Hsp27 Phosphorylation in Experimental Glaucoma

Abstract

purpose. The role of heat shock proteins (Hsp) in injury response has been well established, but it is now becoming apparent that the phosphorylation state of Hsp27 may be a critical determinant of its ability to act in a protective capacity. In this study, the expression of Hsp27 and its phosphorylation were evaluated in an experimental glaucoma model in Brown Norway rats and in a spontaneous model of glaucoma in the DBA/2J mouse.

methods. The intraocular pressure (IOP) of one eye was elevated by injecting 1.9 M saline into the episcleral vein, as previously described in Brown Norway rats. IOP was measured in awake Brown Norway rats before surgery and every other day after saline injection. After 10 days of elevated IOP, the retinas were either removed for Western blot analysis or fixed for immunohistochemistry (IHC). IOP measurement in 7-month-old female DBA/2J mice was performed by direct cannulation. Retinas of eyes with and without elevated IOP were collected for Western blot analysis.

results. Western blot results showed a significant increase in total Hsp27 (3.9-fold; P < 0.05; n = 8) and phosphorylated Hsp27 (pHsp27) (2.1-fold; P < 0.05; n = 6) in high IOP eyes in the experimental rat glaucoma model, and similar increases were observed in DBA/2J mice with elevated IOP (3.1-fold and 2.2-fold for Hsp27 and pHsp27, respectively; P < 0.05; n = 5). In rats, increased Hsp27 and pHsp27 immunoreactivity were observed in the nerve fiber layer of elevated IOP eyes and colocalized with glial fibrillary acidic protein (GFAP) and with vimentin staining, suggesting that glial cells contribute to the increase in Hsp27 and pHsp27 seen in experimental glaucoma. No change in Hsp70 or Hsp90 was observed.

conclusions. These results confirm previous reports of elevated Hsp27 in experimental glaucoma and extend them to the DBA/2J mouse. In addition, a significant increase occurred in Hsp27 phosphorylation with elevated IOP in both models of glaucoma. IHC studies show that the increases in Hsp27 and pHsp27 occur primarily in glial cells.

The small heat shock protein 27 (Hsp27) increases its expression in glial cells and neurons after a wide range of stimuli, including ischemia, epileptic seizure, and hyperthermia. ¹ Small Hsp are characterized by their phosphorylative capacity, and they form multimeric structures. Hsp27 becomes phosphorylated in response to heat shock and various stimuli, including cytokines, growth factors, and peptide hormones. ² The rodent form of Hsp27 can be phosphorylated at two sites, Ser15 and Ser85. ³ Phosphorylation of Hsp27 plays a critical role in the regulation of Hsp27 function and may influence the cells' ability to protect against a variety of cellular stresses. ⁴ Overexpression of Hsp27 can confer thermoresistance, but overexpression of a mutant Hsp27 lacking phosphorylation sites fails to protect cells from heat shock. ^{5 6} Given that protein phosphorylation is one of the most important cellular regulatory components and that Hsp27 is a major target of phosphorylation, investigating the phosphorylation status of Hsp27 may be critical for understanding the defense mechanisms of cells against stress. Hsp27 kinase mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 (MAPKAP-K2), through its association with Akt and activation by p38 MAPK, is reported to phosphorylate Hsp27, ⁷ though recent reports indicate that protein kinase C (PKC) α and δ and cyclic adenosine monophosphate (cAMP)–dependent kinase can also phosphorylate Hsp27. ⁸  

Most investigations of Hsp in glaucoma have focused on characterizing expression changes in the various Hsp forms, but no reports address the phosphorylation state of Hsp27 under conditions of elevated intraocular pressure (IOP). A recent report demonstrates that the heat shock transcription factors HSF-1 and HSF-2 are present in retinal ganglion cells (RGCs) ⁹ and another demonstrates that Akt is activated in experimental glaucoma. ¹⁰ Together with the previously reported observation that Hsp27 levels are increased in glaucoma, ¹¹ our data suggest there may be a role for Hsp27 phosphorylation in glaucoma. 

In this article, we examine the phosphorylation of Hsp27 in experimental glaucoma in rat and in the DBA/2J mouse model of spontaneous glaucoma. These models have distinct ways in which IOP is elevated and different time courses, resulting in RGC death. Changes of Hsp27 phosphorylation have not previously been reported in glaucoma. To further our understanding of the effects of Hsp27 phosphorylation, we used fluorogold (FG) back-labeling and immunohistochemistry to determine the specific cell types expressing Hsp27 in glaucoma. 

Methods

Animals

All procedures concerning animals were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Adult male Brown Norway rats (300–450 g; Charles River Laboratories, Wilmington, MA) and 7-month-old female DBA/2J mice (The Jackson Laboratory, Bar Harbor, ME) were housed in covered cages, fed with a standard rodent diet ad libitum, and kept on a 12-hour light/12-hour dark cycle. 

Experimentally Induced Glaucoma in the Rat

Anesthesia was induced using a mixture of acepromazine maleate (1.5 mg/kg), xylazine (7.5 mg/kg), and ketamine (75 mg/kg; all from Webster Veterinary Supply, Sterling, MA). Injection of 1.9 M hypertonic saline into aqueous veins, as previously described by Morrison, ¹² produced unilateral elevation of IOP. The fellow eye served as control. If the IOP was not elevated within 2 weeks, reinjection was performed in a different episcleral vein. A maximum of three injections was performed. 

Intraocular Pressure Determination

All IOP measurements in rats were performed with animals in the awake state between 10 am and 2 pm to minimize diurnal variability in IOP. After applying a drop of 0.5% proparacaine local anesthesia, IOP was measured with a tonometer (TonoPen XL; Medtronic Ophthalmics, Jacksonville, FL). Fifteen readings were taken for each eye and were averaged. Baseline IOP was obtained on three consecutive days before the first saline injection and three times per week thereafter. We subjected the rats to a 10-day period of elevated pressure exposure. After IOP had been elevated for 10 days, the rats were killed by asphyxiation with carbon dioxide, the eyes were rapidly removed, and the eyecups or retinas were collected, either for immunohistochemistry (IHC) or for Western blot analysis. To assess IOP exposure, we integrated IOP with time, which included both the length and the degree of IOP exposure. Integrated IOP was calculated as the area under the time–pressure curve (experimental eye–control eye), beginning with the day of the first saline injection. 

IOP was measured in DBA/2J mice by direct cannulation, as described. ¹³ After mice were anesthetized with a mixture of 90 mg/kg ketamine and 9 mg/kg xylazine, a glass micropipette tip with an external diameter of 50 μm and a length of 2 mm was introduced into the anterior chamber (taking care to avoid the lens) and was connected to polyethylene tubing. A pressure transducer (CyQ low-pressure transducer; Cybersence, Nicholasville, KY) and a signal conditioner (CyQ104 signal conditioner; Cybersence) were used to determine pressures for a 1-minute period, when the eye had stabilized after micropipette insertion. 

Back-Labeling of Retinal Ganglion Cells in Rats

After the induction of deep anesthesia, as described, rats were placed in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA), and the skin overlying the skull was incised and retracted. The skull was leveled using the lambda and bregma sutures as landmarks. Windows 6 mm wide were made in the skull overlying the superior colliculus 5.3 mm posterior to bregma, 1.5 mm lateral to midline, and 4.8 mm ventral to the skull surface. Two microliters of a 3% fluorogold solution (Fluorogold; Fluorochrome LLC, Denver, CO) in PBS with 10% dimethyl sulfoxide (DMSO) was injected over 10 minutes. The procedure was repeated on the contralateral side of the brain. The skin was sutured closed, antibiotic ointment was applied to the wound, and the animal was returned to its cage after recovery from anesthesia. ¹⁴ Animals were allowed to recover for a week before further experimental interventions to allow for adequate back-labeling of RGCs. 

Tissue Preparation

For IHC, deeply anesthetized animals were perfused with PBS followed by 4% paraformaldehyde (PFA) in PBS. Eyes were enucleated, and anterior segments were rapidly removed and postfixed in 4% PFA for another hour and were cryoprotected with serial sucrose dilutions. Eyecups were frozen in optimal cutting temperature compound (OCT; Tissue- Tek, Miles Inc., Diagnostic Division, Elkhart, IN) and sectioned on a cryostat at 16 μm, mounted on slides (Superfrost Plus; VWR Company, West Chester, PA), and stored at –80°C. 

For Western blot analysis, retinas were homogenized and lysed with buffer containing 1 mM EDTA/EGTA/DTT, 10 mM HEPES (pH 7.6), 0.5% surfactant (Igepal; Sigma, St. Louis, MO), 42 mM KCl, 5 mM MgCl₂, 1 mM PMSF, and 1 tablet of protease inhibitors (Complete Mini; Roche Diagnostics GmbH, Mannheim, Germany) per 10-mL buffer. Samples were incubated for 15 minutes on ice and then centrifuged at 21,000 rpm at 4°C for 30 minutes, and the supernatant was stored at –80°C. 

Western Blot Analysis

Proteins were separated on SDS-PAGE gels (10%–20% Tris-HCl Ready-Gels; Bio-Rad Laboratories, Hercules, CA), 12 μg total retinal protein per lane, transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA), and blocked with 5% nonfat dry milk in 0.1% TBS-T. Proteins were detected by incubating membranes with antibodies against Hsp27 (1:10,000; Stressgen, Victoria, CA), phosphorylated Hsp27 (Ser82) (1:5000; Upstate, Chicago, IL), Hsp 70 (1:10,000; Stressgen), and Hsp 90 (1:1000; Stressgen) overnight at 4°C and incubated for 1 hour in peroxidase-conjugated secondary antibody at 1:20,000 (Jackson ImmunoResearch, West Grove, PA). Labeled protein was detected using SuperSignal reagent (Pierce, Rockford, IL). The membrane was reblotted with an antibody to α-tubulin (1:2000; Abcam, Cambridge, MA) as a loading control. Membranes were exposed to multipurpose film (HyperFilm; Amersham, Little Chalfont, Buckinghamshire, UK), and densitometry was performed (ImageQuant 1.2; Molecular Dynamics, Inc., Sunnyvale, CA). 

Immunohistochemistry

Sixteen-micrometer–thick cryostat sections were incubated with a blocking solution for 1 hour at room temperature. Primary antibodies specific for Hsp27 (1:5000; Stressgen), phosphorylated Hsp27 (Ser82; 1:400; Upstate), GFAP (1:400; Sigma), and vimentin (1:40,000, Sigma) were used. Anti–rabbit and anti–mouse antibodies (Alexa 488 [1:250] and Alexa 594 [1:500]; Molecular Probes, Eugene, OR) were used as secondary antibodies with 1-hour incubation at room temperature. Tissues were evaluated using confocal microscopy (Leica, Deerfield, IL). Positive cells were confirmed as RGCs by detection of FG labeling. 

Statistical Analysis

All data are presented as a mean ± SD. Paired comparisons were performed with the Student's t-test, and significance was assessed at the 0.05 level. 

Results

Rat IOP History

Baseline IOP readings of right and left eyes were 18.2 ± 0.8 mm Hg and 18.1 ± 0.7 mm Hg, respectively (n = 12) before any intervention. Mean peak IOP for experimental eyes was significantly elevated at 44.17 ± 1.49 mm Hg (n = 12; P < 0.05). Rats were monitored for 10 days after the initial increase of IOP. Integrated IOP (area under the pressure–time curve, experimental eye–control eye) was 324.32 ± 53.59 mm Hg-days (n = 12). We have previously reported that this degree of IOP exposure leads to significant RGC death. ^{14 15 16}  

Elevated IOP Leads to Increased Hsp27 and Hsp27 Phosphorylation in Rat Retinas in Experimental Glaucoma

Western blot analysis was used to determine the effect of elevated IOP on the levels of total Hsp27, pHsp27, Hsp70, and Hsp90 in the retina. The Hsp27 antibody recognized a single band with a molecular mass of 27 kDa (Fig. 1A) . A basal level of Hsp27 was expressed in control retinas. Levels were significantly increased in all eyes with elevated IOP (Fig. 1B) . Constitutively expressed pHsp27 in control retinas was not detected by an antibody recognizing only pHsp27 (Ser82). However, in retinas of animals that experienced 10 days of elevated IOP, significant expression of pHsp27 was observed (Fig. 1A) . Hsp70 expression in response to elevated IOP was also evaluated, but no difference was observed between control and elevated IOP eyes. We also investigated the expression level of Hsp90 after inducing elevated IOP. Hsp90 was unchanged in control and elevated IOP eyes (Fig. 1A) . Summary data show a 3.9-fold increase in Hsp27 (P < 0.01; n = 6) and a 1.9-fold increase in pHsp27 (P < 0.01; n = 6) levels after elevated IOP in this rodent model (Fig. 1B) . 

Elevated IOP Leads to Increased Hsp27 and Hsp27 Phosphorylation in a Spontaneous Mouse Glaucoma Model

To further investigate our findings, a second model of glaucoma was examined. We studied the DBA/2J mouse model of spontaneous glaucoma. Eighteen eyes (nine mice) of 7-month-old female DBA/2J mice were screened. Other data from these animals have previously been reported in another paper. ¹⁴ In three mice, IOP was normal in one eye and higher than 17 in the other eye. In another mouse, IOP was higher than 17 in both eyes. This IOP distribution was agreement with what has been reported in the literature for the DBA/21J mouse. ¹⁷ Retinas from these four mice (eight eyes) were harvested for analysis for Hsp27. Eyes classified as having normal IOP (10 ± 1.5 mm Hg; n = 3) or elevated IOP (18.6 ± 0.9 mm Hg; n = 5) were evaluated by Western blot. Hsp27 expression was higher in elevated IOP eyes, and pHsp27 was evident only in eyes with high IOP (Fig. 2A) . Comparing levels between eyes with and without elevated IOP, the summary data show a 3.1-fold (P < 0.05; n = 5) increase in Hsp27 and a 2.2-fold (P < 0.05, n = 5) increase in pHsp27 levels in this mouse model (Fig. 2B) . No change in Hsp70 was observed (Fig. 2) . 

Glial Cells Are Mainly Responsible for the Increases in pHsp27 and Hsp27 in Experimental Glaucoma in Rat

IHC was used to determine the cell types responsible for the changes of Hsp27 expression and phosphorylation in rat eyes with elevated IOP. In control retinas, there was only moderate Hsp27 expression, mainly in the nerve fiber layer (NFL), ganglion cell layer (GCL), and inner nuclear layer (INL) (Figs. 3A 3G) . After 10 days of elevated IOP, Hsp27 immunoreactivity increased in the NFL, GCL, and INL (Figs. 3D 3J) . Expression of the astrocyte marker GFAP was markedly increased in the NFL, and GFAP-labeled cell processes became thicker and were visualized from the GCL to the photoreceptor layer in eyes with high IOP (Fig. 3E) . A similar pattern of GFAP immunoreactivity has been shown in the retina in glaucoma eyes by other researchers. ¹¹ Double staining of GFAP and Hsp27 showed that the increase in Hsp27 expression is principally located in GFAP-positive cells (Fig. 3F) . Additional IHC was performed with the well-established Müller cell marker vimentin. ^{18 19 20} In control retinas, vimentin-positive cells were observed to extend from the inner to the outer retina in a pattern characteristic of Müller cells (Fig. 3H) , and there was little colocalization with Hsp27 (Fig. 3I) . Vimentin staining did not appear to be significantly changed in eyes with high IOP (Fig. 3K) . Double staining for Hsp27 and vimentin did, however, show colocalization in high IOP retinas in the NFL and GCL (Fig. 3L) . 

A similar pattern of staining was seen for pHsp27. In control retinas, there was no phosphorylated Hsp27 signal (Figs. 4A 4G) , but after 10 days of elevated IOP, there was a significant increase in pHsp27 immunoreactivity in the GCL and NFL (Figs. 4D 4J) . The increased pHsp27 signal localized with GFAP (Fig. 4F)and vimentin (Fig. 4L)immunoreactivity in the GCL and NFL, suggesting that astrocytes and Müller cell end-feet are the major source of increased pHsp27. 

No pHsp27 immunoreactivity was seen in FG back-labeled RGCs in retinas with normal IOP (Fig. 5B) . In retinas with high IOP, pHsp27 expression was seen in FG-labeled RGCs and in the NFL (Fig. 5F) . 

Discussion

With the use of Western blot analysis and IHC, we investigated the changes in low molecular–weight Hsp (Hsp27) and the posttranslational modification of Hsp27 (phosphorylation) in two rodent models of glaucoma. These two models differ in the timing of RGC death, with death occurring more slowly in the DBA/2J mouse than in the rat. Our results indicated that elevated IOP induces an increase in Hsp27 and its phosphorylation in both models. We did not detect other Hsp changes in either model of experimental glaucoma. We found that the increases in Hsp27 and pHsp27 colocalize to GFAP- and vimentin-positive cells in the retina. In addition, we found that RGCs in normal IOP eyes do not express pHsp27 but that RGCs in high IOP eyes do express pHsp27. These results suggest that the phosphorylation of Hsp27 is a major Hsp response to elevated IOP exposure and that activated glia are a major source of the increase. 

Hsp are a family of highly homologous chaperone proteins that are induced in response to environmental, physical, and chemical stresses. They are ubiquitously expressed in multiple tissues and are classified as low-molecular–weight Hsp and high-molecular–weight Hsp, based on apparent molecular size. Low-molecular–weight Hsp, such as Hsp27 and αB-crystallin (molecular mass, 10–30 kDa), have high homology in amino acid sequences and play an important role in protection from various insults. ²¹ High-molecular–weight Hsp, such as Hsp70 and Hsp90, act as molecular chaperones in protein folding, oligomerization, and translocation. ²¹ Although the functions of the low-molecular–weight Hsp are less well understood than those of the high-molecular–weight Hsp, several lines of evidence suggest that Hsp27 may have a more potent protective effect in the nervous system. For example, in cultured neurons, overexpression of Hsp27 protects against a variety of stresses, ²² whereas overexpression of Hsp70 can protect against subsequent exposure to thermal or ischemic stress but not against exposure to other stressful stimuli, such as growth factor withdrawal. ²³  

Hsp27 is regulated at the transcriptional and the posttranslational levels. ^{24 25 26 27 28 29} Overexpression confers cellular resistance to stimuli that induce cell death with necrotic or apoptotic features, including physical and chemical stress, growth factor withdrawal, and activation of death receptors. ^{25 30 31} Hsp27 becomes rapidly phosphorylated at two serine sites in rats (Ser15 and Ser85) in response to cytotoxic stress or exposure to cytokines and mitogens. ^{32 33} Under unstimulated conditions, Hsp27 exists as a high-molecular–weight aggregate that dissociates after phosphorylation. ^{34 35}  

Several mechanisms have been proposed for Hsp27 protective activities through actin polymerization, chaperone activity, and regulation of reactive oxygen species. ^{25 36 37} Hsp27 expression and its phosphorylation state can influence the fate of the cell in response to an apoptotic stimulus. ³⁸ Expression of a phosphorylation-defective mutant of Hsp27 enhanced apoptosis. ³⁹ Expression of mutant Hsp27 lacking phosphorylation sites failed to protect Chinese hamster ovary cells from heat shock in vitro ⁴⁰ and failed to protect axotomized peripheral neurons in vivo. ⁴¹ Interestingly, this difference was not observed in a mouse ischemia/reperfusion model, suggesting that differences may exist between Hsp responses in different systems. ² pHsp27 has been shown to interact with an adaptor protein of Daxx to inhibit Fas-mediated apoptosis. ²⁵ Taken together, these data support the importance of the phosphorylation-dependent protective function of Hsp27 during stress. 

Several reports have demonstrated expression changes of multiple Hsp in the retina. In the retinas of monkey eyes with laser-induced glaucoma, the intensity of immunostaining for Hsp27, Hsp60, and Hsp90 was greatly enhanced, especially in the ganglion cell and nerve fiber layers. ⁴² Staining intensity for Hsp70 was also moderately increased, whereas immunoreactivity against Hsp47 remained almost unchanged in glaucomatous retinas. ⁴² In human glaucomatous eye tissue, the intensity of the immunostaining and the number of labeled cells for Hsp27 and Hsp60 were also reported to be greater than in normal eyes of age-matched donors. ¹¹ Retinal immunostaining of Hsp27 was prominent in the nerve fiber layer and ganglion cells and in the retinal vessels. Optic nerve heads of glaucomatous eyes exhibited increased Hsp27 (but not Hsp60) that was primarily associated with astroglial cells in the lamina cribrosa. ¹¹ Tezel and Wax ⁴³ reported elevated serum levels of anti–Hsp27 antibodies in patients with glaucoma and hypothesized that these circulating antibodies would result in an inactivation of endogenous Hsp27, leading to a loss of the Hsp27-protective response to stress in RGCs. ⁴⁴ In addition, we have previously reported the involvement of both the caspase 8 and the caspase 9 pathways in RGCs in experimental glaucoma, ¹⁶ and the upregulation of Hsp27 has been suggested to act as an endogenous protective response to block both cascades. ^{45 46 47}  

Glial cells provide neurons with nourishment, physical support, and protection. GFAP is well known to be a sensitive marker of glial activation in response to neural injury. ⁴⁸ In normal retinas, GFAP is expressed principally by astrocytes in the nerve fiber layer. Müller cells do not normally express GFAP, but they can be induced and can dramatically increase their expression in response to injury. ^{49 50 51} In eyes with normal IOP, GFAP was restricted to astrocytes in the nerve fiber layer. After laser-induced IOP elevation in rats, Woldemussie et al. ⁵² reported that GFAP immunoreactivity was no longer limited to the nerve fiber layer but that it was increased throughout the length of the Müller cells, from the inner to the outer limiting membranes. Optic nerve head astrocytes have been shown to have increased Hsp27 expression in glaucoma and under conditions of elevated hydrostatic pressure. ⁵³ In the present study, increased Hsp27 and pHsp27 immunoreactivity colocalized with GFAP and vimentin in the nerve fiber layer and the ganglion cell layer but did not extend to processes spanning the retina. These results suggest that astrocytes and Müller cells are the major contributors to the increases observed in Hsp27 and pHsp27. Our results are in good agreement with other findings that Hsp27 is principally located in glia in the normal adult retina ^{54 55} and with Salvador-Silva ⁵³ showing increased GFAP and Hsp27 in vitro under conditions of elevated hydrostatic pressure. Although incompletely understood, some recent data suggest that Hsp can be secreted from stressed cells. ^{56 57 58} Taken together, we speculate that glia secrete pHsp27 and Hsp27 to protect RGCs in glaucoma. Accordingly, anti–Hsp27 antibodies might block this action leading to RGC death in glaucoma, supporting and extending the findings of Tezel and Wax. ⁴⁴  

We conclude that in experimental glaucoma, increases in the small heat shock protein Hsp27 are associated with elevated IOP exposure. Hsp27 is also regulated at the posttranslational level and becomes phosphorylated. That this robust increase of Hsp27 occurs in two different models (one induced and the other spontaneous) and in two different species (rat and mouse) suggests that the phosphorylation of Hsp27 is a fundamental biological response to elevated IOP rather than a consequence of a specific model system. We hypothesize that these changes in Hsp27, mainly observed in glial cells, may be part of a larger, self-regulated physiological protection mechanism to prevent neuronal injury, especially under conditions of chronically elevated IOP. 

 Supported by the American Health Assistance Foundation (CLG), the RPB Career Development Award (CLG), the Margolis Fund (CLG), and the Massachusetts Lion Eye Research Fund (CLG).

 Submitted for publication June 5, 2006; revised November 1, 2006, and May 10, 2007; accepted June 19, 2007.

 Disclosure: W. Huang, None; J.B. Fileta, None; T. Filippopoulos, None; A. Ray, None; A . Dobberfuhl, None; C.L. Grosskreutz, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Elevated IOP-induced changes in Hsp27 and Hsp27 phosphorylation in Brown Norway rat retina. (A) After experimentally elevated IOP for 10 days, Hsp27 and pHsp27 (lanes 2 and 4) increased compared with normal retinas (lanes 1 and 3). (B) Quantification of the Western blot analysis further confirmed that Hsp27 and pHsp27 levels increase in elevated IOP retinas. However, Hsp 70 and Hsp 90 levels did not change significantly (n = 6; *P < 0.01).

Figure 2.

 

Elevated IOP-induced changes in Hsp27 and Hsp27 phosphorylation in DBA/2J mice. (A) Hsp27 and pHsp27 (lanes 2 and 4) increased in eyes with high IOP compared with normal retinas (lanes 1 and 3). (B) Quantification of the Western blot analysis further confirmed that Hsp27 and pHsp27 levels increase in elevated IOP retinas. However, the Hsp 70 level did not change significantly (n = 5; *P < 0.01).

Figure 3.

 

Hsp27 and glial cell markers in rat retina after experimentally elevated IOP. (A, G) Hsp27 immunoreactivity (red) in the GCL and INL in control retina. (B) GFAP (green) in the NFL in control retina. (C) Little colocalization of Hsp27 and GFAP (yellow) in normal retina. (D, J) Increased Hsp27 immunoreactivity in the NFL, GCL, and INL in experimental glaucoma. (E) Increased GFAP in the NFL and processes spanning the retina in experimental glaucoma. (F) Colocalization of Hsp27 and GFAP primarily in the NFL (yellow) in experimental glaucoma. (H) Vimentin immunoreactivity (green) in cells spanning the height of the retina. (I) Little colocalization of Hsp27 and vimentin in normal retina. (K) Vimentin staining changes little in experimental glaucoma. (L) Scattered colocalization of Hsp27 and vimentin (yellow) in experimental glaucoma. NFL, nerve fiber layer; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer.

Figure 4.

 

pHsp27 and glial cell markers in rat retina after experimentally elevated IOP. (A, G) Low levels of pHsp27 immunoreactivity (red) in the GCL and INL in control retina. (B) GFAP (green) in the NFL in control retina. (C) No colocalization of pHsp27 and GFAP in normal retina. (D, J) Increased pHsp27 immunoreactivity primarily in the NFL in experimental glaucoma. (E) Increased GFAP in the NFL and processes spanning the retina in experimental glaucoma. (F) Colocalization of pHsp27 and GFAP primarily in the NFL (yellow) in experimental glaucoma. (H) Vimentin immunoreactivity (green) in cells spanning the height of the retina. (I) No colocalization of pHsp27 and vimentin in normal retina. (K) Vimentin staining changes little in experimental glaucoma. (L) Colocalization of pHsp27 and vimentin (yellow) in experimental glaucoma. NFL, nerve fiber layer; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer.

Figure 5.

 

pHsp27 is in RGCs. (A) FG back-labeled RGCs in control retina. (B) Low levels of pHsp27 in RGCs in normal retina. (C) Merged image. (D) FG back-labeled RGCs in eye with high IOP. (E) Increased pHsp27 immunoreactivity in RGCs in high IOP retina. (F) Colocalization of pHsp27 and FG in experimental glaucoma. FG, fluorogold.