IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1167/iovs.07-1597 on June 19, 2008
 (Investigative Ophthalmology and Visual Science. 2008;49:4971-4981.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.07-1597
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
iovs.07-1597v1
49/11/4971    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schmeer, C.
Right arrow Articles by Isenmann, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schmeer, C.
Right arrow Articles by Isenmann, S.

Statins Modulate Heat Shock Protein Expression and Enhance Retinal Ganglion Cell Survival after Transient Retinal Ischemia/Reperfusion In Vivo

Christian Schmeer,1 Adriana Gámez,2 Svetlana Tausch,1 Otto W. Witte,1 and Stefan Isenmann1,3

1From the Department of Neurology, Friedrich-Schiller University, Jena, Germany; 2Laboratorio de Neuroquímica, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela; and 3HELIOS Klinikum Wuppertal, Department of Neurology, University Witten/Herdecke, Wuppertal, Germany.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To evaluate putative mechanisms for the pleiotropic effects of statins, the expression of members of the heat shock family of proteins (HSPs) was compared between normal and ischemic rat retinas after transient retinal ischemia/reperfusion and statin treatment in vivo.

METHODS. Retinal ischemia/reperfusion was induced by transient elevation of intraocular pressure (IOP). Retinal expression of HSPs was evaluated at different time points after drug and solvent injection and retinal ischemia/reperfusion by means of PCR and Western blot analysis. Immunofluorescent staining and confocal laser scanning microscopy were used to localize the expression of HSPs in normal and ischemic retinas.

RESULTS. During the acute phase after retinal ischemia, {alpha}B-crystallin protein and mRNA expression were reduced after statin treatment. After 72 hours of reperfusion, statins increased the expression of {alpha}B-crystallin and reduced the expression of HSP27 in the retina. Increased expression of {alpha}B-crystallin early after lesion and statin delivery correlated with increased expression of the heat shock factors 1 and 2. Statins significantly enhanced retinal ganglion cell (RGC) survival 10 days after transient retinal ischemia in vivo.

CONCLUSIONS. Systemic delivery of statins after a transient period of retinal ischemia significantly modulated HSP expression in the retina and enhanced RGC survival. Together, these results support the notion that statins constitute a feasible therapeutic approach to prevent some of the neuronal damage in the acute and possibly also the delayed phase and have beneficial effects in central nervous system (CNS) disorders directly affecting the visual system.

Retinal ischemia is a serious and common clinical problem. It occurs as a result of acute vascular occlusion and leads to visual loss in several ocular diseases, including glaucoma, diabetic retinopathy, and hypertensive vascular disease. Transient global retinal ischemia shares many similarities with transient global cerebral ischemia.

HSPs are ubiquitous and highly conserved proteins. Their expression is upregulated in response to various physiological imbalances and environmental factors, and they enable cells to survive otherwise lethal insults.1 Families of HSPs are classified according to their molecular masses, namely HSP100, HSP90, HSP70, HSP60, HSP40, and small HSPs (~20 kDa).2

Most HSPs are chaperones involved in proper folding and/or elimination of misfolded proteins. In addition, they protect subcellular structures, particularly mitochondria, which are crucial in antioxidant and antiapoptotic cell protection, and they can directly interfere with apoptotic pathways.3

The vertebrate crystallins belong to the small HSP family of proteins. They are divided into two major families: {alpha}-crystallins and β{gamma}-crystallins. Crystallins constitute approximately 90% of water-soluble lens proteins and contribute to the transparency and refractive properties by a uniform concentration gradient in the lens. In addition, {alpha}-crystallins are molecular chaperones and function as protective proteins against physiological stress. {alpha}B-crystallin, but not {alpha}A, is a stress-inducible protein.4 Crystallins, initially associated only with the lens, now represent a growing family of genes and proteins that are expressed in numerous distinct cell types. In the mouse eye, {alpha}B-crystallin transcripts have been identified in the retinal pigment epithelium, optic nerve (ON), extraocular muscles, iris, ciliary body, cornea, and several nonocular sites such as heart and nasal epithelium.5

Another member of the small HSP family of proteins, HSP27 has a role in cellular repair and protection against cell stress. In particular, it has been shown that ON injury induces the expression of HSP27 in three distinct layers of the rat visual system: sensory RGCs, glial cells of the optic tract, and astrocytes in the optic layer of the superior colliculus.6 Furthermore, HSP27 protects RGCs from ischemic injury.7 {alpha}-Crystallins and HSP27 are closely related family members.8 In the unstressed state, both proteins can exist as a complex, and thus have been suggested to act in concert in response to cellular stress.9

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, generically termed statins, are widely prescribed for their cholesterol-lowering properties. Findings in recent studies suggest that, in addition to inhibiting cholesterol synthesis, statins have pleiotropic effects and may reduce cerebrovascular and cardiovascular risk, even in patients with normal cholesterol levels.10 11 12 Accumulating evidence supports statin therapy for CNS diseases, including neurodegenerative diseases affecting the visual system.13 Previous studies have shown that statins induce overexpression of HSPs in vitro,14 as well as in vivo.15 In the latter study, simvastatin specifically induced an increase in the expression of HSP27 and enhanced RGCs survival 7 and 14 days after an ON axotomy.15

Statins prevent the recruitment of leukocytes to retinal tissue and reduce ischemia/reperfusion injury by blocking the leukocyte adhesion molecules P-selectin and intracellular adhesion molecule (ICAM)-1.16

The purpose of this study was to evaluate the expression of members of the HSP family of proteins in vivo, after induction of transient retinal ischemia/reperfusion, and their putative role in statin-mediated neuroprotective effects in the rat retina.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animal Guidelines
All experiments were performed in accordance with the European Convention for Animal Care and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Use of laboratory animals was approved by the local Animal Care Committee.

Transient Retinal Ischemia
Female Sprague-Dawley rats weighing 220 to 250 g were anesthetized with an intraperitoneal injection of chloral hydrate (420 mg/kg body weight; Fluka, Seelze, Germany). After topical application of oxybuprocaine-hydrochloride (4 mg/mL), transient, unilateral ischemia was induced in one eye.17 Briefly, the anterior chamber of the right eye was cannulated with a 27-gauge needle connected to an elevated normal saline container by silastic tubing. IOP was elevated above systolic pressure for 75 minutes. For sham procedures, a needle attached to a saline reservoir was inserted into the anterior chamber of the contralateral eye, but pressure was not increased and ischemia did not occur. Blanching of the posterior segment of the eye confirmed retinal ischemia. IOP increase and maintenance were evaluated during the procedure by means of an induction–impact tonometer (Tiolat Ltd., Helsinki, Finland). This method has been validated in the rat eye.18 After 75 minutes of ischemia, the needle was withdrawn from the anterior chamber and the IOP normalized. One drop of ofloxacin solution was applied topically to the right eye before and after cannulating the anterior chamber.

Statin Administration
Four commercially available statins, simvastatin, lovastatin, mevastatin, and pravastatin were used in the present study (Calbiochem, Darmstadt, Germany). Simvastatin, lovastatin, and mevastatin were dissolved in ethanol (50 mg/mL) plus 1 N NaOH. Before use, the statins were activated by adding 1 N HCl (pH 7.2). Pravastatin was dissolved in sterile PBS and required no further activation. The amount of statin to be injected was further dissolved in PBS. Statins (0.2–4 mg/kg; 100–150 µL) were injected intraperitoneally or in the subcutis of the flank once daily up to 5 or 10 days after retinal ischemia/reperfusion. To elucidate the role of HSP expression in the neuroprotective effects of statins, quercetin at 4 mg/kg (dissolved in 0.1% DMSO; Sigma-Aldrich, Taufkirchen, Germany)15 was injected in the subcutis in a group of animals 24 hours before statin injection and in combination with it up to 5 days after retinal ischemia/reperfusion. Some groups of animals were treated with a vehicle solution consisting of a mixture of ethanol and NaOH (used to dissolve hydrophilic statins), or DMSO (used to dissolve quercetin).

Evaluation of RGC Densities in the Retina of Normal and Ischemic Animals
Rats were killed by an overdose of chloral hydrate 1, 3, 6, 24, or 72 hours or 10 days after ischemia/reperfusion. RGCs were identified and quantified by means of Brn-3 immunofluorescent staining.19 Briefly, retinas from ischemic and statin- or vehicle-injected animals were dissected and fixed in 4% paraformaldehyde (PFA) in PBS for 30 minutes. Retinas were then washed with PBS to remove residual fixative. Tissue was permeabilized by a 45-minute incubation in 0.3% Triton X-100 (Sigma-Aldrich) in PBS. Nonspecific binding of antibodies was blocked by incubating retinas with 10% normal donkey serum (NDS) plus 3% bovine serum albumin (BSA) in PBS containing 0.3% Triton X-100 for 2 hours at room temperature (RT). Retinas were incubated overnight with 1:50 dilution of goat anti-mouse Brn-3 antiserum (SC6026; Santa Cruz Biotechnology, Heidelberg, Germany), with 5% NDS plus 3% BSA/PBS at 4°C. Unbound antibody was removed by two successive 10-minute washes with PBS. Retinas were then incubated with 1:500 dilution of donkey anti-goat IgG linked to Alexa-fluor 488 (Invitrogen-Molecular Probes, Eugene, OR) with 10% NDS plus 3% BSA/PBS for 1 hour at RT, followed by washes as before. To improve cell permeability and antibody penetration, all reaction solutions contained 0.3% Triton X-100. After the staining procedure, the retinas were flat mounted on glass slides and embedded in a 1:1 glycerol-PBS solution.

RGCs densities were determined by counting labeled RGCs in 12 grid-defined areas of 62,500 µm each (three areas per retinal quadrant at 1/6, 3/6, and 5/6 of the retinal radius).20 21 The area evaluated represents 18% of the total retina. Labeled RGCs were counted in a standardized fashion as thoroughly described and discussed previously.20 21 For each paradigm, at least four retinas were evaluated. RGC quantification was achieved by means of image-analysis software (Axiovision 4.0; Carl Zeiss Meditec, Jena, Germany).

Immunofluorescent Staining for HSPs
Retinal expression of HSPs was evaluated at different time points after drug/solvent injection and retinal ischemia/reperfusion. HSPs were also investigated in unlesioned and contralateral eyes. Eye cups were fixed in 4% PFA/PBS (pH 7.4) for 30 minutes and cryoprotected by infiltration with 30% sucrose/PBS at 4°C. Tissue specimens were then embedded (Tissue-Tek; Shandon, Pittsburgh, PA) and snap frozen in liquid nitrogen. Cryostat transverse sections (16 µm) were permeabilized with 0.3% Triton X-100 in PBS and preincubated with 10% NGS or NDS for 2 hours at RT followed by reaction with antibodies raised against HSP27 (1:500; Santa Cruz Biotechnology), {alpha}B-crystallin (1:250; Stressgen, Victoria, BC, Canada), β-III-tubulin (1:500, Covance, Berkeley, CA), and cellular retinaldehyde binding protein (CRALBP, 1:250; Abcam, Hiddenhausen, Germany) at 4°C overnight (each in 2% NGS or NDS). Specific immunoreactions were detected using the appropriate Cy3-conjugated secondary antibodies (1:500 in 10% NGS; Jackson ImmunoResearch Laboratories, West Grove, PA) or Alexafluor 488-conjugated secondary antibodies (1:500; Invitrogen-Molecular Probes). To improve cell permeability, reaction solutions contained 0.3% Triton X-100 (Sigma-Aldrich) in 1% BSA/PBS. Specificity of the signal was verified omitting primary antisera. To specifically locate immunolabeled cells, retinas were incubated in the presence of 4,6-diaminido-2 phenylindole (DAPI) for 2 minutes and then washed twice with PBS. Immunostaining was evaluated by means of a Laser scanning microscope (LSM510; Carl Zeiss Meditec).

Reverse Transcription–Polymerase Chain Reaction Analysis
At various intervals after ischemia, total RNA was extracted from the retina (RNeasy micro kit; Qiagen, Hilden, Germany). DNA-free total RNA (1 µg per sample) was reversed transcribed into first-strand cDNA (iScript cDNA synthesis kit; Bio-Rad, München, Germany). The cDNA product (1 µL) was then amplified by PCR. The primer pairs were as follows: {alpha}B-crystallin, 5'-AGAGCACCTGTTGGAGTCTG-3' (forward), and 5'-TTCCTTGGTCCATTC-ACAGT-3' (reverse, BLAST, accession number S77138); HSP27, 5'-CCTGGTGTCCTCTTCCCTGT-3' (forward), and 5'-TGGTGATCTCCGCTGATTGTG-3' (reverse, BLAST, accession number M86389); HSF1, 5'-CTGGTGCACTACAC-GGCTCA-3' (forward), and 5'-GTTGTGCTGGCTTGACCTAG-3' (reverse, BLAST, accession number X83094); HSF2, 5'-GTAAGCTTGTCCGCCTGGAA-3' (forward), and 5'-ATATGCCTAGTCAGCCAGCC-3' (reverse, BLAST, accession number NM031694); β-actin, 5'-GTGGGCCGCTC-TAGGCACCA-3' (forward), 5'-CGGTTGGCCTTAGGGTTCA-GGGGGG-3' (reverse, BLAST, accession number X03765).

Western Blot Analysis
Retinas were dissected at 1, 3, 6, 24, or 72 hours or 10 days after ischemia/reperfusion with or without statin or vehicle injection. Tissue was lysed in lysis buffer containing a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Protein concentrations were determined using the Coomassie assay,22 with BSA as the standard protein.

Lysates were probed with antisera against HSP27 (1:500, Santa Cruz Biotechnologies, Heidelberg, Germany), {alpha}B-crystallin (1:10,000, Stressgen), phospho-{alpha}B-crystallin (Ser59, 1:250; Santa Cruz Biotechnology), HSP70 (1:500; Santa Cruz Biotechnology), HSP72 (1:10,000; Stressgen), and HSP90 (1:500; Santa Cruz Biotechnology), and against β-actin as loading control (1:10,000; Abcam) after blocking with 5% nonfat milk powder. Bound antibody was visualized with enhanced chemiluminescence (ECL; GE Healthcare, Little Chalfont, UK). Densitometric analysis was performed on computer (Advanced Image Data Analyzer [AIDA], ver. 3.52; Raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany). Densitometric values obtained were normalized to corresponding β-actin values to correct for differences in loading. Each immunoblot was repeated three times, to confirm the results (n = 3).

Statistical Analysis
Data are given as the mean ± SEM. Statistical significance was evaluated with ANOVA. For expressing survival-promoting effects, we defined the RGC Rescue Rate (RRR) as follows: RRR = (Nther – Ncon)/(Ntot – Ncon) x 100, where Ntot represents the total number of RGCs in unlesioned retinas, Ncon equals the total number of RGCs surviving without therapy, and Nther is the control number of RGCs surviving after a given therapy.23


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effect of Statin Treatment on IOP after Transient Retinal Ischemia
IOP was monitored during and after surgery for up to 10 days after the lesion. With the exception of simvastatin (4 mg/kg) which elicited a transient small increase after 6 days of reperfusion, neither vehicle nor treatment with other statins modified the IOP in the lesioned eye (Fig. 1) . A small increase was also observed for the contralateral unlesioned eye of animals treated with simvastatin after 3 days of reperfusion.


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		FIGURE 1. Temporal patterns of IOP during ischemia and reperfusion. IOP was evaluated during the procedure by means of an induction/impact tonometer. IOP patterns in ischemic and contralateral (sham) eyes (A) after simvastatin treatment (4 mg/kg) and (B) without statin treatment. IOP patterns in ischemic and contralateral (sham) eyes after (C) vehicle or (D) DMSO delivery. *P < 0.005 compared with basal IOP before ischemia. Values represent the mean ± SEM; n = 4 to 6. **P < 0.005 compared with the corresponding time in the ischemic eye. Isch., ischemia; Sim, simvastatin; Vehic., vehicle.

 
Statin Treatment Rescues RGCs after Acute Retinal Ischemia Reperfusion
The mean RGC density in retinas of nonsurgical animals was 1343.9 ± 110.70 RGCs/mm2. RGC density decreased to 516.3 ± 49.15 RGCs/mm2 after 10 days of reperfusion (i.e., 64% less Brn-3-positive cells than in control retinas; Fig. 2 ). The greatest cell loss was in the periphery of the retina, where 79% of the RGCs had disappeared after the lesion (Fig. 2 ; Table 1 ). Daily intraperitoneal administration of simvastatin up to 8 mg/kg did not significantly improve RGC survival 10 days after the lesion (not shown). Subcutaneous delivery of statins at 0.2 and 2 mg/kg up to 5 days during reperfusion did not reduce RGC loss after 10 days of reperfusion (Table 1) . However, at a dose of 4 mg/kg, statins increased the number of surviving RGCs by 11.6% (mevastatin), 35.9% (pravastatin), 18.2% (lovastatin), and 35.6% (simvastatin), resulting in RGC rescue rates of 18.8%, 58.3%, 29.6%, and 57.7%, respectively (Fig. 2 ; Table 1 ), when compared with untreated retinas, at 10 days of reperfusion. The mevastatin-mediated effect was not significant, and lovastatin treatment was significant only in the periphery of the retina (Fig. 2) . Simvastatin- and pravastatin-mediated effects on RGC survival were also higher in RGCs located at 5/6 of the retinal radius (i.e., the peripheral retina) compared with 1/6 (RRR at 5/6 retinal radius, 73.8% and 69.9% vs. 33.54% and 34.7% at 1/6; respectively; Fig. 2 ). This difference may be caused by elevated IOP causing damage to the optic nerve head, including depletion of neurotrophins, which differentially affects RGC survival due to longer axon fibers in the periphery than in the central retina.24 There were no significant differences in RGC survival rates between the four retinal quadrants, confirming that systemic statin treatment rescued RGCs evenly throughout the retina. Injections with vehicle did not affect RGC survival 10 days after retinal ischemia (545.4 ± 56.22 RGCs/mm2; 39.8% of nonsurgical control retinas; Table 1 ).


Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 2. Number of surviving RGCs in different experimental conditions and after treatment with different statins. The number of RGCs was determined at 1/6, 3/6, and 5/6 from the retinal radius. Bars, mean ± SEM; n = 4 to 6. *P < 0.05 compared with untreated retina; +P < 0.05 compared with ischemic vehicle-treated retina. Isch., ischemia; Mev., mevastatin; Prav., pravastatin; Lov, lovastatin; Sim, simvastatin.

 

View this table:
[in this window]
[in a new window]

  		TABLE 1. Statin-Mediated Effect on RGC Survival 10 Days after a Transient Retinal Ischemia/Reperfusion

 
The statin-mediated increase in the number of RGCs surviving 10 days after the lesion was significantly blocked by the flavonoid quercetin when delivered together with simvastatin or pravastatin up to 5 days after the lesion (994.0 ± 97.44 RGCs/mm2 for ischemia+simvastatin 4 mg/kg vs. 637.9 ± 63.83 RGCs/mm2 for ischemia+quercetin+simvastatin 4 mg/kg; 998.7 ± 54.46 RGCs/mm2 for ischemia+pravastatin 4 mg/kg vs. 729.2 ± 55.13 RGCs/mm2 for ischemia+quercetin+pravastatin 4 mg/kg; Table 1 ).

Effect of Statin Treatment on Retinal HSP Expression after Acute Retinal Ischemia/Reperfusion
{alpha}B-crystallin expression levels in untreated retinas were initially low but rapidly increased (1870% compared with untreated retinas) after 3 hours of reperfusion (Figs. 3A 3B) . Six hours after lesioning, the levels were lower than at 3 hours (34%) but still higher than in untreated retinas (650%). The levels increased further at 24 hours (1100%) compared with those in untreated retinas. Simvastatin treatment (4 mg/kg) initially lowered {alpha}B-crystallin protein levels in the retina after 3, 6, and 24 hours of reperfusion (52%, 22%, and 55%, respectively). After 72 hours of reperfusion, {alpha}B-crystallin levels were similar to those in untreated control retinas (Fig. 3C) . At this time point, simvastatin treatment induced an increase in the expression level of {alpha}B-crystallin by 200% compared with ischemic untreated retinas (Fig. 3C) . {alpha}B-crystallin expression after pravastatin treatment (4 mg/kg) showed a very similar pattern (Fig. 3B) , with an initial decrease after 3, 6, and 24 hours of reperfusion (38%, 94%, and 46%, respectively, compared with untreated retinas). Similar to simvastatin, pravastatin induced an increase in {alpha}B-crystallin expression levels at 72 hours by 380% compared with ischemic untreated retinas (Fig. 3C) . Ten days after reperfusion, {alpha}B-crystallin expression levels were 31.4% compared with those in untreated retinas. Daily delivery of simvastatin or pravastatin (4 mg/kg up to 10 days of reperfusion) increased expression levels of {alpha}B-crystallin by 195% and 210%, respectively, compared with ischemic untreated retinas (Fig. 3D) . However, expression levels were only 61% to 76% compared with untreated control retinas.


Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 3. Western blot analysis showing temporal patterns of protein expression for {alpha}B-crystallin in the retina after acute retinal ischemia and statin treatment. Protein expression levels of {alpha}B-crystallin after 3, 6, and 24 hours of reperfusion, with or without (A) simvastatin (4 mg/kg) or (B) pravastatin delivery. Protein expression levels of {alpha}B-crystallin after (C) 72 hours of reperfusion with or without simvastatin or pravastatin delivery or (D) 10 days of reperfusion with or without simvastatin or pravastatin delivery. n.t., no treatment; Isch., ischemia; Sim, simvastatin 4 mg/kg; Prav, pravastatin 4 mg/kg.

 
HSP27 expression levels in untreated retinas were low and were further reduced (12% compared with untreated retinas) after 3 hours of reperfusion (Fig. 4A) . Six hours after the lesion, the levels rose to 57% compared with the control level. The level further increased at 24 hours (300%) and 72 hours (1500%), compared with that in the untreated retinas. Simvastatin treatment initially increased levels of HSP27 expression in the retina after 6 hours of reperfusion by 260% and 250%, respectively, compared with ischemic retinas without treatment. Simvastatin delivery decreased expression levels of HSP27 after 24 and 72 hours of reperfusion to 48% and 53% compared with ischemic retinas. Pravastatin at the same concentration (4 mg/kg) had no effect on HSP27 expression levels after retinal ischemia (not shown).


Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 4. Western blot analysis showing temporal pattern of protein expression for HSP27 and -72 in the retina after acute retinal ischemia and statin treatment. Protein expression levels of HSP27 after (A) 3, 6, 24 and 72 hours of reperfusion with or without simvastatin or (B) 10 days of reperfusion with or without simvastatin delivery. HSP72 expression levels after 3, 6, and 24 hours of reperfusion with or without (C) simvastatin or (D) pravastatin delivery. n.t., no treatment; Isch., ischemia; Sim, simvastatin 4 mg/kg; Prav, pravastatin 4 mg/kg.

 
Expression levels of HSP27 after 10 days of reperfusion were significantly higher (280%) compared with those in untreated retinas and were not further modified by simvastatin treatment (Fig. 4B) . Pretreatment with quercetin reduced {alpha}B-crystallin and HSP27 expression levels after retinal ischemia and also partially blocked simvastatin- and pravastatin-mediated modulation of protein expression (Figs. 5A 5B) .


Figure 5
View larger version (9K):
[in this window]
[in a new window]

  		FIGURE 5. Western blot analysis showing the effect of quercetin on protein expression for {alpha}B-crystallin and HSP27 in the retina after acute retinal ischemia and statin treatment. Protein expression levels of {alpha}B-crystallin after (A) 3 hours of reperfusion with or without statin delivery or (B) 24 hours of reperfusion with or without statin delivery. n.t., no treatment; Isch., Ischemia; Sim, simvastatin 4 mg/kg; Prav, pravastatin 4 mg/kg; Q, quercetin.

 
HSP70, HSP72, and HSP90 in the Ischemic Retina
HSP72 expression levels were significantly increased after 3, 6, and 24 hours of reperfusion (123%) compared with untreated retinas (Figs. 4C 4 D); statin treatment with simvastatin (4 mg/kg; Fig. 4C ) or pravastatin (4 mg/kg; not shown) did not alter HSP72 expression at any time tested. HSP70 expression was significantly increased after 6 hours of reperfusion (150%), and expression remained unaltered after treatment with either simvastatin or pravastatin (4 mg/kg; Figs. 6A 6B ). HSP90 expression was not modified at any time during reperfusion and also not after statin treatment with simvastatin or pravastatin (4 mg/kg; Figs. 6C 6D ).


Figure 6
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 6. Western blot analysis showing temporal patterns of protein expression for HSP70 and HSP90 in the retina after acute retinal ischemia and statin treatment. Protein expression levels of HSP70 after 3 and 6 hours of reperfusion with or without (A) simvastatin or (B) pravastatin delivery. Protein expression levels of HSP90 after 3 and 6 hours of reperfusion with or without (C) simvastatin or (D) pravastatin delivery. n.t., no treatment; Isch., ischemia; Sim, simvastatin 4 mg/kg; Prav, pravastatin 4 mg/kg.

 
Figure 7 shows the temporal changes in the expression of {alpha}B-crystallin, HSP27, HSP72, and HSP90 in ischemic retinas and after statin treatment. The peak in {alpha}B-crystallin expression occurred after 3 hours of reperfusion and in HSP27 on day 3 after the ischemic lesion. HSP72 expression levels were increased 3 and 6 hours after ischemia and remained higher than the controls 24 hours after the lesion. HSP90 expression was not changed after the lesion or after statin treatment.


Figure 7
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 7. Densitometric analysis of the temporal patterns of protein expression for {alpha}B-crystallin and HSP27 after acute retinal ischemia and simvastatin treatment. Expression pattern for (A) {alpha}B-crystallin, (B) HSP27, (C) HSP72, and (D) HSP90. Bars, mean ± SEM; n = 3 to 4. *P < 0.05 compared with untreated retina; +P < 0.05 compared with ischemia alone. n.t., no treatment; Isch., ischemia; Sim, simvastatin.

 
Increased mRNA Expression Levels for {alpha}B-Crystallin and HSP27 after Transient Retinal Ischemia
Expression levels of mRNA for {alpha}B-crystallin were increased in the retina after 24 and 72 hours of reperfusion (Figs. 8A 8B) . Simvastatin treatment reduced expression almost to control levels 24 hours after the injury (Figs. 8A) . On the contrary, after 72 hours of reperfusion, mRNA expression levels were increased by simvastatin treatment compared with control and ischemic eyes (Fig. 8B) . Similarly, mRNA expression levels for HSP27 were increased 24 hours after the ischemic lesion, and simvastatin attenuated this increase (Fig. 8C) . At 72 hours after retinal ischemia, mRNA expression levels were increased compared with control and ischemic expression levels (Fig. 8C) .


Figure 8
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 8. Expression patterns of mRNA for {alpha}B-crystallin and HSP27 in the retina after acute retinal ischemia/reperfusion. (A, B) {alpha}B-crystallin and (C) HSP27 mRNA expression after 24 and 72 hours of reperfusion. n.t., no treatment; Vehic., vehicle; Isch., ischemia; Sim, simvastatin 4 mg/kg.

 
Treatment with quercetin also reduced mRNA expression levels for {alpha}B-crystallin 3 hours after ischemia and for HSP27 24 hours after the lesion. However, expression levels were still higher than the control level and were similar after statin treatment (Figs. 9A 9B) .


Figure 9
View larger version (12K):
[in this window]
[in a new window]

  		FIGURE 9. Expression patterns of mRNA for {alpha}B-crystallin and HSP27 in the retina after quercetin delivery and acute retinal ischemia/reperfusion. (A) {alpha}B-crystallin mRNA expression levels after 3 hours and (B) HSP27 mRNA expression levels after 24 hours of reperfusion. n.t., no treatment; Isch., ischemia; Sim, simvastatin 4 mg/kg; Prav, pravastatin 4 mg/kg; Q, quercetin.

 
{alpha}B-Crystallin Expression in Müller Cells after Transient Retinal Ischemia
Localization of {alpha}B-crystallin in sham-treated and ischemic retinas (3 hours) was evaluated by immunofluorescence analysis (Fig. 10) . Ganglion cells were labeled ex vivo with an antibody raised against microtubules derived from rat brain, which is highly reactive to neuron-specific class III β-tubulin present in neurons but not in glial cells. As shown in Figure 10A , cells in the ganglion cell layer (GCL) immunostained for the class III β-tubulin. {alpha}B-crystallin labeling was localized to the GCL and processes extending between the GCL and the inner plexiform layer, consistent with end feet and processes of Müller glia (Fig. 10B) . RGCs were not colabeled with an antibody recognizing {alpha}B-crystallin (Fig. 10D) . Figure 10E shows the pattern of labeling after immunostaining with an antibody raised against CRALBP present in Müller cells, which are the main store for this protein in the mature retina, 3 hours after an ischemic insult. End feet and processes from Müller cells were specifically stained with this antibody. As shown in Figures 10E 10F 10G 10H , a significant number of processes labeled with the anti-CRALBP antibody were also positive for {alpha}B-crystallin 3 hours after the lesion. Simvastatin treatment (4 mg/kg) significantly reduced the number of {alpha}B-crystallin-labeled processes after 3 hours of reperfusion (Figs. 10I 10J 10K 10L) .


Figure 10
View larger version (77K):
[in this window]
[in a new window]

  		FIGURE 10. Immunolocalization of {alpha}B-crystallin in the rat retina 3 hours after ischemic lesion. Immunostaining for (A) β-III-tubulin (neuronal marker) and (B) {alpha}B-crystallin. (C) DAPI staining to identify retinal layers. (D) Combined micrographs (A–C). (E) Immunostaining for CRALBP (Müller glia) and (F) {alpha}B-crystallin. (G) DAPI staining to identify retinal layers. (H) Combined micrographs (E–G). (I) Immunostaining for CRALBP (Müller glia) and (J) {alpha}B-crystallin after simvastatin delivery (4 mg/kg). (K) DAPI staining to identify retinal layers. (L) Combined micrographs (I–K). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bar represents 50 µm. Magnification, x40.

 
Increased HSF-1 and -2 mRNA Expression after Transient Retinal Ischemia
mRNA expression levels for HSF-1 and -2 were increased at 3 hours after ischemic injury (Fig. 11) . After 72 hours of reperfusion, expression levels were similar to control levels and were increased by simvastatin treatment (4 mg/kg).


Figure 11
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 11. Expression patterns of mRNA for HSF-1 and -2 in the retina after acute retinal ischemia/reperfusion, with or without simvastatin delivery. n.t., no treatment; Vehic., vehicle; Isch., ischemia; Sim, simvastatin 4 mg/kg.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The heat shock response is a conserved response to a variety of environmental and physiological challenges and results in the immediate induction of HSPs.25

Retinal injury caused by stress (e.g., intense light exposure, retinal tearing or detachment) upregulates members of the {alpha}-, β- and {gamma}-crystallin gene families.26 27 Members of the {alpha}- and β{gamma}-crystallin families are dysregulated in certain pathologic conditions, including age-related macular degeneration. {alpha}-Crystallins are also associated with several degenerative diseases, including glaucomatous optic neuropathy.4

After retinal lesions, HSP27 expression is low, whereas after ON injury, expression is upregulated.6 15 In addition, after retinal ischemia/reperfusion, expression levels of HSP70 and HSP72,28 HSP27,29 and {alpha}B-crystallin,29 crystallin30 are also upregulated in vitro. In vivo, in accordance with previous reports, we found a rapid increase in protein expression levels for {alpha}B-crystallin and HSP72 (3 hours after the lesion), -27, and -70 (6 hours after the lesion), but not HSP90. After 24 hours of reperfusion, mRNA and protein expression levels for {alpha}B-crystallin and HSP27 were still elevated. Treatment with simvastatin and pravastatin at the maximum concentration tested in this study (4 mg/kg/d) reduced protein expression levels shortly after onset of the lesion (3–6 hours) and up to 24 hours. Statin treatment significantly increased protein and mRNA expression levels 72 hours after retinal ischemia. Although the highest dose used in our study is four times the dosage commonly recommended for use in clinical medicine (1 mg/kg/d), even with prolonged administration of dosages as high as 20 mg/kg/d, no secondary effects have been reported in other experimental models.

Elevated HSP27 mRNA levels 24 (200%) and 72 hours after a brief period of retinal ischemia (5 minutes) have also been reported.29 In the same study, protein levels were also increased at 24 hours and remained elevated for up to 72 hours after the preconditioning event and then declined at 120 hours. No consistent changes in the levels of HSP70 or -90 were observed. However, as shown herein, a 75-minute period of retinal ischemia induced an increase in expression levels of HSP70, but not of HSP90, which already exhibited rather high levels in untreated retinas, compared with other HSPs.

In eukaryotic cells, the regulation of HSP genes requires the activation and translocation to the nucleus of a transregulatory protein, HSF, which recognizes modular sequence elements referred to as the HSE located within the HSP gene promoters.31 HSF-1 and -2 are present in the rat retina and are predominantly expressed in cells in the GCL, in particular in RGCs.2 We found an increase in mRNA expression levels for both HSF-1 and -2 in the retina after 3 hours of reperfusion, which was reduced after treatment with simvastatin (4 mg/kg). However, at 72 hours of reperfusion, expression levels were similar to untreated retinas. At this time point, treatment with simvastatin after the lesion increased mRNA expression for both HSF-1 and -2. Simvastatin has been shown to induce HSF-1 and increases the steady state levels of HSP70 and -90 and heme oxygenase-1 in human aortic endothelial cells in vitro.32 A similar mechanism may be involved in the statin-mediated effects described herein.

In a previous study, we found that, after ON axotomy, simvastatin increased HSP27 levels in the retina in vivo and thereby enhanced RGC survival.15 Systemic delivery of quercetin partially blocks statin-mediated neuroprotection of RGCs.15 In the present study, we have shown that systemic delivery of quercetin also significantly blocked simvastatin-mediated increase in RGC survival after retinal ischemia/reperfusion. Lesion-induced increase of {alpha}B-crystallin and HSP27 expression and statin-mediated modulation were also downregulated after quercetin delivery both at protein and mRNA levels. Since quercetin specifically inhibits or downregulates transcription factor HSF-1 activation,33 depending on the cell type, these results further support the notion that this mechanism is involved in the statin-mediated increase in {alpha}B-crystallin and the HSP27 expression levels shown in this study.

Ischemia/reperfusion injury to the retina causes degeneration of inner retinal cells including RGCs, and multiple cytokines such as IL-1β, IL-18, TNF, IFN-{gamma}, TGF-β, and IL-6 are upregulated.34 35 Semiquantitative real-time RT-PCR showed that after acute retinal ischemia/reperfusion injury, endogenous IL-6 mRNA levels increase between 2 and 18 hours after injury.36 After elevated hydrostatic pressure in the eye, glial cells secrete TNF as well as other noxious agents such as nitric oxide and facilitate apoptotic death of RGCs.37

Since several lines of evidence suggest that statins may attenuate the inflammatory cytokine responses that accompany cerebral ischemia,38 a similar mechanism could be involved in the modulation of HSPs expression found in this study.

Overexpression of {alpha}B-crystallin protects retinal pigment epithelial cells from stress-induced apoptosis.39 Consistent with these findings, increased levels of {alpha}B-crystallin have been found in the rd mouse, a model of inherited retinal degeneration.40 Increased mRNA levels were localized to glial cells in the degenerative state. In addition, a recent study has indicated that ocular hypertension modulates expression of {alpha}- and β-crystallins, and that expression of these genes is predominantly localized to the cells in the GCL and to a lesser degree in the inner and outer nuclear layers. In the GCL crystallin-positive cells were identified as RGCs.41

In this study, {alpha}B-crystallin expression localized to end feet and processes of Müller cells, but not to retinal neurons. The increased localization of immunoreactive {alpha}B-crystallin to Müller cells after an ischemic insult suggests a rapid onset of glial response to neuronal damage and may consist of a defensive mechanism to the stress insult.

{alpha}B-crystallin is a negative regulator of apoptosis, and studies have shown the central role of this chaperone in linking the differentiation program with apoptosis resistance in muscle cells.42 By overexpressing {alpha}B-crystallin in cells subjected to stress, this protein was found to stall the maturation of caspase-3, a central protease involved in apoptosis.43

At the concentration used in our study, a small transient increase in the IOP after simvastatin delivery was observed in the ischemic and contralateral eyes. The IOP increase in the ischemic eye due to simvastatin delivery was time correlated with the increased {alpha}B-crystallin expression observed at this time point. Because no similar IOP increment was found in control or vehicle treated animals, it raises the question about the possible reason for this increment.

In contrast to the known anti-inflammatory properties of statins, several recent studies have shown that the lipophilic statins such as fluvastatin, simvastatin, atorvastatin, and lovastatin can induce proinflammatory responses in several paradigms.44 Mechanisms involve activation of caspase-1 and enhancement in the secretion of inflammatory cytokines such as IL-1β, IL-18, and the Th1 cytokine IFN-{gamma}. Whether this or a similar mechanism could be involved in the IOP increase after chronic statin treatment is a matter of active research.

Differential effects achieved by statins are probably due to molecular differences that contribute to differences in their efficacy and safety and are also likely to affect differentially the non-LDL pleiotropic effects observed in this and other studies including clinical ones.45 Also, differences in cerebrovascular permeabilities and membrane transport mechanisms of statins have been described.46 The lipophilic statins simvastatin and lovastatin are normally administered as inactive lactone prodrugs of the active hydroxy acid forms, simvastatin acid and lovastatin acid, and can readily enter the brain via a simple diffusion mechanism, whereas the hydrophilic molecule pravastatin cannot. Statins in the acid form (simvastatin acid and lovastatin acid) are transported by a carrier-mediated transport system, for which pravastatin has a low affinity. Mevastatin is also lipophilic, but to a lesser extent than simvastatin. Since simvastatin, lovastatin, and mevastatin were converted to their acid form before delivery, the transport mechanism described cannot account for the differential effects described herein. Another possibility is the efflux mechanism across the blood–brain barrier. Efflux of pravastatin occurs through multiple transporters, including rOat3 and rOatp2, expressed at the blood–brain barrier. Differential effects seen in this study could be related to the rate of influx and efflux from statins across the blood–retina barrier.47

In summary, these results show that systemic delivery of statins after a transient period of retinal ischemia induced by increased IOP significantly enhances RGC survival. The ischemic insult modified the expression of several members of the family of HSPs in the retina including {alpha}B-crystallin and HSP72, -70, and -27. The earliest response after the lesion was observed for {alpha}B-crystallin and HSP72, however, only {alpha}B-crystallin and HSP27 expression levels were modified by statin delivery after retinal ischemia/reperfusion. Upregulated expression of HSP70 and -72 is probably part of the cellular response to stress; however it does not appear to play a role in the statin-mediated neuroprotective effects described herein.

Time-dependent statin-mediated effects fall into two classes: effects during the acute phase after the retinal ischemic insult, probably involving a downregulation of stress signals due to protective actions on the endothelium, stabilization of the blood–retina barrier and attenuation of cytokine inflammatory responses, and effects during reperfusion in the chronic phase which involve upregulation of {alpha}B-crystallin by activation of HSF-1 and -2 and downregulation of HSP27 expression.

Statin-mediated neuroprotective effects on RGCs also remain to be further evaluated at the functional level, to determine whether the surviving cells are still capable of processing visual information, or if the cell death process is simply delayed by the microenvironment-supportive properties of statins, as is the case with many neuroprotective and neurotrophic factors.48 Statins may be useful to prevent some of the neuronal damage in the acute and possibly also delayed phases and may have beneficial effects in several CNS conditions, including those directly affecting the visual system.


   Acknowledgements
 
The authors thank Alexandra Kretz for helpful comments on this manuscript.


   Footnotes
 
Supported by the Bundesministerium für Bildung und Forschung (BMBF) and the IZKF (Interdisciplinary Centre for Clinical Research), the Ernst-Abbe-Foundation, and the Humboldt Foundation.

Submitted for publication December 13, 2007; revised June 7, 2008; accepted August 27, 2008.

Disclosure: C. Schmeer, None; A. Gámez, None; S. Tausch, None; O.W. Witte, None; S. Isenmann, 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.

Corresponding author: Christian Schmeer, Department of Neurology, Neuroregeneration Laboratory, University of Jena Medical School, Erlanger Allee 101, 07747 Jena, Germany; christian.schmeer{at}med.uni-jena.de.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Parcellier A, Gurbuxani S, Schmitt E, Solary E, Garrido C. Heat shock proteins, cellular chaperones that modulate mitochondrial cell death pathways. Biochem Biophys Res Commun. 2003;304:505–512.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  2. Kwong JM, Lalezary M, Nguyen JK, et al. Co-expression of heat shock transcription factors 1 and 2 in rat retinal ganglion cells. Neurosci Lett. 2006;405:191–195.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  3. Beere HM. Death versus survival: functional interaction between the apoptotic and stress-inducible heat shock protein pathways. J Clin Invest. 2005;115:2633–2639.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  4. Andley UP. Crystallins in the eye: function and pathology. Prog Retin Eye Res. 2007;26:78–98.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  5. Robinson ML, Overbeek PA. Differential expression of alphaA- and alphaB-crystallin during murine ocular development. Invest Ophthalmol Vis Sci. 1996;37:2276–2284.[Abstract/Free Full Text]
  6. Krueger-Naug AM, Emsley JG, Myers TL, Currie RW, Clarke DB. Injury to retinal ganglion cells induces expression of the small heat shock protein Hsp27 in the rat visual system. Neuroscience. 2002;110:653–665.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  7. Lewis SE, Mannion RJ, White FA, et al. A role for HSP27 in sensory neuron survival. J Neurosci. 1999;19:8945–8953.[Abstract/Free Full Text]
  8. Arrigo AP. Small stress proteins: chaperones that act as regulators of intracellular redox state and programmed cell death. Biol Chem. 1998;379:19–26.[Web of Science][Medline][Order article via Infotrieve]
  9. Zantema A, Verlaan-De Vries M, Maasdam D, Bol S, van der Eb A. Heat shock protein 27 and alphaB-crystallin can form a complex, which dissociates by heat shock. J Biol Chem. 1992;267:12936–12941.[Abstract/Free Full Text]
  10. Delanty N, Vaughan CJ. Vascular effects of statins in stroke. Stroke. 1997;28:2315–2320.[Abstract/Free Full Text]
  11. Sacks FM, Pfeffer MA, Moye LA, et al. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels: Cholesterol and Recurrent Events Trial investigators. N Engl J Med. 1996;335:1001–1009.[Abstract/Free Full Text]
  12. Schmeer C, Kretz A, Isenmann S. Statin-mediated protective effects in the central nervous system: general mechanisms and putative role of stress proteins. Restor Neurol Neurosci. 2006;24:79–95.[Web of Science][Medline][Order article via Infotrieve]
  13. Schmeer C, Kretz A, Isenmann S. Therapeutic potential of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors for the treatment of retinal and eye diseases. CNS Neurol Disord Drug Targets. 2007;6:282–287.[CrossRef][Medline][Order article via Infotrieve]
  14. Wang X, Tokuda H, Hatakeyama D, et al. Mechanism of simvastatin on induction of heat shock protein in osteoblasts. Arch Biochem Biophys. 2003;415:6–13.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  15. Kretz A, Schmeer C, Tausch S, Isenmann S. Simvastatin promotes heat shock protein 27 expression and Akt activation in the rat retina and protects axotomized retinal ganglion cells in vivo. Neurobiol Dis. 2006;21:421–430.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  16. Honjo M, Tanihara H, Nishijima K, et al. Statin inhibits leukocyte-endothelial interaction and prevents neuronal death induced by ischemia-reperfusion injury in the rat retina. Arch Ophthalmol. 2002;120:1707–1713.[Abstract/Free Full Text]
  17. Kawai SI, Vora S, Das S, Gachie E, Becker B, Neufeld AH. Modeling of risk factors for the degeneration of retinal ganglion cells after ischemia/reperfusion in rats: effects of age, caloric restriction, diabetes, pigmentation, and glaucoma. FASEB J. 2001;15:1285–1287.[Abstract/Free Full Text]
  18. Kontiola AI, Goldblum D, Mittag T, Danias J. The induction/impact tonometer: a new instrument to measure intraocular pressure in the rat. Exp Eye Res. 2001;73:781–785.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  19. Leahy KM, Ornberg RL, Wang Y, et al. Quantitative ex vivo detection of rodent retinal ganglion cells by immunolabeling Brn-3b. Exp Eye Res. 2004;79:131–140.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  20. Isenmann S, Klöcker N, Gravel C, Bähr M. Short communication: protection of axotomized retinal ganglion cells by adenovirally delivered BDNF in vivo. Eur J Neurosci. 1998;10:2751–2756.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  21. Schmeer C, Straten G, Kügler S, Gravel C, Bähr M, Isenmann S. Dose-dependent rescue of axotomized rat retinal ganglion cells by adenovirus-mediated expression of glial cell-line derived neurotrophic factor in vivo. Eur J Neurosci. 2002;15:637–643.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  22. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  23. Klöcker N, Bräunling F, Isenmann S, Bähr M. In vivo neurotrophic effects of GDNF on axotomized retinal ganglion cells. Neuroreport. 1997;8:3439–3442.[Web of Science][Medline][Order article via Infotrieve]
  24. Johnson EC, Deppmeier LM, Wentzien SK, Hsu I, Morrison J. Chronology of optic nerve head and retinal responses to elevated intraocular pressure. Invest Ophthalmol Vis Sci. 2000;41:431–442.[Abstract/Free Full Text]
  25. Morimoto RI, Santoro MG. Stress-inducible responses and heat shock proteins: new pharmacologic targets for cytoprotection. Nat Biotechnol. 1998;16:833–838.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  26. Sakaguchi H, Miyagi M, Darrow RM, et al. Intense light exposure changes the crystallin content in retina. Exp Eye Res. 2003;76:131–133.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  27. Vazquez-Chona F, Song BK, Geisert EE, Jr. Temporal changes in gene expression after injury in the rat retina. Invest Ophthalmol Vis Sci. 2004;45:2737–2746.[Abstract/Free Full Text]
  28. Lewden O, Garcher C, Assem M, Morales C, Rochette L, Bron AM. Changes of the inducible heat shock protein 70 mRNA level in rat retina after ischemia and reperfusion. Ophthalmic Res. 1998;30:291–294.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  29. Li Y, Roth S, Laser M, Ma JX, Crosson CE. Retinal preconditioning and the induction of heat-shock protein 27. Invest Ophthalmol Vis Sci. 2003;44:1299–1304.[Abstract/Free Full Text]
  30. Yu AL, Fuchshofer R, Birke M, et al. Hypoxia/reoxygenation and TGF-beta increase alphaB-crystallin expression in human optic nerve head astrocytes. Exp Eye Res. 2007;84:694–706.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  31. Morimoto RI. Cells in stress: transcriptional activation of heat shock genes. Science. 1993;259:1409–1410.[Free Full Text]
  32. Uchiyama T, Atsuta H, Utsugi T, et al. Simvastatin induces heat shock factor 1 in vascular endothelial cells. Atherosclerosis. 2006;188:265–273.[Medline][Order article via Infotrieve]
  33. Nagai N, Nakai A, Nagata K. Quercetin suppresses heat shock response by down regulation of HSF1. Biochem Biophys Res Commun. 1995;208:1099–1105.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  34. Hangai M, Yoshimura N, Yoshida M, Yabuuchi K, Honda Y. Interleukin-1 gene expression in transient retinal ischemia in the rat. Invest Ophthalmol Vis Sci. 1995;36:571–578.[Abstract/Free Full Text]
  35. Hangai M, Yoshimura N, Honda Y. Increased cytokine gene expression in rat retina following transient ischemia. Ophthalmic Res. 1996;28:248–254.[Web of Science][Medline][Order article via Infotrieve]
  36. Sanchez RN, Chan CK, Garg S, et al. Interleukin-6 in retinal ischemia reperfusion injury in rats. Invest Ophthalmol Vis Sci. 2003;44:4006–4011.[Abstract/Free Full Text]
  37. Tezel G, Wax MB. Increased production of tumor necrosis factor-alpha by glial cells exposed to simulated ischemia or elevated hydrostatic pressure induces apoptosis in cocultured retinal ganglion cells. J Neurosci. 2000;20:8693–8700.[Abstract/Free Full Text]
  38. Vaughan CJ, Delanty N. Neuroprotective properties of statins in cerebral ischemia and stroke. Stroke. 1999;30:1969–1973.[Abstract/Free Full Text]
  39. Yoshimura N, Kikuchi T, Kuroiwa S, Gaun S. Differential temporal and spatial expression of immediate early genes in retinal neurons after ischemia-reperfusion injury. Invest Ophthalmol Vis Sci. 2003;44:2211–2220.[Abstract/Free Full Text]
  40. Jones SE, Jomary C, Grist J, Thomas MR, Neal MJ. Expression of alphaB-crystallin in a mouse model of inherited retinal degeneration. Neuroreport. 1998;9:4161–4165.[Web of Science][Medline][Order article via Infotrieve]
  41. Piri N, Song M, Kwong JM, Caprioli J. Modulation of alpha and beta crystallin expression in rat retinas with ocular hypertension-induced ganglion cell degeneration. Brain Res. 2007;1141:1–9.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  42. Kamradt MC, Chen F, Sam S, Cryns VL. The small heat shock protein alphaB-crystallin negatively regulates apoptosis during myogenic differentiation by inhibiting caspase-3 activation. J Biol Chem. 2002;277:38731–38736.[Abstract/Free Full Text]
  43. Kamradt MC, Lu M, Werner ME, et al. The small heat shock protein alphaB-crystallin is a novel inhibitor of TRAIL-induced apoptosis that suppresses the activation of caspase-3. J Biol Chem. 2005;280:11059–11066.[Abstract/Free Full Text]
  44. Coward WR, Marei A, Yang A, Vasa-Nicotera MM, Chow SC. Statin-induced proinflammatory response in mitogen-activated peripheral blood mononuclear cells through the activation of caspase-1 and IL-18 secretion in monocytes. J Immunol. 2006;176:5284–5292.[Abstract/Free Full Text]
  45. Mason RP. Molecular basis of differences among statins and a comparison with antioxidant vitamins. Am J Cardiol. 2006;98:34P–41P.[Web of Science][Medline][Order article via Infotrieve]
  46. Tsuji A, Saheki A, Tamai I, Terasaki T. Transport mechanism of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors at the blood-brain barrier. J Pharmacol Exp Ther. 1993;267:1085–1090.[Abstract/Free Full Text]
  47. Kikuchi R, Kusuhara H, Abe T, Endou H, Sugiyama Y. Involvement of multiple transporters in the efflux of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors across the blood-brain barrier. J Pharmacol Exp Ther. 2004;311:1147–1153.[Abstract/Free Full Text]
  48. Isenmann S, Kretz A, Cellerino A. Molecular determinants of retinal ganglion cell development, survival, and regeneration. Prog Retin Eye Res. 2003;22:483–543.[CrossRef][Web of Science][Medline][Order article via Infotrieve]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
iovs.07-1597v1
49/11/4971    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schmeer, C.
Right arrow Articles by Isenmann, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schmeer, C.
Right arrow Articles by Isenmann, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS