August 2015
Volume 56, Issue 9
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New Developments in Vision Research  |   August 2015
Reactivation of the PI3K/Akt Signaling Pathway by the Bisperoxovanadium Compound bpV(pic) Attenuates Photoreceptor Apoptosis in Experimental Retinal Detachment
Author Affiliations & Notes
  • Dan Mao
    Department of Ophthalmology Eye Research Institute of Shanghai Jiao Tong University, Shanghai, China
  • Xiaodong Sun
    Department of Ophthalmology Eye Research Institute of Shanghai Jiao Tong University, Shanghai, China
  • Correspondence: Xiaodong Sun, Department of Ophthalmology, Shanghai First People's Hospital, School of Medicine, Shanghai JiaoTong University, 100 Haining Road, Shanghai 200080, P.R. China; xdsun@sjtu.edu.cn
Investigative Ophthalmology & Visual Science August 2015, Vol.56, 5519-5532. doi:10.1167/iovs.15-16757
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      Dan Mao, Xiaodong Sun; Reactivation of the PI3K/Akt Signaling Pathway by the Bisperoxovanadium Compound bpV(pic) Attenuates Photoreceptor Apoptosis in Experimental Retinal Detachment. Invest. Ophthalmol. Vis. Sci. 2015;56(9):5519-5532. doi: 10.1167/iovs.15-16757.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Phosphatase and tensin homology deleted on chromosome 10 (PTEN) is crucial in neuronal apoptosis. This study evaluated the role of PTEN in photoreceptor cell apoptosis caused by retinal detachment (RD).

Methods: A rat model of RD was established, and PTEN expression changes were detected at different time points by Western blotting and immunofluorescence. Some of the rats were given subretinal injections of bisperoxovanadium compound (bpV[pic]) after RD. We documented the expression and distribution of phospho-Akt (p-Akt) and B-cell lymphoma 2 (Bcl-2) in the retina by Western blot analysis and immunofluorescence. Levels of phosph-phosphoinositide–dependent kinase 1 (p-PDK1), phospho–Bcl-2 death promotor (p-BAD), cytosolic cytochrome c (Cyt c), and cleaved Caspase-3 were detected by Western blotting. We measured phosphatidylinositol 3,4,5-triphosphate (PIP3) by ELISA. Apoptosis of photoreceptors was detected using the TUNEL assay. The thickness of the outer nuclear layer (ONL) also was recorded.

Results: The expression of PTEN gradually increased after RD, peaking at 3 days and then decreasing to normal by 7 days after RD. Subretinal injection of bpV(pic) effectively reduced the apoptosis of photoreceptors and preserved the retinal thickness of the ONL after RD. Compared to vehicle-treated RD groups, levels of p-Akt and p-PDK1 were significantly upregulated in bpV-treated RD groups. In addition, bpV treatment increased the levels of p-BAD and Bcl-2, and decreased the expression levels of cytosolic Cyt c and cleaved caspase-3 after RD.

Conclusions: Phosphatase and tensin homology deleted on chromosome 10 (PTEN) participates in the apoptosis of photoreceptors after RD. Blocking PTEN may reactivate the PI3K/Akt pathway and attenuate photoreceptor apoptosis by suppressing the mitochondrial pathway.

Retinal detachment (RD) is a serious disease that may cause blindness. In recent years, with the development of vitreous microsurgery technology, the retinal anatomical reattachment rate has been increasing, but the final visual outcome remains disappointing in a small subset of cases. In patients with macular detachment, after retinal reattachment surgery, 34% of the eyes had a long-term visual outcome of vision poorer than 6/18.1 After surgery to treat macular detachment, although the anatomical reattachment rate reaches 96.1%, postoperative visual acuity of at least 20/40 is only achieved in 28% of cases.2 
Studies have shown that outer segment degeneration, incomplete regeneration, and apoptosis of photoreceptor cells after RD are the main reasons for the poor postoperative visual function after anatomically successful reattachment surgery. Degeneration mainly occurred in outer segments of photoreceptors, because after RD, the five outer layers of the retina lose the oxygen and nutrition supply provided by choroid circulation.3 At the early stage after reattachment, outer segments regenerate at an average speed of 2.5 μm/days.4 After detachment, many rod terminals retract from the outer plexiform layer (OPL) and are found throughout the outer nuclear layer (ONL). In addition, cone terminals, adopting a flattened appearance and losing their deep synaptic invaginations, change their synaptic connections with the second-order neurons, thereby causing visual information transduction disorders.5 Photoreceptor degeneration after RD occurs mainly through apoptosis. Apoptosis occurs early after RD and peaks at 1 to 3 days; as long as the retina is not reattached, apoptosis of photoreceptors will persist.68 
In the developing retina, Phosphatase and tensin homology deleted on chromosome 10 (PTEN) is localized preferentially to ganglion, amacrine, and horizontal cells.9,10 In addition, PTEN is crucial in neuronal apoptosis. It is a dual-specificity phosphatase demonstrating phosphatase activity against protein and lipid substrates,11 and its lipid phosphatase activity can antagonize the activity of the PI3K/Akt signaling pathway by dephosphorylating phosphatidylinositol 3,4,5-triphosphate (PIP3) and accelerating cell apoptosis.12 For example, developing neurons are programmed to undergo apoptosis unless they are protected by growth factors that stimulate prosurvival pathways, among which the PI3K/Akt pathway has a major role.13 Also known as protein kinase B (PKB), Akt is a serine/threonine kinase that is recruited to PIP3 in the membrane, where it is activated by phosphoinositide-dependent kinase 1 (PDK1) and PDK2 at Phospho-Akt (Thr308) and serine 473 (Ser473).14 Phosphorylated Akt (p-Akt) has multiple biological roles by activating or inactivating its downstream target molecules, including Bcl-2–associated death promotor (BAD),1517 caspase-9,18 forkhead transcription factor,19 and NF-κB.20 Inactivation of PTEN significantly promotes the survival of retinal neurons after optic nerve injury.21,22 Deletion of PTEN enhances the regenerative ability of adult corticospinal neurons.23 Deletion of PTEN in the developing mouse brain leads to the overproliferation of progenitors and enhanced neuronal survival.2426 Neural progenitor cells that are PTEN-null show enhanced self-renewal capacity, reduced growth factor dependency, shortened cell cycles and accelerated G0–G1 entry.27 In the adult nervous system, deletion of PTEN enhances neurogenesis through the perpetual self-renewal of endogenous stem cells.28 
In this study, we used the PTEN inhibitor bisperoxovanadium compound (bpV[pic]) to regulate the activity of PTEN to assess whether it contributes to photoreceptor apoptosis after RD in rat retina. We provide evidence that blocking PTEN after RD may reactivate the PI3K/Akt pathway and attenuate photoreceptor apoptosis by suppressing the intrinsic apoptotic pathway. 
Materials and Methods
Animals
All animal experiments adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and the protocols were approved by the Shanghai Jiao Tong University. Adult Sprague-Dawley (SD) rats (200–250 g) were fed standard laboratory chow and allowed free access to water in an air-conditioned room with a 12-hour light/12-hour dark cycle. 
Induction of RD
Rats were anesthetized with 1% pentobarbital sodium (40 mg/kg). The pupils were dilated, and a sclerotomy was created approximately 2 mm posterior to the limbus with a 30-gauge needle. A retinotomy was created in the peripheral retina with the tip of the subretinal injector, and Healon (1.0% sodium hyaluronate; Abbott Medical Optics, Inc., Abbott Park, IL, USA) was slowly injected into the subretinal space (SRS), causing detachment of 90% of the retina. Immediately after RD, bpV(pic) (40 ng/kg, 400 ng/kg, or 4 μg/kg; Sigma-Aldrich Corp., St. Louis, MO, USA) or PBS (vehicle) 4 to 5 μL was injected subretinally. First, bpV(pic) was dissolved in PBS to 200 μg/mL and then diluted in PBS to 20 μg/mL and 2 μg/mL. 
TUNEL Staining
Cryosections (10 μm thick) were incubated in methanol for 15 minutes at room temperature, washed in PBS for 5 minutes, and incubated in a bromodeoxy-UTP/terminal deoxynucleotidyltransferase mixture (Roche Molecular Biochemicals, Indianapolis, IN, USA) at 37°C for 1 hour in a humidified chamber followed by three rinses in PBS. The sections then were incubated with peroxidase converter (Roche Molecular Biochemicals) for 30 minutes and then counterstained with 4′,6-diamidino-2-phenylendole (DAPI), a nuclear stain. TUNEL staining was performed using an in situ cell death-detection kit (Roche Molecular Biochemicals). Cells in the ONL costained by TUNEL and DAPI were counted as apoptotic cells. An apoptotic ratio ([number of TUNEL-positive cells/total ONL area]/[number of DAPI-positive cells/total ONL area]) was obtained for each eye. 
Evaluation of the ONL Thickness Ratio
Retinal sections were deparaffinized, and stained with hematoxylin and eosin (H&E). The ratio of ONL thickness to the total retinal thickness was determined using ImageJ software (available in the public domain at http://imagej.nih.gov/ij/) and standardized to that of the attached retina. Five sections were randomly selected in each eye, and the central area of the detached retina and the midperipheral region of the attached retina were photographed. Then, the ONL thickness ratio was measured at 10 points in each section by blinded observers. The data are expressed as the normalized ONL thickness ratio: ([ONL/neuroretinal thickness in detached retina]/[ONL/neuroretinal thickness in attached retina]). 
Immunohistochemistry
Retinal sections (10 μm) through the optic nerve head were selected for each eye. Deparaffinized retinal sections were immersed in proteinase K solution for antigen retrieval and blocked in 10% goat serum for 1 hour. The sections then were incubated with the following primary antibodies at 4°C overnight: PTEN (1:100, ab154812; Abcam, Cambridge, UK), p-Akt (1:200, 4060S; Cell Signaling Technology, Beverly, MA, USA) and B-cell lymphoma 2 (Bcl-2, 1:100, ab7973; Abcam). After thorough washing with PBS, the sections were exposed to their corresponding secondary Cy3 antibodies (1:300) for 1 hour at room temperature, after which they were counterstained with DAPI, washed again, coverslipped, and examined under a fluorescence microscope. 
Isolation of Total Proteins
Experimental and control retinas from treatment and vehicle groups were separated from vitreous and RPE, and were subsequently homogenized in 150 mL radioimmunoprecipitation assay (RIPA) lysis buffer. In experimental eyes, attached parts of the retinas were manually dissected and excluded from analysis. Samples were centrifuged at 12,000g at 4°C for 25 minutes, and the supernatant was collected as the total protein sample for Western blot analysis. 
Isolation of Cytosolic and Mitochondrial Fractions
The isolation of cytosolic and mitochondrial fractions was performed using a cytosol/mitochondria isolation kit (Applygen Technologies, Beijing, China) according to the manufacturer's instructions. In brief, fresh tissues were removed quickly, chopped into small pieces, and placed in ice-cold mitochondrial isolation buffer. After homogenization, the homogenate was centrifuged at 800g for 5 minutes at 4°C. Next, the supernatant was collected and further centrifuged at 800g for 10 minutes at 4°C. The pellet was gently resuspended in isolation buffer and centrifuged at 12,000g for 10 minutes at 4°C. The supernatant from this step was considered the cytosolic fraction. The cytochrome c level in the cytosolic fraction was analyzed by Western blot. 
Western Blot Analysis
Protein samples were separated by 15% SDS-PAGE and transferred to polyvinylidine fluoride (PVDF) membranes. Blocking was performed with 5% nonfat milk in TBST. The membranes were incubated with primary antibodies against PTEN (1:5000, ab154812; Abcam), p-Akt (1:2000, 4060S; Cell Signaling Technology), Akt (1:1000; Cell Signaling Technology, 4685), p-PDK1 (1:500, ab32800, Abcam), p-BAD (1:500, ab28824; Abcam), Bcl-2 (1:100, ab7973; Abcam), cytochrome c (1 μg/mL, ab90529, Abcam), and caspase-3 (1:200, ab2171; Abcam). β-Actin (1:50000; Sigma-Aldrich Corp.) was used as a loading control. The membranes then were washed three times, for 15 minutes each, with PBS containing 0.5% Tween 20 (PBS-T) and incubated with secondary antibody (1:8000, Alexa Fluor 700 goat anti-mouse IgG [H+L] or Alexa Fluor 800 goat anti-rabbit IgG [H+L]; Invitrogen, Carlsbad, CA, USA) in PBS at room temperature for 1 hour. Densitometry was performed with ImageJ (1.43 μ, National Institutes of Health [NIH], Bethesda, MD, USA), and protein levels were normalized against actin levels. 
ELISA Test
Retinal lipids were extracted as previously described.29,30 Snap-frozen tissue was powdered in liquid nitrogen and dissolved in 0.5 M trichloroacetic acid (TCA), and the precipitate was washed twice in 5% TCA, 1 mM EDTA. Neutral lipids were extracted twice in MeOH:CHCl3 (2:1), and acidic lipids were extracted in MeOH:CHCl3:12 M HCl (80:40:1). Phase splitting was initiated by the addition of CHCl3, 0.1 M HCl (1:2), and the organic phase was dried in a vacuum centrifuge. Dried lipids were diluted in 50 mM HEPES, 150 mM NaCl, 1.5% Na cholate (pH 7.4), sonicated, and stored overnight at 4°C. Then, PIP3 was measured by ELISA according to the manufacturer's instructions (PIP3 Mass ELISA Kit; Echelon Biosciences Salt Lake City, UT, USA). The pellet was redissolved in 6 M guanidine HCl (50 mM HEPES, pH 7.5) for determining the protein concentration. 
Statistical Analysis
Data are presented as the mean ± SEM. Statistical analysis was performed with GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). A 1-way ANOVA with Tukey's test was used for comparisons of multiple groups, and Student's t-test was used for comparisons between two groups. Differences were considered statistically significant at P < 0.05. 
Results
PTEN Is Involved in the Regulation of Photoreceptor Apoptosis After RD
To investigate the potential role of PTEN after RD, we used Western blotting and immunocytochemistry to assess the expression of PTEN in the retina at 1, 2, 3, and 7 days after RD. Western blot analysis confirmed that compared to normal control retina, PTEN expression increased after RD, peaked at 3 days (4.2-fold higher compared to normal control), and then declined at 7 days (1.4-fold higher compared to normal control; Figs. 1A, 1B). Immunofluorescence showed PTEN in the inner nuclear layer (INL) and ganglion cell layer (GCL) in normal control retina (Fig. 2A). We detected PTEN in the ONL, INL, and GCL at 1 day after RD (Fig. 2B). Increased staining of the ONL, INL, and GCL was detected at 2 and 3 days (Figs. 2C, 2D). Immunoreactivity of PTEN was mainly localized to the INL and GCL at 7 days (Fig. 2E). We also assessed neuronal apoptosis after RD at different time points, which revealed that TUNEL-positive cells were not detectable in normal control retina and were present at 1 day (8.29%) after RD, peaked at 2 (21.08%) and 3 (23.14%) days, and then decreased by 7 days (4.62%; Figs. 3A–E). These data showed that PTEN may be involved in the regulation of photoreceptor apoptosis after RD. 
Figure 1
 
Expression of PTEN in rat retina after RD. Western blot and quantitative analysis of PTEN in different groups. Differences in loading were normalized using the levels of β-actin. The increase in PTEN protein expression peaked at 3 days after RD and then decreased at 7 days. The data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, n = 6).
Figure 1
 
Expression of PTEN in rat retina after RD. Western blot and quantitative analysis of PTEN in different groups. Differences in loading were normalized using the levels of β-actin. The increase in PTEN protein expression peaked at 3 days after RD and then decreased at 7 days. The data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, n = 6).
Figure 2
 
Note that PTEN was highly expressed in the ONL, INL, and GCL after RD. (AE) Immunostaining of PTEN. (A) Normal control, (B) 1 day after RD, (C) 2 days after RD, (D) 3 days after RD, (E) 7 days after RD. Immunoreactivity of PTEN was detected in the INL and GCL in normal control retina. After RD, PTEN immunoreactivity in the ONL, INL, and GCL increased. Scale bar: 50 μm, n = 6.
Figure 2
 
Note that PTEN was highly expressed in the ONL, INL, and GCL after RD. (AE) Immunostaining of PTEN. (A) Normal control, (B) 1 day after RD, (C) 2 days after RD, (D) 3 days after RD, (E) 7 days after RD. Immunoreactivity of PTEN was detected in the INL and GCL in normal control retina. After RD, PTEN immunoreactivity in the ONL, INL, and GCL increased. Scale bar: 50 μm, n = 6.
Figure 3
 
Changes in photoreceptor apoptosis in rat retina after RD. (AE) TUNEL (green) and DAPI (blue) staining of sections of attached or detached retina. (A) Normal control, (B) 1 day after RD, (C) 2 days after RD, (D) 3 days after RD, (E) 7 days after RD. TUNEL-positive cells were present at 1 day after RD, peaked at 2 and 3 days, and then decreased by 7 days. Scale bar: 50 μm, n = 6. (F) Quantification of TUNEL-positive photoreceptors. The data are presented as the mean ± SEM (*P < 0.05, ***P < 0.001, n = 6).
Figure 3
 
Changes in photoreceptor apoptosis in rat retina after RD. (AE) TUNEL (green) and DAPI (blue) staining of sections of attached or detached retina. (A) Normal control, (B) 1 day after RD, (C) 2 days after RD, (D) 3 days after RD, (E) 7 days after RD. TUNEL-positive cells were present at 1 day after RD, peaked at 2 and 3 days, and then decreased by 7 days. Scale bar: 50 μm, n = 6. (F) Quantification of TUNEL-positive photoreceptors. The data are presented as the mean ± SEM (*P < 0.05, ***P < 0.001, n = 6).
PTEN Inhibition Attenuates Photoreceptor Apoptosis After RD
To determine whether the PTEN-inhibition effect of bpV(pic) resulted in neuroprotection against RD-induced photoreceptor apoptosis, we investigated the ability of subretinal administration of bpV(pic) to preserve ONL thickness after RD. Compared to the normal control group (Fig. 4A), photoreceptors in the untreated and vehicle-treated RD groups (Figs. 4B, 4C) were disorderly and loosely arranged, the inner segments were partly missing, and the outer segments were mostly detached. The outer segments of the bpV-treated (40 ng/kg) RD group were partly preserved (Fig. 4D). Photoreceptors were arranged closely in the bpV-treated (400 ng/kg and 4 μg/kg) RD groups, and their outer segments were mostly preserved (Figs. 4E, 4F). The standardized ratio of ONL to total thickness in detached versus attached areas was compared in the vehicle and bpV-treated groups. A ratio of 1 represented no loss of ONL thickness, and ratios less than 1 represented loss of ONL thickness. After 3 and 7 days of detachment, the ONL thickness ratio in the vehicle-treated group decreased to 64.3% and 53.8%, respectively, whereas bpV (4 μg/kg) significantly prevented the reduction in the ONL thickness ratio (88.4% and 79.7%, respectively; Fig. 4G). Bisperoxovanadium compound increased the numbers of cells in ONL 3 days (6013.8 ± 677.5 vs. 7380.4 ± 921.9 cells per mm2 in bpV-treated group) and 7 days (3613 ± 838.6 vs. 5569 ± 690 cells per mm2 in bpV-treated group) after RD (Fig. 4H). We also assessed photoreceptor apoptosis after RD by TUNEL staining. There were no TUNEL-positive cells in vehicle-treated control retina (Fig. 5A). However, vehicle-treated RD retina showed TUNEL-positive cells (24.8%) 3 days after RD (Figs. 5B, 5D). In contrast, the proportion of TUNEL-positive cells decreased in bpV-treated RD retina (3.5%; Figs. 5C, 5D). These data demonstrated that inhibiting PTEN with bpV(pic) has a significant neuroprotective effect on RD-induced photoreceptor apoptosis. 
Figure 4
 
Morphological changes and analysis of ONL thickness in the retina after RD. (AF) Representative histopathological images of retina sections. (A) Normal control, (B) untreated (7 days after RD), (C) RD-vehicle (7 days after RD), (D) RD-bpV 40 ng, (E) RD-bpV 400 ng, (F) RD-bpV 4 μg. Scale bar: 50 μm, n = 6. (G) Quantitative data showing the protective effect of bpV (4 μg/kg) on preserving ONL thickness at 3 and 7 days after RD. Data are presented as the mean ± SEM (**P < 0.01, ***P < 0.001, n = 6). (H) Quantification of cells in ONL. The data are presented as the mean ± SEM (**P < 0.01, ***P < 0.001, n = 6).
Figure 4
 
Morphological changes and analysis of ONL thickness in the retina after RD. (AF) Representative histopathological images of retina sections. (A) Normal control, (B) untreated (7 days after RD), (C) RD-vehicle (7 days after RD), (D) RD-bpV 40 ng, (E) RD-bpV 400 ng, (F) RD-bpV 4 μg. Scale bar: 50 μm, n = 6. (G) Quantitative data showing the protective effect of bpV (4 μg/kg) on preserving ONL thickness at 3 and 7 days after RD. Data are presented as the mean ± SEM (**P < 0.01, ***P < 0.001, n = 6). (H) Quantification of cells in ONL. The data are presented as the mean ± SEM (**P < 0.01, ***P < 0.001, n = 6).
Figure 5
 
Apoptosis was significantly reduced by bpV(pic) after RD. (A-D) TUNEL (green) and DAPI (blue) staining of retina sections. (A) Control-vehicle, (B) RD-vehicle (3 days after RD), (C) RD-bpV 4 μg. There were no TUNEL-positive cells in the vehicle-treated control retina. However, the vehicle-treated RD retina showed TUNEL-positive cells 3 days after RD. In contrast, there were fewer TUNEL-positive cells in the bpV-treated RD retina. Scale bar: 50 μm, n = 6. (D) Quantification of TUNEL-positive cells in ONL. The data are presented as the mean ± SEM (*P < 0.05, ***P < 0.001, n = 6).
Figure 5
 
Apoptosis was significantly reduced by bpV(pic) after RD. (A-D) TUNEL (green) and DAPI (blue) staining of retina sections. (A) Control-vehicle, (B) RD-vehicle (3 days after RD), (C) RD-bpV 4 μg. There were no TUNEL-positive cells in the vehicle-treated control retina. However, the vehicle-treated RD retina showed TUNEL-positive cells 3 days after RD. In contrast, there were fewer TUNEL-positive cells in the bpV-treated RD retina. Scale bar: 50 μm, n = 6. (D) Quantification of TUNEL-positive cells in ONL. The data are presented as the mean ± SEM (*P < 0.05, ***P < 0.001, n = 6).
PTEN Inhibition Leads to Reactivation of Decreased PI3K/Akt Activity in Rat Retina After RD
To investigate whether PTEN has a role in RD-induced photoreceptor apoptosis by antagonizing the activity of the PI3K/Akt signaling pathway, antibodies recognizing p-Akt (Ser473) and p-PDK1 (Ser241) were used to monitor Akt and PDK1 phosphorylation 3 days after RD via Western blot analysis. In comparison with the vehicle-treated control retina, we observed decreased expression of p-PDK1 and p-Akt in the vehicle-treated RD retina. Intriguingly, bpV treatment significantly increased the levels of p-PDK1 and p-Akt (by 8.7-fold and 5.9-fold, respectively, compared to the vehicle-treated RD group; Figs. 6B, 6C). Consistent with the reduced Akt phosphorylation in the retina, PIP3 levels were significantly decreased after RD compared to the vehicle-treated control retina (Fig. 6A). We next determined the cellular distribution of p-Akt protein expression in the RD retina. In the vehicle-treated control retina, we observed that p-Akt immunostaining was localized in the ONL as well as in the OS and inner segment (IS; Fig. 7A). The vehicle-treated RD retina showed less p-Akt immunoreactivity in the ONL than did the vehicle-treated control retina (Fig. 7B). Surprisingly, we also found that p-Akt immunoreactivity was significantly increased in the ONL in the bpV-treated RD retina (Fig. 7C). These findings demonstrated that the decreased Akt phosphorylation during photoreceptor apoptosis after RD is a result of reduced PI3K activity and that blocking PTEN may reactivate the PI3K/Akt pathway. 
Figure 6
 
Modulation of PIP3 levels and p-PDK1, p-Akt and Akt protein expression in the rat retina by bpV(pic) after RD. (A) Levels of PIP3 were measured in retinal lipid extracts prepared 3 days after RD. Bar graph shows PIP3 levels expressed per mg protein as the mean ± SEM (*P < 0.05, ***P < 0.001, n = 6). (B, C) Western blot and quantitative analysis of p-PDK1 (B), p-Akt, and Akt (C) in different groups. Differences in loading were normalized using the levels of β-Actin. The data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n = 6).
Figure 6
 
Modulation of PIP3 levels and p-PDK1, p-Akt and Akt protein expression in the rat retina by bpV(pic) after RD. (A) Levels of PIP3 were measured in retinal lipid extracts prepared 3 days after RD. Bar graph shows PIP3 levels expressed per mg protein as the mean ± SEM (*P < 0.05, ***P < 0.001, n = 6). (B, C) Western blot and quantitative analysis of p-PDK1 (B), p-Akt, and Akt (C) in different groups. Differences in loading were normalized using the levels of β-Actin. The data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n = 6).
Figure 7
 
Note that bpV(pic) increased Akt phosphorylation after RD. (AC) Phospho-Akt immunostaining. (A) Control-vehicle, (B) RD-vehicle (3 days after RD), (C) RD-bpV 4 μg. In the vehicle-treated control retina, p-Akt immunostaining was observed in the ONL as well as in the OS and IS. Immunoreactivity for p-Akt in the ONL was reduced in the vehicle-treated RD retina compared to the vehicle-treated control retina. Phospho-Akt immunoreactivity was significantly increased in the ONL in the bpV-treated RD retina. Scale bar: 50 μm, n = 6.
Figure 7
 
Note that bpV(pic) increased Akt phosphorylation after RD. (AC) Phospho-Akt immunostaining. (A) Control-vehicle, (B) RD-vehicle (3 days after RD), (C) RD-bpV 4 μg. In the vehicle-treated control retina, p-Akt immunostaining was observed in the ONL as well as in the OS and IS. Immunoreactivity for p-Akt in the ONL was reduced in the vehicle-treated RD retina compared to the vehicle-treated control retina. Phospho-Akt immunoreactivity was significantly increased in the ONL in the bpV-treated RD retina. Scale bar: 50 μm, n = 6.
The Neuroprotective Effects of PTEN Inhibition Are Associated With Increases in p-BAD and Bcl-2 Expression by Reactivating the PI3K/Akt Pathway
To address whether increased Akt phosphorylation is associated with increased Akt activity, we performed Western blotting using antibodies for p-BAD (Ser136), Bcl-2, cytosolic cytochrome c (Cyt c) and cleaved caspase-3. We found that p-BAD protein expression increased in the vehicle-treated ischemic retina compared to the vehicle-treated control retina (P < 0.05). In contrast, bpV treatment produced a greater increase of p-BAD protein expression (by 2.1-fold compared to vehicle-treated RD group; Fig. 8A). Compared to the vehicle-treated control retina, the vehicle-treated RD retina showed decreased Bcl-2 expression (P < 0.05). In addition, Bcl-2 protein expression was significantly increased (by 7.4-fold compared to vehicle-treated RD group; Fig. 8B) in the bpV-treated RD retina. We also observed that bpV treatment significantly decreased the RD-induced increases in cytosolic Cyt c and cleaved caspase-3 protein (∼15% and ∼12% compared to vehicle-treated group, respectively; Figs. 8C, 8D). We next assessed the cellular distribution of Bcl-2 protein expression in the RD retina. Immunoreactivity of Bcl-2 was detected in the INL, GCL, and ONL in the vehicle-treated control retina (Fig. 9A). However, only slight immunoreactivity was observed in the INL, GCL, and ONL in the vehicle-treated RD retina (Fig. 9B). Interestingly, bpV treatment showed significantly increased Bcl-2 immunoreactivity in the INL, GCL, and ONL in the RD retina (Fig. 9C). 
Figure 8
 
Modulation of p-BAD, Bcl-2, cytosolic Cyt c, and cleaved caspase-3 protein expression in the rat retina by bpV(pic) after RD. Western blot and quantitative analysis of p-BAD (A), Bcl-2 (B), cytosolic Cyt c (C), and cleaved caspase-3 (D) in different groups. Differences in loading were normalized using the levels of β-Actin. The data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n = 6).
Figure 8
 
Modulation of p-BAD, Bcl-2, cytosolic Cyt c, and cleaved caspase-3 protein expression in the rat retina by bpV(pic) after RD. Western blot and quantitative analysis of p-BAD (A), Bcl-2 (B), cytosolic Cyt c (C), and cleaved caspase-3 (D) in different groups. Differences in loading were normalized using the levels of β-Actin. The data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n = 6).
Figure 9
 
Note that bpV(pic) induced upregulation of Bcl-2 protein expression after RD. (AC) Immunostaining of Bcl-2. (A) Control-vehicle, (B) RD-Vehicle, (C) RD-bpV 4 μg. In vehicle-treated control retina, Bcl-2 immunoreactivity was detected in the INL, GCL, and ONL. Only slight immunoreactivity was observed in the INL, GCL, and ONL in the vehicle-treated RD retina. bpV treatment showed a significant increase in Bcl-2 immunoreactivity in the INL, GCL, and ONL (arrows) in the RD retina. Scale bar: 50 μm, n = 6.
Figure 9
 
Note that bpV(pic) induced upregulation of Bcl-2 protein expression after RD. (AC) Immunostaining of Bcl-2. (A) Control-vehicle, (B) RD-Vehicle, (C) RD-bpV 4 μg. In vehicle-treated control retina, Bcl-2 immunoreactivity was detected in the INL, GCL, and ONL. Only slight immunoreactivity was observed in the INL, GCL, and ONL in the vehicle-treated RD retina. bpV treatment showed a significant increase in Bcl-2 immunoreactivity in the INL, GCL, and ONL (arrows) in the RD retina. Scale bar: 50 μm, n = 6.
Discussion
Neuronal apoptosis is a “final common pathway” for a variety of neurologic and retinal diseases, including RD. Inhibition of PTEN has a significant neuroprotective effect in many types of central nervous system (CNS) damage by activating the PI3K/Akt pathway.3135 In this model of RD, we evaluated whether PTEN might contribute to the photoreceptor apoptosis after RD. 
This study provides evidence supporting the involvement of the PTEN/PI3K/Akt pathway in cellular apoptosis after RD. We found that PTEN expression was low in the normal retina of rats; then, during the induction of photoreceptor apoptosis, PTEN expression increased in detached retina, peaking at 3 days, when photoreceptor apoptosis also peaked. PTEN was located in the INL and GCL in normal control retina. After RD, PTEN was highly expressed in the ONL, where most of the apoptosis occurred. These findings demonstrated that PTEN might be involved in mediating photoreceptor apoptosis after RD. 
Pretreatment of rats with bpV(pic), which is a potential inhibitor of PTEN activity,36 decreased photoreceptor apoptosis and increased the numbers of DAPI-positive photoreceptors after RD. This confirm that inhibiting PTEN with bpV(pic) has a significant neuroprotective effect on RD-induced photoreceptor apoptosis. To investigate the specific effects of PTEN inhibition on RD, retinal morphology should be part of the phenotypic analysis. In our study, the ONL was significantly thicker in bpV-treated groups than in the vehicle-treated group after RD. We also showed that there was more preservation of the outer segments when bpV was administered. In our current study, the transient inhibition of PTEN by bpV(pic) leads to neuroprotection as demonstrated by retinal morphology and TUNEL assays in photoreceptor cells. Therefore, the transient inhibition of PTEN immediately after RD is a plausible target for minimizing retinal damage. This approach may offer a promising tool for the pharmacological manipulation of PTEN to study the significance of PTEN in RD-induced photoreceptor apoptosis. 
The in vivo effect of PTEN inhibitor bpV(pic) on photoreceptors was detectable at a dose as low as 40 ng/kg. Treatment with bpV(pic) at 400 ng/kg and 4 μg/kg produced a greater effect than did 40 ng/kg. Thus, PTEN inhibition appears to depend on the dose of bpV(pic), and low-dose bpV(pic) results only in an acute neuroprotective effect on RD-induced photoreceptor apoptosis. This effect is consistent with previous findings that bpV exerts acute neuroprotection when it is administered before or immediately after hypoxia-ischaemia.37,38 However, it remains unclear whether delayed treatment with bpV(pic) also has a neuroprotective effect after RD. At low concentrations, bpV is a relatively specific inhibitor of PTEN.36 It is possible that at higher doses, the activity of PI3K/Akt signaling might be dramatically increased to an uncontrolled high level. The functional consequence of this sudden change could include the rapid modification of the energy metabolism of cells, resulting in increased consumption of oxygen and glucose. The retina normally responds to this change by altering the substrate metabolism to increase energy efficiency. Photoreceptors under ischemic conditions may not function very well; thus, preventing this adaptive response and leading to further injury. This possibility highlights the need for developing specific PTEN inhibitors to achieve safe and reversible manipulation of PTEN function. Furthermore, the highest effective dose was not identified in our study and, therefore, requires further investigation. 
Protein kinase B (Akt) is a downstream factor of PTEN, which has a critical role in controlling the balance between survival and apoptosis by regulating the phosphorylation of its downstream components.39 The major substrate of PTEN is PIP3, a second messenger molecule produced following PI3K activation. Phosphoinositide-dependent kinase 1 (PDK1) was identified by its ability to phosphorylate and activate Akt. Here, consistent with our p-Akt data, we observed significant reductions in the levels of PIP3 and p-PDK1 at 3 days after RD. This suggests that the observed loss of Akt phosphorylation is due to a reduction in PI3K signaling. We also found that bpV(pic) significantly increased the levels of PIP3, p-PDK1, and p-Akt after RD, suggesting that PTEN inhibition reactivates the PI3K/Akt pathway. In addition, some researchers report that Akt transfers from the cytoplasm to the nucleus after its activation.40,41 We found that inhibition of PTEN was accompanied by an increase in p-Akt translocation from the cytoplasm to the nucleus. This behavior may occur because p-Akt's transcription factor substrates, such as CREB and Forkhead, are located in the nucleus. Inactive Akt travels from the plasma membrane to the nucleus after its activation, and the substrates of its serine/threonine phosphorylation have roles in antiapoptosis and cell protection.4244 Our finding that p-Akt-positive cells were confined to the ONL in the normal control and bpV-treated groups suggests that the PI3K/Akt pathway may be involved in the regulation of apoptosis only in photoreceptors and not in other retinal cells after RD. 
Retinal detachment–induced ischemic hypoxic injury may cause functional changes to mitochondria, thus activating the intrinsic mitochondrial pathway. The dysfunction and loss of mitochondria are likely to profoundly affect the ability of the highly active photoreceptors to regenerate, despite surgical reattachment of the retina.45 Mitochondria release many proapoptotic proteins and have a central role in regulating apoptosis.4651 In RD-induced photoreceptor apoptosis, we detected changes in the expression of p-BAD, Bcl-2, or cleaved caspase-3. Similarly, increased cytoplasmic Cyt c also was detected after RD. Treatment with bpV(pic) induced overexpression of p-BAD and Bcl-2, and significantly reduced the levels of cytosolic Cyt c and cleaved caspase-3. B-cell lymphoma-2–associated death promotor (BAD) forms heterodimers with Bcl-2, thereby inactivating Bcl-2. Phosphorylation of BAD eliminates this dimerization, thereby activating Bcl-2.52 The higher levels of p-BAD after RD may represent an endogenous repair mechanism against RD-induced retinal injury. Expression of antiapoptotic genes, such as Bcl-2 has been found to be downregulated after induction of apoptosis.5356 In contrast, expression of apoptosis-promoting genes, such as the cysteine proteases (caspases), are upregulated following pro-apoptotic stimuli.5759 Overexpression of Bcl-2 has protective effects against retinal apoptosis.6066 A major function of Bcl-2 is the regulation of Cyt c release from mitochondria,6769 which also may induce the apoptotic execution cascade by activating caspase-3,70,71 a central executioner in many apoptotic systems. An important mechanism of the cytoprotective effect of p-Akt is thought to come from its inhibition of the proapoptotic BH3-only protein BAD by phosphorylating BAD at Ser-136, leading to the inhibition of caspase-3 activation.7274 Additionally, p-Akt can induce the expression of Bcl-2, limiting the depolarization of the mitochondrial membrane and the release of Cyt c, which also induces antiapoptotic effects.75,76 Together, our results indicate that blocking PTEN may protect photoreceptor cells against the mitochondria-related apoptotic pathway after RD by increasing the expression of Bcl-2 and the phosphorylation of BAD. 
Taken together, these data clearly demonstrated that PTEN inhibition has an antiapoptotic role by reactivating the PI3K/Akt signaling pathway after RD, resulting in suppression of the activation of the mitochondrial apoptotic pathway. Thus, reactivation of the PI3K/Akt pathway with PTEN inhibitors may open up a new therapeutic avenue for photoreceptor protection after RD. 
Acknowledgments
Supported by the Basic Research Program of China “973 Program” (2011CB707506), the National Natural Science Foundation of China (81170861 and 81271030), a Shanghai Key Basic Research Grant (11JC141601), and a Shanghai Key Medical Research Grant (1341195400). The authors alone are responsible for the content and writing of this paper. 
Disclosure: D. Mao, None; X. Sun, None 
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Figure 1
 
Expression of PTEN in rat retina after RD. Western blot and quantitative analysis of PTEN in different groups. Differences in loading were normalized using the levels of β-actin. The increase in PTEN protein expression peaked at 3 days after RD and then decreased at 7 days. The data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, n = 6).
Figure 1
 
Expression of PTEN in rat retina after RD. Western blot and quantitative analysis of PTEN in different groups. Differences in loading were normalized using the levels of β-actin. The increase in PTEN protein expression peaked at 3 days after RD and then decreased at 7 days. The data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, n = 6).
Figure 2
 
Note that PTEN was highly expressed in the ONL, INL, and GCL after RD. (AE) Immunostaining of PTEN. (A) Normal control, (B) 1 day after RD, (C) 2 days after RD, (D) 3 days after RD, (E) 7 days after RD. Immunoreactivity of PTEN was detected in the INL and GCL in normal control retina. After RD, PTEN immunoreactivity in the ONL, INL, and GCL increased. Scale bar: 50 μm, n = 6.
Figure 2
 
Note that PTEN was highly expressed in the ONL, INL, and GCL after RD. (AE) Immunostaining of PTEN. (A) Normal control, (B) 1 day after RD, (C) 2 days after RD, (D) 3 days after RD, (E) 7 days after RD. Immunoreactivity of PTEN was detected in the INL and GCL in normal control retina. After RD, PTEN immunoreactivity in the ONL, INL, and GCL increased. Scale bar: 50 μm, n = 6.
Figure 3
 
Changes in photoreceptor apoptosis in rat retina after RD. (AE) TUNEL (green) and DAPI (blue) staining of sections of attached or detached retina. (A) Normal control, (B) 1 day after RD, (C) 2 days after RD, (D) 3 days after RD, (E) 7 days after RD. TUNEL-positive cells were present at 1 day after RD, peaked at 2 and 3 days, and then decreased by 7 days. Scale bar: 50 μm, n = 6. (F) Quantification of TUNEL-positive photoreceptors. The data are presented as the mean ± SEM (*P < 0.05, ***P < 0.001, n = 6).
Figure 3
 
Changes in photoreceptor apoptosis in rat retina after RD. (AE) TUNEL (green) and DAPI (blue) staining of sections of attached or detached retina. (A) Normal control, (B) 1 day after RD, (C) 2 days after RD, (D) 3 days after RD, (E) 7 days after RD. TUNEL-positive cells were present at 1 day after RD, peaked at 2 and 3 days, and then decreased by 7 days. Scale bar: 50 μm, n = 6. (F) Quantification of TUNEL-positive photoreceptors. The data are presented as the mean ± SEM (*P < 0.05, ***P < 0.001, n = 6).
Figure 4
 
Morphological changes and analysis of ONL thickness in the retina after RD. (AF) Representative histopathological images of retina sections. (A) Normal control, (B) untreated (7 days after RD), (C) RD-vehicle (7 days after RD), (D) RD-bpV 40 ng, (E) RD-bpV 400 ng, (F) RD-bpV 4 μg. Scale bar: 50 μm, n = 6. (G) Quantitative data showing the protective effect of bpV (4 μg/kg) on preserving ONL thickness at 3 and 7 days after RD. Data are presented as the mean ± SEM (**P < 0.01, ***P < 0.001, n = 6). (H) Quantification of cells in ONL. The data are presented as the mean ± SEM (**P < 0.01, ***P < 0.001, n = 6).
Figure 4
 
Morphological changes and analysis of ONL thickness in the retina after RD. (AF) Representative histopathological images of retina sections. (A) Normal control, (B) untreated (7 days after RD), (C) RD-vehicle (7 days after RD), (D) RD-bpV 40 ng, (E) RD-bpV 400 ng, (F) RD-bpV 4 μg. Scale bar: 50 μm, n = 6. (G) Quantitative data showing the protective effect of bpV (4 μg/kg) on preserving ONL thickness at 3 and 7 days after RD. Data are presented as the mean ± SEM (**P < 0.01, ***P < 0.001, n = 6). (H) Quantification of cells in ONL. The data are presented as the mean ± SEM (**P < 0.01, ***P < 0.001, n = 6).
Figure 5
 
Apoptosis was significantly reduced by bpV(pic) after RD. (A-D) TUNEL (green) and DAPI (blue) staining of retina sections. (A) Control-vehicle, (B) RD-vehicle (3 days after RD), (C) RD-bpV 4 μg. There were no TUNEL-positive cells in the vehicle-treated control retina. However, the vehicle-treated RD retina showed TUNEL-positive cells 3 days after RD. In contrast, there were fewer TUNEL-positive cells in the bpV-treated RD retina. Scale bar: 50 μm, n = 6. (D) Quantification of TUNEL-positive cells in ONL. The data are presented as the mean ± SEM (*P < 0.05, ***P < 0.001, n = 6).
Figure 5
 
Apoptosis was significantly reduced by bpV(pic) after RD. (A-D) TUNEL (green) and DAPI (blue) staining of retina sections. (A) Control-vehicle, (B) RD-vehicle (3 days after RD), (C) RD-bpV 4 μg. There were no TUNEL-positive cells in the vehicle-treated control retina. However, the vehicle-treated RD retina showed TUNEL-positive cells 3 days after RD. In contrast, there were fewer TUNEL-positive cells in the bpV-treated RD retina. Scale bar: 50 μm, n = 6. (D) Quantification of TUNEL-positive cells in ONL. The data are presented as the mean ± SEM (*P < 0.05, ***P < 0.001, n = 6).
Figure 6
 
Modulation of PIP3 levels and p-PDK1, p-Akt and Akt protein expression in the rat retina by bpV(pic) after RD. (A) Levels of PIP3 were measured in retinal lipid extracts prepared 3 days after RD. Bar graph shows PIP3 levels expressed per mg protein as the mean ± SEM (*P < 0.05, ***P < 0.001, n = 6). (B, C) Western blot and quantitative analysis of p-PDK1 (B), p-Akt, and Akt (C) in different groups. Differences in loading were normalized using the levels of β-Actin. The data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n = 6).
Figure 6
 
Modulation of PIP3 levels and p-PDK1, p-Akt and Akt protein expression in the rat retina by bpV(pic) after RD. (A) Levels of PIP3 were measured in retinal lipid extracts prepared 3 days after RD. Bar graph shows PIP3 levels expressed per mg protein as the mean ± SEM (*P < 0.05, ***P < 0.001, n = 6). (B, C) Western blot and quantitative analysis of p-PDK1 (B), p-Akt, and Akt (C) in different groups. Differences in loading were normalized using the levels of β-Actin. The data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n = 6).
Figure 7
 
Note that bpV(pic) increased Akt phosphorylation after RD. (AC) Phospho-Akt immunostaining. (A) Control-vehicle, (B) RD-vehicle (3 days after RD), (C) RD-bpV 4 μg. In the vehicle-treated control retina, p-Akt immunostaining was observed in the ONL as well as in the OS and IS. Immunoreactivity for p-Akt in the ONL was reduced in the vehicle-treated RD retina compared to the vehicle-treated control retina. Phospho-Akt immunoreactivity was significantly increased in the ONL in the bpV-treated RD retina. Scale bar: 50 μm, n = 6.
Figure 7
 
Note that bpV(pic) increased Akt phosphorylation after RD. (AC) Phospho-Akt immunostaining. (A) Control-vehicle, (B) RD-vehicle (3 days after RD), (C) RD-bpV 4 μg. In the vehicle-treated control retina, p-Akt immunostaining was observed in the ONL as well as in the OS and IS. Immunoreactivity for p-Akt in the ONL was reduced in the vehicle-treated RD retina compared to the vehicle-treated control retina. Phospho-Akt immunoreactivity was significantly increased in the ONL in the bpV-treated RD retina. Scale bar: 50 μm, n = 6.
Figure 8
 
Modulation of p-BAD, Bcl-2, cytosolic Cyt c, and cleaved caspase-3 protein expression in the rat retina by bpV(pic) after RD. Western blot and quantitative analysis of p-BAD (A), Bcl-2 (B), cytosolic Cyt c (C), and cleaved caspase-3 (D) in different groups. Differences in loading were normalized using the levels of β-Actin. The data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n = 6).
Figure 8
 
Modulation of p-BAD, Bcl-2, cytosolic Cyt c, and cleaved caspase-3 protein expression in the rat retina by bpV(pic) after RD. Western blot and quantitative analysis of p-BAD (A), Bcl-2 (B), cytosolic Cyt c (C), and cleaved caspase-3 (D) in different groups. Differences in loading were normalized using the levels of β-Actin. The data are presented as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n = 6).
Figure 9
 
Note that bpV(pic) induced upregulation of Bcl-2 protein expression after RD. (AC) Immunostaining of Bcl-2. (A) Control-vehicle, (B) RD-Vehicle, (C) RD-bpV 4 μg. In vehicle-treated control retina, Bcl-2 immunoreactivity was detected in the INL, GCL, and ONL. Only slight immunoreactivity was observed in the INL, GCL, and ONL in the vehicle-treated RD retina. bpV treatment showed a significant increase in Bcl-2 immunoreactivity in the INL, GCL, and ONL (arrows) in the RD retina. Scale bar: 50 μm, n = 6.
Figure 9
 
Note that bpV(pic) induced upregulation of Bcl-2 protein expression after RD. (AC) Immunostaining of Bcl-2. (A) Control-vehicle, (B) RD-Vehicle, (C) RD-bpV 4 μg. In vehicle-treated control retina, Bcl-2 immunoreactivity was detected in the INL, GCL, and ONL. Only slight immunoreactivity was observed in the INL, GCL, and ONL in the vehicle-treated RD retina. bpV treatment showed a significant increase in Bcl-2 immunoreactivity in the INL, GCL, and ONL (arrows) in the RD retina. Scale bar: 50 μm, n = 6.
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