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1.  Alterations of the Tunica Vasculosa Lentis in the Rat Model of Retinopathy of Prematurity 
Purpose
To study the relation between retinal and tunica vasculosa lentis (TVL) disease in ROP. Although the clinical hallmark of retinopathy of prematurity (ROP) is abnormal retinal blood vessels, the vessels of the anterior segment, including the TVL, are also altered.
Methods
ROP was induced in Long Evans pigmented and Sprague-Dawley albino rats; room-air-reared (RAR) rats served as controls. Then, fluorescein angiographic images of the TVL and retinal vessels were serially obtained with a scanning laser ophthalmoscope (SLO) near the height of retinal vascular disease, ∼20 days-of-age, and again at 30 and 64 days-of-age. Additionally, electroretinograms (ERGs) were obtained prior to the first imaging session. The TVL images were analyzed for percent coverage of the posterior lens. The tortuosity of the retinal arterioles was determined using Retinal Image multiScale Analysis (RISA; Gelman et al., 2005).
Results
In the youngest ROP rats, the TVL was dense, while in RAR rats, it was relatively sparse. By 30 days, the TVL in RAR rats had almost fully regressed, while in ROP rats it was still pronounced. By the final test age, the TVL had completely regressed in both ROP and RAR rats. In parallel, the tortuous retinal arterioles in ROP rats resolved with increasing age. ERG components indicating postreceptoral dysfunction, the b-wave and oscillatory potentials (OPs), were attenuated in ROP rats.
Conclusions
These findings underscore the retinal vascular abnormalities and, for the first time, show abnormal anterior segment vasculature in the rat model of ROP. There is delayed regression of the TVL in the rat model of ROP. This demonstrates that ROP is a disease of the whole eye.
doi:10.1007/s10633-013-9392-z
PMCID: PMC3775643  PMID: 23748796
2.  Targeted Ablation of Crb1 and Crb2 in Retinal Progenitor Cells Mimics Leber Congenital Amaurosis 
PLoS Genetics  2013;9(12):e1003976.
Development in the central nervous system is highly dependent on the regulation of the switch from progenitor cell proliferation to differentiation, but the molecular and cellular events controlling this process remain poorly understood. Here, we report that ablation of Crb1 and Crb2 genes results in severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. We show that the levels of CRB1 and CRB2 proteins are crucial for mouse retinal development, as they restrain the proliferation of retinal progenitor cells. The lack of these apical proteins results in altered cell cycle progression and increased number of mitotic cells leading to an increased number of late-born cell types such as rod photoreceptors, bipolar and Müller glia cells in postmitotic retinas. Loss of CRB1 and CRB2 in the retina results in dysregulation of target genes for the Notch1 and YAP/Hippo signaling pathways and increased levels of P120-catenin. Loss of CRB1 and CRB2 result in altered progenitor cell cycle distribution with a decrease in number of late progenitors in G1 and an increase in S and G2/M phase. These findings suggest that CRB1 and CRB2 suppress late progenitor pool expansion by regulating multiple proliferative signaling pathways.
Author Summary
Mutations in the human CRB1 gene lead to one of the most severe forms of retinal dystrophies, called Leber congenital amaurosis. Here, we report that ablation of CRB1 and the second family member CRB2 are crucial for proper retinal development. These mice display severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. The thickening of the retina is due to increased cell proliferation during late retinal development leading to an increased number of late-born retinal cells. We describe in these CRB1 Leber congenital amaurosis mouse models the molecular and cellular events involving CRB proteins during the development of the retina.
doi:10.1371/journal.pgen.1003976
PMCID: PMC3854796  PMID: 24339791
3.  Lack of the Sodium-Driven Chloride Bicarbonate Exchanger NCBE Impairs Visual Function in the Mouse Retina 
PLoS ONE  2012;7(10):e46155.
Regulation of ion and pH homeostasis is essential for normal neuronal function. The sodium-driven chloride bicarbonate exchanger NCBE (Slc4a10), a member of the SLC4 family of bicarbonate transporters, uses the transmembrane gradient of sodium to drive cellular net uptake of bicarbonate and to extrude chloride, thereby modulating both intracellular pH (pHi) and chloride concentration ([Cl−]i) in neurons. Here we show that NCBE is strongly expressed in the retina. As GABAA receptors conduct both chloride and bicarbonate, we hypothesized that NCBE may be relevant for GABAergic transmission in the retina. Importantly, we found a differential expression of NCBE in bipolar cells: whereas NCBE was expressed on ON and OFF bipolar cell axon terminals, it only localized to dendrites of OFF bipolar cells. On these compartments, NCBE colocalized with the main neuronal chloride extruder KCC2, which renders GABA hyperpolarizing. NCBE was also expressed in starburst amacrine cells, but was absent from neurons known to depolarize in response to GABA, like horizontal cells. Mice lacking NCBE showed decreased visual acuity and contrast sensitivity in behavioral experiments and smaller b-wave amplitudes and longer latencies in electroretinograms. Ganglion cells from NCBE-deficient mice also showed altered temporal response properties. In summary, our data suggest that NCBE may serve to maintain intracellular chloride and bicarbonate concentration in retinal neurons. Consequently, lack of NCBE in the retina may result in changes in pHi regulation and chloride-dependent inhibition, leading to altered signal transmission and impaired visual function.
doi:10.1371/journal.pone.0046155
PMCID: PMC3467262  PMID: 23056253
4.  PGC-1α Determines Light Damage Susceptibility of the Murine Retina 
PLoS ONE  2012;7(2):e31272.
The peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) proteins are key regulators of cellular bioenergetics and are accordingly expressed in tissues with a high energetic demand. For example, PGC-1α and PGC-1β control organ function of brown adipose tissue, heart, brain, liver and skeletal muscle. Surprisingly, despite their prominent role in the control of mitochondrial biogenesis and oxidative metabolism, expression and function of the PGC-1 coactivators in the retina, an organ with one of the highest energy demands per tissue weight, are completely unknown. Moreover, the molecular mechanisms that coordinate energy production with repair processes in the damaged retina remain enigmatic. In the present study, we thus investigated the expression and function of the PGC-1 coactivators in the healthy and the damaged retina. We show that PGC-1α and PGC-1β are found at high levels in different structures of the mouse retina, most prominently in the photoreceptors. Furthermore, PGC-1α knockout mice suffer from a striking deterioration in retinal morphology and function upon detrimental light exposure. Gene expression studies revealed dysregulation of all major pathways involved in retinal damage and apoptosis, repair and renewal in the PGC-1α knockouts. The light-induced increase in apoptosis in vivo in the absence of PGC-1α was substantiated in vitro, where overexpression of PGC-1α evoked strong anti-apoptotic effects. Finally, we found that retinal levels of PGC-1 expression are reduced in different mouse models for retinitis pigmentosa. We demonstrate that PGC-1α is a central coordinator of energy production and, importantly, all of the major processes involved in retinal damage and subsequent repair. Together with the observed dysregulation of PGC-1α and PGC-1β in retinitis pigmentosa mouse models, these findings thus imply that PGC-1α might be an attractive target for therapeutic approaches aimed at retinal degeneration diseases.
doi:10.1371/journal.pone.0031272
PMCID: PMC3278422  PMID: 22348062
5.  PARP1 Gene Knock-Out Increases Resistance to Retinal Degeneration without Affecting Retinal Function 
PLoS ONE  2010;5(11):e15495.
Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness in humans. Previously, excessive activation of enzymes belonging to the poly-ADP-ribose polymerase (PARP) group was shown to be involved in photoreceptor degeneration in the human homologous rd1 mouse model for RP. Since there are at least 16 different PARP isoforms, we investigated the exact relevance of the predominant isoform - PARP1 - for photoreceptor cell death using PARP1 knock-out (KO) mice. In vivo and ex vivo morphological analysis using optic coherence tomography (OCT) and conventional histology revealed no major alterations of retinal phenotype when compared to wild-type (wt). Likewise, retinal function as assessed by electroretinography (ERG) was normal in PARP1 KO animals. We then used retinal explant cultures derived from wt, rd1, and PARP1 KO animals to test their susceptibility to chemically induced photoreceptor degeneration. Since photoreceptor degeneration in the rd1 retina is triggered by a loss-of-function in phosphodiesterase-6 (PDE6), we used selective PDE6 inhibition to emulate the rd1 situation on non-rd1 genotypes. While wt retina subjected to PDE6 inhibition showed massive photoreceptor degeneration comparable to rd1 retina, in the PARP1 KO situation, cell death was robustly reduced. Together, these findings demonstrate that PARP1 activity is in principle dispensable for normal retinal function, but is of major importance for photoreceptor degeneration under pathological conditions. Moreover, our results suggest that PARP dependent cell death or PARthanatos may play a major role in retinal degeneration and highlight the possibility to use specific PARP inhibitors for the treatment of RP.
doi:10.1371/journal.pone.0015495
PMCID: PMC2990765  PMID: 21124852
6.  Novel Rodent Models for Macular Research 
PLoS ONE  2010;5(10):e13403.
Background
Many disabling human retinal disorders involve the central retina, particularly the macula. However, the commonly used rodent models in research, mouse and rat, do not possess a macula. The purpose of this study was to identify small laboratory rodents with a significant central region as potential new models for macular research.
Methodology/Principal Findings
Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli, laboratory rodents less commonly used in retinal research, were subjected to confocal scanning laser ophthalmoscopy (cSLO), fluorescein and indocyanine green angiography, and spectral-domain optical coherence tomography (SD-OCT) using standard equipment (Heidelberg Engineering HRA1 and Spectralis™) adapted to small rodent eyes. The existence of a visual streak-like pattern was assessed on the basis of vascular topography, retinal thickness, and the topography of retinal ganglion cells and cone photoreceptors. All three species examined showed evidence of a significant horizontal streak-like specialization. cSLO angiography and retinal wholemounts revealed that superficial retinal blood vessels typically ramify and narrow into a sparse capillary net at the border of the respective area located dorsal to the optic nerve. Similar to the macular region, there was an absence of larger blood vessels in the streak region. Furthermore, the thickness of the photoreceptor layer and the population density of neurons in the ganglion cell layer were markedly increased in the visual streak region.
Conclusions/Significance
The retinal specializations of Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli resemble features of the primate macula. Hence, the rodents reported here may serve to study aspects of macular development and diseases like age-related macular degeneration and diabetic macular edema, and the preclinical assessment of therapeutic strategies.
doi:10.1371/journal.pone.0013403
PMCID: PMC2955520  PMID: 20976212
8.  Noninvasive, In Vivo Assessment of Mouse Retinal Structure Using Optical Coherence Tomography 
PLoS ONE  2009;4(10):e7507.
Background
Optical coherence tomography (OCT) is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration.
Methodology/Principal Findings
We achieved to adapt a commercial 3rd generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Leber's congenital amaurosis), retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE) due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified.
Conclusions/Significance
We found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical assessment of therapeutic strategies.
doi:10.1371/journal.pone.0007507
PMCID: PMC2759518  PMID: 19838301
9.  Vasoregression Linked to Neuronal Damage in the Rat with Defect of Polycystin-2 
PLoS ONE  2009;4(10):e7328.
Background
Neuronal damage is correlated with vascular dysfunction in the diseased retina, but the underlying mechanisms remain controversial because of the lack of suitable models in which vasoregression related to neuronal damage initiates in the mature retinal vasculature. The aim of this study was to assess the temporal link between neuronal damage and vascular patency in a transgenic rat (TGR) with overexpression of a mutant cilia gene polycystin-2.
Methods
Vasoregression, neuroglial changes and expression of neurotrophic factors were assessed in TGR and control rats in a time course. Determination of neuronal changes was performed by quantitative morphometry of paraffin-embedded vertical sections. Vascular cell composition and patency were assessed by quantitative retinal morphometry of digest preparations. Glial activation was assessed by western blot and immunofluorescence. Expression of neurotrophic factors was detected by quantitative PCR.
Findings
At one month, number and thickness of the outer nuclear cell layers (ONL) in TGR rats were reduced by 31% (p<0.001) and 17% (p<0.05), respectively, compared to age-matched control rats. Furthermore, the reduction progressed from 1 to 7 months in TGR rats. Apoptosis was selectively detected in the photoreceptor in the ONL, starting after one month. Nevertheless, TGR and control rats showed normal responses in electroretinogram at one month. From the second month onwards, TGR retinas had significantly increased acellular capillaries (p<0.001), and a reduction of endothelial cells (p<0.01) and pericytes (p<0.01). Upregulation of GFAP was first detected in TGR retinas after 1 month in glial cells, in parallel with an increase of FGF2 (fourfold) and CNTF (60 %), followed by upregulation of NGF (40 %) at 3 months.
Interpretation
Our data suggest that TGR is an appropriate animal model for vasoregression related to neuronal damage. Similarities to experimental diabetic retinopathy render this model suitable to understand general mechanisms of maturity-onset vasoregression.
doi:10.1371/journal.pone.0007328
PMCID: PMC2752170  PMID: 19806208
10.  Rb-Mediated Neuronal Differentiation through Cell-Cycle–Independent Regulation of E2f3a 
PLoS Biology  2007;5(7):e179.
It has long been known that loss of the retinoblastoma protein (Rb) perturbs neural differentiation, but the underlying mechanism has never been solved. Rb absence impairs cell cycle exit and triggers death of some neurons, so differentiation defects may well be indirect. Indeed, we show that abnormalities in both differentiation and light-evoked electrophysiological responses in Rb-deficient retinal cells are rescued when ectopic division and apoptosis are blocked specifically by deleting E2f transcription factor (E2f) 1. However, comprehensive cell-type analysis of the rescued double-null retina exposed cell-cycle–independent differentiation defects specifically in starburst amacrine cells (SACs), cholinergic interneurons critical in direction selectivity and developmentally important rhythmic bursts. Typically, Rb is thought to block division by repressing E2fs, but to promote differentiation by potentiating tissue-specific factors. Remarkably, however, Rb promotes SAC differentiation by inhibiting E2f3 activity. Two E2f3 isoforms exist, and we find both in the developing retina, although intriguingly they show distinct subcellular distribution. E2f3b is thought to mediate Rb function in quiescent cells. However, in what is to our knowledge the first work to dissect E2f isoform function in vivo we show that Rb promotes SAC differentiation through E2f3a. These data reveal a mechanism through which Rb regulates neural differentiation directly, and, unexpectedly, it involves inhibition of E2f3a, not potentiation of tissue-specific factors.
Author Summary
The retinoblastoma protein (Rb), an important tumor suppressor, blocks division and death by inhibiting the E2f transcription factor family. In contrast, Rb is thought to promote differentiation by potentiating tissue-specific transcription factors, although differentiation defects in Rb null cells could be an indirect consequence of E2f-driven division and death. Here, we resolve different mechanisms by which Rb controls division, death, and differentiation in the retina. Removing E2f1 rescues aberrant division of differentiating Rb-deficient retinal neurons, as well as death in cells prone to apoptosis, and restores both normal differentiation and function of major cell types, such as photoreceptors. However, Rb-deficient starburst amacrine neurons differentiate abnormally even when E2f1 is removed, providing an unequivocal example of a direct role for Rb in neuronal differentiation. Rather than potentiating a cell-specific factor, Rb promotes starburst cell differentiation by inhibiting another E2f, E2f3a. This cell-cycle–independent activity broadens the importance of the Rb–E2f pathway, and suggests we should reassess its role in the differentiation of other cell types.
The retinoblastoma protein (Rb), a tumor suppressor, promotes the differentiation of starburst amacrine cells in the retina by inhibiting the transcription factor E2f3a, whereas it suppresses retinal cell division and death by inhibiting E2f1.
doi:10.1371/journal.pbio.0050179
PMCID: PMC1914394  PMID: 17608565
11.  Independent degeneration of photoreceptors and retinal pigment epithelium in conditional knockout mouse models of choroideremia 
Journal of Clinical Investigation  2006;116(2):386-394.
Choroideremia (CHM) is an X-linked degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid, caused by loss of function of the CHM/REP1 gene. REP1 is involved in lipid modification (prenylation) of Rab GTPases, key regulators of intracellular vesicular transport and organelle dynamics. To study the pathogenesis of CHM and to develop a model for assessing gene therapy, we have created a conditional mouse knockout of the Chm gene. Heterozygous-null females exhibit characteristic hallmarks of CHM: progressive degeneration of the photoreceptors, patchy depigmentation of the RPE, and Rab prenylation defects. Using tamoxifen-inducible and tissue-specific Cre expression in combination with floxed Chm alleles, we show that CHM pathogenesis involves independently triggered degeneration of photoreceptors and the RPE, associated with different subsets of defective Rabs.
doi:10.1172/JCI26617
PMCID: PMC1326146  PMID: 16410831

Résultats 1-11 (11)