10 research outputs found
Genotype distribution for both chemokine knockout alleles in the F2 generation obtained from three independent F1 breeding pairs during the backcross of the original <i>CCDKO</i> mice with <i>C57Bl/6</i> mice.
<p>The number of animals with respective genotype combinations for both chemokine loci is shown in the field at crossing of columns (<i>Ccl2</i> genotype) and rows (<i>Cx3cr1</i> genotype). Founder animals for newly re-derived <i>Ccl2</i>, <i>Cx3cr1</i> single and <i>Ccl2/Cx3cr1</i> double knockout lines are underlined.</p
Microglia are recruited to the primary retinal lesions in the outer retina of <i>CCDKO</i> mice.
<p>Confocal maximum projection images of a subset of images at different depth of the z-stack (A,C,D,F) and images of 3D-reconstructions (IMARIS) of corresponding complete z-stacks (B,E). Z-stack images were taken from the inferior area of retinal flat mount preparations from <i>CCDKO</i> and <i>C57Bl/6</i> mice at 2 months of age. The samples were labeled with Tritc-lectinB4 (vascular marker, red), Iba1 (microglia marker, green) and DAPI as a nuclear counter stain (blue). OPL: outer plexiform layer, ONL: outer nuclear layer, IS: inner segments. (A+D) At the level of OPL ramified microglia (green) are located between vessels of the deep capillary plexus (red). Microglia of <i>CCDKO</i> mice (white arrows, D) show a slightly bigger cell bodies than those in <i>C57Bl/6</i> mice (A). Microglia were also observed in the outer retina of <i>CCDKO</i> mice in the 3D Imaris reconstruction (E) and in confocal z-projections from the outer retina (F) while they were not observed in the outer retina of <i>C57Bl/6</i> mice (B, C). microglia in the outer retina of <i>CCDKO</i> mice surround circular lesions (F, white circle) that may correspond well with the disciform shape of autofluorescent lesion observed in AF-SLO fundus images.</p
The distribution of the inferior autofluorescent fundus lesions in phenotyped animals of the F2 generation suggests that this phenotype segregates independently from either of the chemokine loci in an autosomal recessive fashion.
<p>The distribution of the inferior autofluorescent fundus lesions in phenotyped animals of the F2 generation suggests that this phenotype segregates independently from either of the chemokine loci in an autosomal recessive fashion.</p
<i>Ccl2</i> and <i>Cx3cr1</i> as well as the genetic <i>C57Bl/6</i> background differentially modulate the autosomal recessive early onset, inferior retinal degeneration caused by the RD8 mutation in exon 9 of the <i>Crb1</i> gene.
<p><i>In vivo</i> phenotyping by autofluorescent fundus imaging of the two parental (P) mouse lines (<i>CCDKO</i> and <i>C57Bl/6</i>) (A) as well as in the newly established chemokine knockout mouse lines (<i>Ccl2<sup>−/−</sup>, Cx3cr1<sup>−/−</sup></i> and <i>Ccl2<sup>−/−</sup>/Cx3cr1<sup>−/−</sup></i>) at the age of 2 months (2 M, B) and 6 months (6 M, C). Autofluorescent fundus lesions were observed independently from the chemokine genotypes at the age of 2 months and 6 months suggesting that an independent genomic locus is responsible for the early onset, inferior retinal degeneration. (D) Quantification of the number of autofluorescent lesions per fundus image for all parental, affected and unaffected chemokine knockout mouse lines indicated significant differences in the number of autofluorescent fundus lesions between the groups. The individual number of autofluorescent lesions per fundus and animal is shown together with the mean ± standard deviation. *: (One-way-ANOVA p<0.0001, Tukey posthoc test for multiple comparison (p<0.05)). This data indicate an attenuating influence of increasing <i>C57Bl/6</i> genetic background in all offspring obtained from the F2 generation relative to the parental <i>CCDKO</i> mouse line and thus were labelled as F3 generation. A differential modulatory effect of both chemokine signalling pathways on the severity of the retinal degeneration at 8 weeks of age was also observed by comparing the three affected lines within the F3 generation. N (<i>CCDKO</i> original line P) = 7, N(<i>C57Bl/6 wt</i> P) = 19, N(<i>Ccl2−/−</i> affected) = 42, N(<i>Ccl2−/−</i> unaffected F3) = 23, N(<i>Cx3cr1−/−</i> affected F3) = 16, N(<i>Cx3cr1−/−</i> unaffected F3) = 71, N(new Ccl2−/−/<i>Cx3cr1−/−</i> affected F3) = 19, N(new Ccl2−/−/<i>Cx3cr1−/−</i> unaffected F3) = 32.</p
The primary degenerative event in the course of the degeneration is located in the outer nuclear layer of <i>CCDKO</i> mice and leads to the secondary development of RPE damage.
<p>(A) Superior-inferior oriented sagittal semithin histology of the outer retina of <i>CCDKO</i> mice and age-matched <i>C57Bl/6</i> wildtype mice at the level of the optic disc between 2 weeks and over 20 months of age. A localized drop-out of nuclei from the ONL (white arrowheads) as early as 2 weeks of age in <i>CCDKO</i> mice suggests a primary pathological event in the retina that initiates the progressive inferior retinal degeneration in this model finally leading to the complete loss of retinal layers from 8 months of age onwards. The RPE underneath these lesions is secondarily affected. White arrows: drop out of photoreceptor columns, black arrowheads: descending retinal vessels, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, IS: inner segments, OS: outer segments, RPE: retinal pigment epithelium. (B) Quantitative morphometry of RPE damage on the same sections. We observed an normal age-related increase of RPE damage in wildtype mice (<i>C57Bl/6</i>: Pearson r<sup>2</sup> = 0.5562, p<0.0001, N = 37) and observed that the age-related increase of RPE damage in <i>CDDKO</i> mice (<i>CCDKO</i>: Pearson r<sup>2</sup> = 0.7115, p<0.0001; N = 43) was significantly higher compared to <i>C57Bl/6</i> mice (linear regression analysis, p<0.0001),which was significantly more pronounced in <i>CCDKO</i> mice (Pearson r<sup>2</sup> = 0.465,p = 0.0007; slope difference: p = 0.01). Nevertheless the RPE damage becomes only significantly higher during late stages of the degeneration suggesting a secondary involvement of the RPE during the degenerative process. Scale bars: 25 µm.</p
Modulatory effect of the <i>Ccl2</i> locus genotype (<i>Ccl2<sup>+/+</sup>, Ccl2<sup>+/−</sup>, Ccl2<sup>−/−</sup></i>) on the appearance of autofluorescent fundus lesions in <i>Crb1<sup>RD8/RD8</sup> mice.</i>
<p>Quantification of number of autofluorescent lesions/fundus image and littermate (n = ) according to the <i>Ccl2</i> genotype and in comparison to the parental strains (<i>CCDKO</i> and <i>C57Bl/6</i> mice). These findings indicate that the <i>Ccl2</i> locus does not significantly modulate the severity of RD8 induced retinal degeneration although we can not exclude a mild influence due to the intermediate position of the <i>Ccl2<sup>−/−</sup>/Crb1<sup>RD8/RD8</sup></i> group. *: indicates significant difference between the indicated groups (One-way-ANOVA p<0.0001, Tukey's posthoc test for multiple comparison (p<0.05)). n.s.: not significant. N (<i>CCDKO</i> original line P) = 7, N(<i>C57Bl/6 wt</i> P) = 14, N(<i>Ccl2<sup>+/+</sup>/Crb1<sup>RD8/RD8</sup></i>) = 15, N(<i>Ccl2<sup>+/−</sup>/Crb1<sup>RD8/RD8</sup></i>) = 31, N(<i>Ccl2<sup>−/−</sup>/Crb1<sup>RD8/RD8</sup></i>) = 10.</p
<i>CCDKO</i> mice show an early onset, focal inferior retinal degeneration starting in the outer retina which subsequently leads to a breakdown of retinal layering in the affected area with age.
<p><i>In vivo</i> assessment of the retinal degeneration of <i>CCDKO</i> mice and age-matched <i>C57Bl/6</i> wildtype mice at 1,4,8,12,15 and older than 20 months of age using autofluorescence scanning laser ophthalmoscopy (A) and optical coherence tomography (B). Note the weak autofluorescent fundus lesions in <i>CCDKO</i> mice visible as early as 1 month (A) which correlate with the observation of an abnormal signal in the inferior OCT image at the same age (B) and suggests an early pathological event in the outer retina as the basis of the degeneration in this model. Corresponding signals in AF-SLO and OCT images are labelled identically. White arrowheads: small early inner retinal autofluorescent lesion, black arrowheads: discrete disciform autofluorescent lesion, white arrow: “confluent” autofluorescent lesion in AF-SLO represents loss of retinal layering and severe retinal degeneration, black arrow: discrete autofluorescent spot-like lesion. GCL: ganglion cell layer, OPL: outer plexiform layer, ONL: outer nuclear layer, RPE: retinal pigment epithelium.</p
PCR primer for genotyping of <i>Ccl2</i> and <i>Cx3cr1</i> alleles and for amplification of the <i>RD8</i> locus for sequencing.
<p>PCR primer for genotyping of <i>Ccl2</i> and <i>Cx3cr1</i> alleles and for amplification of the <i>RD8</i> locus for sequencing.</p
The inferior retinal degeneration in <i>CCDKO</i> mice is not dependent on light.
<p>(A) Comparison of autofluorescent SLO fundus images of <i>CCDKO</i> mice at 8 weeks of age that were raised from birth either in complete darkness (24 h dark, luminescence <0.5 lx) or in normal 12 h/12 h light/dark cycle (light 12 h/12 h, luminescence = 33±28 lx). As a comparison an AF-SLO fundus image from an aged-matched <i>C57Bl/6</i> wildtype mouse raised under normal 12 h/12 h light/dark cycle is shown. (B) Quantitative assessment of the number of autofluorescent lesions/per fundus image for the three animal groups. The number of autofluorescent lesions in <i>CCDKO</i> (24 h dark, n = 9) and <i>CCDKO</i> (12 h/12 h light/dark, n = 9) was not significantly different, but was significantly higher compared to that in <i>C57Bl/6</i> mice (12 h/12 h light/dark, n = 14). *: One-way-ANOVA (p<0.0001), Tukey's posthoc test for multiple comparison (p<0.05). This suggests that light is not necessary for the manifestation of the early onset inferior retinal degeneration in <i>CCDKO</i> mice and that ambient light (33+−28 lx) does not augment the number of inferior retinal autofluorescent lesions in <i>CCDKO</i> mice within the first 8 weeks of their life.</p
Vascular lesions in <i>CCDKO</i> mice develop secondarily to the retinal degeneration and show similarities to retinal telangiectasia rather than choroidal neovascularisation.
<p>(A) Late phase fluorescein angiography images of <i>CCDKO</i> mice and age-matched wildtype mice between 1 month and over 20 months of age reveals a late phase (∼7 min post i.p. injection) hyperfluorescence of fluorescein (white arrowheads) secondary to the focal early onset, inferior retinal degeneration. (B) Quantification of the penetrance (%) of late stage hyperfluorescence in <i>CCDKO</i> mice at different ages in comparison to <i>C57Bl/6</i> mice. The number of animals (N) analysed per age group and genotype are shown in the graph (N = eyes with late FFA hyperfluorescence/total number of imaged eyes). (C) DIC bright field images of retinal flat mount preparations of <i>CCDKO</i> and age-matched wildtype controls at 1 months, 12 months and 20 months, scale bar: 750 µm. (D) Magnified DIC bright field images and corresponding immunohistochemistry staining for the vascular marker isolectin B4 (TRITC-lectin-B4, red) taken from the affected inferior degenerate areas (black squares in (C)).At 1 month, early separate grey spots (5D, black arrowhead) were observed inferior to the optic disc, which later grow bigger, become confluent and involve pigmented cell attached to the focal inferior retinal lesion. Lectin-positive vessels within the area of degeneration show vascular anomalies secondary to the retinal degeneration, including vascular tortuosity (5D, white arrowhead, at 12 months) and dilation of retinal capillaries (5D, white arrowhead, at 20 months).Scale 150 µm. (E) Overlay of a bright field and immunohistochemistry image taken from the apical side of a lectin-stained RPE/choroidal flat mount from a <i>CCDKO</i> mouse at 20 months of age. The retinal vasculature within the degenerate area (white arrow) was closely attached to the underlying apical side of the RPE. Scale: 100 µm. (F–H): Different views of a three dimensional reconstruction from a tissue block from the centre of an inferior retinal lesion of a <i>CCDKO</i> mouse at 20 months of age using serial block-face scanning electron microscopy with a Gatan 3-View system. Connected vascular lumens are labeled by an identical color, while the location of Bruch's membrane in each ultrathin section is outlined by a pink line. Retinal vessels collapse onto the RPE and induce a migratory response of RPE cells to grow along these vessels into the retina (black arrows). Retinal vessels grow underneath the RPE and become positioned in close vicinity to Bruch's membrane within a RPE/vascular complex, but they never have been observed to grow through Bruch's membrane (Representative 3D reconstruction images from a tissue block with the dimensions; x-axis 176.42 µm, y-axis 176.42 µm, z-axis: 39.9 µm, Scale (G&H): 50 µm.</p