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Механизмы регенерации и развития ткани у позвоночных

Журнальные статьи

1. U03047
Antwis R.E. et al. Tagging Frogs with Passive Integrated Transponders Causes Disruption of the Cutaneous Bacterial Community and Proliferation of Opportunistic Fungi // Applied and Environmental Microbiology. 2014. Vol. 80, № 15. P. 4779–4784.

Symbiotic bacterial communities play a key role in protecting amphibians from infectious diseases including chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis. Events that lead to the disruption of the bacterial community may have implications for the susceptibility of amphibians to such diseases. Amphibians are often marked both in the wild and in captivity for a variety of reasons, and although existing literature indicates that marking techniques have few negative effects, the response of cutaneous microbial communities has not yet been investigated. Here we determine the effects of passive integrated transponder (PIT) tagging on culturable cutaneous microbial communities of captive Morelet's tree frogs (Agalychnis moreletii) and assess the isolated bacterial strains for anti-B. dendrobatidis activity in vitro. We find that PIT tagging causes a major disruption to the bacterial community associated with the skin of frogs (similar to 12-fold increase in abundance), as well as a concurrent proliferation in resident fungi (up to similar to 200-fold increase). Handling also caused a disruption the bacterial community, although to a lesser extent than PIT tagging. However, the effects of both tagging and handling were temporary, and after 2 weeks, the bacterial communities were similar to their original compositions. We also identify two bacterial strains that inhibit B. dendrobatidis, one of which increased in abundance on PIT-tagged frogs at 1 day postmarking, while the other was unaffected. These results show that PIT tagging has previously unobserved consequences for cutaneous microbial communities of frogs and may be particularly relevant for studies that intend to use PIT tagging to identify individuals involved in trials to develop probiotic treatments.

2.
Bachleda A.R., Pevny L.H., Weiss E.R. Sox2-Deficient Muller Glia Disrupt the Structural and Functional Maturation of the Mammalian Retina // Invest. Ophthalmol. Vis. Sci. 2016. Vol. 57, № 3. P. 1488–1499.

PURPOSE. Muller glia (MG), the principal glial cells of the vertebrate retina, display quiescent progenitor cell characteristics. They express key progenitor markers, including the high mobility group box transcription factor SOX2 and maintain a progenitor-like morphology. In the embryonic and mature central nervous system, SOX2 maintains neural stem cell identity. However, its function in committed Muller glia has yet to be determined. METHODS. We use inducible, MG-specific genetic ablation of Sox2 in vivo at the peak of MG genesis to analyze its function in the maturation of murine MG and effects on other cells in the retina. Histologic and functional analysis of the Sox2-deficient retinas is conducted at key points in postnatal development. RESULTS. Ablation of Sox2 in the postnatal retina results in disorganization of MG processes in the inner plexiform layer and mislocalized cell bodies in the nuclear layers. This disorganization is concurrent with a thinning of the neural retina and disruption of neuronal processes in the inner and outer plexiform layers. Functional analysis by electroretinography reveals a decrease in the b-wave amplitude. Disruption of MG maturation due to Sox2 ablation therefore negatively affected the function of the retina. CONCLUSIONS. These results demonstrate a novel role for SOX2 in glial process outgrowth and adhesion, and provide new insights into the essential role Muller glia play in the development of retinal cytoarchitecture. Prior to this work, SOX2 was known to have a primary role in determining cell fate. Our experiments bypass cell fate conversion to establish a new role for SOX2 in a committed cell lineage.

3.
Bolleyn J. et al. Effect of Trichostatin A on miRNA expression in cultures of primary rat hepatocytes // Toxicol. Vitro. 2011. Vol. 25, № 6. P. 1173–1182.

In the present study, the effect of Trichostatin A (TSA), a histone deacetylase inhibitor, was investigated on the microRNA (miR, miRNA) expression profile in cultured primary rat hepatocytes by means of microarray analysis. Simultaneously, albumin secretory capacity and morphological features of the hepatocytes were evaluated throughout the culture time. In total, 25 out of 348 miRNAs were found to be differentially expressed between freshly isolated hepatocytes and 7-day cultured cells. Nineteen of these miRNAs were connected with 'general metabolism'. miR-21 and miR-126 were shown to be the most up and down regulated miRs upon cultivation and could be linked to the proliferative response triggered in the hepatocytes upon their isolation from the liver. miR-379 and miR-143, on the other hand, were found to be the most up and down regulated miRs upon TSA treatment. Together with the higher expression of miR-122 observed in TSA-treated versus non-treated cultures, we hypothesize that the changes observed for miR-122, miR-143 and miR-379 could be related to the inhibitory effects of TSA on hepatocellular proliferation. (C) 2011 Elsevier Ltd. All rights reserved.

4. U04533
Chavira-Suarez E. et al. Expression and high glucose-mediated regulation of K+ channel interacting protein 3 (KChIP3) and K(V)4 channels in retinal Muller glial cells // Biochem. Biophys. Res. Commun. 2011. Vol. 404, № 2. P. 678–683.

Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Muller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Muller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K+ channels K(V)4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Muller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Muller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the K(V)4 channel and KChIP3 in Muller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K+ current and an alteration in the inactivation of the macroscopic outward K+ currents expressed in high glucose-cultured Muller cells. Our data support the notion that induction of KChIP3 and functional changes of K(V)4 channels in Muller cells could exert a physiological role in the onset of diabetic retinopathy. (C) 2010 Elsevier Inc. All rights reserved.

5.
Chen C., Koutalos Y. Rapid formation of all-trans retinol after bleaching in frog and mouse rod photoreceptor outer segments // Photochem. Photobiol. Sci. 2010. Vol. 9, № 11. P. 1475–1479.

All-trans retinol is formed in the outer segments of vertebrate rod photoreceptors from the reduction of the all-trans retinal released by photoactivated rhodopsin. The reduction requires NADPH and is therefore dependent on metabolic input. In metabolically intact photoreceptors, a large increase in rod outer segment fluorescence, attributed to the fluorescence of all-trans retinol, follows rhodopsin photoactivation. The fluorescence increase is biphasic, including a rapid and a slow component. In metabolically compromised cells, there is a much smaller fluorescence increase following rhodopsin photoactivation, but it too contains a rapid component. We have measured the fluorescence signal in single living frog and mouse rod photoreceptors, and have characterized its dependence on the wavelengths of light selected for excitation and for collecting emission. We find that in metabolically intact cells, the excitation and emission properties of both the rapid and slow components of the fluorescence signal are in close agreement with those of all-trans retinol fluorescence. In metabolically compromised cells, however, the signal can only partially be due to all-trans retinol, and most of it is consistent with all-trans retinal. The results suggest that in the outer segments of living rod photoreceptors there is rapid release of all-trans retinal, which in metabolically intact cells is accompanied by rapid conversion to all-trans retinol.

6.
Chen X., Tolkovsky A.M., Herbert J. Cell Origin and Culture History Determine Successful Integration of Neural Precursor Transplants into the Dentate Gyrus of the Adult Rat // PLoS One. 2011. Vol. 6, № 2. P. e17072.

The success of transplants of neural tissue into the adult dentate gyrus in generating mature neurons is highly variable. Here we address the roles of the origin of the tissue and its pre-implantation preparation, and show that both are critical. We transplanted neonatal cultured or primary rat cells from either the ventral subventricular zone (vSVZ) or the dentate gyrus (DG) into the adult rat DG. Only primary DG cells robustly generated DG neurons (80% NeuN and Prox1-positive cells at 6 weeks), substantially repaired the damaged DG, and formed glutamatergic projections to the target CA3 region. Cultured DG cells expanded for 7 days showed limited neuronal differentiation after transplantation (10% NeuN and Prox1-positive cells) whereas cultured or primary vSVZ cells failed to make any Prox1-positive DG granular neurons. We found that a specific population of postmitotic young neurons (triple doublecortin/NeuN/Prox1-positive) were particularly abundant in primary DG cells, but were markedly reduced in the cultured DG cells and were absent in the cultured and primary vSVZ cells. Labelling of primary DG cells with the mitotic marker BrdU suggested that postmitotic young neurons are the source of the transplanted mature neurons in-vivo. We conclude that both the origin and pre-transplantation history of donor cells are key factors that determine the outcome of transplantation. These findings may be of therapeutic interest for cell replacement therapy in treating the damaged hippocampus.

7.
Del Debbio C.B. et al. Notch Signaling Activates Stem Cell Properties of Muller Glia through Transcriptional Regulation and Skp2-mediated Degradation of p27(Kip1) // PLoS One. 2016. Vol. 11, № 3. P. e0152025.

"Notch Signaling Activates Stem Cell Properties of Muller Glia through Transcriptional Regulation and Skp2-mediated Degradation of p27(Kip1) By: Del Debbio, Carolina Beltrame; Mir, Qulsum; Parameswaran, Sowmya; et al. PLOS ONE Volume: 11 Issue: 3 Article Number: e0152025 Published: MAR 24 2016 Full Text from Publisher Close AbstractClose Abstract Muller glia (MG), the sole glial cells generated by retinal progenitors, have emerged as a viable cellular target for therapeutic regeneration in degenerative blinding diseases, as they possess dormant stem cell properties. However, the mammalian MG does not display the neurogenic potential of their lower vertebrate counterparts, precluding their practical clinical use. The answer to this barrier may be found in two interlinked processes underlying the neurogenic potential, i.e., the activation of the dormant stem cell properties of MG and their differentiation along the neuronal lineage. Here, we have focused on the former and examined Notch signaling-mediated activation of MG. We demonstrate that one of the targets of Notch signaling is the cyclin-dependent kinase inhibitor (CKI), p27(Kip1), which is highly expressed in quiescent MG. Notch signaling facilitates the activation of MG by inhibiting p27(Kip1) expression. This is likely achieved through the Notch-p27(Kip1) and Notch-Skp2-p27(Kip1) axes, the former inhibiting the expression of p27(Kip1) transcripts and the latter levels of p27(Kip1) proteins by Skp2-mediated proteasomal degradation. Thus, Notch signaling may facilitate re-entry of MG into the cell cycle by inhibiting p27(Kip1) expression both transcriptionally and post-translationally."

8.
Digilio L., Yap C.C., Winckler B. Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19) // PLoS One. 2015. Vol. 10, № 10. P. e0140010.

Background: Retinal degeneration in transgenic rats that express a mutant cilia gene polycystin-2 (CMV-PKD2(1/ 703) HA) is characterized by initial photoreceptor degeneration and glial activation, followed by vasoregression and neuronal degeneration (Feng et al., 2009, PLoS One 4: e7328). It is unknown whether glial activation contributes to neurovascular degeneration after photoreceptor degeneration. We characterized the reactivity of Muller glial cells in retinas of rats that express defective polycystin-2. Methods: Age-matched Sprague-Dawley rats served as control. Retinal slices were immunostained for intermediate filaments, the potassium channel Kir4.1, and aquaporins 1 and 4. The potassium conductance of isolated Muller cells was recorded by whole-cell patch clamping. The osmotic swelling characteristics of Muller cells were determined by superfusion of retinal slices with a hypoosmotic solution. Findings: Muller cells in retinas of transgenic rats displayed upregulation of GFAP and nestin which was not observed in control cells. Whereas aquaporin-1 labeling of photoreceptor cells disappeared along with the degeneration of the cells, aquaporin-1 emerged in glial cells in the inner retina of transgenic rats. Aquaporin-4 was upregulated around degenerating photoreceptor cells. There was an age-dependent redistribution of Kir4.1 in retinas of transgenic rats, with a more even distribution along glial membranes and a downregulation of perivascular Kir4.1. Muller cells of transgenic rats displayed a slight decrease in their Kir conductance as compared to control. Muller cells in retinal tissues from transgenic rats swelled immediately under hypoosmotic stress; this was not observed in control cells. Osmotic swelling was induced by oxidativenitrosative stress, mitochondrial dysfunction, and inflammatory lipid mediators. Interpretation: Cellular swelling suggests that the rapid water transport through Muller cells in response to osmotic stress is altered as compared to control. The dislocation of Kir4.1 will disturb the retinal potassium and water homeostasis, and osmotic generation of free radicals and inflammatory lipids may contribute to neurovascular injury.

9. U03047
Eisenberg T. et al. Isolation of Potentially Novel Brucella spp. from Frogs // Applied and Environmental Microbiology. 2012. Vol. 78, № 10. P. 3753–3755.

Bacterial isolates from frogs were phenotypically identified as Ochrobactrum anthropi, but 16S rRNA sequencing showed up to 100% identity with Brucella inopinata. Further analysis of recA, omp2a, omp2b, bcsp31, and IS711 and multilocus sequence analysis (MLSA) verified a close relationship with Brucella, suggesting the isolates may actually represent novel members of this growing genus of zoonotic pathogens.

10.
Fischer A.J., Bongini R. Turning Muller Glia into Neural Progenitors in the Retina // Mol. Neurobiol. 2010. Vol. 42, № 3. P. 199–209.

11.
Fischer A.J., Bosse J.L., El-Hodiri H.M. The ciliary marginal zone (CMZ) in development and regeneration of the vertebrate eye // Exp. Eye Res. 2013. Vol. 116. P. 199–204.

The ciliary marginal zone (CMZ) is a circumferential ring of cells found at the extreme periphery of the maturing and mature neural retina that consists of retinal stem and progenitor cells. It functions to add retinal neurons to the periphery of the neural retina in larval and adult fish, larval frogs, and birds. Additionally, the CMZ may contribute to regeneration of the damaged retina in frogs and fish. In mammals, cells from the ciliary epithelium can be induced to express retinal stem cell-like characteristics in culture but may not comprise a classically defined CMZ. (C) 2013 Published by Elsevier Ltd.

12.
Frippiat J.-P. Contribution of the urodele amphibian Pleurodeles waltl to the analysis of spaceflight-associated immune system deregulation // Mol. Immunol. 2013. Vol. 56, № 4. P. 434–441.

Immune system deregulation has been demonstrated to occur during and immediately following spaceflight. Several animal models have been used to study this phenomenon because of the limited availability of human subjects in space as well as of the need to carry out experiments requiring samples and experimental conditions that cannot be performed using humans. Here, we review major spaceflight-induced microbial and immunological modifications, some of the existing hardware developed to host amphibians in a space station and immunological information provided by space experiments performed with Pleurodeles waitl as an animal model. These data show that the urodele amphibian P. waftl fulfills many technical requirements associated with spaceflight experimentation and that this model is interesting to improve our understanding of the immunosuppressive effects of spaceflight, data required for the preparation of future deep-space missions. (C) 2013 Elsevier Ltd. All rights reserved.

13.
Gaddini L. et al. Muller glia activation by VEGF-antagonizing drugs: An in vitro study on rat primary retinal cultures // Exp. Eye Res. 2016. Vol. 145. P. 158–163.

The effects of the anti-Vascular Endothelial Growth Factor (VEGF) drugs ranibizumab and aflibercept were studied in Muller glia in primary mixed cultures from rat neonatal retina. Treatment with both agents induced activation of Muller glia, demonstrated by increased levels of Glial Fibrillary Acidic Protein. In addition, phosphorylated Extracellular-Regulated Kinase 1/2 (ERK 1/2) showed enhanced immunoreactivity in activated Muller glia. Treatment with aflibercept induced an increase in K+ channel (Kir) 4.1 levels and both drugs upregulated Aquaporin 4 (AQP4) in activated Muller glia. The results show that VEGF-antagonizing drugs influence the homeostasis of Muller cells in primary retinal cultures, inducing an activated phenotype. Upregulation of Kir4.1 and AQP4 suggests that Muller glia activation following anti-VEGF drugs may not depict a detrimental gliotic reaction. Indeed, it could represent one of the mechanisms able to contribute to the therapeutic effects of these drugs, particularly in the presence of macular edema. (C) 2015 Elsevier Ltd. All rights reserved.

14.
Gallina D., Todd L., Fischer A.J. A comparative analysis of Muller glia-mediated regeneration in the vertebrate retina // Exp. Eye Res. 2014. Vol. 123. P. 121–130.

This article reviews the current state of knowledge regarding the potential of Muller glia to become neuronal progenitor cells in the avian retina. We compare and contrast the remarkable proliferative and neurogenic capacity of Muller glia in the fish retina to the limited capacity of Muller glia in avian and rodent retinas. We summarize recent findings regarding the secreted factors, signaling pathways and cell intrinsic factors that have been implicated in the formation of Miller glia-derived progenitors. We discuss several key similarities and differences between the fish, rodent and chick model systems, highlighting several of the key transcription factors and signaling pathways that regulate the formation of Muller glia-derived progenitors. (C) 2013 Elsevier Ltd. All rights reserved.

15.U04533
Jin K., Xiang M. Ebf1 deficiency causes increase of Muller cells in the retina and abnormal topographic projection at the optic chiasm // Biochem. Biophys. Res. Commun. 2011. Vol. 414, № 3. P. 539–544.

The Ebf transcription factors play important roles in the developmental processes of many tissues. We have shown previously that four members of the Ebf family are expressed during mouse retinal development and are both necessary and sufficient to specify multiple retinal cell fates. Here we describe the changes in cell differentiation and retinal ganglion cell (RGC) projection in Ebf1 knockout mice. Analysis of marker expression in Ebf1 null mutant retinas reveals that loss of Ebf1 function causes a significant increase of Muller cells. Moreover, there is an obvious decrease of ipsilateral and retinoretinal projections of RGC axons at the optic chiasm, whereas the contralateral projection significantly increases in the mutant mice. These data together suggests that Ebf1 is required for suppressing the Muller cell fate during retinogenesis and important for the correct topographic projection of RGC axons at the optic chiasm. (C) 2011 Elsevier Inc. All rights reserved.

16.
Kim Y. et al. Role of Prox1 in the Transforming Ascending Thin Limb of Henle’s Loop during Mouse Kidney Development // PLoS One. 2015. Vol. 10, № 5. P. e0127429.

The homeobox transcription factor Prox1 is critical to the development of many embryonic organs and tissues, although current understanding of its expression in the developing renal medulla is limited. We examined the functional role of Prox1 during mouse kidney development with particular emphasis on the developing loop of Henle. Our data show that Prox1 is expressed in the transdifferentiating region from the NKCC2-positive thick ascending limb, into the CLC-K1-positive ascending thin limb of Henle's loop beginning at embryonic day 18. From 1 to 14 days of age, Prox1-positive cells gradually disappeared from the papillary tip, and remained in the initial part of inner medulla after 21 days. In this transforming area, no Prox1 was observed in cells undergoing apoptosis but was expressed strongly in the remaining cells, which differentiated into ascending thin limb epithelial cells. In vitro and in vivo approaches showed that Prox1 expression increases where the osmolality is near optimal range, but decreases at below-or above-optimal ranges. Renal hypoosmolality induced by furosemide (NKCC2 inhibitor) inhibited Prox1 expression and delayed maturation of the ascending limb of Henle's loop. Together, these studies suggest that Prox1 appears to be a critical stage specific regulator of specifying ascending thin limb cell fate and that its expression is regulated by osmolality.

17.
Mark P. Gundersona,1, Nik Veldhoena, Rachel C. Skirrowb, Magnus K. Macnaba, Wei Dinga, Graham van Aggelenb, Caren C. Helbinga. Effect of low dose exposure to the herbicide atrazine and its metabolite on cytochrome P450 aromatase and steroidogenic factor-1 mRNA levels in the brain of premetamorphic bullfrog tadpoles (Rana catesbeiana) // Aquatic Toxicology. 2011. Vol. 102, P.31–38

"The transcriptional regulator steroidogenic factor 1 (SF-1) and the enzyme cytochrome P450 aromatase (CYP19) play a central role in modulation of a broad range of tissue-specific developmental processes associated with hormone homeostasis that includes differentiation of the central nervous system. SF- 1 and CYP19 expression may be targeted by a variety of endocrine disruptive agents prevalent within the environment. In the present study, we cloned and characterized partial sequences for bullfrog (Rana catesbeiana) SF-1 and CYP19 and examined the effects of a 48 h exposure to 1 and 100 g/l of the herbicide atrazine (ATZ) and its major metabolite desethylatrazine (DEA), as well as 5 ng/l of the estrogenic chemical, 17 -ethynylestradiol (EE2), and 673 ng/l of the thyroid hormone, 3,5,3 -triiodothyronine (T3), on SF-1 and CYP19 mRNA abundance in the brains of premetamorphic bullfrog tadpoles. Quantitative RT-PCR analysis showed an increase in CYP19 mRNA following a 48 h exposure to EE2 but not T3 while no significant changes in SF-1 transcript levels occurred. We observed a strong positive correlation between CYP19 and SF-1 transcript abundance in the ATZ-exposed animals which was not evident with DEA- or hormone-exposed tadpoles. Our results are intriguing in light of reported behavioral changes in ATZexposed frogs and suggest that further research is warranted to examine the relationship and role of CYP19 and SF-1 in amphibian brain development. ©"

18.
Nadal-Nicolas F.M. et al. Retino-retinal projection in juvenile and young adult rats and mice // Exp. Eye Res. 2015. Vol. 134. P. 47–52.

Identification of retino-retinal projecting RGCs (ret-ret RGCs) has been accomplished by tracing RGCs in one retina after intravitreal injection of different tracers in the other eye. In mammals, rabbit and rat, ret-ret RGCs are scarce and more abundant in newborn than in adult animals. To our knowledge, ret-ret RGCs have not been studied in mice. Here we purpose to revisit the presence of ret-ret RGCs in juvenile and young adult rats and mice by using retrograde tracers applied to the contralateral optic nerve instead of intravitreally. In P20 (juvenile) and P60 (young adult) animals, the left optic nerve was intraorbitally transected and Fluorogold (rats) or its analogue OHSt (mice) were applied onto its distal stump. P20 animals were sacrificed 3 (mice) or 5 (rats) days later and adult animals at 5 (mice) or 7 (rats) days. Right retinas were dissected as flat-mounts and double immunodetected for Brn3a and melanopsin. Ret-ret RGCs were those with tracer accumulation in their somas. Out of them some expressed Brn3a and/or melanopsin, while other were negative for both markers. In young adult rats, we found 2 ret-ret RGCs displaced to the inner nuclear layer. In both species, ret-ret RGCs are quite scarce and found predominantly in the nasal retina. In juvenile animals there are significantly more ret-ret RGCs (9 +/- 3, rats, 13 +/- 3 mice) than in young adult ones (5 6 rats, 7 3 mice). Finally, juvenile and young adult mice have more ret-ret RGCs than rats. (C) 2015 The Authors. Published by Elsevier Ltd.

19.
Ola M.S., Hosoya K.-I., LaNoue K.F. Regulation of glutamate metabolism by hydrocortisone and branched chain keto acids in cultured rat retinal Muller cells (TR-MUL) // Neurochem. Int. 2011. Vol. 59, № 5. P. 656–663.

Glutamate released from retinal neurons during neurotransmission is taken up by retinal Muller cells, where much of the amino acid is subsequently amidated to glutamine or transaminated to alpha-ketoglutarate for oxidation. Muller cell glutamate levels may have to be carefully maintained at fairly low concentrations to avoid excesses of glutamate in extracellular spaces of the retina that would otherwise cause excitotoxicity. We employed a cultured rat retinal Muller cell line in order to study the metabolism and the role of Muller cell specific enzymes on the glutamate disposal pathways. We found that the TR-MUL cells express the glial specific enzymes, glutamine synthetase, the mitochondrial isoform of branched chain aminotransferase (BCATm) and pyruvate carboxylase, all of which are involved in glutamate metabolism and homeostasis in the retina. Hydrocortisone treatment of TR-MUL cells increased glutamine synthetase expression and the rate of glutamate amidation to glutamine. Addition of branched chain keto acids (BCKAs) increased lactate and aspartate formation from glutamate and also oxidation of glutamate to CO(2) and H(2)O. The two glutamate disposal pathways (amidation and oxidation) did not influence each other. When glutamate levels were independently depleted within TR-MUL cells, the uptake of glutamate from the extracellular fluid increased compared to uptake from control (undepleted) cells suggesting that the level of intracellular glutamate may influence clearing of extracellular glutamate. (C) 2011 Elsevier B.V. All rights reserved.

20.
Powell C. et al. Zebrafish Muller glia-derived progenitors are multipotent, exhibit proliferative biases and regenerate excess neurons // Sci Rep. 2016. Vol. 6. P. 24851.

Unlike mammals, zebrafish can regenerate a damaged retina. Key to this regenerative response are Muller glia (MG) that respond to injury by reprogramming and adopting retinal stem cell properties. These reprogrammed MG divide to produce a proliferating population of retinal progenitors that migrate to areas of retinal damage and regenerate lost neurons. Previous studies have suggested that MG-derived progenitors may be biased to produce that are lost with injury. Here we investigated MG multipotency using injury paradigms that target different retinal nuclear layers for cell ablation. Our data indicate that regardless of which nuclear layer was damaged, MG respond by generating multipotent progenitors that migrate to all nuclear layers and differentiate into layer-specific cell types, suggesting that MG-derived progenitors in the injured retina are intrinsically multipotent. However, our analysis of progenitor proliferation reveals a proliferative advantage in nuclear layers where neurons were ablated. This suggests that feedback inhibition from surviving neurons may skew neuronal regeneration towards ablated cell types.

21.
Sharma R. et al. In Vivo Two-Photon Fluorescence Kinetics of Primate Rods and Cones // Invest. Ophthalmol. Vis. Sci. 2016. Vol. 57, № 2. P. 647–657.

PURPOSE. The retinoid cycle maintains vision by regenerating bleached visual pigment through metabolic events, the kinetics of which have been difficult to characterize in vivo. Two-photon fluorescence excitation has been used previously to track autofluorescence directly from retinoids and pyridines in the visual cycle in mouse and frog retinas, but the mechanisms of the retinoid cycle are not well understood in primates. METHODS. We developed a two-photon fluorescence adaptive optics scanning light ophthalmoscope dedicated to in vivo imaging in anesthetized macaques. Using pulsed light at 730 nm, two-photon fluorescence was captured from rods and cones during light and dark adaptation through the eye's pupil. RESULTS. The fluorescence from rods and cones increased with light exposure but at different rates. During dark adaptation, autofluorescence declined, with cone autofluorescence decreasing approximately 4 times faster than from rods. Rates of autofluorescence decrease in rods and cones were approximately 4 times faster than their respective rates of photopigment regeneration. Also, subsets of sparsely distributed cones were less fluorescent than their neighbors immediately following bleach at 565 nm and they were comparable with the S cone mosaic in density and distribution. CONCLUSIONS. Although other molecules could be contributing, we posit that these fluorescence changes are mediated by products of the retinoid cycle. In vivo two-photon ophthalmoscopy provides a way to monitor noninvasively stages of the retinoid cycle that were previously inaccessible in the living primate eye. This can be used to assess objectively photoreceptor function in normal and diseased retinas.

22.
Unterlauft J.D. et al. Enhanced survival of retinal ganglion cells is mediated by Muller glial cell-derived PEDF // Exp. Eye Res. 2014. Vol. 127. P. 206–214.

The death of retinal ganglion cells (RGC) leads to visual impairment and blindness in ocular neurodegenerative diseases, primarily in glaucoma and diabetic retinopathy; hence, mechanisms that contribute to protecting RGC from ischemia/hypoxia are of great interest. We here address the role of retinal glial (Muller) cells and of pigment-epithelium-derived factor (PEDF), one of the main neuroprotectants released from the glial cells. We show that the hypoxia-induced loss in the viability of cultured purified RGC is due to apoptosis, but that the number of viable RGC increases when co-cultured with Muller glial cells suggesting that glial soluble mediators attenuate the death of RGC. When PEDF was ablated from Muller cells a significantly lower number of RGC survived in RGC-Muller cell co-cultures indicating that PEDF is a major survival factor allowing RGC to escape cell death. We further found that RGC express a PEDF receptor known as patatin-like phospholipase domain-containing protein 2 (PNPLA2) and that PEDF exposure, as well as the presence of Muller cells, leads to an activation of nuclear factor (NF)-kappa B in RGC. Furthermore, adding an NF-kappa B inhibitor (SN50) to PEDF-treated RGC cultures reduced the survival of RGC. These findings strongly suggest that NF-kappa B activation in RGC is critically involved in the pro-survival action of Muller-cell derived PEDF and plays an important role in maintaining neuronal survival. (C) 2014 Elsevier Ltd. All rights reserved.

23. S17054
Ursprung E. et al. Toe regeneration in the neotropical frog Allobates femoralis // Herpetolog. J. 2011. Vol. 21, № 1. P. 83–86.

Toe-clipping is a standard method for marking and tissue sampling in amphibians, and in most adult anurans such marks are permanent. Here we document the consistent regeneration of toes in the aromobatid frog Allobates femoralis during a three-year population study. The emergence of new toe discs was observed after about two months. After one year the regrown toes had recovered to 65.6%/63.8% (males/females) of the size of unclipped toes and after two years they had attained 74.0%/69.0%. Whereas toe discs before amputation were white dorsally, all but one regenerated toe discs were dark. We did not detect any malformations or infections of the digits. Recapture rates of toe-clipped individuals were indiscernible from those of a nearby population where no toe clips were taken. We discuss a possible link between toe regeneration ability and life-history attributes.

24.
Vega-Melendez G.S., Blagburn J.M., Blanco R.E. Ciliary Neurotrophic Factor and Fibroblast Growth Factor Increase the Speed and Number of Regenerating Axons After Optic Nerve Injury in Adult Rana pipiens // J. Neurosci. Res. 2014. Vol. 92, № 1. P. 13–23.

Neurotrophins such as ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) and growth factors such as fibroblast growth factor (FGF-2) play important roles in neuronal survival and in axonal outgrowth during development. However, whether they can modulate regeneration after optic nerve injury in the adult animal is less clear. The present study investigates the effects of application of these neurotrophic factors on the speed, number, and distribution of regenerating axons in the frog Rana pipiens after optic nerve crush. Optic nerves were crushed and the factors, or phosphate-buffered saline, were applied to the stump or intraocularly. The nerves were examined at different times after axotomy, using anterograde labeling with biotin dextran amine and antibody against growth-associated protein 43. We measured the length, number, and distribution of axons projecting beyond the lesion site. Untreated regenerating axons show an increase in elongation rate over 3 weeks. CNTF more than doubles this rate, FGF-2 increases it, and BDNF has little effect. In contrast, the numbers of regenerating axons that have reached 200 m at 2 weeks were more than doubled by FGF-2, increased by CNTF, and barely affected by BDNF. The regenerating axons were preferentially distributed in the periphery of the nerve; although the numbers of axons were increased by neurotrophic factor application, this overall distribution was substantially unaffected. (c) 2013 Wiley Periodicals, Inc.

25.
Vogler S. et al. Muller Cell Reactivity in Response to Photoreceptor Degeneration in Rats with Defective Polycystin-2 // PLoS One. 2013. Vol. 8, № 6. P. e61631.

Background: Retinal degeneration in transgenic rats that express a mutant cilia gene polycystin-2 (CMV-PKD2(1/ 703) HA) is characterized by initial photoreceptor degeneration and glial activation, followed by vasoregression and neuronal degeneration (Feng et al., 2009, PLoS One 4: e7328). It is unknown whether glial activation contributes to neurovascular degeneration after photoreceptor degeneration. We characterized the reactivity of Muller glial cells in retinas of rats that express defective polycystin-2. Methods: Age-matched Sprague-Dawley rats served as control. Retinal slices were immunostained for intermediate filaments, the potassium channel Kir4.1, and aquaporins 1 and 4. The potassium conductance of isolated Muller cells was recorded by whole-cell patch clamping. The osmotic swelling characteristics of Muller cells were determined by superfusion of retinal slices with a hypoosmotic solution. Findings: Muller cells in retinas of transgenic rats displayed upregulation of GFAP and nestin which was not observed in control cells. Whereas aquaporin-1 labeling of photoreceptor cells disappeared along with the degeneration of the cells, aquaporin-1 emerged in glial cells in the inner retina of transgenic rats. Aquaporin-4 was upregulated around degenerating photoreceptor cells. There was an age-dependent redistribution of Kir4.1 in retinas of transgenic rats, with a more even distribution along glial membranes and a downregulation of perivascular Kir4.1. Muller cells of transgenic rats displayed a slight decrease in their Kir conductance as compared to control. Muller cells in retinal tissues from transgenic rats swelled immediately under hypoosmotic stress; this was not observed in control cells. Osmotic swelling was induced by oxidativenitrosative stress, mitochondrial dysfunction, and inflammatory lipid mediators. Interpretation: Cellular swelling suggests that the rapid water transport through Muller cells in response to osmotic stress is altered as compared to control. The dislocation of Kir4.1 will disturb the retinal potassium and water homeostasis, and osmotic generation of free radicals and inflammatory lipids may contribute to neurovascular injury.

26.
Wang H. et al. Short Hairpin RNA-Mediated Knockdown of VEGFA in Muller Cells Reduces Intravitreal Neovascularization in a Rat Model of Retinopathy of Prematurity // Am. J. Pathol. 2013. Vol. 183, № 3. P. 964–974.

Vascular endothelial growth factor (VEGF) A is implicated in aberrant angiogenesis and intravitreous neovascularization (IVNV) in retinopathy of prematurity (ROP). However, VEGFA also regulates retinal vascular development and functions as a retinal neural survival factor. By using a relevant ROP model, the 50/10 oxygen-induced retinopathy (OIR) model, we previously found that broad inhibition of VEGFA bioactivity using a neutralizing antibody to rat VEGF significantly reduced IVNV area compared with control IgG but also significantly reduced body weight gain in the pups, suggesting an adverse effect. Therefore, we propose that knockdown of up-regulated VEGFA in cells that overexpress it under pathological conditions would reduce IVNV without affecting physiological retinal vascular development or overall pup growth. Herein, we determined first that the VEGFA mRNA signal was located within the inner nuclear layer corresponding to CRALBP-labeled Muller cells of pups in the 50/10 OIR model. We then developed a lentiviral-delivered miR-30-embedded shRNA against VEGFA that targeted Muller cells. Reduction of VEGFA by lentivector VEGFA-shRNA-targeting Muller cells efficiently reduced 50/10 OIR up-regulated VEGFA and IVNV in the model, without adversely affecting physiological retinal vascular development or pup weight gain. Knockdown of VEGFA in rat Muller cells by lentivector VEGFA-shRNA significantly reduced VEGFR2 phosphorylation in retinal vascular endothelial cells. Our results suggest that targeted knockdown of overexpressed VEGFA in Muller cells safely reduces IVNV in a relevant ROP model.

27.
Wang J.-S., Kefalov V.J. The Cone-specific visual cycle // Prog. Retin. Eye Res. 2011. Vol. 30, № 2. P. 115–128.

Cone photoreceptors mediate our daytime vision and function under bright and rapidly-changing light conditions. As their visual pigment is destroyed in the process of photoactivation, the continuous function of cones imposes the need for rapid recycling of their chromophore and regeneration of their pigment. The canonical retinoid visual cycle through the retinal pigment epithelium cells recycles chromophore and supplies it to both rods and cones. However, shortcomings of this pathway, including its slow rate and competition with rods for chromophore, have led to the suggestion that cones might use a separate mechanism for recycling of chromophore. In the past four decades biochemical studies have identified enzymatic activities consistent with recycling chromophore in the retinas of cone-dominant animals, such as chicken and ground squirrel. These studies have led to the hypothesis of a cone-specific retina visual cycle. The physiological relevance of these studies was controversial for a long time and evidence for the function of this visual cycle emerged only in very recent studies and will be the focus of this review. The retina visual cycle supplies chromophore and promotes pigment regeneration only in cones but not in rods. This pathway is independent of the pigment epithelium and instead involves the Muller cells in the retina, where chromophore is recycled and supplied selectively to cones. The rapid supply of chromophore through the retina visual cycle is critical for extending the dynamic range of cones to bright light and for their rapid dark adaptation following exposure to light. The importance of the retina visual cycle is emphasized also by its preservation through evolution as its function has now been demonstrated in species ranging from salamander to zebrafish, mouse, primate, and human. (C) 2010 Elsevier Ltd. All rights reserved.

28.U21308
Wei H.-F. et al. BDNF and GAP43 contribute to dynamic transhemispheric functional reorganization in rat brain after contralateral C7 root transfer following brachial plexus avulsion injuries // Neurosci. Lett. 2011. Vol. 500, № 3. P. 187–191.

It is known that contralateral seventh cervical nerve (C7) root transfer after brachial plexus avulsion injuries (BPAI) causes interhemispheric cortical functional reorganization. However, the potential mechanisms and the role of neurotrophic factors and/or growth-associated protein expression in the process of cerebral reorganization are not well understood. The present study identified the expression of brain-derived neurotrophic factor (BDNF) and growth-associated protein 43 (GAP43) mRNA in primary motor cortex after contralateral C7 root transfer following BPAI. BDNF and GAP43 mRNA levels were significantly increased in brain samples at both 6 and 9 months after contralateral C7 root transfer following BPAI, in comparison with the samples from the rats with BPAI only. These findings indicate that BDNF and GAP43 may play an important role during the dynamic transhemispheric functional reorganization. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

29.U01107
Zhelev Z. et al. Assessment of Ecological Status of Two Rivers with Different Types of Anthropogenic Pollution in Southern Bulgaria Based on the Level of Fluctuating Asymmetry in the Populations of Marsh Frog Rana ridibunda (Amphibia: Ranidae) // Acta Zoologica Bulgarica. 2012. P. 229–235.

The degree of manifestation of the indicators of fluctuating asymmetry (FA) was studied in the populations of Rana ridibunda in two of the most polluted river ecosystems in Southern Bulgaria. The integral indicator of developmental stability, evaluation of the ecological condition parallel to the data of physicochemical analysis was performed. It was found that the variations in genetic homeostasis in the populations of R. ridibunda in a habitat under conditions of anthropogenic pollution occur independently on the type of toxicants, but the degree of violation expression in the form of FA depends on the nature of the pollution.

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