Quantitative Microbial Risk Assessment of Contracting COVID-19 Derived from Measured and Simulated Aerosol Particle Transmission in Aircraft Cabins.

BACKGROUND: SARS-CoV-2 can be effectively transmitted between individuals located in close proximity to each other for extended durations. Aircraft provide such conditions. Although high attack rates during flights were reported, little was known about the risk levels of aerosol transmission of SARS-CoV-2 in aircraft cabins. OBJECTIVES: The major objective was to estimate the risk of contracting COVID-19 from transmission of aerosol particles in aircraft cabins. METHODS: In two single-aisle and one twin-aisle aircraft, dispersion of generated aerosol particles over a seven-row economy class cabin section was measured under cruise and taxi conditions and simulated with a computational fluid dynamic model under cruise conditions. Using the aerosol particle dispersion data, a quantitative microbial risk assessment was conducted for scenarios with an asymptomatic infectious person expelling aerosol particles by breathing and speaking. Effects of flight conditions were evaluated using generalized additive mixed models. RESULTS: Aerosol particle concentration decreased with increasing distance from the infectious person, and this decrease varied with direction. On a typical flight with an average shedder, estimated mean risk of contracting COVID-19 ranged from 1.3×10-3 to 9.0×10-2. Risk increased to 7.7×10-2 with a super shedder (<3% of cases) on a long flight. Risks increased with increasing flight duration: 2-23 cruise flights of typical duration and 2-10 flights of longer duration resulted in at least 1 case of COVID-19 due to onboard aerosol transmission by one average shedder, and in the case of one super shedder, at least 1 case in 1-3 flights of typical duration cruise and 1 flight of longer duration. DISCUSSION: Our findings indicate that the risk of contracting COVID-19 by aerosol transmission in an aircraft cabin is low, but it will not be zero. Testing before boarding may help reduce the chance of a (super)shedder boarding an aircraft and mask use further reduces aerosol transmission in the aircraft cabin. https://doi.org/10.1289/EHP11495.

Calpain and PARP activation during photoreceptor cell death in P23H and S334ter rhodopsin mutant rats.

Retinitis pigmentosa (RP) is a heterogeneous group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness. Many human cases are caused by mutations in the rhodopsin gene. An important question regarding RP pathology is whether different genetic defects trigger the same or different cell death mechanisms. To answer this question, we analysed photoreceptor degeneration in P23H and S334ter transgenic rats carrying rhodopsin mutations that affect protein folding and sorting respectively. We found strong activation of calpain and poly(ADP-ribose) polymerase (PARP) in both mutants, concomitant with calpastatin down-regulation, increased oxidative DNA damage and accumulation of PAR polymers. These parameters were strictly correlated with the temporal progression of photoreceptor degeneration, mirroring earlier findings in the phosphodiesterase-6 mutant rd1 mouse, and suggesting execution of non-apoptotic cell death mechanisms. Interestingly, activation of caspases-3 and -9 and cytochrome c leakage-key events in apoptotic cell death--were observed only in the S334ter mutant, which also showed increased expression of PARP-1. The identification of the same metabolic markers triggered by different mutations in two different species suggests the existence of common cell death mechanisms, which is a major consideration for any mutation independent treatment.

Evidence of severe mitochondrial oxidative stress and a protective effect of low oxygen in mouse models of inherited photoreceptor degeneration.

The role of oxidative stress within photoreceptors (PRs) in inherited photoreceptor degeneration (IPD) is unclear. We investigated this question using four IPD mouse models (Pde6b(rd1/rd1), Pde6b(atrd1/atrd1), Rho(-/-) and Prph2(rds/rds)) and compared the abundance of reduced glutathione (GSH) and the activity of mitochondrial NADH:ubiquinone oxidoreductase (complex I), which is oxidative stress sensitive, as indirect measures of redox status, in the retinas of wild type and IPD mice. All four IPD mutants had significantly reduced retinal complex I activities (14-29% of wild type) and two showed reduced GSH, at a stage prior to the occurrence of significant cell death, whereas mitochondrial citrate synthase, which is oxidative stress insensitive, was unchanged. We orally administered the mitochondrially targeted anti oxidant MitoQ in order to reduce oxidative stress but without any improvement in retinal complex I activity, GSH or rates of PR degeneration. One possible source of oxidative stress in IPDs is oxygen toxicity in the outer retina due to reduced consumption by PR mitochondria. We therefore asked whether a reduction in the ambient O(2) concentration might improve PR survival in Pde6b(rd1/rd1) retinal explants either directly, by reducing reactive oxygen species formation, or indirectly by a neuroprotective mechanism. Pde6b(rd1/rd1) retinal explants cultured in 6% O(2) showed 31% less PR death than normoxic explants. We conclude that (i) mitochondrial oxidative stress is a significant early feature of IPDs; (ii) the ineffectiveness of MitoQ may indicate its inability to reduce some mediators of oxidative stress, such as hydrogen peroxide; and (iii) elucidation of the mechanisms by which hypoxia protects mutant PRs may identify novel neuroprotective pathways in the retina.

A key role for cyclic nucleotide gated (CNG) channels in cGMP-related retinitis pigmentosa.

The rd1 natural mutant is one of the first and probably the most commonly studied mouse model for retinitis pigmentosa (RP), a severe and frequently blinding human retinal degeneration. In several decades of research, the link between the increase in photoreceptor cGMP levels and the extremely rapid cell death gave rise to a number of hypotheses. Here, we provide clear evidence that the presence of cyclic nucleotide gated (CNG) channels in the outer segment membrane is the key to rod photoreceptor loss. In Cngb1(-/-) × rd1 double mutants devoid of regular CNG channels, cGMP levels are still pathologically high, but rod photoreceptor viability and outer segment morphology are greatly improved. Importantly, cone photoreceptors, the basis for high-resolution daylight and colour vision, survived and remained functional for extended periods of time. These findings strongly support the hypothesis of deleterious calcium (Ca(2+))-influx as the cause of rapid rod cell death and highlight the importance of CNG channels in this process. Furthermore, our findings suggest that targeting rod CNG channels, rather than general Ca(2+)-channel blockade, is a most promising symptomatic approach to treat otherwise incurable forms of cGMP-related RP.

Photoreceptor rescue and toxicity induced by different calpain inhibitors.

Photoreceptor degeneration is the hallmark of a group of inherited blinding diseases collectively termed retinitis pigmentosa (RP); a major cause of blindness in humans. RP is at present untreatable and the underlying neurodegenerative mechanisms are largely unknown, even though the genetic causes are often established. The activation of calpain-type proteases may play an important role in cell death in various neuronal tissues, including the retina. We therefore tested the efficacy of two different calpain inhibitors in preventing cell death in the retinal degeneration (rd1) human homologous mouse model for RP. Pharmacological inhibition of calpain activity in rd1 organotypic retinal explants had ambiguous effects on photoreceptor viability. Calpain inhibitor XI had protective effects when applied for short periods of time (16 h) but demonstrated substantial levels of toxicity in both wild-type and rd1 retina when used over several days. In contrast, the highly specific calpain inhibitor calpastatin peptide reduced photoreceptor cell death in vitro after both short and prolonged exposure, an effect that was also evident after in vivo application via intravitreal injection. These findings highlight the importance of calpain activation for photoreceptor cell death but also for photoreceptor survival and propose the use of highly specific calpain inhibitors to prevent or delay RP.

Antioxidants rescue photoreceptors in rd1 mice: Relationship with thiol metabolism.

We have previously shown that the use of a combination of antioxidants delayed the degeneration process in rd1 mouse retina. In an effort to understand the mechanism of action of these substances (zeaxanthin, lutein, alpha-lipoic acid, glutathione, and Lycium barbarum extract) the changes in the levels of several proteins and oxidative stress markers in the rd1 retina have been studied. The treatment increased glutathione peroxidase activity and glutathione levels and decreased cystine concentrations in rd1 retinas. Considering all the results obtained from treated and untreated animals, a high correlation was present between glutathione concentration and glutathione peroxidase activity, and there was a negative correlation between glutathione retinal concentration and number of TUNEL-positive cells. No difference was observed between the numbers of nNOS- and NADPH-diaphorase-positive cells in treated and untreated rd1 mice. Thiol contents and thiol-dependent peroxide metabolism seem to be directly related to the survival of photoreceptors in rd1 mouse retina.

Study of gene-targeted mouse models of splicing factor gene Prpf31 implicated in human autosomal dominant retinitis pigmentosa (RP).

PURPOSE: Pre-mRNA processing factor 31 (PRPF31) is a ubiquitous protein needed for the assembly of the pre-mRNA splicing machinery. It has been shown that mutations in this gene cause autosomal dominant retinitis pigmentosa 11 (RP11), which is characterized by rod-cell degeneration. Interestingly, mutations in this ubiquitously expressed gene do not lead to phenotypes other than retinal malfunction. Furthermore, the dominant inheritance pattern has shown incomplete penetrance, which poses interesting questions about the disease mechanism of RP11. METHODS: To characterize PRPF31 function in the rod cells, two animal models have been generated. One was a heterozygous knock-in mouse (Prpf31(A216P/+)) carrying a point mutation p.A216P, which has previously been identified in RP11 patients. The second was a heterozygous knockout mouse (Prpf31(+/-)). Retinal degeneration in RP11 mouse models was monitored by electroretinography and histology. RESULTS: Generation of the mouse models is presented, as are results of ERGs and retinal morphology. No degenerative phenotype on fundus examination was found in Prpf31(A216P/+) and Prpf31(+/-) mice. Prpf31(A216P/A216P) and Prpf31(-/-) genotypes were embryonic lethal. CONCLUSIONS: The results imply that Prpf31 is necessary for survival, and there is no compensation mechanism in mouse for the lack of this splicing factor. The authors suggest that p.A216P mutation in Prpf31 does not exert a dominant negative effect and that one Prpf31 wild-type allele is sufficient for maintenance of the healthy retina in mice.

PKG activity causes photoreceptor cell death in two retinitis pigmentosa models.

Photoreceptor degeneration in retinitis pigmentosa is one of the leading causes of hereditary blindness in the developed world. Although causative genetic mutations have been elucidated in many cases, the underlying neuronal degeneration mechanisms are still unknown. Here, we show that activation of cGMP-dependent protein kinase (PKG) hallmarks photoreceptor degeneration in rd1 and rd2 human homologous mouse models. When induced in wild-type retinae, PKG activity was both necessary and sufficient to trigger cGMP-mediated photoreceptor cell death. Target-specific, pharmacological inhibition of PKG activity in both rd1 and rd2 retinae strongly reduced photoreceptor cell death in organotypic retinal explants. Likewise, inhibition of PKG in vivo, using three different application paradigms, resulted in robust photoreceptor protection in the rd1 retina. These findings suggest a pivotal role for PKG activity in cGMP-mediated photoreceptor degeneration mechanisms and highlight the importance of PKG as a novel target for the pharmacological intervention in RP.

Photoreceptor cell death mechanisms in inherited retinal degeneration.

Photoreceptor cell death is the major hallmark of a group of human inherited retinal degenerations commonly referred to as retinitis pigmentosa (RP). Although the causative genetic mutations are often known, the mechanisms leading to photoreceptor degeneration remain poorly defined. Previous research work has focused on apoptosis, but recent evidence suggests that photoreceptor cell death may result primarily from non-apoptotic mechanisms independently of AP1 or p53 transcription factor activity, Bcl proteins, caspases, or cytochrome c release. This review briefly describes some animal models used for studies of retinal degeneration, with particular focus on the rd1 mouse. After outlining the major features of different cell death mechanisms in general, we then compare them with results obtained in retinal degeneration models, where photoreceptor cell death appears to be governed by, among other things, changes in cyclic nucleotide metabolism, downregulation of the transcription factor CREB, and excessive activation of calpain and PARP. Based on recent experimental evidence, we propose a putative non-apoptotic molecular pathway for photoreceptor cell death in the rd1 retina. The notion that inherited photoreceptor cell death is driven by non-apoptotic mechanisms may provide new ideas for future treatment of RP.

Sialoadhesin expression in intact degenerating retinas and following transplantation.

PURPOSE: Resident microglial cells normally do not express sialoadhesin (Sn; a sialic acid-binding receptor), whereas recruited inflammatory macrophages have been shown to do so. The expression of Sn was examined in the course of photoreceptor cell degeneration and after transplantation. METHODS: Sn expression was analyzed in retinas of rd1 and rds mice. For transplantation studies, neonatal (P2) retinal cells derived from GFP mice were injected intraocularly in adult rd1 mice and control mice. Antibodies recognizing different Sn epitopes, CD11b, and MHC-II were used to identify activated microglial cells in intact retinas and 21 days after transplantation. RESULTS: In rd1 mice, a few CD11b-positive cells were observed in the outer nuclear layer in the central retina at postnatal day (P)11 and in increasing numbers between P12 to P21. In rds mice, CD11b-expressing cells were found from P16 onward. No Sn-expressing cells were observed within the rd1 or rds mouse retinas at any of the ages examined (up to P150). Specific staining was observed only in cells found in the vitreous margin of the retina and in surrounding tissues (sclera, cornea, ciliary body, choroid). After transplantation to normal and rd1 mice, a variable number of Sn-positive cells were detected within the grafts, in the graft-host interface, and in the subretinal space. CONCLUSIONS: The significant activation of microglia/macrophages observed in the various stages of degeneration in rd1 and rds mouse retinas is not accompanied by Sn expression. However, Sn-expressing cells are observed after transplantation. The occurrence of such cells could be of significance for the integration and long-term survival of retinal grafts, as the expression of Sn could facilitate other phagocytic receptors.

rd1 Mouse retina shows an imbalance in the activity of cysteine protease cathepsins and their endogenous inhibitor cystatin C.

PURPOSE: To compare in vivo levels, spatial localization, and in vitro secretion of cysteine protease cathepsins and cystatin C (cysC) in the retinal degeneration 1 (rd1) mouse model of retinitis pigmentosa and control (wt) mouse retinas. METHODS: The spatial localization, protein contents, cysC levels and cathepsin-B, -S, and -L activities in wt and rd1 retinas at postnatal (PN) days 2, 7, 14, 21, and 28 were analyzed by immunostaining, spectrophotometry, ELISA, and fluorescence spectrophotometry. The in vitro secretion of cysC and cysteine proteases by PN7 retinal explants into the conditioned medium (RCM) was quantified. RESULTS: The pigment epithelium, photoreceptors, and inner retinal and ganglion cell layers of both wt and rd1 retinas showed cysC and cathepsin-B labeling. CysC immunostaining was extensive in the optic nerve head fibers. The rd1 explants secreted higher amounts of cysteine protease into the RCM. The protein content in wt and rd1 retinal extracts increased up to PN14, then decreased in rd1 but not in wt. In rd1 extracts at PN14 to -28, cathepsin activity was higher and increased with age, but the cysC level was higher and constant. The ratios of cathepsin activity to cysC (cathepsin-L at PN2 and total, -B, and -L at PN14 to -28) were higher in rd1 extracts. CONCLUSIONS: Similar localization of both cathepsin-B and cysC in wt and rd1 retinas along with lower proteins and higher cathepsin activity in rd1 retinal extracts and RCM are consistent with their localization in extracellular matrix and a role in physiopathologic remodeling in wt and rd1 retinas.

Excessive activation of poly(ADP-ribose) polymerase contributes to inherited photoreceptor degeneration in the retinal degeneration 1 mouse.

Retinitis pigmentosa (RP) is an inherited blinding disease for which there is no treatment available. It is characterized by a progressive and neurodegenerative loss of photoreceptors but the underlying mechanisms are poorly understood. Excessive activation of the enzyme poly(ADP-ribose) polymerase (PARP) has recently been shown to be involved in several neuropathologies. To investigate the possible role of PARP in retinal photoreceptor degeneration, we used the retinal degeneration 1 (rd1) mouse RP model to study PARP expression, PARP activity, and to test the effects of PARP inhibition on photoreceptor viability. PARP expression was found to be equal between rd1 and wild-type counterpart retinas. In contrast to this, a dramatic increase in both PARP activity per se and PARP product formation was detected by in situ assays in rd1 photoreceptors actively undergoing cell death. Furthermore, PARP activity colabeled with oxidatively damaged DNA and nuclear translocation of AIF (apoptosis-inducing factor), suggesting activation of PARP as a bridge between these events in the degenerating photoreceptors. The PARP-specific inhibitor PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide x HCl[ reduced the number of cells exhibiting death markers in a short-term retinal culture paradigm, a protective effect that was translated into an increased number of surviving photoreceptors when the inhibitor was used in a long-term culture setting. Our results thus demonstrate an involvement of PARP activity in rd1 photoreceptor cell death, which could have a bearing on the understanding of neurodegenerations as such. The findings also suggest that the therapeutical possibilities of PARP inhibition should include retinal diseases like RP.

Low glutathione peroxidase in rd1 mouse retina increases oxidative stress and proteases.

Malondialdehyde, reduced glutathione, glutathione peroxidase, glutathione reductase and cysteine protease cathepsins at postnatal (PN) days 2, 7, 14, 21 and 28 in controls (wt) and the retinal degeneration 1 (rd1) mouse model for retinitis pigmentosa retinas were measured to determine oxidative stress. In PN28 wt and PN2 rd1 retinas, elevated malondialdehyde and low glutathione peroxidase activity indicate higher oxidative load, despite higher reduced glutathione in PN2 rd1 retinas. This is due to physiological exposure to light and retinal vascular/neural restructuring, respectively. Compared with wt retinas, relatively high malondialdehyde at PN2 and cathepsin levels at PN14, 21 and 28 in rd1 retinas indicate that cells of the residual inner retina also contribute to the oxidative stress and retinal degeneration.

CNTF+BDNF treatment and neuroprotective pathways in the rd1 mouse retina.

The rd1 mouse is a relevant model for studying the mechanisms of photoreceptor degeneration in retinitis pigmentosa. Treatment with ciliary neurotrophic factor (CNTF) in combination with brain derived neurotrophic factor (BDNF) is known to rescue photoreceptors in cultured rd1 retinal explants. To shed light on the underlying mechanisms, we studied the effects of 9 days (starting at postnatal day 2) in vitro CNTF+BDNF treatment on the endogenous production of CNTF, BDNF, fibroblast growth factor 2 (FGF2), or the activation of extracellular signal-regulated kinase (ERK), Akt and cAMP-response-element-binding protein (CREB) in retinal explants. In rd1 explants, CNTF+BDNF decreased the number of TUNEL-positive photoreceptors. The treatment also increased endogenous rd1 levels of CNTF and BDNF, but lowered the level of FGF2 expression in rd1 explants. When wild-type explants were treated, endogenous CNTF was similarly increased, while BDNF and FGF2 levels remained unaffected. In addition, treatment of rd1 retinas strongly increased the phosphorylation of ERK, Akt and CREB. In treated wild-type explants, the same parameters were either unchanged (ERK) or decreased (Akt and CREB). The results suggest a role for Akt, ERK and CREB in conveying the neuroprotective effect of CNTF+BDNF treatment in rd1 retinal explants.

Up-regulation and increased phosphorylation of protein kinase C (PKC) delta, mu and theta in the degenerating rd1 mouse retina.

The rd1 mouse serves as a model for inherited photoreceptor degeneration: retinitis pigmentosa. Microarray techniques were employed to compare the transcriptomes of rd1 and congenic wild-type retinas at postnatal day 11, when degenerative processes have started but most photoreceptors are still present. Of the several genes that were differentially expressed, focus was put on those associated with the protein kinase C (PKC) signaling pathway, in particular PKCdelta, mu and theta. Microarray identified these as being up-regulated in the rd1 retina, which was confirmed by QRT-PCR. Western blotting and immunostaining, using antibodies against either total or phosphorylated variants of the PKC isoforms, revealed increased expression and phosphorylation of PKCdelta, mu and theta in the rd1 retina at the protein level as well. Our results suggest that these PKC isoforms are involved in rd1 degeneration.

Calpain is activated in degenerating photoreceptors in the rd1 mouse.

The retinal degeneration (rd)1 mouse displays an inherited retinal degeneration and therefore allows studies of the molecular mechanisms behind the blinding disease retinitis pigmentosa. Activation of the calcium-dependent protease calpain has been suggested to play an important role in cell death in various tissues, but little is known about the expression and activity of calpain during inherited retinal degeneration. Using microarray techniques, transcript levels of cyclic AMP response element-binding protein (CREB)-1, calpastatin and of various calpain genes were analysed in the rd1 mouse compared with its wild-type control. Expression of distinct calpain isoforms and calpastatin was investigated using immunofluorescence and immunoblotting. Gene transcription and protein expression levels were compared with calpain activity using an enzymatic assay that allowed monitoring of calpain activity at the cellular level. We found that CREB-1 and calpastatin expression was reduced in rd1 retinas, whereas calpain activity was substantially increased in rd1 photoreceptors. Calpain activity peaked at postnatal day 13, together with rd1 photoreceptor cell death. Calpain-specific inhibitors decreased calpain activity in situ. These results indicate that activation of calpains correlates with rd1 photoreceptor cell death, which raises the possibility of using calpain inhibitors to prevent or delay photoreceptor degeneration.

rd1 mouse retina shows imbalance in cellular distribution and levels of TIMP-1/MMP-9, TIMP-2/MMP-2 and sulfated glycosaminoglycans.

BACKGROUND: The rd1 mouse retina displays fast degeneration of photoreceptors resulting in a depletion of almost all rod photoreceptors by postnatal day 21 (PN21). To evaluate the role of proteinases in the pathophysiology of this animal model of retinitis pigmentosa, C3H rd1 and congenic wild-type (wt) mice retinas were analyzed. MATERIAL AND METHODS: The cellular localization and levels of proteins, matrix metalloproteinases (MMPs), their endogenous inhibitors (TIMPs), total sulfated glycosaminoglycans (sGAG) and nature of saccharides in rd1 and wt retinal extracts were compared. RESULTS: MMP-2/TIMP-2 and MMP-9/TIMP-1 were predominantly localized in the interphotoreceptor matrix (IPM) of both genotypes, but MMP-2/TIMP-2 also appeared in the Muller cell fibers of rd1 retina. In rd1 retinal extracts the levels of total proteins were lower and those of active MMP-9, MMP-2, TIMP-1 and total sGAG were higher than those of wt extracts. Despite an increase in TIMP-1, active MMP-9/MMP-2 were disproportionately elevated in rd1 compared to wt retina. With increasing age, MMPs in wt retinas were decreased but were increased in rd1. The sialylation of proteoglycans in PN2 and PN7 rd1 retinas was lower, and galactosylation was higher than that in wt retinas. CONCLUSIONS: MMP-9/MMP-2 and TIMP-1/TIMP-2 are associated with IPM, possibly after secretion by retinal pigmented epithelial cells. In degenerating rd1 retina, MMP-2/TIMP-2 are associated with the Muller cell fibers, which apparently play a central role in modifying the balance between MMPs and TIMPs. Elevated sGAG and proteolysis due to an imbalance in the levels of TIMPs and active MMP-9/MMP-2 in rd1 retina possibly contribute to retinal degeneration in the rd1 mouse.

Differential modification of phosducin protein in degenerating rd1 retina is associated with constitutively active Ca2+/calmodulin kinase II in rod outer segments.

Retinitis pigmentosa comprises a heterogeneous group of incurable progressive blinding diseases with unknown pathogenic mechanisms. The retinal degeneration 1 (rd1) mouse is a retinitis pigmentosa model that carries a mutation in a rod photoreceptor-specific phosphodiesterase gene, leading to rapid degeneration of these cells. Elucidation of the molecular differences between rd1 and healthy retinae is crucial for explaining this degeneration and could assist in suggesting novel therapies. Here we used high resolution proteomics to compare the proteomes of the rd1 mouse retina and its congenic, wild-type counterpart at postnatal day 11 when photoreceptor death is profound. Over 3000 protein spots were consistently resolved by two-dimensional gel electrophoresis and subjected to a rigorous filtering procedure involving computer-based spot analyses. Five proteins were accepted as being differentially expressed in the rd1 model and subsequently identified by mass spectrometry. The difference in one such protein, phosducin, related to an altered modification pattern in the rd1 retina rather than to changed expression levels. Additional experiments showed phosducin in healthy retinae to be highly phosphorylated in the dark- but not in the light-adapted phase. In contrast, rd1 phosducin was highly phosphorylated irrespective of light status, indicating a dysfunctional rd1 light/dark response. The increased rd1 phosducin phosphorylation coincided with increased activation of calcium/calmodulin-activated protein kinase II, which is known to utilize phosducin as a substrate. Given the increased rod calcium levels present in the rd1 mutation, calcium-evoked overactivation of this kinase may be an early and long sought for step in events leading to photoreceptor degeneration in the rd1 mouse.

Differential Akt activation in the photoreceptors of normal and rd1 mice.

Retinitis pigmentosa is a blinding disease in which unknown mechanisms cause the degeneration of retinal photoreceptors. The retinal degeneration (rd1) mouse is a relevant model for this condition, since it carries a mutation also found in some forms of retinitis pigmentosa. To understand the degenerative process in the rd1 mouse, we must identify the survival and apoptosis-related signaling pathways in its photoreceptors and determine whether signaling differs from that in normal mice. The phosphatidylinositol 3-kinase/Akt kinase pathway promotes survival in several different cell types. The purpose of the present study has been to compare Akt activity in retinal cells of normal and rd1 mice. We have found that, in normal mice, Akt becomes activated in the retina in a developmentally regulated and cell-type-specific fashion, encompassing essentially all retinal cells. In most cell types, once Akt activation has begun, it remains in this state throughout life. An exception is seen in the rod photoreceptors, in which Akt is activated only transiently during their development. The rd1 retina behaves identically in all but one respect, namely that the activation of Akt in rod photoreceptors persists until these cells undergo apoptosis. Thus, Akt may participate in constitutive survival processes in retinal neurons, except in rod photoreceptors in which the role of this pathway may be restricted to the developmental period. However, Akt activation in the rods may be part of a defense mechanism initiated in response to insults, such as the retinal degeneration seen in the rd1 mouse.

Integration between abutting retinas: role of glial structures and associated molecules at the interface.

PURPOSE: Integration between subretinal grafts and the host retina is limited in part by the presence of a barrier at the graft-host interface. This study was conducted to identify factors that may contribute to this barrier, by examining the distribution of glial structures and associated molecules in different setups of overlapping retinal pieces. METHODS: Neuroretinal tissue derived from mice that express green fluorescent protein (GFP) was fragmented and transplanted into the subretinal space of adult rd1 mice. In an in vitro system, two retinal pieces, derived from GFP and rd1 mice, respectively, were placed overlapping each other and forming either laminar-laminar pairs or fragment-laminar pairs. The glia-associated markers analyzed included glial fibrillary acidic protein (GFAP), cellular retinaldehyde-binding protein (CRALBP), and two molecules known to inhibit neurite outgrowth: CD44 and neurocan. Bridging fibers and migrated cells were visualized with GFP fluorescence and retinal cell markers. RESULTS: A thick CRALBP-immunolabeled band was observed in the interface in cultured laminar-laminar pairs, whereas a thinner band was seen in cultured fragment-laminar pairs and in transplants. Accumulation of CD44 and neurocan was also observed in the interface between abutting retinal pieces in all setups. GFP(+) bridging fibers and GFP(+) cells (some of which coexpressed neuronal markers) were observed within the abutting rd1 retina in some areas. However, such integration occurred exclusively where CRALBP, CD44, and neurocan immunolabeling appeared disrupted in the interface, but coincided with high GFAP expression within the rd1 retina. CONCLUSIONS: The results demonstrate that, on the one hand, an accumulation of glial-associated inhibitory molecules in the interface correlates with limited integration between overlapping retinal pieces. On the other hand, glial reactivity within the rd1 retina does not appear to be incompatible with integration.