In vivo requirement of protein prenylation for maintenance of retinal cytoarchitecture and photoreceptor structure

Recent studies have demonstrated that inhibition of mevalonate synthesis in cultured cells leads to altered cell morphology due to inhibition of protein prenylation. To investigate the effects in vivo of mevalonate deprivation in nondividing, terminally differentiated neural cells, we have analyzed the effects on retinal tissue of intravitreal injection of lovastatin, a potent inhibitor of the mevalonate-producing enzyme, HMG-CoA reductase. A single injection of lovastatin (0.25 mumol) produced profound dysplastic-like changes in adult rat retinas primarily involving the photoreceptor layer. Within 2 d after injection, photoreceptor nuclei migrated in a circular pattern resulting in the formation of rosette-like structures by 4 d. Also during this period, photoreceptor inner and outer segment degeneration was evident. By 21 d, intact photoreceptor nuclei with remnants of inner and outer segments were dispersed throughout all retinal layers. To investigate the biochemical specificity of the lovastatin-induced alterations, and to distinguish the relative importance of the various branches of the mevalonate pathway, the incorporation of [3H]acetate into retinal lipids was examined in the presence and absence of metabolic inhibitors. HPLC analysis of lovastatin-treated retinas revealed a dramatic reduction in the incorporation of intravitreally injected [3H]acetate into nonsaponifiable lipids, compared with controls. In contrast, intravitreal injection of NB-598, a specific inhibitor of squalene epoxidase, eliminated the conversion of newly synthesized squalene to sterols without obvious pathology. Hence, involvement to the sterol branch of isoprenoid metabolism in the lovastatin-induced morphologic disruption was obviated. Intravitreal injection of 0.27 mumol of N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), an inhibitor of carboxyl methyltransferase activity and prenylated protein function, produced morphologic changes that were virtually indistinguishable from those induced with lovastatin. These results implicate a defect in protein prenylation in the lovastatin- induced retinal degeneration, and suggest the presence of a dynamic pathway in the retina that requires isoprenylated proteins to maintain retinal cytoarchitecture.     

Study of pharmacological effects of nilvadipine on RCS rat retinal degeneration by microarray analysis

In our recent study, we found that the Ca(2+) antagonist, nilvadipine caused significant preservation of photoreceptor cells in The Royal College of Surgeons (RCS) rats [Invest. Ophthalmol. Vis. Sci. 43 (2002) 919]. Here, to elucidate the mechanisms of nilvadipine-induced effects we analyzed altered gene expression of 1101 genes commonly expressed in rodent by DNA microarray analysis in the retinas of nilvadipine-treated and untreated RCS rats and SD rat. In the total number of genes, the expression of 30 genes was altered upon administration of nilvadipine to RCS rats, including several genes related to the apoptotic pathway and other mechanisms. Remarkably, neurotrophic factors, FGF-2 and Arc, known to suppress the apoptosis in the central nervous system, were up-regulated. These changes were also confirmed by real-time quantitative (Taqman) RT-PCR and Western blot analysis. Therefore, our present data suggested that administration of nilvadipine to RCS rats increases the expression of endogenous FGF-2 and Arc in retina, and potentially has a protective effect against retinal degeneration.     

New bioengineering insights into human neural precursor cell expansion in culture

Understanding initial cell growth, interactions associated with the process of expansion of human neural precursor cells (hNPCs), and cellular events pre- and postdifferentiation are important for developing bioprocessing protocols to reproducibly generate multipotent cells that can be used in basic research or the treatment of neurodegenerative disorders. Herein, we report the in vitro responses of telencephalon hNPCs grown in a serum-free growth medium using time-lapse live imaging as well as cell-surface marker, aggregate size, and immunocytochemical analyses. Time-lapse analysis of hNPC initial expansion indicated that cell-surface attachment in stationary culture and the frequency of cell-cell interaction in suspension conditions are important for subsequent aggregate formation and hNPC growth. In the absence of cell-surface attachment in low-attachment stationary culture, large aggregates of cells were formed and expansion was adversely affected. The majority of the telencephalon hNPCs expressed CD29, CD90, and CD44 (cell surface markers involved in cell-ECM and cell-cell interactions to regulate biological functions such as proliferation), suggesting that cell-surface attachment and cell-cell interactions play a significant role in the subsequent formation of cell aggregates and the expansion of hNPCs. Before differentiation, about 90% of the cells stained positive for nestin and expressed two neural precursor cells surface markers (CD133 and CD24). Upon withdrawal of growth cytokines, hNPCs first underwent cell division and then differentiated preferentially towards a neuronal rather than a glial phenotype. This study provides key information regarding human NPC behavior under different culture conditions and favorable culture conditions that are important in establishing reproducible hNPC expansion protocols.     

Bioreactor expansion of human neural precursor cells in serum‐free media retains neurogenic potential

Human neural precursor cells (hNPCs), harvested from somatic tissue and grown in vitro, may serve as a source of cells for cell replacement strategies aimed at treating neurodegenerative disorders such as Parkinson's disease (PD), Huntington's disease (HD), and intractable spinal cord pain. A crucial element in a robust clinical production method for hNPCs is a serum‐free growth medium that can support the rapid expansion of cells while retaining their multipotency. Here, we report the development of a cell growth medium (PPRF‐h2) for the expansion of hNPCs, achieving an overall cell‐fold expansion of 10¹³ over a period of 140 days in stationary culture which is significantly greater than other literature results. More importantly, hNPC expansion could be scaled‐up from stationary culture to suspension bioreactors using this medium. Serial subculturing of the cells in suspension bioreactors resulted in an overall cell‐fold expansion of 7.8 × 10¹³ after 140 days. These expanded cells maintained their multipotency including the capacity to generate large numbers of neurons (about 60%). In view of our previous studies regarding successful transplantation of the bioreactor‐expanded hNPCs in animal models of neurological disorders, these results have demonstrated that PPRF‐h2 (containing dehydroepiandrosterone, basic fibroblast growth factor and human leukemia inhibitory factor) can successfully facilitate the production of large quantities of hNPCs with potential to be used in the treatment of neurodegenerative disorders. Biotechnol. Bioeng. 2010. 105: 823–833. © 2009 Wiley Periodicals, Inc.     

Chromosome 7 and 19 Trisomy in Cultured Human Neural Progenitor Cells

Background

Stem cell expansion and differentiation is the foundation of emerging cell therapy technologies. The potential applications of human neural progenitor cells (hNPCs) are wide ranging, but a normal cytogenetic profile is important to avoid the risk of tumor formation in clinical trials. FDA approved clinical trials are being planned and conducted for hNPC transplantation into the brain or spinal cord for various neurodegenerative disorders. Although human embryonic stem cells (hESCs) are known to show recurrent chromosomal abnormalities involving 12 and 17, no studies have revealed chromosomal abnormalities in cultured hNPCs. Therefore, we investigated frequently occurring chromosomal abnormalities in 21 independent fetal-derived hNPC lines and the possible mechanisms triggering such aberrations.

Methods and Findings

While most hNPC lines were karyotypically normal, G-band karyotyping and fluorescent in situ hybridization (FISH) analyses revealed the emergence of trisomy 7 (hNPC⁺⁷) and trisomy 19 (hNPC⁺¹⁹), in 24% and 5% of the lines, respectively. Once detected, subsequent passaging revealed emerging dominance of trisomy hNPCs. DNA microarray and immunoblotting analyses demonstrate epidermal growth factor receptor (EGFR) overexpression in hNPC⁺⁷ and hNPC⁺¹⁹ cells. We observed greater levels of telomerase (hTERT), increased proliferation (Ki67), survival (TUNEL), and neurogenesis (β_III-tubulin) in hNPC⁺⁷ and hNPC⁺¹⁹, using respective immunocytochemical markers. However, the trisomy lines underwent replicative senescence after 50–60 population doublings and never showed neoplastic changes. Although hNPC⁺⁷ and hNPC⁺¹⁹ survived better after xenotransplantation into the rat striatum, they did not form malignant tumors. Finally, EGF deprivation triggered a selection of trisomy 7 cells in a diploid hNPC line.

Conclusions

We report that hNPCs are susceptible to accumulation of chromosome 7 and 19 trisomy in long-term cell culture. These results suggest that micro-environmental cues are powerful factors in the selection of specific hNPC aneuploidies, with trisomy of chromosome 7 being the most common. Given that a number of stem cell based clinical trials are being conducted or planned in USA and a recent report in PLoS Medicine showing the dangers of grafting an inordinate number of cells, these data substantiate the need for careful cytogenetic evaluation of hNPCs (fetal or hESC-derived) before their use in clinical or basic science applications.     

The localization of glutathione peroxidase in the photoreceptor cells and the retinal pigment epithelial cells of Wistar and Royal College of Surgeons dystrophic rats

INTRODUCTION: If degenerating photoreceptor outer segments not phagocytized by RPE cells in the retina of Royal College Surgeons (RCS) rats were to undergo peroxidation, the distribution of glutathione peroxidase (GSH-PO) in the mitochondria or cytoplasm of the retina might be altered. We evaluated the immunocytochemical localization of GSH-PO to identify subcellular organelles in sections of the retinas of RCS rats. METHODS: Immunoblot analysis confirmed the presence of GSH-PO molecules in the retinas of RCS and Wistar rats aged 3 weeks. Sections were reacted with the F(ab) fragment of anti-rat alphaGSH-PO and then examined by laser scanning microscopy (LSM) and transmission electron microscopy (TEM). RESULTS: The size of the GSH-PO molecule in the retina was about 21 KD in the mitochondria and 23 KD in the cytosol in both strains of rats. LSM revealed fluorescent granules in the photoreceptor inner segments of the Wistar rats, and immunohistochemical TEM revealed GSH-PO in the mitochondria of their photoreceptor inner segments and retinal pigment epithelial (RPE) cells. In the RCS rats, the degenerating photoreceptor outer segments were clearly seen to be positive for anti-GSH-PO by conventional light microscopy (CLM). However, the photoreceptor inner segments of the RCS rats were negative for staining with anti-GSH-PO by LSM, and no GSH-PO could be detected in the mitochondria of the photoreceptor inner segments or RPE cells by immuno-TEM. CONCLUSION: Degeneration of the photoreceptor outer segments induced mitochondrial damage in the photoreceptor inner segments, and as a result GSH-PO shifted from the photoreceptor inner segments to the degenerating outer segments.     

小分子化合物诱导干细胞向视网膜感光细胞分化的研究进展

干细胞是一类具有多向分化潜能的细胞群,如胚胎干细胞(embryonic stem cell,ESC)、诱导多潜能干细胞(induced pluripotent stem cell,i PSC)等,可在特定的条件下向包括视网膜感光细胞在内的多种细胞分化。小分子化合物是一类由组织细胞合成、分泌的小分子多肽类因子,特定的小分子化合物可作用于干细胞诱导其向视网膜感光细胞分化。目前,对干细胞体外培养,通过使用不同的诱导培养方案,探索干细胞向视网膜感光细胞分化的研究成为热点。早期,研究者们主要在共培养条件下采用小分子化合物诱导ESC向视网膜感光细胞分化,随着研究的进展,逐渐开始探索在无共培养条件下小分子化合物诱导ESC向视网膜感光细胞的分化以及小分子化合物诱导i PSC向视网膜感光细胞的分化。本文主要就小分子化合物促进ESC和i PSC向视网膜感光细胞分化的研究进展进行综述。     

Effect of human patient plasma ex vivo treatment on gene expression and progenitor cell activation of primary human liver cells in multi‐compartment 3D perfusion bioreactors for extra‐corporeal liver support

Cultivation of primary human liver cells in innovative 3D perfusion multi‐compartment capillary membrane bioreactors using decentralized mass exchange and integral oxygenation provides in vitro conditions close to the physiologic environment in vivo. While a few scale‐up bioreactors were used clinically, inoculated liver progenitors in these bioreactors were not investigated. Therefore, we characterized regenerative processes and expression patterns of auto‐ and paracrine mediators involved in liver regeneration in bioreactors after patient treatment. Primary human liver cells containing parenchymal and non‐parenchymal cells co‐cultivated in bioreactors were used for clinical extra‐corporeal liver support to bridge to liver transplantation. 3D tissue re‐structuring in bioreactors was studied; expression of proteins and genes related to regenerative processes and hepatic progenitors was analyzed. Formation of multiple bile ductular networks and colonies of putative progenitors were observed within parenchymal cell aggregates. HGF was detected in scattered cells located close to vascular‐like structures, expression of HGFA and c‐Met was assigned to biliary cells and hepatocytes. Increased expression of genes associated to hepatic progenitors was detected following clinical application. The results confirm auto‐ and paracrine interactions between co‐cultured cells in the bioreactor. The 3D bioreactor provides a valuable tool to study mechanisms of progenitor activation and hepatic regeneration ex vivo under patient plasma treatment. Biotechnol. Bioeng. 2009;103: 817–827. © 2009 Wiley Periodicals, Inc.     

Pre-induced adult human peripheral blood mononuclear cells migrate widely into the degenerative retinas of rd1 mice

Background aimsRecent advances in stem cell research have raised the possibility of stem cells repairing or replacing retinal photoreceptor cells that are either dysfunctional or lost in many retinal diseases. Various types of stem cells have been used to replace retinal photoreceptor cells. Recently, peripheral blood stem cells, a small proportion of pluripotent stem cells, have been reported to mainly exist in the peripheral blood mononuclear cells (PBMCs).MethodsIn this study, the effects of pre-induced adult human PBMCs (hPBMCs) on the degenerative retinas of rd1 mice were investigated. Freshly isolated adult hPBMCs were pre-induced with the use of the conditioned medium of rat retinas for 4 days and were then labeled with chloromethyl-benzamidodialkylcarbocyanine (CM-DiI) and then transplanted into the subretinal space of the right eye of rd1 mice through a trans-scleral approach. The right eyes were collected 30 days after transplantation. The survival and migration of the transplanted cells in host retinas were investigated by whole-mount retinas, retinal frozen sections and immunofluorescent staining.ResultsAfter subretinal transplantation, pre-induced hPBMCs were able to survive and widely migrate into the retinas of rd1 mice. A few CM-DiI–labeled cells migrated into the inner nuclear layer and the retinal ganglion cell layer. Some transplanted cells in the subretinal space of rd1 host mice expressed the human photoreceptor–specific marker rhodopsin.ConclusionsThis study suggests that pre-induced hPBMCs may be a potential cell source of cell replacement therapy for retinal degenerative diseases.     

An extracellular retinol-binding glycoprotein in the eyes of mutant rats with retinal dystrophy: development, localization, and biosynthesis

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein in the interphotoreceptor matrix of bovine, human, monkey, and rat eyes. It may transport retinol between the retinal pigment epithelium and the neural retina. In light-reared Royal College of Surgeons (RCS) and RCS retinal dystrophy gene (rdy)+ rats, the amount of IRBP in the interphotoreceptor matrix increased in corresponding proportion to the amount of total rhodopsin through postnatal day 22 (P22). In the RCS-rdy+ rats, the amount increased slightly after P23. However, in the RCS rats there was a rapid fall in the quantity of IRBP as the photoreceptors degenerated between P23 and P29. No IRBP was detected by immunocytochemistry in rats at P28. The amount of rhodopsin fell more slowly. Although retinas from young RCS and RCS-rdy+ rats were able to synthesize and secrete IRBP, this ability was lost in retinas from older RCS rats (P51, P88) but not their congenic controls. The photoreceptor cells have degenerated at these ages in the RCS animals, and may therefore be the retinal cells responsible for IRBP synthesis. The putative function of IRBP in the extracellular transport of retinoids during the visual cycle is consistent with a defect in retinol transport in the RCS rat reported by others.     

Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells

Purpose

Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins.

Testing the biocompatibility of a glutathione-containing intra-ocular irrigation solution by using an isolated perfused bovine retina organ culture model - an alternative to animal testing

The effects of a glutathione-containing intra-ocular irrigation solution, BSS Plus?, on retinal function and on the survival of ganglion cells in whole-mount retinal explants were studied. Evidence is provided that the perfused ex vivo bovine retina can serve as an alternative to in vivo animal testing. Isolated bovine retinas were prepared and perfused with an oxygen-saturated standard irrigation solution, and an electroretinogram was recorded to assess retinal function. After stable b-waves were detected, the isolated retinas were perfused with BSS Plus for 45 minutes. To investigate the effects of BSS Plus on photoreceptor function, 1mM aspartate was added to the irrigation solution in order to obtain a-waves, and the ERG trace was monitored for 75 minutes. For histological analysis, isolated whole retinal mounts were stored for 24 hours at 4°C, in the dark. The percentages of cell death in the retinal ganglion cell layer and in the outer and inner nuclear layers were estimated by using an ethidium homodimer-1 stain and the TUNEL assay. General swelling of the retina was examined with high-resolution optical coherence tomography. During perfusion with BSS Plus, no significant changes in a-wave and b-wave amplitudes were recorded. Retinas stored for 24 hours in BSS Plus showed a statistically significant smaller percentage (52.6%, standard deviation [SD] = 16.1%) of cell death in the retinal ganglion cell layer compared to the control group (69.6%, SD = 3.9, p = 0.0031). BSS Plus did not seem to affect short-term retinal function, and had a beneficial effect on the survival of retinal ganglion cells. This method for analysing the isolated perfused retina represents a valuable alternative for testing substances for their retinal biocompatibility and toxicity.     

Enhancing survival of photoreceptor cells in vivo using the synthetic progestin Norgestrel

Retinal degenerations such as Retinitis Pigmentosa remain difficult to treat given the diverse array of genes responsible for their aetiology. Rather than concentrate on specific genes, our focus is on identifying therapeutic avenues for the treatment of retinal disease that target general survival mechanisms or pathways. Norgestrel is a synthetic progestin commonly used in hormonal contraception. Here, we report a novel anti-apoptotic role for Norgestrel in diseased mouse retinas in vivo. Dosing with Norgestrel protects photoreceptor cells from undergoing apoptosis in two distinct models of retinal degeneration; the light damage model and the Pde6b(rd10) model. Photoreceptor rescue was assessed by analysis of cell number, structural integrity and function. Improvements in cell survival of up to 70% were achieved in both disease models, indicating that apoptosis had been halted or at least delayed. A speculative mechanism of action for Norgestrel involves activation of survival pathways in the retina. Indeed, Norgestrel increases the expression of basic fibroblast growth factor which is known to both promote cell survival and inhibit apoptosis. In summary, our results demonstrate significant protection of photoreceptor cells which may be attributed to Norgestrel mediated activation of endogenous survival pathways within the retina.     

Ocular distribution of 70-kDa heat-shock protein in rats with normal and dystrophic retinas

Summary Stress proteins are thought to play an important role in cellular development and in survival mechanisms. We compared the immunolocalization of the 70-kDa stress protein (SP70) in the ocular tissue of the normal Sprague-Dawley (SD) rat with that in the Royal College of Surgeons (RCS) rat with retinal dystrophy. SP70 was present in the maturing ocular tissues of both rat strains. However, once retinal degeneration began in the RCS rat, the retinal pigment epithelium and photoreceptor cells showed increased immunostaining for SP70 over that observed in age-matched SD rats. In late stages of retinal degeneration, immunostaining for SP70 was considerably reduced in the RCS retina, whereas normal distribution of immunostaining for SP70 in the SD retina was preserved, albeit decreased, through postnatal day 180. The optic nerve, ciliary body, and corneal epithelium were also influenced by the dystrophic disease condition, although the pattern of changes in SP70 immunostaining differed for each tissue. These results suggest that the genetic defect in the RCS rat produces a state of metabolic stress in all ocular tissues as the degeneration progresses, but that the subsequent rise in ocular SP70 is insufficient to prevent progression of the disease.     

Light exposure causes functional changes in the retina: increased photoreceptor cation channel permeability, photoreceptor apoptosis, and altered retinal metabolic function

Light exposure induces retinal photoreceptor degeneration and retinal remodeling in both the normal rat retina and in animal models of retinal degeneration. Although cation entry is one of the triggers leading to apoptosis, it is unclear if this event occurs in isolation, or whether a number of pathways lead to photoreceptor apoptosis following light exposure. Following light exposure, we investigated the characteristics of cation entry, apoptotic markers [using terminal deoxynucleotidyl transferase (EC 2.7.7.31) dUTP nick-end labeling (TUNEL) labeling] and metabolic properties of retina from Sprague-Dawley (SD) rats and a rat model of retinitis pigmentosa [proline-23-histidine (P23H) rat]. Assessment of cation channel permeability using agmatine (AGB) labeling showed that excessive cation gating accompanied the series of anomalies that occur prior to photoreceptor loss. Increased AGB labeling in photoreceptors was seen in parallel with the appearance of apoptotic photoreceptors detected by TUNEL labeling with only a smaller proportion of cells colocalizing both markers. However, SD and P23H retinal photoreceptors differed in the amounts and colocalization of AGB gating and TUNEL labeling as a function of light exposure. Finally, reduced retinal lactate dehydrogenase levels were found in SD and P23H rat retinas after a 24-h light exposure period. Short-term (2 h) exposure of the P23H rat retina caused an increase in lactate dehydrogenase activity suggesting increased metabolic demand. These results suggest that energy availability may be exacerbated during the early stages of light exposure in susceptible retinas. Also, the concomitant observation of increased ion gating and TUNEL labeling suggest the existence of at least two possible mechanisms in light-damaged retinas in both SD and the P23H rat retina.     

Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells

The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning and in vivo genetic complementation to demonstrate that Mertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. We found that Mertk protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat Mertk to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. Mertk protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated Mertk accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that Mertk likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion.     

Cellular interactions determine neuronal phenotypes in rodent retinal cultures

Progenitor cells isolated from early rat embryo retinas differentiate into phenotypes normally generated early in retinal development (e.g., ganglion cells), whereas progenitors isolated from postnatal retinas differentiate into later-generated retinal cell types (e.g., rod photoreceptors; Reh and Kljavin, J. Neurosci. 9:4179–4189; 1989; Adler and Hatlee, 1989; Science 243:391–393; Sparrow, Hicks, and Barnstable, 1990, Dev. Brain Res. 51:69–84). To determine whether this change in committment is intrinsic to the progenitor cells, or alternatively can be modified by interactions with their developing environment, I co-cultured mouse and rat retinal cells, from different developmental stages, and identified the resulting phenotypes with species-specific and cell class-specific antibodies. I found that the phenotypes into which mouse neuroepithelial cells differentiate depends on the phenotypes of the rat cells that surround them. Retinal precursor cells from embryonic day (E) 10–12 will adopt the rod photoreceptor phenotype only when close to cells expressing this phenotype. By contrast, when the E10–12 retinal progenitor cells are cultured with cells from the cerebral cortex, they differentiate primarily into large multipolar neurons, similar in their morphology and antigen expression to retinal ganglion cells. These results indicate that interactions among the cells of the developing retina are important in the determination of cell fate. © 1992 John Wiley & Sons, Inc.     

Docosahexaenoic Acid Is Required for the Survival of Rat Retinal Photoreceptors In Vitro

Abstract: The effect of docosahexaenoic acid (DHA) on neuronal survival was studied in cultured cells isolated from newborn rat retina. In vivo, the content of DHA in the retina increased nearly fourfold from days 2 to 12 after birth, whereas in retinal cells in culture it remained constant. Unlike amacrine cells, the photoreceptor cells in control cultures underwent a selective degeneration, starting at day 7, that led to their massive death by day 11. The addition of DHA at day 7 led to its active incorporation by the cultures, increasing from 6 to 21% of total fatty acids in cell lipids, and completely prevented photo-receptor cell death. When other fatty acids were tested, both neuronal fatty acid composition and photoreceptor death were the same as in control cultures. These results indicate that DHA is specifically required for the survival of retinal photoreceptors.     

Distinct effects of inflammation on gliosis, osmohomeostasis, and vascular integrity during amyloid beta-induced retinal degeneration

In normal retinas, amyloid-β (Aβ) accumulates in the subretinal space, at the interface of the retinal pigment epithelium, and the photoreceptor outer segments. However, the molecular and cellular effects of subretinal Aβ remain inadequately elucidated. We previously showed that subretinal injection of Aβ(1-42) induces retinal inflammation, followed by photoreceptor cell death. The retinal Müller glial (RMG) cells, which are the principal retinal glial cells, are metabolically coupled to photoreceptors. Their role in the maintenance of retinal water/potassium and glutamate homeostasis makes them important players in photoreceptor survival. This study investigated the effects of subretinal Aβ(1-42) on RMG cells and of Aβ(1-42)-induced inflammation on retinal homeostasis. RMG cell gliosis (upregulation of GFAP, vimentin, and nestin) on day 1 postinjection and a proinflammatory phenotype were the first signs of retinal alteration induced by Aβ(1-42). On day 3, we detected modifications in the protein expression patterns of cyclooxygenase 2 (COX-2), glutamine synthetase (GS), Kir4.1 [the inwardly rectifying potassium (Kir) channel], and aquaporin (AQP)-4 water channels in RMG cells and of the photoreceptor-associated AQP-1. The integrity of the blood-retina barrier was compromised and retinal edema developed. Aβ(1-42) induced endoplasmic reticulum stress associated with sustained upregulation of the proapoptotic factors of the unfolded protein response and persistent photoreceptor apoptosis. Indomethacin treatment decreased inflammation and reversed the Aβ(1-42)-induced gliosis and modifications in the expression patterns of COX-2, Kir4.1, and AQP-1, but not of AQP-4 or GS. Nor did it improve edema. Our study pinpoints the adaptive response to Aβ of specific RMG cell functions.     

Spinal cord tissue affects sprouting from aortic fragments in ex vivo co‐culture

It is a well‐known fact, that there is a close interconnection between vascular and neural structures in both embryonic development and postnatal life. Different models have been employed to dissect the mechanisms of these interactions, ranging from in vitro systems (e.g., co‐culture of neural and endothelial cells) to in vivo imaging of central neural system recovery in laboratory animals after artificially induced trauma. Nevertheless, most of these models have serious limitations. Here, we describe an ex vivo model, representing an organotypic co‐culture of aortic fragments (AF) with longitudinal slices of mouse neonatal spinal cord (SC) or dorsal root ganglia (DRG). The samples were co‐cultured in a medium adapted for SC tissue and lacking any pro‐angiogenic or neurotrophic growth factors. It was found, that cultivation of AFs in the SC injury zone (transversal dissection of a SC slice) resulted in the initiation of active aortic sprouting. Remarkably, the endothelial cells exiting the AFs never invaded the SC tissue, concentrating in a nearby area (negative taxis). In contrast, the DRGs, while also promoting the sprouting, were a target of active endothelial CD31⁺ cell invasion (positive taxis). Thus, the tissues of both central and peripheral nervous systems have a prominent positive effect on aortic sprouting, while the vector of endothelial cell expansion is strictly nervous‐tissue‐type dependent. The ex vivo AF co‐culture with SC or DRG appeared to be a useful and promising model for a further endeavor into the mechanisms driving the complex interactions between neural and endothelial tissues.