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Animal Model |
From the Center for Ophthalmology and Visual Science* and the Department of Pathology, The University of Western Australia, Crawley; the Lions Eye Institute, Nedlands; and the Vision Touch and Hearing Research Center, University of Queensland, Brisbane, Queensland, Australia
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Abstract |
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AMD has been proposed to be caused by a primary failure of the retinal pigment epithelial (RPE) cells because of abnormal build-up of photoreceptor outer segment (POS) breakdown products.14 The non-renewable RPE cells form a single multifunctional cell layer responsible for phagocytosis and lysosomal digestion of the continuously growing POS.15 Although presence of POS breakdown products in the RPE cells or its proximity cannot be directly linked to AMD development, there is compelling evidence to implicate lipofuscin, drusen and/or basal laminar and linear deposits as risk factors.4,16 Accumulation of abnormal POS breakdown product in the RPE cells can be the result of a variety of impairments, ie, 1) RPE cell lysosomal abnormalities including loss or decrease of enzymatic activity,17 2) changes in or related to POS structure,18 or 3) obstruction of exocytosis because of changes in Bruch’s membrane (BM).19 The exact role of different types of debris in AMD development is unknown but they seem to leadto a common pathway—apoptotic photoreceptor cell death.20
In developing an animal model that might reproduce at least some features of AMD, the modulation of RPE cell lysosomal enzyme activity is an attractive approach. RPE cells have been shown to express strikingly high levels of cathepsin D (CatD),21,22 suggesting an important role for CatD in POS digestion. Inactive proCatD accumulation seems to be an abnormal condition and has been associated with breast and ovarian cancers,23 24 and an age-related increase in the amount of proCatD has been demonstrated in several organs.25-27 Recently, we demonstrated in vitro and in vivo that presence of proCatD impairs POS proteolysis, resulting in POS-derived breakdown product accumulation in the RPE cells of heterozygous transgenic mice (mcd) that express a form of CatD lacking the Glu44p and Gly1 cleavage site.28
We hypothesize that any change, genetic or environmental, that might lead to an accelerated POS breakdown product accumulation in the RPE layer will compromise RPE cell function, initiating changes similar to those observed in AMD. To test this hypothesis we produced a homozygous (mcd/mcd) transgenic mouse line and demonstrated that progressive POS-derived breakdown product accumulation could induce hypo- and hyperpigmentary changes in the fundus, the development of basal laminar and linear deposits and photoreceptor and RPE cell proliferation/loss in an age-dependent manner.
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Materials and Methods |
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Animal experimentation was performed in compliance with the Association for Research in Vision and Opthmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. The 3.2-kb fragment containing the mutated CatD (CatDm1) gene with a two-amino acid deletion (Glu44p-Gly1) corresponding to the first cleavage site, and driven by the human cytomegalovirus promoter was released from pMCD. After two rounds of gel purification, the DNA fragment was microinjected into fertilized single-cell stage C57BL/6 embryos. The resultant offspring were screened for the presence of CatDm1 by polymerase chain reaction (PCR) and Southern blot analysis of mouse-tail DNA. First generation mcd mice28 from the same founder were mated and the resultant mcd/mcd mice were bred to establish a stable line. Mcd/mcd and age-matched control C57BL/6 mice (6 to 18 months old) were used for subsequent characterization. All animals were maintained in cages at a constant temperature of 22°C and with a 12:12 hour light/dark cycle (light on at 0800 hours). Food and water were available ad libitum.
Fundus Photography
Mice were anesthetized by intraperitoneal injection of ketamine (50 mg/kg body weight) and xylazine (8 mg/kg body weight) before pupil dilation with a drop of 1.0% mydriacyl and 2.5% phenylephrine hydrochloride. Fundus photography of mice was performed using a small animal fundus camera (Kowa Genesis, Tokyo, Japan).
Electroretinography
Ten-month-old mcd/mcd and C57BL/6 mouse eyes were assessed using electroretinography as previously described,29 but using flash stimulus at 0.25 Hz and four consecutive responses. Stimulus-response characteristics were generated by attenuating the flash intensity with neutral density filters. The a-wave amplitude was measured from the baseline to the trough of the a-wave response and the b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave. Data were expressed as the mean wave amplitude ±SEM (µV). Two-factor repeated measures analysis of variance was performed to compare the responses from retinas of mcd/mcd mice (n = 4) with those of control mice (n = 4) over the flash stimulus range. The b-wave data were fitted with a Naka-Rushton equation [R/Rmax = I/(I + K)] using SigmaPlot (SPSS, Chicago, IL.) to determine Rmax (maximum amplitude) and K (semisaturation constant) from the response amplitude (R) and the flash intensity (I) over the range of -6 to -1.2 log neutral density units.
Reverse Transcriptase (RT)-PCR
CatDm1 expression in the transgenic mice was detected by RT-PCR. Total retinal RNA from C57BL/6, mcd, and mcd/mcd were isolated using Trizol Reagent (Life Technologies, Inc., Gaithersburg, MD). A primer-specific one-step RT-PCR was performed with Qiagen One-Step RT-PCR Kit (Qiagen, Hilden, Germany) using 1 µg of total RNA plus a pair of CatDm1-specific primers, 5'-ATGCAGCCCTCCAGCCTTCT-3' and 5'-TACTTGTGGTGGATCCAGCA-3'. A mouse hypoxanthine phosphoribosyl-transferase (HPRT) primer pair, 5'-CACAGGACTAGAACACCTGC-3' and 5'-GCTGGTGAAAAGGACCTCT-3', was used as positive control for quality of the RT and the amount of cDNA added to each PCR reaction. The cycles used were: 1 cycle at 50°C for 30 minutes and 95°C for 15 minutes; followed by 35 cycles of 94°C for 1 minute, 62°C for 1 minute, and 72°C for 1 minute.
Western Blot Analysis
Western blot analysis was performed as previously described.22 Briefly, the mouse retina was homogenized in phosphate-buffered saline and loading buffer before being used for polyacrylamide gel electrophoresis. A mouse anti-human CatD mAb (Calbiochem, San Diego, CA), diluted 1/200, was used as the primary antibody and a goat anti-mouse IgG-horseradish peroxidase (Amersham, Uppsala, Sweden), diluted 1/2000, as the secondary antibody. The blots were developed with the ECL Western blotting analysis system (Amersham).
Histology and Immunohistochemistry
Enucleated mouse eyes were fixed in 4% paraformaldehyde (pH 7.4) for 4 hours and embedded in paraffin. Five-µm-thick sections of eyes from 11- to 14-month-old mcd/mcd and C57BL/6 mice were stained with hematoxylin and eosin (H&E) for light microscopy. Some eyes were embedded in OCT compound for frozen section preparation for fluorescence microscopy. For immunohistochemistry, sections were deparaffinized, rehydrated, and bleached to remove melanin by incubation of sections in 0.25% potassium permanganate for 20 minutes and in 1% oxalic acid for 5 minutes before incubation with rabbit anti-bovine rod outer segment (ROS) polyclonal IgG, rabbit anti-bovine CatD polyclonal IgG, mouse anti-human CatD mAb (Calbiochem) or control nonimmune serum (rabbit or mouse) diluted 1/200. Sections were incubated with goat anti-rabbit IgG conjugated with alkaline phosphatase (Life Technologies, Inc.) or goat anti-mouse IgG conjugated with alkaline phosphatase (Sigma, St. Louis, MO) diluted 1/100, followed by alkaline phosphatase substrate (Fast Red, Sigma). Sections were counterstained with Mayer’s hematoxylin for light microscopy.
Terminal dUTP Nick-End Labeling (TUNEL) Staining
Eyes from C57BL/6 (n = 4) and mcd/mcd (n = 4) mice were fixed in 10% neutral buffered formalin for 4 hours for paraffin embedding, followed by section preparation. The TUNEL technique was performed using ApopTag Plus Peroxidase in Situ Apoptosis Detection Kit (Intergen Discovery Products, Purchase, NY) according to the manufacturer’s protocol. From each control C57BL/6 and mcd/mcd retina, five 60-µm-long sections of the outer nuclear layer (ONL) were randomly selected under a light microscope (Leica DM RBE, Solms, Germany) using a x100 objective lens and the images captured using an attached video camera (Olympus DP-10, Tokyo, Japan) and digitized. The total number of nuclei and total number of TUNEL-positive nuclei was counted using the Scion Image package (Scion Corp., Frederick, MA).
Electron Microscopy
Enucleated eyes from 12- to 18-month-old mcd/mcd and C57BL/6 mice were fixed in 2.5% glutaraldehyde in 0.05 mol/L of cacodylate buffer (pH 7.4). The eyes were then trimmed into 1-mm3 blocks and re-immersed into a fresh fixative for a further 24 hours. After postfixing in 1% osmium tetroxide, the tissues were processed for transmission electron microscopy by conventional methods and embedded in Araldite. Semithin sections (1 µm thick) were stained with 0.5% toluidine blue in 5% borax and examined with a light microscope. After selecting the areas of interest, the blocks were trimmed under a dissecting microscope and ultra-thin sections (70 nm thick) prepared on an ultramicrotome (LKB Nova, Sweden), stained with Reynolds lead citrate and examined in a Philips 410LS Transmission Electron Microscope at an accelerating voltage of 80 kV.
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Results |
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Five transgenic founders, designated D2, D8, D28, D29, and D30, were generated and identified by PCR and Southern blot analysis from a total of 47 pups produced from microinjected eggs. D8, however, did not contain the cytomegalovirus promoter region, and D28, D29, and D30 failed to produce a positive transgenic offspring. Hence, only D2 was used to established stable mcd and mcd/mcd transgenic lines and only mcd/mcd mice were used in the subsequent characterization.28
The level of CatDm1 expression in the retina was assessed by RT-PCR, immunohistochemistry, and Western blot analysis. From RT-PCR analysis using human CatDm1 transgene-specific primers, a 379-bp cDNA fragment of the human CatD was amplified in mcd/mcd mice, but not in age-matched C57BL/6 mice (Figure 1A)
. The 260-bp HPRT bands had similar intensities, demonstrating equal loading of sample. As expected, the mRNA level of CatDm1 in mcd/mcd mice was higher than in mcd mice and no band was detected in the C57BL/6 mice. Western blot analysis demonstrated the presence of human proCatD, appearing at 52 kd, in mcd and mcd/mcd mice (Figure 1B
, lanes 2 and 3). In addition, bands probably representing the human proCatD-opsin-like complex appeared at 75 kd in mcd and mcd/mcd mice. There were no human proCatD-immunoreactive bands present in the control C57BL/6 mice (Figure 1B
, lane 1) demonstrating the lack of human CatD expression in these animals. Transgene expression was also confirmed by immunohistochemistry. Using the rabbit anti-bovine CatD polyclonal antibody, CatD-positive immunostaining was detected in the RPE cell layer of mcd/mcd and C57BL/6 mice (data not shown). However, using a mouse anti-human CatD mAb that was specific to human CatD, CatD-positive immunostaining was not observed in the C57BL/6 (Figure 1C)
but was detected in mcd/mcd mice where it was localized to the cytoplasm of the RPE cells (Figure 1C)
.
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Fundus photography was performed on mcd/mcd (n = 17) and C57BL/6 (n = 17) mice every 1 to 2 months from the age of 6 months. In C57BL/6 mice, the fundus appeared normal and no changes were detected with progress in age (data not shown). In mcd/mcd mice, the fundus appeared normal up to 9 months (Figure 2A)
after which light, pinkish-yellowish patches (Figure 2B)
, suggestive of retinal atrophy (Figure 4, C and D)
, were detected in 16 (94%) of the mcd/mcd mice. A typical atrophy emerged at the peripheral part of the retina. The size of these patches increased with age, reaching a maximum size covering between 3 to 10% of the fundus at 10 to 12 months and remaining stable thereafter (Figure 2C
, Table 1
). In addition to these patches, multiple small yellowish spots (probably pigmentary disturbance) in the periphery and at the posterior pole were also observed and their numbers increased with age around 18 months. In three 18-month-old mice, these small yellowish spots were scattered throughout the retina (Figure 2C
, Table 1
). At the same time, signs of hyperpigmentary changes were also noted (Figure 2, B and C)
.
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As a consequence of the geographic distribution of photoreceptor degeneration and the constant rod/cone ratio throughout the mcd/mcd mouse retinas, retinal function/dysfunction could be assessed easily by full-field electroretinography in this transgenic mouse model. Typical flash ERGs recorded from four dark-adapted 12-month-old mcd/mcd and four C57BL/6 eyes are shown in Figure 3A
. The negative a-wave was generated by hyperpolarization of the photoreceptors and the positive b-wave resulted from depolarization of outer nuclear bipolar cells. The waveform of the experimental response was similar to that of C57BL/6 mice but the amplitude of the response was much smaller. A two-way repeated measures analysis of variance of the stimulus-response characteristics demonstrated a significant (P < 0.01) difference in the a-wave amplitude between the C57BL/6 and mcd/mcd mice. Post hoc Bonferroni tests revealed a significant (P < 0.001, n = 4) suppression of the a-wave response at all flash intensities greater than -1.2 log neutral density units (Figure 3B)
. Figure 3C
shows the significant difference (P < 0.01) between the stimulus intensity b-wave response characteristics of the C57BL/6 and mcd/mcd mice. The b-wave amplitude was significantly attenuated (P < 0.002, n = 4) in mcd/mcd mice at all flash intensities greater than -3 log neutral density units.
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, inset) for flash intensities up to -1.2 log neutral density units.30
The Rmax value (±SEM) of 370 ± 29 µV for mcd/mcd mice was significantly lower than the C57BL/6 mice value of 740 ± 73 µV (P < 0.005). Similarly, a significant reduction in retinal sensitivity of 0.85 log neutral density units was seen in mcd/mcd mice. The calculated values of log K were -3.30 ± 0.08 (C57BL/6, n = 4) and -2.45 ± 0.23 neutral density units (mcd/mcd, P < 0.05, n = 4). The kinetic properties of the maximum intensity, dark-adapted a-waves recorded from four control and four mcd/mcd eyes were analyzed to assess rod phototransduction. The leading edges of the a-waves were normalized to the deepest a-wave trough31
and the averaged responses plotted in Figure 3D
. The averaged response recorded from mcd/mcd mice overlaps the averaged response recorded in control mice. Histological Studies
Light microscopy of H&E-stained paraffin sections and toluidine blue-stained resin sections from 10- to 18-month-old mcd/mcd mice demonstrated that while the RPE cell layer appeared normal and continuous throughout the retina of C57BL/6 mice (data not shown) and most parts of the retina in mcd/mcd mice (Figure 4A)
, areas of atrophy and hypertrophy were evident in mcd/mcd mice (Table 1)
. These include clumps of pigmented cells in the subretinal space (Figure 4B
, arrowheads) in the central or peripheral retina. The putative RPE cells beneath these pigmented cells were attenuated, enlarged, and sometimes less pigmented (Figure 4, B and C)
. Obvious changes in the peripheral and central parts of the retina (Figure 4, C and D
, respectively) were present. In some eyes, there were also regions where RPE cells were disorganized, swollen (Figure 4, C and D)
, clumped (Figure 4C)
, atrophic, or absent. The ONLs in these regions were also significantly thinner or sometimes missing (Figure 4D)
with large stunted inner segments. In some parts, the outer segments were shortened or missing (Figure 4; C, D, and F)
. Progressive degeneration of photoreceptors, thus sometimes aggressive thinning of the ONL became visible by 12 months with subsequent disappearance of the ONL by 18 months. In these animals some disorganization in the inner nuclear layers accompanied the disappearance of the ONL (Figure 4D)
. However at 18 months of age the ganglion cell layer remained normal (data not shown).
ROS immunohistochemistry was performed to determine whether or not there was any abnormal ROS-immunoreactive breakdown product accumulation in the RPE cell layer of mcd/mcd mice. In C57BL/6 mice, ROS-positive immunostaining was localized predominantly to the ROS with weak immunostaining in the RPE (Figure 4E)
. In mcd/mcd mice, the ROS were shortened (Figure 4, E and F
, double-headed arrow) but they continued to demonstrate ROS-positive immunostaining (Figure 4F)
. In addition, strong ROS-positive immunostaining was also detected in the cytoplasm of the RPE cells (Figure 4F
, arrow). Fluorescence microscopy of frozen sections showed autofluorescent deposits resembling lipofuscin in the C57BL/6 and mcd/mcd mouse eyes (Figure 4, G and H)
. Some increase in autofluorescent signal was detected in RPE cell layers of mcd/mcd mice (Figure 4H
, white arrow) and the abnormal regions containing clumped, enlarged RPE cells were also autofluorescent (Figure 4H
, white arrowheads).
Measurement of Photoreceptor Cell Death
There was a decrease in the average number of photoreceptor cell nuclei in mcd/mcd mice (131.33 ± 8.5) when compared to control age-matched C57BL/6 mice (145.50 ± 12.5) but this decrease was not statistically significant (P 
0.05). However, compared to C57BL/6 mice (0.25 ± 0.16), there was a statistically significant increase in TUNEL-positive cells in the mcd/mcd mice (5.0 ± 0.81, P 
0.001). Although the sections for counting were randomly selected, the number of TUNEL-positive cells varied between regions of normal and abnormal RPE cells in the mcd/mcd mice. The number of TUNEL-positive cells ranged from 0 to 4 in normal and 5 to 20 in abnormal RPE regions in mcd/mcd mice.
Transmission Electron Microscopy
Distinct differences between RPE cells of mcd/mcd and C57BL/6 mice were revealed by transmission electron microscopy. In unaffected areas of mcd/mcd mouse retinas, the cytoplasmic concentration and morphology of melanosomes in RPE cells was similar to those of control mice. Similarly, the convoluted border at the base of these cells did not deviate from normal. There were no deposits between the plasma membrane and the basal lamina of RPE cells while components of BM showed only minor focal undulations (Figure 5A)
. The most obvious change in the RPE cells from the histologically affected areas in mcd/mcd mice was an increase in the number of cytoplasmic vacuoles, some of which possessed membranous aggregates at the periphery. Although melanosomes were more numerous in more than a few RPE cells, their concentration was obviously reduced in others when compared to those from C57BL/6 mice (data not shown). In a few instances, relatively large foci of poorly melanized granules were also found (Figure 5B)
. The latter, moreover, were usually smaller than those seen in RPE cells from control mice. Lastly, several RPE cells displayed focal aggregates of lipid globules (data not shown). The most striking feature from mcd/mcd mice retinas was the presence of extensive deposits, which were situated between the plasma membrane and the basal lamina of RPE cells. These regions contained substantial subepithelial deposits whereas the basal in-foldings of the RPE cells were often distorted and the space between them expanded. These deposits, measuring from 50 nm to several µm in their maximal diameter, were often dissected by thin attenuated processes that extended from RPE cells (Figure 5C)
. They contained bundles of filaments measuring 15 nm in diameter, collections of vesicles (50 to 90 nm in diameter), banded fibers with the periodicity of wide-spaced collagen (100 nm) (Figure 5D)
, and membranous whorls (Figure 5, D and E)
containing electron-dense granules and vesicles. Spherules of size varying from 0.2 to 9.5 µm were also seen in the central region of some of these deposits and many of these had coalesced into large aggregates and on occasion were insinuated between adjoining cells. The periphery of these spherules contained numerous electron dense spicules suggestive of calcium salts (Figure 5F)
. The ultrastructural integrity of the components of BM appeared normal in most regions (Figure 5A)
. The BM at the site of the basal laminar deposits, sometimes containing whorls (Figure 5E)
, was often thickened because of increased collagenous matrix deposits. In these areas, vesicular structures (Figure 5E)
and wide-spaced collagen were also observed.
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. Detailed studies of the BM revealed a close correlation between the presence of atypical RPE cells and BM abnormalities. Some RPE cells demonstrated the beginning of debris accumulation between the plasma membrane and the basal lamina. This debris, resembling basal laminar deposit could reach a thickness up to 5 µm (Figure 5, C and E
, and Figure 6B
). There were significant changes observed in the BM beneath the abnormal RPE cells demonstrating basal laminar deposits (Figure 6, B and C)
. Sometimes these changes were limited to the loss of layered structure in the BM (Figure 6B
, black arrows) but frequently the thickened membrane contained granular and vesicular material (Figures 5E and 6C
, small arrows) resembling basal linear deposit.
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Discussion |
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There are several retinal features associated with and preceding AMD development such as RPE cell attenuation with depigmentation, hypertrophy and atrophy,37,38 lipofuscin accumulation,39,40 the presence of drusen,5,41,42 basal laminar5 and linear4 deposits, and BM19 and choroidal changes.43 Recently several authors demonstrated the occurrence of debris accumulation similar to basal lamina in mice on high-fat diets.44-46 However, these animals did not present other features of AMD such as pigmentary changes and the appearance of this debris did not result in progressive retinal degeneration.44 One of the most significant features of the mcd/mcd model is a change in the regions of BM. Basal laminar and linear deposits have been strongly associated with AMD.37,47 Basal laminar deposits consist of fibrillar, amorphous, and banded deposits located between the plasma membrane and the basal lamina of the RPE cells. The earliest manifestation of the disease is the appearance of fibrillar deposits. The banded form resembles long spacing collagen and it may be interspersed with membranous material.5 Basal linear deposits are composed of vesicular material and granular material and are located between the basal lamina and the inner collagenous zone of the BM.37 Data presented by Curcio and Millican47 suggest that membranous debris is a lesion specific to age-related maculopathy and that soft drusen is the result of basal linear deposit build-up. There was a strong correlation between basal laminar deposit and the appearance of basal linear deposit in the mcd/mcd mouse. Both basal laminar and basal linear deposits were present in the mcd/mcd mouse model, although the BM thickening was less than that observed in human age-related maculopathy and there were no obvious changes in the choriocapillaris up to 12 months of age. To our knowledge the mcd/mcd mouse model is the first to demonstrate photoreceptor breakdown product-related development of basal laminar and linear deposits. Hence, the mcd/mcd mouse provides an excellent model to study the processes and pathways that drive the RPE cell changes seen.
In mcd/mcd mice, changes in the fundus appeared to be age-dependent. At around 10 to 12 months, pigmentary changes including mottled areas of hypopigmentation were detected in all animals, with some variation in size and location. These changes were strikingly similar to fundus changes observed in patients with GA in whom sharp areas of RPE cells atrophy were seen.2,4 Although the presence of a few small hard drusen is not considered to be a pathological condition, it has been suggested that in large numbers they may lead to larger drusen development and subsequent complications, especially GA.41 In our animal model, small hard drusen development did not precede the retinal changes observed with fundus photography and small hard drusen could not be detected up to the age of 18 month. In this respect the photoreceptor and RPE changes seen in the mcd/mcd mice are similar to those observed in some younger age-related maculopathy patients in whom hyperpigmentation appears without drusen (S and J Sarks, personal communication). It is thought that photoreceptor breakdown product accumulation in the RPE cells of mcd/mcd mice simply do not contain the specific materials that contribute to the formation of small hard drusen. The accumulation of autofluorescent photoreceptor-derived breakdown products, or lipofuscin, has been implicated in AMD development,39,40 and recently a lysosomotropic amine which is a component of lipofuscin,48 has been shown to induce the formation of cellular age pigments. In the normal RPE cells of the mcd/mcd mouse there was an increased accumulation of autofluorescent debris that was also present in abnormal RPE cells.
The wider distribution of retinal features has facilitated ERG examination of the mcd/mcd model. The relatively few studies available on ERG of AMD patients demonstrated abnormal rod-dominated ERGs.49 Analysis of the dark-adapted electroretinogram of experimental mice revealed a significant reduction in the photoreceptor-generated a-wave amplitude.49,50 This attenuation indicates the degeneration of photoreceptors that coincides with the increase in apoptotic photoreceptor nuclei. The presence of apoptotic photoreceptor nuclei and subsequent death of photoreceptor cells results in thinning of the ONL at 12 months. The thickness of the ONL at this age varied significantly. In one animal the ONL was completely missing whereas in some almost all layers were present. However, by the age of 18 months, the loss of photoreceptor cells was complete in the regions affected by changes in the RPE layer in all mcd/mcd eyes examined to date. Considering that fundus photography and histology did not show abnormalities in the mcd/mcd retinas up to the age of 10 to 12 months, these data tentatively support the threshold theory14 in which it is proposed that a variety of gradual changes such as photoreceptor breakdown product accumulation, basal laminar and linear deposit formation, suddenly results in a cascading functional breakdown.
Interestingly, the similarity in the kinetics of the control and mcd/mcd a-waves suggests that the phototransduction mechanisms in the remaining photoreceptors are not affected by the accumulation of ROS debris. Similar findings have been reported in other models of mouse retinal degeneration including the nervous mutant30
rds heterozygotes50
and a transgenic mouse with a triple substitution in the rhodopsin molecule.51
Figure 4C
shows that a number of the outer segments of surviving photoreceptors are shortened in the mcd/mcd retina. Shortened outer segments would result in reduced photon capture. This histological observation is reflected in the decrease in retinal sensitivity (increased log K) of 0.85 log neutral density units that was seen in the experimental mice compared with the controls when a Naka-Rushton curve fit was applied to the b-wave amplitude data. Decreased rod sensitivity has also been described in abcr+/- mutant mouse model of AMD.52
Moreover, abnormal rod-dominated electroretinograms have been reported in some patients with AMD.49
These abnormalities include decreased b-wave amplitudes and increased log K values indicative of a reduction in retinal sensitivity similar to that observed in mcd/mcd mice.
In summary, the mcd/mcd model presents several features of AMD, including changes in the pigment epithelium and BM, which occur progressively with aging, and thus it will prove a valuable tool in the investigation of the underlying biological processes and pathomechanism of AMD.
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Acknowledgements |
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Footnotes |
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Supported by the National Health and Medical Research Council of Australia and Retina Australia.
The Lions Eye Institute is affiliated with The University of Western Australia.
Accepted for publication June 20, 2002.
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Y. Imamura, S. Noda, K. Hashizume, K. Shinoda, M. Yamaguchi, S. Uchiyama, T. Shimizu, Y. Mizushima, T. Shirasawa, and K. Tsubota Drusen, choroidal neovascularization, and retinal pigment epithelium dysfunction in SOD1-deficient mice: A model of age-related macular degeneration PNAS, July 25, 2006; 103(30): 11282 - 11287. [Abstract] [Full Text] [PDF]
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F. Doonan, M. Donovan, and T. G. Cotter Activation of Multiple Pathways during Photoreceptor Apoptosis in the rd Mouse Invest. Ophthalmol. Vis. Sci., October 1, 2005; 46(10): 3530 - 3538. [Abstract] [Full Text] [PDF]
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D. Zhang, M. Brankov, M. T. Makhija, T. Robertson, E. Helmerhorst, J. M. Papadimitriou, and P. E. Rakoczy Correlation between Inactive Cathepsin D Expression and Retinal Changes in mcd2/mcd2 Transgenic Mice Invest. Ophthalmol. Vis. Sci., September 1, 2005; 46(9): 3031 - 3038. [Abstract] [Full Text] [PDF]
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 A. G. Marneros and B. R. Olsen Physiological role of collagen XVIII and endostatin FASEB J, May 1, 2005; 19(7): 716 - 728. [Abstract] [Full Text] [PDF]
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 E. F. Nandrot, Y. Kim, S. E. Brodie, X. Huang, D. Sheppard, and S. C. Finnemann Loss of Synchronized Retinal Phagocytosis and Age-related Blindness in Mice Lacking {alpha}v{beta}5 Integrin J. Exp. Med., December 20, 2004; 200(12): 1539 - 1545. [Abstract] [Full Text] [PDF]
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C. Tardy, H. Autefage, V. Garcia, T. Levade, and N. Andrieu-Abadie Mannose 6-Phosphorylated Proteins Are Required for Tumor Necrosis Factor-induced Apoptosis: DEFECTIVE RESPONSE IN I-CELL DISEASE FIBROBLASTS J. Biol. Chem., December 17, 2004; 279(51): 52914 - 52923. [Abstract] [Full Text] [PDF]
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N. Strunnikova, C. Zhang, D. Teichberg, S. W. Cousins, J. Baffi, K. G. Becker, and K. G. Csaky Survival of Retinal Pigment Epithelium after Exposure to Prolonged Oxidative Injury: A Detailed Gene Expression and Cellular Analysis Invest. Ophthalmol. Vis. Sci., October 1, 2004; 45(10): 3767 - 3777. [Abstract] [Full Text] [PDF]
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I. Chowers, Y. Kim, R. H. Farkas, T. L. Gunatilaka, A. S. Hackam, P. A. Campochiaro, S. C. Finnemann, and D. J. Zack Changes in Retinal Pigment Epithelial Gene Expression Induced by Rod Outer Segment Uptake Invest. Ophthalmol. Vis. Sci., July 1, 2004; 45(7): 2098 - 2106. [Abstract] [Full Text] [PDF]
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H. Ida, K. Ishibashi, K. Reiser, L. M. Hjelmeland, and J. T. Handa Ultrastructural Aging of the RPE-Bruch's Membrane-Choriocapillaris Complex in the D-Galactose-Treated Mouse Invest. Ophthalmol. Vis. Sci., July 1, 2004; 45(7): 2348 - 2354. [Abstract] [Full Text] [PDF]
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 H. Ida, S. A. Boylan, A. L. Weigel, and L. M. Hjelmeland Age-related changes in the transcriptional profile of mouse RPE/choroid Physiol Genomics, November 11, 2003; 15(3): 258 - 262. [Abstract] [Full Text] [PDF]
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M. Danciger, J. Lyon, D. Worrill, M. M. LaVail, and H. Yang A Strong and Highly Significant QTL on Chromosome 6 that Protects the Mouse from Age-Related Retinal Degeneration Invest. Ophthalmol. Vis. Sci., June 1, 2003; 44(6): 2442 - 2449. [Abstract] [Full Text] [PDF]
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