September 2008
Volume 49, Issue 9
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Retinal Cell Biology  |   September 2008
Attenuation of Vision Loss and Delay in Apoptosis of Photoreceptors Induced by Proinsulin in a Mouse Model of Retinitis Pigmentosa
Author Affiliations
  • Silvia Corrochano
    From the 3D Lab (Development, Differentiation, and Degeneration), Department of Cellular and Molecular Physiopathology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain; the
  • Rima Barhoum
    Department of Physiology, Universidad de Alcalá, Alcalá de Henares, Spain; and
  • Patricia Boya
    From the 3D Lab (Development, Differentiation, and Degeneration), Department of Cellular and Molecular Physiopathology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain; the
  • Ana I. Arroba
    From the 3D Lab (Development, Differentiation, and Degeneration), Department of Cellular and Molecular Physiopathology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain; the
  • Natalia Rodríguez-Muela
    From the 3D Lab (Development, Differentiation, and Degeneration), Department of Cellular and Molecular Physiopathology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain; the
  • Violeta Gómez-Vicente
    From the 3D Lab (Development, Differentiation, and Degeneration), Department of Cellular and Molecular Physiopathology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain; the
  • Fátima Bosch
    Department of Biochemistry and Molecular Biology, Center of Animal Biotechnology and Gene Therapy, Universitat Autònoma de Barcelona, Bellaterra, Spain.
  • Flora de Pablo
    From the 3D Lab (Development, Differentiation, and Degeneration), Department of Cellular and Molecular Physiopathology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain; the
  • Pedro de la Villa
    Department of Physiology, Universidad de Alcalá, Alcalá de Henares, Spain; and
  • Enrique J. de la Rosa
    From the 3D Lab (Development, Differentiation, and Degeneration), Department of Cellular and Molecular Physiopathology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain; the
Investigative Ophthalmology & Visual Science September 2008, Vol.49, 4188-4194. doi:10.1167/iovs.08-2182
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      Silvia Corrochano, Rima Barhoum, Patricia Boya, Ana I. Arroba, Natalia Rodríguez-Muela, Violeta Gómez-Vicente, Fátima Bosch, Flora de Pablo, Pedro de la Villa, Enrique J. de la Rosa; Attenuation of Vision Loss and Delay in Apoptosis of Photoreceptors Induced by Proinsulin in a Mouse Model of Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2008;49(9):4188-4194. doi: 10.1167/iovs.08-2182.

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      © 2015 Association for Research in Vision and Ophthalmology.

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purpose. Retinitis pigmentosa (RP) is a heterogeneous group of inherited conditions that lead to blindness and for which there is no effective therapy. Apoptosis of photoreceptors is a common feature in animal models of the disease. Thus, the authors studied the therapeutic potential of proinsulin, an antiapoptotic molecule active during retinal development.

methods. Transgenic mice expressing human proinsulin (hPi) in the skeletal muscle were generated in a mixed C57BL/6:SJL background and were back-crossed to a C57BL/6 background. Two independent lineages of transgenic mice were established in which hPi production in muscle was constitutive and not regulated by glucose levels. hPi levels in serum, muscle, and retina were determined with a commercial ELISA kit, visual function was evaluated by electroretinographic (ERG) recording, and programmed cell death was assessed by TUNEL. Immunohistochemistry was used to evaluate retinal structure preservation and oxidative damage.

results. Transgenic expression of hPi in the rd10 retinal degeneration mouse model led to prolonged vision, as determined by ERG recording, in a manner that was related to the level of transgene expression. This attenuation of visual deterioration was correlated with a delay in photoreceptor apoptosis and with the preservation of retinal cytoarchitecture, particularly that of the cones.

conclusions. These results provide a new basis for possible therapies to counteract retinitis pigmentosa and a new tool to characterize the mechanisms involved in the progress of retinal neurodegeneration.

Programmed cell death is a self-destructive physiological process essential for the correct development and homeostasis of an organism. 1 Consequently, unregulated cell death underlies many diseases, including neurodegenerative disorders. 2 Thus, understanding how cell death is regulated during neural development may help to establish new therapeutic approaches to prevent neurodegeneration. 3  
Retinitis pigmentosa (RP) constitutes a large, heterogeneous group of inherited retinal neurodegenerative conditions that involve the progressive loss of retinal function in parallel with the loss of photoreceptors through apoptosis. 4 5 Thus, attenuation of apoptosis represents a good therapeutic target in RP, especially considering the vast heterogeneity of the disease. 6 The most extensively studied animal model of RP is the rd1 mouse, which carries a recessive nonsense mutation in the Pde6b gene. 7 Indeed, a number of mutations have been found in the catalytic domain of the human PDE6B gene in patients with autosomal recessive RP. 8 9 The rd1 mouse is characterized by early onset of photoreceptor loss and apoptosis peaking around postnatal day (P)14 to 15. 4 10 As a consequence, electroretinographic (ERG) recordings of visual function never reach notable values, even when attenuation has been reported. A more recently characterized animal model, the rd10 mouse, also carries a recessive mutation in the Pde6b gene, which produces a delayed phenotype when compared with the rd1 mouse. Therefore, electroretinograms can be readily obtained before degeneration in this model 11 12 ; accordingly, this situation better resembles the course of the human disease. 
Molecules that promote survival, such as brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor, fibroblast growth factor, glial-derived neurotrophic factor (GDNF), and pigment epithelium-derived factor, are moderately successful in preventing disease progression when used in animal models. 13 14 15 16 17 18 Antioxidants also provide modest protection, probably by reducing the oxidative damage found in the rd1 mouse retinas. 19 20 Members of the insulin family are well-characterized neuroprotective molecules active in development and aging. 21 22 23 We have previously reported that locally expressed proinsulin acts as a survival factor during embryonic retinal development in the chick and the mouse. 24 25 26 27 In the present study we show that transgenic human proinsulin expression in rd10 mice attenuates retinal degeneration, as assessed by the maintenance of electroretinogram and the histologic preservation of photoreceptors. Systemic proinsulin was able to reach retinal tissue, delay apoptotic death of photoreceptors, and decrease oxidative damage. Thus, proinsulin represents a possible new therapy for RP and a new tool to characterize the mechanisms involved in pathologic cell death. 
Materials and Methods
Animals
All procedures were approved by the respective local ethics committee for animal experimentation, and experiments were carried out in accordance with the European Union guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Control wild-type (wt) C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The mouse model of retinal degeneration, Pde6b rd10 (rd10), also on a C57BL/6J background, was kindly provided by Bo Chang (The Jackson Laboratory). 
Transgenic mice expressing human proinsulin (hPi) driven by the myosin light chain (MCL)-I promoter and a muscle-specific enhancer were generated on a mixed C57BL/6:SJL background and back-crossed to a C57BL/6 background. Two lines of transgenic mice, L1 and L2, were established in which hPi production in muscle was constitutive and not regulated by glucose levels. Expression of hPi in transgenic mice was confirmed by ELISA of muscle and serum samples, whereas hPi was undetectable in nontransgenic animals. 
The rd10 mice used in this study were homozygous for the Pde6b rd10 mutation, whereas the transgenic mice were either homozygous or hemizygous for the hPi transgene, as indicated. 
The hPi genotype was determined by Southern blotting using specific dCT32P-labeled probes for hPi and for mouse S16 as a reference gene (Random Primer Kit; Stratagene, La Jolla, CA), hybridized at the same time. Phosphorimager IP software (Fuji Film, Kanagawa, Japan) was used to quantify the signal corresponding to the respective specific bands, and the ratio between both bands was used to establish the zygosity of each transgenic animal. The rd10 genotypes were assessed as recommended by The Jackson Laboratory. 11  
Selected animals were weighed monthly over a 13-month period (GM-100; Gram Precision, L'Hospitalet de Llobregat, Spain). After each weighing, food was removed from the cage, and a blood sample was taken 18 hours later to determine the extent of glycemia (Roche Accu-Chek Sensor; Roche Diagnostic, Mannheim, Germany). 
ERG Recordings
Mice were dark adapted overnight, and subsequent manipulations and ERG recordings were performed in dim red light. Mice were anesthetized with intraperitoneal injections of a ketamine (95 mg/kg) and xylazine (5 mg/kg) solution and maintained on a heating pad at 37°C. Pupils were dilated by applying a topical drop of 1% tropicamide (Colircusí Tropicamida, Alcon Cusí; El Masnou, Barcelona, Spain). To optimize electrical recording, a topical drop (2% Methocel; Ciba Vision, Hetlingen, Switzerland) was instilled on each eye immediately before situating the corneal electrode. Flash-induced ERG responses were recorded from the right eye in response to light stimuli produced with a Ganzfeld stimulator. The intensity of the light stimuli was measured with a photometer at the level of the eye (Mavo Monitor USB; Gossen, Nürenberg, Germany). At each light intensity, 4 to 64 consecutive stimuli were averaged with an interval between light flashes in scotopic conditions of 10 seconds for dim flashes and of up to 60 seconds for the highest intensity. In contrast, under photopic conditions, the interval between light flashes was fixed at 1 second. ERG signals were amplified and band filtered between 0.3 and 1000 Hz with an amplifier (CP511 AC amplifier; Grass Instruments, Quincy, MA). Electrical signals were digitized at 20 kHz with a power laboratory data acquisition board (AD Instruments, Chalgrove, UK). Bipolar recording was performed between an electrode fixed on a corneal lens (Burian-Allen electrode; Hansen Ophthalmic Development Laboratory, Coralville, IA) and a reference electrode located in the mouth, with a ground electrode located in the tail. Under dark adaptation, rod-mediated responses were recorded to light flashes ranging from −4 to −1.5 log cd · s/m2, whereas mixed rod- and cone-mediated responses were recorded in response to light flashes ranging from −1.5 to 1.5 log cd · s/m2. Oscillatory potential (OP) was isolated using white flashes of 0.48 log cd · s/m2 in a recording frequency range of 100 to 10,000 Hz., and cone-mediated responses to light flashes ranging from 0.5 to 2 log cd · s/m2 on a rod-saturating background of 30 cd/m2 were recorded. Flicker responses (20 Hz) to light flashes of 1.5 log cd · s/m2 were also recorded on a rod-saturating background. Amplitudes of the a-wave and b-wave were measured off-line, and results were averaged. Measurements were performed by an observer masked to the experimental condition of the animal. 
Detection of Transgenic Proinsulin
Human proinsulin production in transgenic mice was determined in serum and muscle using an ELISA kit (human total proinsulin ELISA kit; Linco Research, St. Louis, MO) according to manufacturer’s instructions. For serum determination, 20 μL serum was assayed in duplicate. For muscle determination, quadriceps muscles were first dissected and homogenized in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% (wt/vol) Triton X-100 buffer. Duplicates containing 30 μg protein, as determined by the BCA method (Pierce, Rockford, IL), were assayed. To determine the access of proinsulin to the retina, litters of P25 wt and rd10 mice were injected subcutaneously with 5 μg hPi (in 100 μL phosphate-buffered saline, P-4672 [Sigma, St. Louis, MO] or as kindly provided by Eli-Lilly). Retinal extracts were prepared 2 hours later by dissection and homogenization of one retina in 50 μL Tris-HCl buffer, as described, and duplicates of 20 μL were assayed. 
Cell Death Progression
Programmed cell death was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Mice of the indicated genotype and age were decapitated, their eyes were enucleated, and the retinas removed and flat mounted onto nitrocellulose filter (Sartorius, Göttingen, Germany) photoreceptor layer side up. Retinas were then fixed in 4% (wt/vol) paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4°C overnight and subsequently were processed for TUNEL staining, as described previously (Promega, Madison, WI 27 ). After labeling, the retinas were mounted in medium (Vectashield; Vector Laboratories, Burlingame, CA) containing DAPI (Vectashield; Vector Laboratories) and were analyzed on a laser confocal microscope (TCS SP2; Leica Microsystems, Wetzlar, Germany). Serial optical sections were acquired with a 10× objective every 5 μm in depth around the optic nerve head. To ensure that TUNEL-positive nuclei were located in the outer nuclear layer (ONL), the depth of analysis was set according to ONL thickness, as determined in retinal sections. The number of total TUNEL-positive cells per square millimeter was quantified in compiled projections from at least four retinas per experimental group. The central retina area quantified represents approximately 15% of the retinal surface. 
Histochemistry
Animals from the different experimental groups were decapitated, and their eyes were enucleated, immediately embedded (Tissue-Tek; Sakura Finetek, Torrance, CA), and frozen in dry ice. Sections measuring 12 μm were cut with a cryostat (CM 1900; Leica Microsystems) and mounted on poly-lysine–coated glass slides (Fisher Biotech, Pittsburgh, PA). Cryosections were then dried at room temperature, fixed for 15 minutes with 4% (wt/vol) paraformaldehyde in phosphate-buffered saline, and stained with Alexa 488-conjugated peanut agglutinin (1:500; Molecular Probes, Eugene, OR) or a rabbit anti–acrolein antibody (1:150; AbD Serotec, Oxford, UK) followed by Alexa 488-conjugated goat anti–rabbit immunoglobulin (1:200; Molecular Probes). After staining, sections were mounted in medium (Vectashield; Vector Laboratories) containing DAPI to counterstain the nuclei. 
Results
Preservation of Visual Function in hPi/rd10 Mice by Transgenic Proinsulin Expression
The antiapoptotic activity of proinsulin during retinal development 25 prompted us to investigate a possible effect of proinsulin in the photoreceptor apoptosis associated with retinal degeneration. We crossed two hPi transgenic mice lineages (L1 and L2) with the retinal degeneration model rd10 and analyzed the visual function of hPi/rd10 homozygous animals. Electroretinography provided a comprehensive evaluation of visual function, both in scotopic (nighttime, rod selective) and photopic (daytime, cone selective) conditions. Although ERG waves could be recorded before degeneration in the rd10 mouse, as previously described, 11 12 28 the ERG amplitudes diminished as retinal degeneration occurred, and they became negligible by P30 (Fig. 1) . By contrast, visual activity could still be measured at P30 in the two different hPi/rd10 mice lines (Fig. 1) , and it was most prominent in the cones, whose degeneration was secondary to that of rods. 29 Thus, transgenic expression of human proinsulin in muscle was able to attenuate the loss of vision in rd10 mice. 
To rule out potential metabolic effects of transgenic hPi expression, glucose levels in the sera of transgenic mice and their weights were compared with those of wt animals over a period of 13 months. Transgenic hPi animals were in normal ranges of glycemia and weight (Fig. 2)  
Correlation of Visual Function Preservation in hPi/rd10 Mice with Proinsulin Levels
We assessed the correlation between hPi expression levels and the preservation of visual function, a particularly relevant issue when contemplating the therapeutic potential of any factor. As such, we evaluated visual function by recording electroretinograms from homozygous and hemizygous hPi/rd10 animals of lineage 1. ERG responses were recorded at P30 in dark- and light-adapted conditions to reflect rod- and cone-mediated vision, and standard ERG-wave amplitudes were determined. As a result, we identified significant differences in the averaged ERG amplitude values of bdim, OP, amax, bmax, bphot, and flicker responses in each experimental group (Supplementary Fig. S1). Indeed, each of these ERG parameters was higher in homozygous hPi/rd10 mice than in rd10 mice (P < 0.001), and, notably, the mean amplitude values of OP, bphot, and flicker (Supplementary Figs. S1B, S1E, and S1F, respectively) were 26%, 37%, and 60% of the respective values from wt mice. Furthermore, OP, amax, and flicker values from hemizygous hPi/rd10 animals were also significantly higher than those from rd10 mice (P < 0.01). 
Once we had established a correlation between visual function preservation and transgenic hPi gene dosage, we examined the correlation between hPi protein levels and visual function. The measured hPi concentrations in serum were below 15 pM, close to the inferior limit of the hPi ELISA kit used for their determination. Because the hPi transgene was under the control of the MLC-I muscle promoter, we determined the levels of hPi produced in muscle instead. Significant correlation between muscle hPi protein and several ERG parameters was found. Indeed, there was a hyperbolic correlation between hPi muscle content and the amplitudes of bmax, OP, bphot, and flicker (Fig. 3) . Therefore, a dose-response relationship between the production of transgenic hPi in muscle and induced neuroprotection in the retina supported the potential therapeutic use of proinsulin. 
In addition, we evaluated the duration of the neuroprotective effect and functional preservation afforded by proinsulin by comparing electroretinograms from wt, rd10, and hPi/rd10animals up to P55 (Fig. 4and Supplementary Fig. S2). Scotopic and photopic b-waves (bmax and bphot) were partially preserved in hPi/rd10 mice even at P55, a period that doubled the period of vision in this model (Supplementary Fig. S2). Remarkably, cone function was better preserved and longer lasting than rod function. Together, these experiments demonstrated that sustained low levels of systemic proinsulin actually delayed visual loss in rd10 dystrophic animals. 
Delay of Photoreceptor Cell Death by Systemic Proinsulin
The effects of transgenic hPi reflected in the electroretinograms indicated that it acted directly or indirectly on retinal tissue, extending visual function. However, we were unable to detect hPi in retinal extracts from hPi/rd10 mice. Thus, to assess whether systemic proinsulin did indeed reach the retinal tissue, we subcutaneously injected a single dose of hPi (5 μg) and 2 hours later determined the proinsulin content in serum and retinal extracts. In this manner we could detect proinsulin in the retina; interestingly, retinal levels roughly correlated with proinsulinemia (Fig. 5) . Thus, the inability to detect proinsulin in hPi/rd10 retinas probably reflected the low amounts of proinsulin produced or accumulated by the animals, which nevertheless were sufficient to attenuate the loss of vision. 
The loss of visual function in rd10 mice is associated with massive loss of photoreceptors in the ONL. 11 12 28 Given that we observed the attenuation of vision loss in transgenic hPi/rd10 animals, we investigated whether proinsulin expression was able to specifically interfere with photoreceptor apoptosis. Cell death, visualized by TUNEL staining of whole-mount retinas, was delayed in hPi/rd10 compared with rd10 mice (Fig. 6) , an observation that paralleled the delay in the loss of visual function already described (Fig. 4) . Therefore, systemic proinsulin was able to exert an antiapoptotic effect on photoreceptors. 
Preservation of Retinal Structure and Decrease of Oxidative Damage by Transgenic Proinsulin
We further examined the histopathologic aspects of the rd10 mouse retina in comparison with hPi/rd10. In agreement with our previous observations, transgenic hPi/rd10 animals displayed more photoreceptor rows than rd10 animals at P32 (Fig. 7) . This histologic feature also correlated with transgene dosage, as described for electroretinography (Supplementary Fig. S1) and proinsulinemia. We studied cone preservation in more detail by staining the retina with the specific peanut agglutinin cone marker, which labels cone outer segments and synaptic terminals. A remarkable preservation of these cells was observed in hPi/rd10 retinas, sustaining the ERG cone responses described in Figure 4and Supplementary Figure S2. 
Increased lipid oxidation has been demonstrated in the retinas of rd1 and rd10 mouse models, 19 30 and oxidative damage appears to trigger cell death in physiological and pathologic conditions. 31 32 Thus, we determined whether proinsulin might interfere at this level of the neurodegenerative process. There was strong immunoreactivity for acrolein, a product of lipid oxidation, in the rd10 mouse retina (Fig. 8) , in accordance with previous observations of Komeima et al. 30 Interestingly, the immunoreactivity for acrolein was weaker in hPi/rd10 transgenic animals than in rd10 at P32, suggesting that the prevention of oxidative damage may be one of the primary effects of proinsulin (Fig. 8)
Discussion
Together our results show that proinsulin preserves photoreceptor number and structure and visual function in the degenerating rd10 mouse retina, fulfilling antiapoptotic and antioxidant roles. Partial preservation of visual function in electroretinograms has been achieved by treatment with certain survival molecules, such as GDNF in the transgenic S334ter-4 rat, 17 BDNF in the rhodopsin mutant mouse, 18 and antioxidants in the rd1 mouse. 19 Nevertheless, the preservation of ERG amplitudes reported in those studies was lower than that observed here with proinsulin. Given that different animal models and delivery methods were used, we can only claim at the present that proinsulin displays a promising neuroprotective effect. 
Another interesting difference in the approach adopted here is the effectiveness of systemic delivery. Most experimental treatments for RP involve direct retinal delivery by viral transfer, 16 17 retinal transgene expression, 18 or intravitreal encapsulated cells. 33 However, systemic administration of antioxidants has also been shown to provoke a modest neuroprotective effect. 19 30 Although systemic delivery would perhaps not be the primary choice in retinal therapy, this aspect raises new questions about the biological availability of proinsulin and other members of this family in nerve tissue and their usefulness in neuroprotective therapies. Indeed, systemic IGF-I seems to be able to pass the blood-brain barrier and to induce neuroprotection. 34  
We have found a hyperbolic correlation between the amount of proinsulin expressed in the muscle, where the transgene is transcriptionally active, and the preservation of visual function parameters (Fig. 3) . This correlation is reminiscent of a receptor-mediated effect of proinsulin. Proinsulin has only weak affinity for the classic insulin receptor, and it has poor metabolic potential (5%–10% that of insulin 35 ), which was reflected by the fact that all animals remained in the normal ranges for glycemia and weight (Fig. 2and data not shown). An atypical, hybrid insulin/IGF-I receptor is present in the developing retina when proinsulin exerts its survival role with efficiency similar to that for insulin or IGF-I. 27 36 Proinsulin seems to block developmental cell death at various levels, including activation of the PI3K/Akt and ERK pathways, stimulation of prosurvival chaperones, and interference with caspases and cathepsins. 27 37 38 39 40 This pleiotropic effect may underlie its neuroprotective action throughout, blocking the multiple death pathways described in the rd mouse models. 41 42 43 To determine whether proinsulin survival pathways coincide under physiological and pathologic conditions requires further study. 
Cell death and visual loss still occur and eventually lead to blindness in hPi/rd10 mice. However, proinsulin provides an extended window of visual function, especially with respect to daylight vision, which may be a useful therapeutic approach to ameliorate the dramatic impact of RP in human patients. 
Figure 1.
 
Electroretinography demonstrates the preservation of visual function in hPi/rd10 mice. Electroretinograms were obtained from wt, rd10, and hPi/rd10 mice at P30. Examples from two different hPi/rd10 lineages (L1 and L2) are shown. Rod and mixed responses were recorded under scotopic conditions to light flashes of −2.0 log cd · s/m2 and 1.5 log cd · s/m2, respectively. Cone-mediated responses were recorded to light flashes of 1.5 log cd · s/m2 on a rod-saturating background of 30 cd/m2. Standard ERG wave amplitudes are indicated in the wt mice panels for identification. Amplitudes of ERG recordings in the two hPi/rd10 animal lineages were higher than in rd10 animals. The same amplitude and time calibration are displayed for all recording traces.
Figure 1.
 
Electroretinography demonstrates the preservation of visual function in hPi/rd10 mice. Electroretinograms were obtained from wt, rd10, and hPi/rd10 mice at P30. Examples from two different hPi/rd10 lineages (L1 and L2) are shown. Rod and mixed responses were recorded under scotopic conditions to light flashes of −2.0 log cd · s/m2 and 1.5 log cd · s/m2, respectively. Cone-mediated responses were recorded to light flashes of 1.5 log cd · s/m2 on a rod-saturating background of 30 cd/m2. Standard ERG wave amplitudes are indicated in the wt mice panels for identification. Amplitudes of ERG recordings in the two hPi/rd10 animal lineages were higher than in rd10 animals. The same amplitude and time calibration are displayed for all recording traces.
Figure 2.
 
Glucose levels and weight in hPi transgenic and wt mice. Paired groups of C57BL/6J and hPi transgenic mice (n = 4) were used to determine the evolution of glucose levels (A) and weight (B) over time. Mean values (±SEM) and the regression lines are shown.
Figure 2.
 
Glucose levels and weight in hPi transgenic and wt mice. Paired groups of C57BL/6J and hPi transgenic mice (n = 4) were used to determine the evolution of glucose levels (A) and weight (B) over time. Mean values (±SEM) and the regression lines are shown.
Figure 3.
 
Preservation of ERG wave amplitudes in hPi/rd10 mice correlates with proinsulin levels in muscle. ERG wave amplitudes for bmax (A), OP (B), bphot (C), and flicker (D) are represented as a function of muscular proinsulin levels measured at P32. Each circle represents a single animal. Parametric Pearson testing was performed, and a significant hyperbolic correlation was found for bmax (r2 = 0.455, n = 16, P < 0.005), OP (r2 = 0.612, n = 9, P < 0.05), bphot (r2 = 0.504, n = 16, P < 0.005), and flicker (r2 = 0.498, n = 13, P < 0.01).
Figure 3.
 
Preservation of ERG wave amplitudes in hPi/rd10 mice correlates with proinsulin levels in muscle. ERG wave amplitudes for bmax (A), OP (B), bphot (C), and flicker (D) are represented as a function of muscular proinsulin levels measured at P32. Each circle represents a single animal. Parametric Pearson testing was performed, and a significant hyperbolic correlation was found for bmax (r2 = 0.455, n = 16, P < 0.005), OP (r2 = 0.612, n = 9, P < 0.05), bphot (r2 = 0.504, n = 16, P < 0.005), and flicker (r2 = 0.498, n = 13, P < 0.01).
Figure 4.
 
Visual function in hPi/rd10 mice is maintained for longer than in rd10 animals. Mixed (A) and cone (B) responses were recorded in wt, rd10, and hPi/rd10 animals with respect to light flashes of 1.5 log cd · s/m2 on P25, P35, P45, and P55 (indicated at the top of the recording traces). Cone-mediated responses were recorded on a rod-saturating background of 30 cd/m2. The complete disappearance of the ERG waves was observed in the rd10 control animals by p35, whereas in the hPi/rd10 animals, the ERG responses persisted even at P55.
Figure 4.
 
Visual function in hPi/rd10 mice is maintained for longer than in rd10 animals. Mixed (A) and cone (B) responses were recorded in wt, rd10, and hPi/rd10 animals with respect to light flashes of 1.5 log cd · s/m2 on P25, P35, P45, and P55 (indicated at the top of the recording traces). Cone-mediated responses were recorded on a rod-saturating background of 30 cd/m2. The complete disappearance of the ERG waves was observed in the rd10 control animals by p35, whereas in the hPi/rd10 animals, the ERG responses persisted even at P55.
Figure 5.
 
Systemic hPi is able to reach the retinal tissue. Litters of wt (closed circles) and rd10 mice (open circles) were subcutaneously injected at P25 with 5 μg hPi or vehicle (crosses). Proinsulin concentration was determined 2 hours later in serum and retinal extracts. Each symbol represents a single animal, and proinsulin levels in the retinal tissue are represented as a function of proinsulinemia. Nonparametric Spearman Rho correlation with proinsulin-injected mice (n = 9) rendered rho = 0.669 and P < 0.05.
Figure 5.
 
Systemic hPi is able to reach the retinal tissue. Litters of wt (closed circles) and rd10 mice (open circles) were subcutaneously injected at P25 with 5 μg hPi or vehicle (crosses). Proinsulin concentration was determined 2 hours later in serum and retinal extracts. Each symbol represents a single animal, and proinsulin levels in the retinal tissue are represented as a function of proinsulinemia. Nonparametric Spearman Rho correlation with proinsulin-injected mice (n = 9) rendered rho = 0.669 and P < 0.05.
Figure 6.
 
Photoreceptor cell death is delayed in hPi/rd10 mice. Cell death was determined by TUNEL in whole-mount retinas of rd10 and hPi/rd10 mice at the ages indicated. Representative labeled retinas are shown at P25 (A, B). The total number of TUNEL-positive nuclei was quantified in confocal images of equivalent central retina fields, similar to those shown in (A, B), from at least four retinas per experimental group and was represented as the mean ± SD in (C). Scale bar, 200 μm.
Figure 6.
 
Photoreceptor cell death is delayed in hPi/rd10 mice. Cell death was determined by TUNEL in whole-mount retinas of rd10 and hPi/rd10 mice at the ages indicated. Representative labeled retinas are shown at P25 (A, B). The total number of TUNEL-positive nuclei was quantified in confocal images of equivalent central retina fields, similar to those shown in (A, B), from at least four retinas per experimental group and was represented as the mean ± SD in (C). Scale bar, 200 μm.
Figure 7.
 
Preservation of retinal structure in hPi/rd10 mice. Retinal sections from rd10 (A), hPi/rd10 (B, C), and wt (D) mice at P32 were stained with DAPI (blue) to reveal retinal cytoarchitecture and with Alexa 488-peanut agglutinin (green) to visualize the state of cones. Preservation of photoreceptor rows in the ONL (vertical bars) and preservation of cone outer segments (upper arrowhead) and synaptic terminals (lower arrowhead) correlate with proinsulin gene dosage and with proinsulinemia (1.3 pM in the shown hemizygous; 2.4 pM in the shown homozygous). INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 25 μm.
Figure 7.
 
Preservation of retinal structure in hPi/rd10 mice. Retinal sections from rd10 (A), hPi/rd10 (B, C), and wt (D) mice at P32 were stained with DAPI (blue) to reveal retinal cytoarchitecture and with Alexa 488-peanut agglutinin (green) to visualize the state of cones. Preservation of photoreceptor rows in the ONL (vertical bars) and preservation of cone outer segments (upper arrowhead) and synaptic terminals (lower arrowhead) correlate with proinsulin gene dosage and with proinsulinemia (1.3 pM in the shown hemizygous; 2.4 pM in the shown homozygous). INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 25 μm.
Figure 8.
 
Reduction of lipid oxidation in hPi/rd10 mice. Retinal sections from rd10 (A), hPi/rd10 (B), and wt (C) mice at P32 were stained with DAPI (blue) to reveal retinal cytoarchitecture and with anti–acrolein antibody (green) to visualize oxidative damage. The increased lipid oxidation observed in rd10 retina is markedly reduced in hPi/rd10 animals. INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 25 μm.
Figure 8.
 
Reduction of lipid oxidation in hPi/rd10 mice. Retinal sections from rd10 (A), hPi/rd10 (B), and wt (C) mice at P32 were stained with DAPI (blue) to reveal retinal cytoarchitecture and with anti–acrolein antibody (green) to visualize oxidative damage. The increased lipid oxidation observed in rd10 retina is markedly reduced in hPi/rd10 animals. INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 25 μm.
 
Supplementary Materials
The authors thank the Retina Madrid Association for encouraging and supporting this work; and Patrick S. Fitze, Lluis Montoliu, Nuria Forns, Ana Robles, Mayte Seisdedos, Silvia Hernández, and the Centro de Investigaciones Biológicas animal facilities staff for conceptual and technical support. 
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Supplementary Figure S1
Supplementary Figure S2
Copyright 2008 The Association for Research in Vision and Ophthalmology, Inc.
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