Reduction of Apoptosis in Ischemic Retinas of Two Mouse Models Using Hyperbaric Oxygen Treatment

Abstract

Purpose.: To investigate the effect of hyperbaric oxygen (HBO) chamber treatment in mouse models of retinal ischemia.

Methods.: Unilateral central retinal artery occlusion (CRAO) or optic nerve crush (ONC) was induced in 50 mice each, of which 30 were treated with 100% oxygen at 2 atm for 90 minutes immediately after injury and then daily for up to 14 days. Mice were euthanatized on days 1, 3, and 21 for histologic analysis, apoptosis assay, and quantitative real-time polymerase chain reaction test. Findings were analyzed by injury and by treatment.

Results.: HBO treatment reduced cell loss from 58% to 30% in the CRAO model and from 52% to 32% in the ONC model. In both models, it was associated with significantly increased cell survival in the retinal ganglion cell layer. Expression levels of the proapoptosis genes (bax, caspase-3) decreased minimally in the HBO-treated CRAO mice on day 1, but this trend was reversed on day 3. In the ONC group, levels of caspase-3, bax, and bcl-x increased on day 1 and dropped below baseline on day 3. The pattern of changes in the expression levels of the ischemia- and oxidative-stress–related genes (HO-1, SOD-1, GPX-1, NOX-2) and the effectiveness of HBO treatment varied by model. Overall, however, gene expression levels that increased in the untreated mice increased further with HBO treatment and levels that decreased, decreased further with treatment.

Conclusions.: HBO treatment protects injured neuronal cells from apoptosis. Response to treatment differs molecularly after ONC or CRAO. These results should prompt clinical trials of acute ischemic retinal damage.

Central retinal artery occlusion (CRAO) is one of the few emergency situations in clinical ophthalmology associated with acute vascular occlusion. ¹ It usually manifests acutely as a severe, painless, monocular visual loss. ^1,2 The visual prognosis is poor and there is no available treatment. ² Histologically, the affected cells undergo necrosis and apoptosis, after which these processes then progress to adjacent cells, resulting in secondary spread of the damage. ³  

Animal models of CRAO have been used to investigate the mechanisms underlying acute retinal ischemia and its possible treatment. In an early study, Hayreh and Jonas ⁴ mechanically induced CRAO in monkeys. Later, Iliaki et al. ⁵ laser-activated an injected dye in rabbits to cause photothrombosis. A similar model was described by Daugeliene et al. ⁶ in rats. Recently, our group characterized a mouse model of CRAO wherein rose bengal dye was injected intravenously (IV), and a laser beam was directed over the optic nerve head to activate the dye, which then released free radicals, injuring the microvascular endothelium and leading to thrombosis. ⁷ This caused neuronal cell loss, partly by apoptosis, in the inner retinal layers and retinal thinning. 

Other researchers induced neuronal cell loss in the inner retina by compressing the optic nerve posterior to the globe. This resulted in a combined mechanical injury to the axons with an ischemic component due to transient obliteration of the central retinal artery. As in the CRAO model, histologic study revealed processes of necrosis and apoptosis, ⁸ mainly in the retinal ganglion cell (RGC) layer. 

In molecular and genetic studies of animal models of retinal ischemia, ^{6,9 –12} researchers reported an upregulation of the proapoptotic protein bax in response to apoptosis signals, leading to an increase in the expression of caspase-3 and other caspases downstream in the final common pathway. ^13,14 The antiapoptotic proteins bcl-2 and bcl-xL inhibited apoptosis by preventing the formation of the bax-induced cascade. 

The identification of genes differentially regulated during ischemia can improve understanding of the cell death pathways. ¹⁵ The most relevant ischemia-related genes are heme-oxygense-1 (HO-1) and hypoxia-inducible factor 1a (HIF-1a). Important oxidative-stress–related genes are CuZn–superoxide dismutase-1 (SOD-1), glutathione peroxidase-1 (GPX-1), and gp91 nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase organizer 1 (NOX-2). HO-1 metabolizes heme to carbon monoxide and antioxidants and exerts a neuroprotective effect by regulating blood perfusion under ischemic conditions. ^16,17 Its expression is closely related to changes in HIF-1a, ^18,19 an oxygen-regulated transcriptional activator that plays a pivotal role in mammalian physiology and disease. The transcription and synthesis of HIF-1a are constitutive and not affected by oxygen levels. ^{20 –22} SOD-1 converts the superoxide radical to hydrogen peroxide, which in turn is converted to water and oxygen by catalase and GPX-1 in the presence of glutathione. ^23,24 All these processes are necessary for complete antioxidation to take place. The HO-1, SOD-1, and GPX-1 genes are located downstream to the NADPH oxidase cascade, a major enzymatic source of superoxide anion production in the brain, leading to the generation of reactive oxygen species. The NADPH oxidases are comprised of a membrane-bound catalytic subunit that transfers electrons from NADPH to molecular oxygen (Nox-1 to Nox-5). At least three isoforms of NADPH oxidase are expressed in the blood vessel wall. They differ from one another in both the Nox homolog that they use (Nox-1 to Nox-4) and their reliance on specific regulatory subunits. Nox-1– and Nox-2–containing NADPH oxidases display a restricted pattern of localization: Nox-1 is confined to vascular smooth muscle cells and, possibly, endothelial cells, and Nox-2 is expressed in endothelial cells and adventitial fibroblasts and invading inflammatory cells of developing atherosclerotic lesions. They are normally expressed in low levels in the vasculature, but are upregulated in cardiovascular risk settings, such as hypertension, diabetes, and hyperlipidemia. ²⁵  

The administration of oxygen at high atmospheric pressures increases oxygen transport into tissues. Hyperbaric oxygen (HBO) chamber treatment is used in various acute and chronic diseases, such as decompression sickness and air embolism, and for chronic skin wounds in diabetic patients. It was recently found to have a neuroprotective effect in ischemic conditions of the central nervous system, such as stroke, ²⁶ acute cerebral ischemia, ²⁷ and cardiovascular ischemic diseases. ^28,29  

In the eye, HBO chamber treatment has been limited to retinal edema and scleral necrosis. ³⁰ Weinberger et al. ³¹ and Beiran et al. ³² suggested that it may be applicable for ischemic CRAO. However, the current lack of efficacy data prevents its clinical use. The aim of this study was to investigate the neuroprotective effect of HBO for the treatment for ischemic retina in mouse models of CRAO and optic nerve crush (ONC). 

Methods

Animals

All protocols were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research and were approved and monitored by the Animal Care Committee of Rabin Medical Center. 

In all, 109 adult male C57bl/6 mice aged 6 weeks and weighing 25 to 30 kg were purchased from Harlan Laboratories, Jerusalem, Israel; 6 adult Thy1 transgenic mice labeled with cyan fluorescent protein (CFP) were kindly provided by Steven Bernstein, University of Maryland, Baltimore, MD. All animals were housed under a 14-hour-light/10-hour-dark cycle with standard chow and water without restriction. 

Study Design

A total of 100 C57bl/6 mice were used in the main experiments. CRAO and ONC were induced in 50 mice each, and 30 mice in each group were treated with HBO. Another 9 C57b1/6 mice underwent ONC followed by treatment with air. ONC was also induced in 6 CFP-Thy-1 transgenic mice, 3 of which were treated with HBO. In all cases, injury was induced in the right eye and the left undamaged eye served as an internal control. Mice were euthanatized on days 1, 3, and 21 for histologic and molecular analysis and measurements of apoptosis-related genes by quantitative real-time polymerase chain reaction (RT-qPCR). The breakdown of the groups and timing of the analyses are shown in Table 1. Findings were compared by injury (yes/no), type of injury (CRAO/ONC), treatment (HBO yes/no), and type of treatment (HBO/air). 

Table 1.

 

Study Design

Model	Treatment	Analysis	Number of Mice
			Day 1	Day 3	Day 21
C57b1/6 mice
CRAO	No HBO	H&E	3	3	3
		TUNEL	3	3
		CASP-3	3	3
		Molecular	12	5
	HBO	H&E	3	3	3
		TUNEL	3	3
		CASP-3	3	3
		Molecular	5	4
ONC	No HBO	H&E	3	3	6
		TUNEL	3	3
		CASP-3	3	3
		Molecular	4	4
	HBO	H&E	3	3	3
		TUNEL	3	3
		CASP-3	3	4
		Molecular	6	4
CFP-Thy-1 transgenic mice
ONC	No HBA	Flat-mount			3
	HBA	Flat-mount			3

 HBA, hyperbaric air; CASP, caspase; H&E, hematoxylin and eosin.

Induction of CRAO

The CRAO model has been previously described. ⁷ Briefly, mice were anesthetized with intramuscular ketamine chloride 80 mg/kg (Fort Dodge Laboratories, Fort Dodge, IA) and xylazine 4 mg/kg (Veterinary Medicines Directorate [VMD], Surrey, UK). A fluorescein photosensitizing pink dye (rose bengal dye; Sigma, St. Louis, MO) dissolved in 0.9% saline at a concentration of 20 mg/mL was injected IV (2.5 mM in PBS, 1 mL/kg) and activated (using a contact lens) by a laser beam (532 nm, 200 mm spot size, 150 mW power) directed toward the optic nerve head. 

Induction of ONC

The induction of ONC has been previously described. ⁸ In brief, mice were anesthetized with 80 mg/kg ketamine (Fort Dodge Laboratories) and 4 mg/kg xylazine (VMD), and the right optic nerve was crushed by applying forceps at a point 2.5–3.0 mm posterior to the globe for 7 seconds. This procedure was repeated three times. 

HBO Chamber Treatment

Treatment was performed in a hyperbaric chamber with 100% oxygen at 2 atm for 90 minutes, administered twice during the first day after injury induction (CRAO or ONC), with a 3-hour interval between sessions, and once daily thereafter for up to 14 days. 

Hyperbaric Air Chamber Treatment

Hyperbaric air treatment was performed with 20% oxygen at 2 atm for 90 minutes, twice during the first day after injury induction (ONC), at a 3-hour interval, and once daily thereafter for up to 14 days. 

Histologic Examination

Both eyes were enucleated and embedded in paraffin, and 5-mm-thick sagittal sections were stained with hematoxylin and eosin and examined under a light microscope. Cells were counted in the RGC layer (horizontal counting) and inner and outer nuclear layers (vertical counting) in three sections (5 μm) of every 30 consecutive sections per slide, for a total of 7 to 10 slides (30 sections per eye). Total retinal thickness was measured from the internal to the external limiting membrane. 

Flat-mount Retina Analysis

Twenty-one days after induction of ONC, the eyes of the CFP-Thy-1 transgenic mice (three HBO-treated) were enucleated and fixed immediately in 4% paraformaldehyde in 0.1 M PBS for 2 hours at room temperature. After the cornea and lens were removed, four radial relaxing incisions were made. The retina was prepared as a flattened whole-mount on a glass slide with a coverslip, and an image was obtained with a fluorescence microscope (Fluoview X; Olympus, Tokyo, Japan) using appropriate filters. Eight square areas of 370 × 370 μm were selected, four at a distance of 0.5 mm from the optic nerve head and four at distance of approximately 2 mm, and the cells were counted manually. ³³ Results for the four quadrant areas were averaged and the number of RGCs was quantified. To verify the findings, the retinas were stained with 4′,6′-diamidino-2-phenindole (DAPI) nuclear dye (Vector Laboratories, Burlingame, CA) and the RGC number was assessed under a fluorescence microscope by averaging the RGC counts from at least three fields. 

Apoptosis Assay

Longitudinal cross-sections from paraffin-embedded eyes were cut 5 μm thick for in situ TdT-mediated dUTP nick end-labeling (TUNEL) assay (Roche Diagnostics, Mannheim, Germany). Staining was performed with the fluorescein-tagged apoptosis detection system. DAPI stain was used to identify nuclear changes. Results were analyzed with a fluorescence microscope (Fluoview X) at 580-nm wavelength. The mean number of TUNEL-positive cells per slide was determined in consecutive sections; special attention was paid to each retinal layer. 

Caspase-3 Immunostaining

Cross-sections of paraffin-embedded tissue were deparaffinized, blocked, and incubated with polyclonal rabbit antiactive caspase-3 antibody (MBL International Corp., Woburn, MA), which specifically recognizes the active form of caspase-3 (at a dilution of 1:150) in PBS. Cy-3–labeled goat anti-rabbit antibody (ab6939; Abcam, Cambridge, UK) was used as a secondary antibody at 1:200 dilutions in PBS. Counterstaining with DAPI was performed to visualize nuclear morphology. For quantitative analysis, active caspase-3–positive cells were counted in three consecutive sections at high-power magnification (×40). 

RT-qPCR

RT-qPCR was used to study the expressions of the apoptosis-related genes bax, bcl-2, bcl-xL, and caspase-3, and the ischemia- and stress-related genes HO-1, SOD-1, GPX-1, and NOX-2. Immediately after euthanatization, the retinas were frozen in liquid nitrogen. Total RNA was extracted with a commercial reagent (TRIzol; Invitrogen, Life Technologies, Carlsbad, CA), followed by reverse-transcription into cDNA using random hexamers, according to the manufacturer's protocol (Amersham Biosciences, Cardiff, UK), and Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). Thereafter, cDNA was analyzed using a sequence detection system (ABI Prism 7900; Applied Biosystems, Inc., Foster City, CA). To measure gene expression, cDNA input levels were normalized against mouse beta actin, a housekeeping gene known to be stable under ischemic conditions. ²³ The primer list is shown in Table 2. Reactions were performed in a 20 mL volume containing 4 mL cDNA, 1 mL each of forward and reverse primers, and buffer included in a master mix (SYBR Green I; Applied Biosystems). PCR cycling conditions were as follows: initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 1-minute denaturation at 95°C and 1 minute of annealing and extension at 60°C. Duplicate transcriptase-based quantitative PCR reactions were performed for each gene to minimize individual tube variability and an average was taken for each time point. Standard curves were obtained using untreated mouse cDNA for each gene PCR assay. The results were quantified by a comparative C _t method (2ddC _t method), ^34,35 where ddC _t = dC _t (sample) − dC _t (reference gene). 

Table 2.

 

Primer Pairs Used for Molecular Analysis

Gene	Forward	Reverse
ACTB	TAG GCA CCA GGG TGT GAT GGT	CAT GTC GTC CCA GTT GGT AAC A
Caspase-3	ATG GGA GCA AGT CAG TGG AC	CGT ACC AGA GCG AGA TGA CA
Bax	CTG AGC TGA CCT TGG AGC	GAC TCC AGC CAC AAA GAT G
Bcl-2	CCT GTG GAT GAC TGA GTA CCT	GAG CAG GGT CTT CAG AGA CA
Bcl-xL	GCA TCG TGG CCT TTT TCT CC	CGA CTG AAG AGT GAG CCC AG
SOD-1	GCC CGG CGG ATG AAG A	CGT CCT TTC CAG CAG TCA CA
GPX-1	CGG TTT CCC GTG CAA TC	GAG GGA ATT CAG AAT CTC TTC AT
NOX-2	TTG CAA GTG AAC ACC CTA ACA C	TGG CAG CAT ACA CTG GTT TC
HO-1	CAG GTG TCC AGA GAA GGC TTT	TCT TCC AGG GCC GTG TAG AT

Statistical Analysis

All data are expressed as mean ± SD. The results were statistically analyzed with analytical software (SPSS for Windows, version 15.0.1; SPSS Inc., Chicago, IL), using two-way ANOVA with group and time as the independent variables. Differences between groups were estimated by Student's t-test. Because of the relatively small size of the sample, we applied the nonparametric Mann–Whitney U test for two independent samples. A value of P < 0.05 was considered statistically significant. 

Results

Hematoxylin and Eosin Staining

Cell Loss.

The rate of cell loss on day 21 in the injured untreated (right) eyes relative to the intact (left) eyes was 58% in the CRAO model (P < 0.05) and 52% in the ONC model (P < 0.05) (Table 3, Fig. 1). On day 1 after HBO treatment (n = 30 in each group), there was no cell loss in either model. On day 3, the cell loss in the injured treated (right) eyes relative to the intact (left) eyes was 18% in the CRAO model and 6% in the ONC model. These values rose to 30% and 32%, respectively, on day 21 (Table 3, Fig. 1). 

Table 3.

 

Mean RGC Loss and Mean Retinal Thickness on Day 21 after Injury with and without HBO Treatment

Model	Cell Loss in RGC Layer	Retinal Thinning	RGC Loss on Flat-mount
			Center	Periphery
C57b1/6 mice
CRAO
No treatment	58 ± 7%	26.6%
HBO treatment	30 ± 11%*	7.5%*
ONC
No treatment	52 ± 10%	16.9%
HBO treatment	32 ± 2%*	2.5%*
HBA treatment	23 ± 2%*
CFP-Thy-1 transgenic mice
ONC
No treatment			78 ± 9%	68 ± 4%
HBO treatment			24 ± 6%*	15 ± 5%*

 Values are mean ± SD.

*   P < 0.05 (a significant protective effect on survival of RGCs and preservation of retinal thickness).

Figure 1.

 

Histologic study on day 21 after injury induction with and without HBO treatment. (C, F) Increased cell loss in the RGC layer in the untreated HBO eyes. (B, E) HBO-treated eyes. (A, D) Normal retina of uninjured left control eye. Arrows show areas of cell loss. Hematoxylin and eosin staining (×20).

Retinal Thickness.

In the CRAO model, mean retinal thickness on day 21 was 152 μm in the treated injured eyes and 165 μm in the intact (left) eyes, for a 7.5% reduction in retinal thickness, compared with a 26.6% reduction in the untreated eyes (91 μm in right eye vs. 124 μm in left eye). This difference was statistically significant (P < 0.05) (Table 3). In the ONC model, mean retinal thickness on day 21 was 157 μm in the treated injured (right) eyes and 161.5 μm in the intact (left) eyes, representing a 2.5% reduction, compared with 16.9% in the untreated mice (133.8 μm in right eye vs. 160 μm in left eye) (P = 0.05). 

Flat-mounted Retinas

RGC quantification in the untreated CFP-Thy-1 transgenic mice on day 21 after ONC revealed a 78% cell loss in the central area of the retina and a 68% cell loss in peripheral areas. In the HBO-treated CFP-Thy-1 transgenic mice, there was a 24% RGC loss in the central area (P < 0.005) and a 15% loss in the periphery (P < 0.005) (Table 3, Fig. 2). 

Figure 2.

 

Flat-mounted retinas of CFP-Thy-1 transgenic mice on day 21 after induction of ONC. (A) HBO untreated eye after induction of ONC injury. Approximately 75% RGC loss was detected. (B) HBO-treated eye shows RGC loss of approximately 20%. (C, D) Immunohistochemistry of activated caspase-3 in flat-mount retina 3 days after induction of ONC (×10, ×40). Note the positive staining (red) in diffuse areas of the retina.

Apoptosis (TUNEL) Assay

In the CRAO model, the mean apoptosis values on TUNEL assay in the untreated injured eyes relative to the intact (left) eyes were 4.1% on day 1 and 13.2% on day 3. Corresponding rates for the treated mice were 0% (NS) and 0.6% (P < 0.05) (Table 4). In the ONC model, mean apoptosis values in the untreated injured eyes relative to the intact (left) eyes were 3.6% on day 1 and 14.8% on day 3. Corresponding values in the treated mice were 8.1% (NS) and 0.4% (P = 0.08). In both models, on day 3, the treated eyes had fewer apoptotic cells than those of the untreated injured eyes (Table 4, Fig. 3). 

Table 4.

 

Apoptosis Measured by TUNEL Assays on Day 1 and Day 3 after Injury and Treatment

Model	Cell Apoptosis
	Day 1	Periphery
C57b1/6 mice
CRAO
No treatment	4.1 ± 2.3%	13.2 ± 0.3%*
HBO treatment	0.0 ± 0%	0.6 ± 0.4%*
ONC
No treatment	3.6 ± 2.4%	14.8 ± 11%
HBO treatment	8.1 ± 4.2%	0.4 ± 0.5%
CFP-Thy-1 transgenic mice
ONC
No treatment
HBA treatment	6.0 ± 2.6% #	4.9 ± 3.7%

 Values are mean ± SD.

*   P < 0.05;

#   P < 0.05 in comparison with ONC injury group only.

Figure 3.

 

TUNEL staining for apoptosis in the ONC (A, B, C) and CRAO (D, E, F) models at 3 days after induction of injury. (C, F) Abundant positively stained apoptotic cells in the ONC and CRAO HBO-untreated eyes. (B, E) Limited number of apoptotic cells in the HBO-treated retinas of injured eyes. (A, D) No apoptotic cells are detected in the control uninjured left eye, without HBO treatment. Arrows indicate apoptotic cells (red, ×40).

On day 21, no apoptosis was detected in the retinas of any animals in either model. 

Caspase-3 Immunostaining

On day 1, HBO-treated mice in the ONC model had cells positive for caspase-3 staining. On day 3, fewer positive cells were noted in the HBO-treated than those in the untreated (Figs. 2C, 2D) eyes in both models. 

Gene Expression

The expression levels of the pro- and antiapoptotic genes are shown in Table 5. In the CRAO group, caspase-3 mRNA levels in the untreated HBO eyes stayed at baseline on days 1 and 3 after injury, and bax, bcl-2, and bcl-xL mRNA levels were decreased. In the HBO-treated mice, levels of all examined genes decreased on day 1, and bax, bcl-2, and bcl-xL levels increased on day 3. There was an increase in the bax/bcl-2 and bax/bcl-xL ratios on day 1 with or without treatment. On day 3, bax/bcl-2 decreased and bax/bcl-xL remained high, but the difference was not statistically significant. In the HBO-untreated ONC group, there was a decrease in expression of caspase-3, bax, and bcl-xL mRNA on day 1, and an increase on day 3. The HBO-treated mice showed an increase in caspase-3, bax, and bcl-xL expression on day 1 and a decrease on day 3. On days 1 and 3, bcl-2 levels decreased in both the HBO-treated and -untreated ONC mice. The untreated mice showed no change in bax/bcl-2 or bax-bcl-xL ratios on day 1, with an increase on day 3. The HBO-treated mice showed an increase in bax/bcl-2 on day 1 and a further increase on day 3, but no change in bax/bcl-xL. 

Table 5.

 

Molecular Analysis of Apoptosis-Related Gene Expression in Both Models with and without HBO Treatment

Apoptosis-Related Gene/Time	CRAO		ONC
	No Treatment	HBO Treatment	No Treatment	HBO Treatment
Caspase-3
Day 1	0.99 ± 0.37	0.71 ± 0.36	0.70 ± 0.45	1.49 ± 0.67
Day 3	0.97 ± 0.43	1.09 ± 0.42	1.13 ± 0.55	0.83 ± 0.28
Bax
Day 1	0.88 ± 0.42	0.73 ± 0.30	0.88 ± 0.41	1.48 ± 1.01
Day 3	0.71 ± 0.31	2.40 ± 1.05	1.16 ± 0.58	0.69 ± 0.15
Bcl-2
Day 1	0.83 ± 0.50	0.47 ± 0.16	0.80 ± 0.25	0.88 ± 0.38
Day 3	0.81 ± 0.48	2.08 ± 1.25	0.66 ± 0.39	0.53 ± 0.14
Bcl-xL
Day 1	0.69 ± 0.38	0.65 ± 0.27	0.79 ± 0.49	1.33 ± 0.78
Day 3	0.85 ± 0.27	1.47 ± 0.53	0.89 ± 0.61	0.62 ± 0.29
Bax/Bcl-2
Day 1	1.27	1.57	1.10	1.11
Day 3	0.87	1.15	1.76	1.30
Bax/Bcl-x
Day 1	1.45	1.14	1.11	1.11
Day 3	0.83	1.63	1.30	1.11

 Values are mean ± SD.

Analysis of the levels of expression of the ischemia- and oxidative-stress–related genes yielded the following changes (Table 6, Fig. 4): CRAO model, untreated mice, day 1: retinal mRNA levels of HO-1 and GPX-1 increased, SOD-1 showed no change, whereas NOX-2 decreased; day 3: HO-1 and GPX-1 decreased from day 1, SOD-1 remained at baseline, and NOX-2 increased. CRAO model, treated mice, day 1: HO-1 and SOD-1 levels increased, NOX-2 decreased, and GPX remained at baseline; day 3: HO-1, SOD-1, and GPX-1 all dropped below baseline, and NOX-2 increased. ONC model, untreated mice, day 1: retinal mRNA expression levels of HO-1 and NOX-2 increased, and GPX-1 and SOD-1 decreased; day 3: HO-1 remained stable, NOX-2 increased further, GPX-1 and SOD-1 return to baseline. ONC model, treated mice: HO-1, NOX-2, GPX-1, and SOD-1 increased; day 3: HO-1 increased significantly, NOX-2 and GPX-1 increased, SOD-1 remained stable. 

Table 6.

 

Molecular Analysis of Ischemia- and Oxidative-Stress-Related Gene Expression in Both Models with and without HBO Treatment

Genes/Time	CRAO		ONC
	No Treatment (n = 6)	HBO Treatment (n = 4)	No Treatment (n = 6)*	HBO Treatment (n = 4)
Ischemia-related
HO-1
Day 1	3.42 ± 3.9	8.03 ± 1.8† ‡	1.74 ± 1.9	2.10 ± 1.1†
Day 3	1.60 ± 1.2	0.64 ± 0.2‡	1.33 ± 0.6	9.96 ± 11.8
Oxidative-stress-related
SOD-1
Day 1	1.10 ± 0.6	1.74 ± 1.2	0.81 ± 0.3	1.35 ± 1.0
Day 3	1.00 ± 0.6	0.51 ± 0.3§	1.33 ± 0.9	1.29 ± 0.4§
GPX-1
Day 1	3.27 ± 5.7	1.22 ± 0.8	0.67 ± 0.2	1.63 ± 2.2
Day 3	0.91 ± 0.5	0.71 ± 0.2	1.13 ± 0.5	2.12 ± 2.3
NOX-2
Day 1	0.53 ± 0.6‖	0.46 ± 0.6	1.99 ± 1.4	1.37 ± 1.4
Day 3	5.37 ± 5.1‖	5.50 ± 8.3	3.41 ± 2.6	3.93 ± 3.2

 Values (injured retinae, right eye) are relative to the control retinae (left eye).

*  ONC day 1 no treatment (n = 3).

†   HO-1 levels of expression were elevated in CRAO-HBO treated versus ONC-HBO treated (P < 0.05) mice.

‡   HO-1 levels of expression in CRAO-HBO treated mice decreased on day 3 versus day 1 (P < 0.05).

§   SOD-1 levels of expression were elevated in the ONC-HBO treated day 3 versus CRAO-HBO treated mice (P < 0.05).

‖   NOX-2 levels of expression were elevated in CRAO treated day 3 versus day 1 (untreated, P < 0.05).

Figure 4.

 

Molecular analysis of ischemia and oxidative-related gene expression in both models with and without HBO treatment. (A) HO-1: CRAO model: both groups (HBO-treated and -untreated) showed increased mRNA levels on day 1, decreasing on day 3 (P < 0.05 for HBO-treated). ONC model: both groups (HBO-treated and -untreated) showed increased mRNA levels on day 1, whereas the untreated demonstrated slightly decreased levels on day 3 and HBO-treated further increased. (B) SOD-1: in both models, untreated mice on day 1 had baseline level, whereas the HBO-treated showed increased levels. On day 3, the HBO-treated groups showed decreased levels compared with the untreated groups (P < 0.05, CRAO versus ONC, day 3 treated). (C) GPX-1: on day 1, mRNA levels in the CRAO model increased, whereas these levels increased to a lesser extent in the HBO-treated group, decreasing below baseline on day 3. Untreated mice in the ONC model had decreased levels on day 1, which returned to baseline on day 3, whereas HBO-treated mice showed increased levels on day 1, with a further increase on day 3. (D) NOX-2 levels: there were decreased levels in the CRAO model in the HBO-treated and -untreated groups on day 1, with significant elevation on day 3 (P < 0.05, untreated group). ONC model: increased levels in the HBO-treated and -untreated groups on day 1, with a further increase on day 3. *Error bar was calculated as SEM.

Effect of Hyperbaric Air Treatment

On day 21 after ONC injury, cell survival in the RGC layer was higher in the air-treated mice (23% RGC loss) than that in either the HBO-treated mice (32% RGC loss) or the untreated ONC mice (52% RGC loss; P < 0.005). Mean apoptosis in the RGC was 6% on day 1 (compared with 1.6% in the HBO-untreated ONC mice, P < 0.05) and 5% on day 3 (compared with 14.8% in the HBO-untreated mice, NS) (Tables 3, 4). 

Discussion

This study reveals a significant neuroprotective effect of HBO treatment in ischemic mouse retinas. Animals with CRAO or ONC injury showed increased cell survival in the RGC layer when treated immediately by HBO. These results are consistent with previous studies of the use of HBO for brain and eye diseases. Wang et al. ³⁶ reported a decrease in RGC loss in rats treated 2 days before ONC injury. Our experiment expanded on this finding, measuring the effect of postinjury treatment for up to 14 days, more closely resembling the clinical setting. 

Apoptosis is the main mechanism of RGC loss after induction of ischemic injury to the eye. ^{3,6,9 –11} The reduction in apoptosis after HBO treatment in our study, in both models, was demonstrated by TUNEL assay, caspase-3 immunoassay, and molecular analysis. Flat-mount retinal studies of CFP-Thy-1 transgenic mice confirmed that apoptosis was reduced in the neuronal cells of the inner retinal layer, specifically the RGC, after HBO treatment. 

The main genes involved in apoptosis are bax, bcl-2, bcl-xL, and caspase-3. ³⁷ Bax is a prominent proapoptosis gene and bcl-2 is an antiapoptosis gene. Surprisingly, in the present study, there was only a minimal reduction in the levels of bax and caspase-3 in the HBO-treated CRAO mice on day 1 (whereas in the ONC treated group, the levels actually increased). This finding may have been due to the effect of HBO on reperfusion, which hastened the death of the already injured cells. This trend was reversed on day 3, when the levels of the proapoptotic genes increased in the HBO-treated CRAO mice. In the ONC group, the levels were already decreased below baseline on day 3. 

Although bcl-2 is expressed in the mouse retina, it appears to be principally restricted to the macroglia (probably Müller cells). ³⁸ Studies have shown that in the RGCs, bcl-xL is 16-fold more abundant that bcl-2, which may make it the major antiapoptotic gene in these cells and other cells of the retina. ³⁸ However, in our study, the expression levels of bcl-2 and bcl-xL were similar. It is noteworthy that in both models of injury, mainly cells in the RGC layer were damaged. 

The bax/bcl-2 ratio has been suggested to be a useful measure of apoptotic potential. ^39,40 Researchers have shown that the bax/bcl-2 and bax/bcl-xL ratios are not modified for up to 24 hours after injury. ⁴¹ In the present study, HBO chamber treatment in both models was associated with an increase in the proapoptotic bax/bcl-2 ratio on day 1. In the CRAO model, this was followed by a decrease on day 3, partially in accordance with the immunoassay results. The finding suggests that the mechanism underlying the neuroprotective effect of HBO treatment involves a reduction in the degeneration of the neighboring cells secondary to the increased death of injured cells, noted immediately after injury. ⁴² The differences between the bax/bcl-2 ratio and bax/bcl-xL ratio in this study were minor, and were noted mainly in the HBO-treated mice with CRAO, where reperfusion might play a role (increase in the proapoptotic ratio on day 3). The high level of the bax/bcl-xL ratio on day 3 may suggest that because the cells in the RGC layer were those mainly damaged in the CRAO model, and because bcl-xL is more specific for RGC, the reduced level of bcl-xL might better represent the response in the RGC layer. 

Some recent studies have questioned the importance of the bax/bcl-2 ratio because neither of these compounds can form dimers, the proposed inhibitory mechanism under physiologic conditions. Other possible intervening factors might be apoptotic pathways other than the bax/bcl-2 pathway. This might be especially important in the ONC model, where the damage is not purely ischemic. 

Additionally, we measured changes in gene expression in the retina that would reflect the ischemic and oxidative stress to the neuronal and neighboring glial cells and the changes caused by the effect of the HBO treatment. The specific genes selected were based on findings in previous studies. ⁴³ The results showed that although the effectiveness of HBO varied by the model and time of examination from damage, the trend in gene expression was enhanced by HBO treatment: levels of the relevant genes that rose without treatment further increased by several-fold with treatment, leading to few folds higher from the increased levels, or reduced from the decreased expression of the relevant genes. 

Our findings of an early upregulation of HO-1 expression levels after CRAO induction (3.4-fold in the untreated mice, 8.0-fold in the treated mice), followed by a decrease on day 3 (to 1.6-fold of baseline in the untreated mice and 0.64-fold in the treated mice), support those of earlier studies using a similar model of retinal ischemia reperfusion. ^16,17 By contrast, in the ONC model, HO-1 showed an early increase (approximately two-fold in both treated and untreated mice) and then further increased, especially in the treated mice (9.96-fold of baseline vs. 1.3-fold in the untreated mice). The differences between the models might be a product of the traumatic but not ischemic nature of the ONC injury. 

The increase in HO-1 expression is indicative of HIF-1a activation. Thus, it is possible that HBO treatment exerts a protective effect indirectly, leading to an early (days 1–3) increase in HIF-1a levels that, in turn, lead to an increase in HO-1 levels. 

The lack of major changes in expression levels of SOD-1 in the untreated mice in both models of injury are in line with a previous study by our group using a model of anterior ischemic optic neuropathy. ⁴⁴ HBO treatment slightly increased expression of the SOD-1 and GPX-1 in both models on day 1 (but less than the levels detected in the untreated CRAO on day 1) and a further increase of GPX-1 on day 3 in the ONC model. Accordingly, earlier in vitro and in vivo experiments in primary neuronal cultures from fetuses overexpressing human GPX-1 reported better protection against oxidative stress, and transgenic rats overexpressing SOD-1 showed reduced neuronal damage from oxidative stress. ²⁴  

The increase in NOX-2 expression levels on day 1 and again on day 3 in the ONC model was similar to the increased neuronal immunoreactivity of gp91phox at 24 hours reported in a rat model of subarachnoid hemorrhage. ⁴⁵ In that study, HBO decreased both NADPH oxidase expression and the level of oxidative stress. An improvement was noted in functional performance throughout the observation period. HBO did not affect NOX-2 levels of expression in any of the models. 

In our study, the uninjured left (control) eyes did not show any toxic side effect of HBO treatment. Nevertheless, prolonged exposure to high inspired fractions of oxygen has been found to cause oxidative damage to the retina. ^46,47 For example, hyperoxic myopia was reported in closed-circuit oxygen rebreather divers ^48,49 and patients undergoing repeated HBO treatments. ^49,50 Nachman-Clewner et al. ⁵¹ found a selective degeneration of central photoreceptors in mice after HBO chamber use. However, in our study, the treatment parameters were less powerful and the treatment time was shorter. 

To better understand the role of oxygen in the treatment of ischemic injury, we included a control group treated with hyperbaric air (20% oxygen) after ONC. Surprisingly, this group showed better results than those of the HBO-treated mice. We speculate that the high pressure is the major factor in neuroprotection, not the high oxygenation. ^{46 –49} Our air-treated group was limited in size and additional studies are needed to further clarify the effectiveness of 20% oxygen and of different levels of hyperbaric oxygen. 

Recent studies in humans and animal models of brain ischemia ^26,27 and brain infarction ^31,52 also reported a protective antiapoptotic effect of HBO treatment, ⁵³ and some suggested that HBO treatment may be effective for retinal ischemia in humans. ^30,32 Weinberger et al. ³¹ reported a significant 83% improvement in visual acuity in HBO-treated patients compared with a 30% improvement in the control group. Treatment was apparently more effective when performed within 2 to 12 hours after appearance of the first symptoms. In the pilot study of Aisenbrey et al., ⁵⁴ HBO proved beneficial in improving visual acuity in eyes with minor branch retinal artery occlusion, where the initial damage is limited but the protective effect on the neighboring cells is most important. 

The effect of HBO treatment might be related to the time of its administration from the acute ischemic event and the extent of necrosis that occurs immediately after injury. Necrosis, as opposed to apoptosis, is an irreversible process that does not respond to any treatment. In our study, as in others, ^31,32 treatment was started immediately after injury induction. However, this might not happen in the clinical setting, where a delay in diagnosis or treatment could reduce its success rate. 

In conclusion, HBO chamber treatment given immediately after injury induction in a mouse model protects neuronal retinal cells from apoptosis, as shown both histologically and molecularly, without toxic side effects. These findings should prompt clinical trials of acute ischemic retinal damage. 

Footnotes

 Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2009; The Israel Society for Eye and Vision Research, Neve Ilan, Israel, March 2009; Rabin Research Day, Petah Tiqwa, Israel, May 2009; and The Israel Ophthalmology Association–Retina Day, Ramat Gan, Israel, July 2009.

Footnotes

 Supported in part by a grant from the Zanvyl and Isabelle Krieger Fund (Baltimore, MD), the Eldor-Metzner Clinician Scientist Award, Chief Scientist, Israel Ministry of Health Grant 3-3741 (NG-C), and the Young Investigator Award, Rabin Medical Center, Petah Tiqwa, Israel (DE).

Footnotes

 Disclosure: V. Gaydar, None; D. Ezrachi, None; O. Dratviman-Storobinsky, None; S. Hofstetter, None; B.C.R. Avraham-Lubin, None; N. Goldenberg-Cohen, None

The authors thank Israel Shreger, Senior Technician, for designing and building the hyperbaric oxygen chamber; and Asher Sheinberg of the Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, for supervising the studies of Vera Gaydar toward the MSc degree, the study of which was performed in partial fulfillment of the MSc requirements.