Neuronal and Glial Alterations in Complex Long-Term Rhegmatogenous Retinal Detachment.

Purpose: To explore neuronal and glial alterations in eyes with complex long-term rhegmatogenous retinal detachment (RRD). Methods: Morphological analysis was performed on eight retinal specimens derived from patients treated with peripheral retinectomy for RRD complicated by retinal shortening or retinal thinning. All eyes had undergone previous surgeries including silicone oil tamponade, and had a median total detachment time of 2.5 months (range 2-12). Specimens were examined with hematoxylin and eosin staining and immunohistochemistry directed against activated Müller cells, ganglion cells, rod bipolar cells, and photoreceptors. Results: Retinal specimens displayed severe loss of photoreceptor and rod bipolar cells. Remaining neuronal cells exhibited disorganized perikarya and neurites with disruption of the normal retinal lamination. Müller cell activation was evident in all specimens with subretinal and epiretinal hypertrophy present in tissue derived from shortened retinal detachments. Conclusion: Long-term RRD leads to retinal remodeling characterized by loss of first and second order retinal neurons, disruption of the entire retinal lamination and gliosis. The severity of histopathological changes indicates that anatomical as well as functional recovery of the involved retina is precarious. The findings may be important when devising surgical strategies to avoid permanent retinal detachment

Protein kinase C expression in the rabbit retina after laser photocoagulation.

Background: Laser photocoagulation is a well-established treatment for diabetic retinopathy but the mechanism behind its effectiveness has not been elucidated. The protein kinase C (PKC) family is a group of enzymes which has been the subject of extensive interest in clinically related research since the advent of its role in the pathogenesis of diabetic retinopathy. With this study we wanted to explore whether PKC expression is altered in the retina after laser photocoagulation. Methods: Normal rabbit eyes were treated with laser photocoagulation of varying intensity and examined after 30 min to 7 weeks. Treated and untreated regions of the retina were investigated histologically with the MC5 monoclonal antibody against PKC. Labeling for glial fibrillary acidic protein (GFAP), as well as hematoxylin and eosin (H&E) staining was also performed to assess the laser-induced trauma. Results: In the normal retina, the MC5 antibody labeled rod bipolar cells and photoreceptor outer segments corresponding to PKC alpha. A translocated PKC expression with labeling concentrated in the rod bipolar terminals was seen in specimens examined 30 min after laser treatment, and after 1 week, no expression was seen in any part of the retina. After 2 weeks, PKC expression again indicated a translocated labeling pattern. After 5 weeks, labeling was found only in rod bipolar terminals in the peripheral retina. When comparing high- and low-intensity laser treatment 7 weeks postoperatively, no labeling was found in the high intensity-treated retinas, whereas low intensity-treated eyes displayed a near-normal labeling pattern. H&E staining revealed focal neuroretinal edema immediately after laser treatment, also in untreated areas. At later stages, destruction of the outer nuclear layer and migration of pigment epithelial cells in laser-lesioned areas was seen. GFAP-labeled Muller cells were seen 1 week postoperatively in the entire retina. Labeling after this time decreased, but was still present in laser spots after 5 and 7 weeks. Conclusions: Laser photocoagulation alters the expression of PKC in the entire normal rabbit retina. The response follows a temporal pattern and is also related to laser intensity. These findings may help to explain the high efficacy of laser treatment in diabetic retinopathy

Specific inhibition of TRPV4 enhances retinal ganglion cell survival in adult porcine retinal explants

Signaling through the polymodal cation channel Transient Receptor Potential Vanilloid 4 (TRPV4) has been implicated in retinal neuronal degeneration. To further outline the involvement of this channel in this process, we here explore modulation of Transient Receptor Potential Vanilloid 4 (TRPV4) activity on neuronal health and glial activation in an in vitro model of retinal degeneration. For this purpose, adult porcine retinal explants were cultured using a previously established standard protocol for up to 5 days with specific TRPV4 agonist GSK1016790A (GSK), or specific antagonist RN-1734, or culture medium only. Glial and neuronal cell health were evaluated by a battery of immunohistochemical markers, as well as morphological staining. Specific inhibition of TRPV4 by RN-1734 significantly enhanced ganglion cell survival, improved the maintenance of the retinal laminar architecture, reduced apoptotic cell death and attenuated the gliotic response as well as preserved the expression of TRPV4 in the plexiform layers and ganglion cells. In contrast, culture controls, as well as specimens treated with GSK, displayed rapid remodeling and neurodegeneration as well as a downregulation of TRPV4 and the Müller cell homeostatic mediator glutamine synthetase. Our results indicate that TRPV4 signaling is an important contributor to the retinal degeneration in this model, affecting neuronal cell health and glial homeostasis. The finding that pharmacological inhibition of the receptor significantly attenuates neuronal degeneration and gliosis in vitro, suggests that TRPV4 signaling may be an interesting pharmaceutical target to explore for treatment of retinal degenerative disease

In vitro biomechanical modulation-retinal detachment in a box.

To illustrate the importance of biomechanical impact on tissue health within the central nervous system (CNS), we herein describe an in vitro model of rhegmatogenous retinal detachment (RRD) in which disruption and restoration of physical tissue support can be studied in isolation

Autophagy and ER-stress contribute to photoreceptor degeneration in cultured adult porcine retina.

The aim of this study was to investigate rod and cone photoreceptor degeneration in organotypic cultures of adult porcine retina. Our hypothesis was that the photoreceptors accumulate opsins, which, together with exposure to cyclic dim light illumination, induce autophagy and endoplasmic reticulum stress (ER-stress) to overcome damaging protein overload. For this purpose, retinas were cultured for 48h and 72h during which they were illuminated with dim light for 8h/day; specimens were analyzed by means of immunohistochemistry, Western blot, real-time polymerase chain reaction (PCR) and transmission electron microscopy. ER-stress and photoreceptor degeneration was observed in conventionally cultured retinas. The additional stress in the form of dim light illumination for 8h/day resulted in increased levels of the ER-stress markers GRP78/BiP and CHOP, as well as increased level of active caspase-12. Increased autophagic processes in cone and rod photoreceptors were detected by LC3B-II increases and occurrence of autophagosomes at the ultrastructural level. Illumination also resulted in altered protein expression for autophagy inducers such as p62 and Beclin-1. Moreover, there was a decrease in phosphorylated mammalian target of rapamycin (mTOR), which further indicate an increase of autophagy. Rod and cone photoreceptors in retinas from a diurnal animal that were exposed to dim light illumination in vitro displayed autophagy and ER-stress processes. As no alteration of rhodopsin mRNA was observed, autophagy and ER-stress are suggested to decrease rhodopsin protein at the posttranscriptional level

Protein kinase C in porcine retinal arteries and neuroretina following retinal ischemia-reperfusion

PURPOSE: Identification of the intracellular signal-transduction pathways activated in retinal ischemia may be important in revealing novel pharmacological targets. To date, most studies have focused on identifying neuroprotective agents. The retinal blood vessels are key organs in circulatory failure, and this study was therefore designed to examine the retinal vasculature separately from the neuroretina. METHODS: Retinal ischemia was induced by elevating the intraocular pressure in porcine eyes, followed by 5, 12, or 20 h of reperfusion. Protein kinase C (PKC)alpha, PKCbeta1, and PKCbeta2 mRNA levels, and protein expression were determined using real-time PCR, western blot, and immunofluorescence staining techniques. RESULTS: The retinal arteries could easily be dissected free and studied separately from the neuroretina in this porcine model. The PKCalpha, PKCbeta1, and PKCbeta2 mRNA levels tended to be lower in ischemia-reperfused than in sham-operated eyes in both the retinal arteries and the neuroretina. This was most prominent after 5 h, and less pronounced after 12 h and 20 h of reperfusion. Likewise, the protein levels of PKCalpha, PKCbeta1, and PKCbeta2 were slightly lower following ischemia-reperfusion when compared to sham-operated eyes. PKCalpha, PKCbeta1, and PKCbeta2 immunostaining were observed in bipolar cells of the neuroretina and in endothelial cells, and to a low extent in the smooth muscle layer, of the retinal arteries. CONCLUSIONS: Retinal ischemia followed by reperfusion results in lower levels of PKC in both the neuroretina and retinal arteries. New targets for pharmacological treatment may be found by studying the retinal vasculature so as to identify the intracellular signal-transduction pathways involved in the development of injury following retinal circulatory failure

A new model for in vitro testing of vitreous substitute candidates

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®), a two component hydrogel of 20 wt.% poly (ethylene glycol) in phosphate buffered saline (PEG) and a cross-linked sodium hyaluronic acid hydrogel (Healaflow®). 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®-exposed explants displayed severe thinning and disruption of retinal layers with massive cell death. Healaflow®-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® 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®-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® 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® shows protection from culture-induced trauma indicating favorable biocompatibility.Swedish Research CouncilUniversity of Lund. Medical FacultyPrincess Margaret's Foundation for Blind ChildrenKnut and Alice Wallenberg FoundationGeneral Sir John Monash Foundation (Scholarship)In Vivo Therapeutics Corporatio

Estimating excess mortality and economic burden of <i>Clostridioides difficile</i> infections and recurrences during 2015–2019:The RECUR England study

Objective To generate real-world evidence on all-cause mortality and economic burden of Clostridioides difficile infections (CDIs) and recurrences (rCDIs) in England. Methods We conducted a cohort study using retrospective data from Clinical Practice Research Datalink linked to Hospital Episode Statistics. Patients diagnosed with CDI in hospital and community settings during 2015–2018 were included and followed for ≥1year. All-cause mortality was described at 6-, 12-, and 24-months. Healthcare resource usage (HCRU) and associated costs were assessed at 12-months of follow-up. A cohort of non-CDI patients, matched by demographic and clinical characteristics including Charlson Comorbidity Index score, was used to assess excess mortality and incremental costs of HCRU. Results All-cause mortality among CDI patients at 6-, 12-, and 24-months was 15.87%, 20.37%, and 27.03%, respectively. A higher proportion of rCDI patients died at any point during follow-up. Compared with matched non-CDI patients, excess mortality was highest at 6-months with 1.81 and 2.53 deaths per 100 patient-months among CDI and ≥1 rCDI patients. Hospitalisations were the main drivers of costs, with an incremental cost of £1,209.21 per CDI patient. HCRU and costs increased with rCDIs. Conclusions CDI poses a substantial mortality and economic burden, further amplified by rCDIs