Maurice Menasche

Quantitative analysis of intravitreal injections in the rat

Intravitreal injections are currently used in the rat to introduce a therapeutic factor in the eye, especially for experimental treatments of retinal degenerations. The injected volume and its location can influence the quantification of results. We have investigated the quantitative effect of a single intravitreal injection in rats at different ages and for different volumes. Albinos rats aged three weeks or two months received intravitreal injections of 1, 3, 5 or 10 µl China ink. Animals were sacrificed immediately after injection, eyes were enucleated, fixated, embedded in paraffin and microtomy was performed in a sagittal plane. Regularly spaced sections were analyzed to reconstruct the vitreous and injected dye volumes. The measured vitreous volume was 6.76 ± 0.37 mm 3 in three weeks old rats and 13.36 ± 0.64 mm 3 in two months old rats. Mean intravitreal ink volumes immediately after injection were 0.8 mm 3 for 1 µl injections, 2 mm 3 for 3 µl, 2.3 to 2.6 mm 3 for 5 µl and 3.2 mm 3 for 10 µl. The percentage of vitreous volume involved by the injection ranged from 4.4% to 33.2%. The injected volume is limited by the large lens size of the rat. Extraocular loss of injected solution increases for higher injected volumes, with larger standard deviations. In this model, the dye tends to localize behind the lens. A 3 or 5 µl volume appears to have the best reproducibility with minimum loss of solution.

Sirt1 Involvement in rd10 Mouse Retinal Degeneration

PURPOSE Sirtuin1 (Sirt1) is an NAD(+)-dependent deacetylase involved in development, cell survival, stress resistance, energy metabolism, and aging. It is expressed in the mammalian central nervous system (CNS) and is activated during processes associated with neuroprotection. The retinal degeneration 10 (rd10) mouse model of retinitis pigmentosa (RP) was used to investigate the possible role of Sirt1 in this type of retinal degeneration. METHODS Eyes from control and rd10 mice were used. Sirt1 mRNA was detected by in situ hybridization, and its abundance was estimated by semiquantitative RT-PCR. The presence of Sirt1 protein was investigated by immunohistofluorescence and Western blot analysis. The apoptosis of photoreceptor cells was analyzed by terminal dUTP transferase nick-end labeling (TUNEL). Immunolabeling for Sirt1, apoptosis-inducing factor (Aif), and caspase-12 (Casp-12) was performed on retinal tissue sections. RESULTS Sirt1 mRNA and immunoreactivity were observed in normal adult mouse eyes. In the control retina, Sirt1 was immunolocalized mostly to the nucleus. In rd10 mice with retinal degeneration, changes in Sirt1 immunolabeling were observed only in the retinal outer nuclear layer (ONL). The pathologic pattern of Sirt1 immunoreactivity correlated with the start of retinal degeneration in rd10 mice. CONCLUSIONS The results suggest a link between Sirt1 production and retinal degeneration in rd10 mice. The anti-apoptotic, neuroprotective role of Sirt1 in the mouse retina is based on the involvement of Sirt1 in double DNA strand-break repair mechanisms and in maintaining energy homeostasis in photoreceptor cells. The results suggest that the neuroprotective properties of Sirt1 may gradually weaken in rd10 mouse photoreceptor cells.

Ocular Cell Transfection with the Human Basic Fibroblast Growth Factor Gene Delays Photoreceptor Cell Degeneration in RCS Rats

Based on the K8/JTS-1-mediated transfection technique, we developed an in vivo protocol for an efficient transfer of plasmid DNA to ocular cells. As determined with condensed plasmids containing reporter genes for either beta-galactosidase (pcDNA-lacZ) or enhanced green fluorescent protein (pREP-EGFP), the immortalized human retinal epithelial cells RPE D407 and human embryonic kidney 293 cells can be transfected with typical efficiencies of 11 and 19%, respectively. Unlike 293 cells, RPE D407 cells had a reduced viability on transfection with both plasmids. In vivo, subretinal injections of DNA-K8/JTS-1 complexes revealed reporter gene expression in choroidal and RPE cells of normal pink-eyed Royal College of Surgeons (RCS) rats. The validity of this transfection technique in terms of retinal cell survival in RCS rats was then examined by using pREP-hFGF2 plasmid, which encodes the human basic fibroblast growth factor isoforms (hFGF2). Subretinal injection of pREP-hFGF2-K8/JTS-1 complexes into 3-week-old dystrophic RCS rat eyes reveals a delayed photoreceptor cell degeneration 60 days postinjection. In this case, the average analyzed field points with delayed cell dystrophy represent 14 to 17% of the retinal surface as compared with 2.6 and 4% in pREP5beta and vehicle-injected eyes, respectively. Peptide-mediated in oculo transfection thus appears to be a promising technique for the treatment of retinal cell and photoreceptor degenerations.

Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet.

PURPOSE To evaluate functional and ultrastructural changes in the retina of scavenger receptor B1 (SR-BI) knockout (KO) mice consuming a high fat cholate (HFC) diet. METHODS Three-month-old male KO and wild-type (WT) mice were fed an HFC diet for 30 weeks. After diet supplementation, plasma cholesterol levels and electroretinograms were analyzed. Neutral lipids were detected with oil red O, and immunohistochemistry was performed on cryostat ocular tissue sections. The retina, Bruchs membrane (BM), retinal pigment epithelium (RPE), and choriocapillaris (CC) were analyzed by transmission electron microscopy. RESULTS Using the WT for reference, ultrastructural changes were recorded in HFC-fed SR-BI KO mice, including lipid inclusions, a patchy disorganization of the photoreceptor outer segment (POS) and the outer nuclear layer (ONL), and BM thickening with sparse sub-RPE deposits. Within the CC, there was abnormal disorganization of collagen fibers localized in ectopic sites with sparse and large vacuolization associated with infiltration of macrophages in the subretinal space, reflecting local inflammation. These lesions were associated with electroretinographic abnormalities, particularly increasing implicit time in a- and b-wave scotopic responses. Abnormal vascular endothelial growth factor (VEGF) staining was detected in the outer nuclear layer. CONCLUSIONS HFC-fed SR-BI KO mice thus presented sub-RPE lipid-rich deposits and functional and morphologic alterations similar to some features observed in dry AMD. The findings lend further support to the hypothesis that atherosclerosis causes retinal and subretinal damage that increases susceptibility to some forms of AMD.

Novel Mouse Model of Monocular Amaurosis Fugax

Background and Purpose— Retinal ischemia is a major cause of visual impairment and is associated with a high risk of subsequent ischemic stroke. The retina and its projections are easily accessible for experimental procedures and functional evaluation. We created and characterized a mouse model of global and transient retinal ischemia and provide a comprehensive chronologic profile of some genes that display altered expression during ischemia. Methods— Ischemia and reperfusion were assessed by observing flat-mounted retinas after systemic fluorescein injection. The temporal pattern of gene expression modulation was evaluated by quantitative reverse transcription–polymerase chain reaction from the occurrence of unilateral 30-minute pterygopalatine artery occlusion until 4 weeks after reperfusion. Electroretinograms evaluated functional sequelae 4 weeks after the ischemic episode and were correlated with histologic lesions. Results— This model is the first to reproduce the features of transient monocular amaurosis fugax resulting from ophthalmic artery occlusion. The histologic structure was roughly conserved, but functional lesions affected ganglion cells, inner nuclear layer cells, and photoreceptor cells. We observed an early and strong upregulation of c-fos, c-jun, Cox-2, Hsp70, and Gadd34 gene expression and a late decrease in Hsp70 transcript levels. Conclusions— A murine model of transient retinal ischemia was successfully developed that exhibited the characteristic upregulation of immediate-early genes and persistent functional deficits. The model should prove useful for investigating mechanisms of injury in genetically altered mice and for testing novel neuroprotective drugs.

Identification of novel genes and altered signaling pathways in the retinal pigment epithelium during the Royal College of Surgeons rat retinal degeneration

Shed photoreceptor outer segments (POS) are phagocytosed by RPE cells in a circadian manner. The homozygous deletion of the c-mer gene abolishes the ingestion phase of this phagocytosis in the Royal College of Surgeons (RCS) rat strain, which in turn leads to the death of photoreceptor cells. We identified RPE transcripts for which the expression is modulated by the abrogation of POS phagocytosis. A microarray approach and the differential display (DDRT-PCR) technique revealed 116 modulated known genes, 4 modulated unknown genes, and 15 expressed sequenced tags (ESTs) corresponding to unknown genes. The microarray and DDRT-PCR analyses detected alterations in signaling pathways such as the phosphatidylinositol 3-kinase-Akt-mTOR pathway and the DLK/JNK/SAPK pathway. The abrogation of POS phagocytosis caused a decrease in endomembrane biogenesis and altered endocytosis, exocytosis, transcytosis, and several metabolic and signaling pathways in RCS RPE cells. We also found differential levels of transcripts encoding proteins involved in phagocytosis, vesicle trafficking, the cytoskeleton, retinoic acid, and general metabolism.

Mutational analysis of the OA1 gene in ocular albinism.

Ocular albinism type 1 (OA1) is an X-linked disorder, mainly characterized by a severe reduction in visual acuity, foveal hypoplasia, nystagmus, hypopigmentation of the retina, the presence of macromelanosomes in the skin and eyes, and the misrouting of optic pathways, resulting in the loss of stereoscopic vision. We screened the OA1 gene for mutations in three unrelated Canadian and French families and in two isolated patients with OA1. We found three different missense mutations and two different nonsense mutations, three of which were novel. To date, 41 mutations (including missense mutations, insertions, and deletions) have been reported in the OA1 gene. Mutation and polymorphism data for this gene are available from the international albinism center albinism database website: http://www.cbc.umn.edu/tad/oa1map.htm.

Eight previously unidentified mutations found in the OA1 ocular albinism gene

BackgroundOcular albinism type 1 (OA1) is an X-linked ocular disorder characterized by a severe reduction in visual acuity, nystagmus, hypopigmentation of the retinal pigmented epithelium, foveal hypoplasia, macromelanosomes in pigmented skin and eye cells, and misrouting of the optical tracts. This disease is primarily caused by mutations in the OA1 gene.MethodsThe ophthalmologic phenotype of the patients and their family members was characterized. We screened for mutations in the OA1 gene by direct sequencing of the nine PCR-amplified exons, and for genomic deletions by PCR-amplification of large DNA fragments.ResultsWe sequenced the nine exons of the OA1 gene in 72 individuals and found ten different mutations in seven unrelated families and three sporadic cases. The ten mutations include an amino acid substitution and a premature stop codon previously reported by our team, and eight previously unidentified mutations: three amino acid substitutions, a duplication, a deletion, an insertion and two splice-site mutations. The use of a novel Taq polymerase enabled us to amplify large genomic fragments covering the OA1 gene. and to detect very likely six distinct large deletions. Furthermore, we were able to confirm that there was no deletion in twenty one patients where no mutation had been found.ConclusionThe identified mutations affect highly conserved amino acids, cause frameshifts or alternative splicing, thus affecting folding of the OA1 G protein coupled receptor, interactions of OA1 with its G protein and/or binding with its ligand.

Truncation of PITX2 differentially affects its activity on physiological targets

The bicoid-like transcription factor PITX2 has been previously described to interact with the pituitary-specific POU homeodomain factor POU1F1 (human ortholog of PIT-1) to achieve cell-specific expression of prolactin (PRL) and GH in pituitary somatolactotroph cells. In this work, we have investigated the functional properties of three PITX2 mutants reported in Axenfeld-Rieger syndrome patients relative to the regulation of these genes, using reporter genes under the control of human PRL (hPRL), hGH, or POU1F1 promoters transfected in nonpituitary and pituitary cell lines. Among the three mutations studied, Y167X and E101X introduce a premature stop codon, and F104L leads to an amino acid substitution. While PITX2(E101X) is not expressed in the cells following transfection, and PITX2(F104L) is functionally inactive, the PITX2(Y167X) mutant keeps its DNA-binding capacity and displays a markedly enhanced activation of the hPRL and POU1F1 promoters, but not of the hGH promoter. Y167X is the first mutation of PITX2 described to result in a differential effect on the activation of its different physiological targets, hPRL and POU1F1 on one hand and hGH on the other hand. The differential effect of the Y167X mutation might be linked to an interaction of PITX2 with different transcription factors or cofactors when bound to the hPRL and POU1F1 or the hGH promoters. These results might form the basis for the identification of the PITX2 protein complex necessary for the differential GH or PRL expression.

Use of denaturing HPLC and automated sequencing to screen the VMD2 gene for mutations associated with Best's vitelliform macular dystrophy

We identified three novel VMD2 mutations in patients with Bests macular dystrophy. DHPLC analysis of the 11 VMD2 exons revealed abnormal profiles in exon 8. Direct sequencing showed that these abnormal profiles were due to monoallelic transitions and trans-versions. We also found three polymorphic sequence changes that have been reported previously and annotated to an online database (http://www.uni-wuerzburg.de/humangenetics/vmd2.html)