates that the vessels throughout the path lesion are located within the neuroretina while in the choroid (after separation of the neural retina) only GFAP-positive but no lectin-positive cells can be seen. The involvement of infiltrating inflammatory cells in these remodeling and healing processes remained minimal throughout the study period. During the 4 weeks following krypton laser photocoagulation in the mouse eye, processes of wound healing and remodeling appear to be driven by cells (and vessels) originating from the retina.

High-resolution ultrasonography of subretinal structure and assessment of retina degeneration in rat.

The purpose of this work was to evaluate the ability of 80 MHz ultrasonography to differentiate intra-retinal layers and quantitatively assess photoreceptor dystrophy in small animal models. Four groups of 10 RCS rats each (five dystrophic and five controls) were explored at 25, 35, 45 and 55 days post-natal (PN). A series of retina cross-sections were obtained ex vivo from outside intact eyes using an 80 MHz three-dimensional ultrasound backscatter microscope (20-microm-axial resolution). Ultrasound features of normal retina were correlated to those of corresponding histology and thickness measurements of photoreceptor segment and nuclear layers were performed on all groups. To show the ability of 80 MHz ultrasonography to distinguish the retinal degeneration in vivo, one RCS rat was explored at 25 and 55 days post-natal. Ultrasound image of normal retina displayed four distinct layers marked by reflections at neurites/nuclei interfaces and permitted to differentiate the photoreceptor segment and nuclear layers. The backscatter level from the retina was shown to be related to the size, density and organization of the intra-layer structure. Ultrasound thickness measurements highly correlated with histologic measurements. A thinning (p<0.05) of outer nuclear layer (ONL) was detected over time for controls and was thought to be assigned to retina maturation. Retinal degeneration started at PN35 and resulted in a more pronounced ONL thinning (p<0.05) over time. ONL degeneration was accompanied by segment layer thickening (p<0.05) at PN35 and thinning thereafter. These changes may indicate accumulation of outer segment debris at PN35 then progressive destruction. In vivo images of rat intra-retinal structure showed the ability of the method to distinguish the photoreceptor layer changes. Our results indicate that 80 MHz ultrasonography reveals intra-retinal layers and is sensitive to age and degenerative changes of photoreceptors. This technique has great potential to follow-up retinal dystrophy and therapeutic effects in vivo.

[The use of liposomes as intravitreal drug delivery system]. / Administration intra-vitréenne de liposomes pour la vectorisation de principes actifs.

Liposomes are vesicular lipidic systems allowing encapsulation of drugs. This article reviews the relevant issues in liposome structure (composition and size), and their influence on intravitreal pharmacokinetics. Liposome-mediated drug delivery to the posterior segment of the eye via intravitreal administration has been addressed by several authors and remains experimental. Liposomes have been used for intravitreal delivery of antibiotics, antivirals, antifungal drugs, antimetabolites, and cyclosporin. Encapsulation of these drugs within liposomes markedly increased their intravitreal half-life, and reduced their retinal toxicity. Liposomes have also shown an attractive potential for retinal gene transfer by intravitreal delivery of plasmids or oligonucleotides.

The dentino-enamel junction revisited.

The dentino-enamel junction is not an simple inert interface between two mineralized structures. A less simplistic view suggests that the dentino-enamel junctional complex should also include the inner aprismatic enamel and the mantle dentin. At early stages of enamel formation, fibroblast growth factor (FGF)-2 is stored in and released from the inner aprismatic enamel, possibly under the control of matrix metalloproteinase (MMP)-3. The concentration peak for MMP-2 and -9 observed in the mantle dentin coincided with a very low labeling for TIMP-1 and -2, favoring the cross-talk between mineralizing epithelial and connective structures, and as a consequence the translocation of enamel proteins toward odontoblasts and pulp cells, and vice versa, the translocation of dentin proteins toward secretory ameloblasts and cells of the enamel organ. Finally, in X-linked hypophosphatemic rickets, large interglobular spaces in the circumpulpal dentin were the major defect induced by the gene alteration, whereas the mantle dentin was constantly unaffected. Altogether, these data plead for the recognition of the dentino-enamel junctional complex as a specific entity bearing its own biological characteristics.