Neuroprotective Effects of d-Allose against Retinal Ischemia–Reperfusion Injury

Abstract

purpose. To investigate the effect of d-allose, a rare sugar, against ischemia reperfusion injury in the rat retina.

methods. Retinal ischemia was induced by increasing intraocular pressure to 130 mm Hg and maintaining that level for 45 minutes. Morphometric studies were performed to study the effect of d-allose on the histologic changes induced by ischemia in the rat retina. Glutamate release from the rat retina and intravitreal Po ₂ profiles were monitored during and after ischemia with a microdialysis biosensor and oxygen-sensitive microelectrodes. The release of hydrogen peroxide stained with diaminobenzidine hydrochloride was monitored by an in vitro retinal ischemia model.

results. Seven days after the ischemia, significant reductions in both the number of ganglion cells and the thickness of the inner plexiform layer were observed. Pretreatment with d-allose significantly inhibited the ischemic injury of the inner retina. A large release of glutamate occurred during the ischemia. After the recirculation, glutamate levels were increased again and reached a maximum in approximately 20 minutes. The increases in extracellular glutamate during and after ischemia tend to be suppressed by administration of d-allose. d-Allose attenuated the increase in intravitreal Po ₂ during reperfusion. After the ischemia, production of hydrogen peroxide was detected within approximately 30 minutes. d-Allose suppressed the production of hydrogen peroxide.

conclusions. These results suggest that d-allose may protect neurons by decreasing extracellular glutamate and attenuating oxidative stress in ischemic insult.

Arare aldo-hexose sugar, d-allose, is derived from d-psicose by microorganisms and their enzymes. ¹ Rare sugars have received increasing attention in recent years for a variety of usages such as low-calorie carbohydrate sweeteners and bulking agents. ² Arnold and Silady ³ have reported that d-allose substantially inhibits segmented neutrophil production and lowers platelet count without other detrimental clinical effects. Hossain et al. ⁴ has recently studied the immunosuppressive effect of d-allose and compared it with that of FK506 on the basis of neutrophil count and animal survival in liver transplantation experiments using rats. Their study showed that the rate of allograft survival was significantly increased with less tissue damage when low-dose FK506 was administered in combination with d-allose compared with the administration of each drug separately. ⁴ They also performed a series of experiments using hepatic ischemia-reperfusion in a rat model to evaluate the protective effect of d-allose and found that the ameliorative effect was achieved mainly by reducing the number of total neutrophils during or after reperfusion. ⁵  

Ischemic injury to the retina is a major cause of visual loss and morbidity. Some studies have demonstrated that oxygen-derived free radicals generated during ischemia and on reperfusion may trigger ischemic cell damage in various organs, such as the brain, heart, kidney, liver, bowel, and retina. ^{6 7 8} In the central nervous system, free radicals lead to the hypersecretion of excitatory amino acids, such as glutamate and aspartate, ⁹ which bind to receptor sites and augment cellular destruction by increasing membrane permeability to calcium and sodium ions and water. ¹⁰ Injured neurons release massive amounts of glutamate, which induce neuronal cell death by continuous overexcitation of postsynaptic receptors. ^{10 11} In this way, the initial trauma is amplified and causes the damage to spread to neighboring cells. During or after ischemia, reactive oxygen species can be produced in large quantity and act as cytotoxic metabolites. ¹² The species of primary concern include superoxide anion (O₂ÿ⁻), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH^.). It appears that reactive oxygen species can provoke cell death, either by reacting with cell components, leading to necrosis, or by activating specific targets and triggering apoptosis. 

The purpose of the present study was to investigate the mechanism of the protective effects of d-allose on neuronal death in retinal ischemia. 

Material and Methods

Animals

Female Sprague-Dawley rats, weighing 200 to 250 g, were obtained from Charles River Japan (Yokohama, Japan). Transient retinal ischemia was induced for 45 minutes in the right eye of each rat. Rats were anesthetized by intraperitoneal injection of pentobarbital (40–50 mg/kg). Animal care and experiments were approved by the standard guidelines for animal experimentation of the Kagawa University Faculty of Medicine and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. d-Allose was administered at a dose of 50, 100, 200, and 400 mg/kg. The animals were divided into two groups according to treatment: a d-allose group and a control group (saline instead of d-allose). In the d-allose group, intraperitoneal injection of d-allose was administered 30 minutes before ischemia was induced. In the control group, an intraperitoneal injection of 0.9% normal saline was administered as a vehicle 30 minutes before ischemia was induced. 

Ischemia

After anesthesia, the anterior chamber of the right eye was cannulated with a 27-gauge infusion needle connected to a reservoir containing normal saline. The intraocular pressure (IOP) was raised to 130 mm Hg for 45 minutes by elevating the saline reservoir. Retinal ischemia was confirmed by the whitening of the iris and fundus. Sham-treated control left eyes underwent a similar procedure, but without the elevation of the saline bag, so that normal ocular tension was maintained. The 45-minute duration of ischemia was chosen on the basis of previous studies. ^{13 14} Because body temperature may influence ischemia-induced retinal ganglion cell death, ¹⁵ rectal and tympanic temperatures were maintained at approximately 37°C, using a feedback-controlled heating pad (CMA, Stockholm, Sweden) during the operation. After restoration of blood flow, temperature was still maintained at 37°C. 

Histologic Examination

For histologic examination, rats were anesthetized by intraperitoneal injection of pentobarbital (40–50 mg/kg) 1 week after ischemia and perfused intracardially with phosphate-buffered saline (PBS), followed by perfusion with 4% paraformaldehyde in PBS. The retinas were removed and embedded in paraffin, and thin sections (5 μm thickness) were cut with a microtome. Each retina was mounted on a silane-coated glass slide and then stained with hematoxylin and eosin (HE). 

Morphometric analysis was performed to quantify ischemic injury. These sections were selected randomly in each eye. A light microscopic examination was performed by a person with no prior knowledge of the groups. A microscopic image of each section within 0.5 to 1 mm of the optic disc was scanned. In each computer image, the number of cells in the ganglion cell layer (GCL) was counted. The thickness of the inner plexiform layer (IPL), inner nuclear layer (INL), and outer nuclear layer (ONL) at the entire frame were measured. The number of cells in the GCL was normalized as linear cell density (cells per millimeter). Finally, in each eye, the thicknesses of the IPL, INL, and ONL were obtained as the mean values of the four measurements, and the linear cell density in the GCL was defined as the mean value of the four measurements. For each animal, these parameters in the right eye were normalized to those in the intact left eye and shown as a percentage. 

Measurements of Glutamate in the Vitreous Body

The measurements were performed as described elsewhere. ¹⁶ Briefly, a dialysis electrode (Microdialysis Biosensor; Applied Neuroscience, London, UK) was used. ¹⁷ The electrode was composed of a platinum wire located inside a hollow semipermeable dialysis membrane (500 Da) with an outside diameter of 230 μm. Two additional electrodes, a reference electrode (Ag/AgCl) and a counter electrode (Ag), comprised the electrochemical cell, and these were collectively installed in a glass capillary, away from the main sensing area. For the measurement of glutamate, the probe was filled with 10 mM phosphate-buffered saline (PBS; pH 7.4), with or without glutamate oxidase (100 U/mL; Yamasa Co. Ltd., Chiba, Japan), and perfused at a flow rate of 0.2 μL/min by means of a microinfusion pump (IP-2 microinfusion pump; Bio Research Center Co. Ltd., Nagoya, Japan). Each substance diffuses across the dialysis membrane into the electrode, and the respective oxidase produces hydrogen peroxide which is detected by the electrode. The current detected by the electrode is sent to an amplifier and is recorded on a polygraph in real time. In each experiment before and after in vivo measurements, glutamate and H₂O₂ were determined with and without glutamate oxidase solution in the test tube respectively and a regression line was obtained from known concentration. The current detected by the electrode was sent to the amplifier (EPS-800; Eicom, Kyoto, Japan). The change of 1 nA was equivalent to 8 μM of glutamate. d-Allose (200 mg/kg) or saline was administered 30 minutes before ischemia, and glutamate was monitored in the d-allose and control groups (n = 4 per group) before, during, and after ischemia. Body temperature was maintained at approximately 37°C during measurement. In each experiment, a regression line was obtained from the known glutamate concentration. 

Measurements of Po ₂ in the Vitreous Body

Intravitreal oxygen was monitored with a Po ₂ probe (Intermedical Co. Ltd., Nagoya, Japan) of 0.1-mm diameter. After the fiber-optic probe was dipped into the oxygen-saturated PBS solution, the value of the amplifier was adjusted to 155 mm Hg. The probe was inserted into the vitreous in the same manner as for glutamate measurement (n = 4 per group). 

In Vitro Histologic Detection of Released H₂O₂

Rats were killed after 200 mg/kg d-allose or saline was administered. For in vitro detection of released hydrogen peroxide, the retina was quickly removed and immersed in ice-cold artificial cerebrospinal fluid (ACSF) with 10 mM glucose bubbled in a gaseous mixture of 95% O₂ and 5% CO₂. The composition of ACSF was as follows: 124 mM NaCl, 4.4 mM KCl, 2.5 mM CaCl₂, 1.3 mM MgSO₄, 1 mM NaH₂PO₄, and 26 mM NaHCO₂. The retina was incubated in an interface-recording chamber maintained at 37°C for at least 30 minutes before the experiment and constantly infused with gas-saturated ACSF with 10 mM glucose at 1.2 mL/min. The retina was then put in an observation chamber and continuously bathed in a circulating fluid of ACSF with 10 mM glucose bubbled in a gaseous mixture of 95% O₂ and 5% CO₂. In vitro detection was performed by applied immunohistochemical staining with horseradish peroxidase (HRP) (anti-mouse IgG, peroxidase-linked species-specific F(ab′)2 fragment from sheep; GE Healthcare, Piscataway, NJ) and diaminobenzidine (DAB) solution (1 mg/mL; Vector Laboratories Inc., Burlingame, CA). A colorized solution was made by adding equal amounts of HRP and DAB solution. Ten microliters of this solution was added to the retina, followed by observation under a stereoscopic microscope for the development of brown color as an indicator of the release of H₂O_2. The DAB solution, on binding to H₂O₂, undergoes a polymerization reaction to yield a brown color. ¹⁶ For ischemia induction, the circulating solution was changed to ACSF bubbled with 100% N₂ gas. After circulating for 45 minutes, the solution was changed back to the original solution. A stereoscopic microscope (MZ FL II; Leica Microsystems, Tokyo, Japan) was used with acharge-coupled device digital camera (DP70; Olympus, Inc., Tokyo, Japan) and analytical software (DP70-WPCP; Olympus, Inc.). 

Appearance of d-Allose in Vitreous

d-[1-¹⁴C]allose was obtained from GE Healthcare Biosciences (Buckinghamshire, UK). Four female Sprague-Dawley rats were used. d-[1-¹⁴C]allose (100 mg/1.11 MBq/2.0 mL PBS/kg body weight) was intraperitoneally injected 30 minutes before enucleation of the eye. Rats were deeply anesthetized with pentobarbital. To minimize contamination of the intraocular tissue and blood, enucleation, and dissection of the eye were performed by the following procedure. A cut was made along the edge of the orbit. After enucleation with surrounding tissue, the eye was immersed immediately in PBS for 5 minutes. Vitreous was exfoliated from the vitreous side of the posterior cup. Radioactivity was measured by a liquid scintillation counter (Tri-Carb Liquid Scintillation Analyzer 2500TR; PerkinElmer, Meriden, CT). 

Statistical Analysis

All statistical values are presented as the mean ± SEM. Data were analyzed using an independent Student’s t-test where appropriate. P < 0.05 was considered statistically significant. 

Results

Histologic Change in the Retina after Ischemia with and without d-Allose

Figure 1A shows a normal retina. Light microscopic photographs were taken 7 days after ischemia and treatment with saline (Fig. 1B)or d-allose (Figs. 1C 1D 1E 1F) . In animals pretreated with the saline control, significant reductions in the number of cells in the GCL and the thickness of the IPL were observed. The number of cells in the GCL was reduced to 52.5% ± 1.4% of the control (P < 0.01) and the thicknesses of the IPL was reduced to 71.5% ± 8.4% (P = 0.02), of the INL to 93.0% ± 9.6% (P = 0.5), and of the ONL to 95.0% ± 4.5% (P = 0.3; n = 4) of the control (Fig. 2) . In animals pretreated with 200 mg/kg d-allose, the number of cells in the GCL was 95.0% ± 2.0% of the control (P = 0.05) and the thickness of the IPL was 95.8% ± 2.3% (P = 0.7), of the INL was 100.4% ± 3.0% (P = 0.9), and of the ONL was 102.4% ± 5.0% (P = 0.7; n = 4) of the control (Fig. 2) . The co-injection of glucose (200 mg/kg) with d-allose had no protective effect against retinal ischemia reperfusion injury (data not shown). 

Effect of d-Allose on Extracellular Glutamate by Ischemia

The time course of glutamate efflux in the vitreous body during the 45-minute ischemia and reperfusion in the control and d-allose groups is shown in Figure 3 . A remarkable increase in glutamate was observed during ischemia. After recirculation, glutamate levels were increased again and reached a maximum in approximately 20 minutes. d-Allose suppressed increased extracellular glutamate by ischemia. The increase in glutamate efflux in the vitreous body during reperfusion tended to be suppressed by the administration of d-allose. 

Effect of d-Allose on Po ₂ in the Vitreous Body after Ischemia

The time course of Po ₂ in the vitreous body during the 45-minute ischemia and reperfusion in the control and d-allose groups is shown in Figure 4 . After recirculation, Po ₂ levels were increased and, in approximately 10 minutes, reached the same levels as before ischemia. During reperfusion, d-allose enhanced Po ₂ levels and reached a maximum in approximately 15 minutes. 

Effect of d-Allose on Released H₂O₂

Figure 5A shows a normal flat-mounted retina. Light-microscopic photographs were taken of treatment without d-allose (Fig. 5B)and with d-allose (Fig. 5C) . Without d-allose treatment, brown color was first observed 30 minutes after ischemia and was stronger 75 minutes later (Fig. 5B) . However, in the presence of d-allose, a brown color was first observed 60 minutes after ischemia (15 minutes after recirculation). Decreased brown staining was observed compared with the ischemic retina (Fig. 5C) . 

The specificity for H₂O₂ was determined by DAB solution without hydrogen peroxide. No specific color development was observed (data not shown). This observation does not exclude the possibility of other reactive oxygen species such as superoxide, nitric oxide, peroxinitrite. However, at least the color development must be dependent on the release of H₂O₂. 

Appearance of d-Allose in Vitreous

When 555 Bq/μL d-[1-¹⁴C]allose was intraperitoneally injected, it appeared in the vitreous and blood at 46.4 ± 5.2 Bq/μL and 42.8 ± 3.0 Bq/μL, respectively, at 30 minutes (n = 4; Table 1 ). The concentration of d-allose in vitreous was shown to be almost same as that in blood. 

Discussion

In the present study, the release of glutamate from the rat retina was observed during the ischemic period, and a larger increase occurred during reperfusion. Similar observations were reported in rabbit ¹⁸ and feline ¹⁹ retinas. A delayed increase in the efflux of glutamate was observed during the recirculation period in the CA1 field of the hippocampus where most presynaptic fibers were eliminated, and the extracellular glutamate level returned very gradually to the baseline range in the setting. ²⁰ Mitani et al. ²¹ suggested that presynaptic terminals in the CA1 field play a major role in the uptake of glutamate after ischemia. The prolonged release of glutamate after ischemia in the retina may reflect such a defective glutamate uptake system in the retinal neurons, because retinal neurons did not show a significant affinity for glutamate uptake. ²¹ Glutamate receptors have been subdivided into ionotropic and metabotropic receptors. ²² Although the details of the pathway are not clear, there is compelling evidence to suggest that neurons that contain ionotropic glutamate receptors are particularly susceptible to ischemia-reperfusion. The neurons in the retina that express such receptors are the ganglion cells and a subset of amacrine cells. ²³ In ischemia-reperfusion, neurotransmitters are related, and they overactivate their appropriate receptors. Such overstimulation, particularly of ionotropic glutamate receptors, generally leads to cell death. ^{24 25 26 27} In this study, d-allose prevented retinal damage by reducing extracellular glutamate levels. 

To clarify the role of d-allose as an antioxidant, a research group at Kagawa University Faculty of Medicine and the Nation Agriculture Research Center for Western Region, Kagawa, has examined the scavenging activities of d-allose and other sugars using electron spin resonance. They found scavenging activities in rare sugars, although the activities were much weaker than those of other common scavengers such as superoxide dismutase and carotinoids. ²⁸ Significant inhibition of reactive oxygen species production was detected only when d-allose was added, although the inhibition was found to be dose dependent. ²⁸ The ameliorative effect of d-allose has been observed after liver transplantation ⁴ and ischemia–reperfusion injury of the liver. ⁵  

A considerable amount of reactive oxygen species is produced in ischemia, especially during reperfusion, due to the increase in oxygen supply and metabolism, and this exacerbates neuronal cell damage (reperfusion injury). ²⁹ In a recent study, using a technique based on electron spin resonance trapping analysis of the signals obtained in microdialysates of the retina, Muller et al. ³⁰ directly showed that OH^. radicals were generated during the ischemic episode itself and remained elevated at reperfusion. This OH^. production was inhibited by superoxide dismutase/catalase and deferoxamine, suggesting that H₂O₂ is an intermediate in radical formation. Molecular oxygen (O₂) can be reduced by various enzymatic reactions, mainly by oxidases and an oxygenase cascade. ³¹ Superoxide is dismutated to H₂O₂. Ullrey and Kalckar ³² previously reported that d-allose may inhibit hexose transport. d-Allose could reduce the production of H₂O₂ by modulating the glycolytic response. In the present study, d-allose affected increased Po ₂ levels in the vitreous body by reperfusion. d-Allose may enhance Po ₂ levels in the vitreous body after ischemia due to a decrease in the production of H₂O₂, consequently preventing reperfusion injury. 

In conclusion, preischemic administration of d-allose suppressed glutamate release during and after ischemia. Furthermore, d-allose reduced the production of H₂O₂ during recirculation. The present study suggests that d-allose may protect neurons by decreasing extracellular glutamate and attenuating oxidative stress in ischemic insult. 

 Supported by the Cooperative Link of Unique Science and Technology for Economy Revitalization (CLUSTER) of Japan.

 Submitted for publication August 3, 2005; revised November 22, 2005; accepted February 8, 2006.

 Disclosure: K. Hirooka, None; O. Miyamoto, None; P. Jinming, None; Y. Du, None; T. Itano, None; T. Baba, None; M. Tokuda, None; F. Shiraga, None

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

 

Figure 1.

 

Light micrographs of a cross-section through normal rat retina (A) and 7 days after ischemia and treatment without d-allose (B) or with 50 (C), 100 (D), 200 (E), or 400 (F) mg/kg d-allose. Bar, 10 μm.

Figure 2.

 

Percentage of change relative to control values in the number of GCL cells and the thicknesses of the IPL, INL, and ONL 7 days after ischemia without d-allose (▪) or with 200 mg/kg d-allose (▒). Results are expressed as the mean ± SEM (*P < 0.05).

Figure 3.

 

Effect of d-allose on the release of glutamate from rat retina. (•) Control; (▴) d-allose. Ischemia was induced by elevating the intraocular pressure for 45 minutes. d-Allose (200 mg/kg, intraperitoneally) was administered 30 minutes before ischemia in the d-allose group, and glutamate was measured by an electroenzymatic method of microdialysis. Glutamate was significantly increased by ischemia. Glutamate during both ischemia and reperfusion was suppressed by d-allose. Data represent the mean ± SEM.

Figure 4.

 

Effect of d-allose on Po ₂ levels. (•) Control; (▴) d-allose. Ischemia was produced by elevating intraocular pressure for 45 minutes d-allose (200 mg/kg, intraperitoneally) was administered 30 minutes before ischemia in the d-allose group and Po ₂ was measured by a Po ₂ probe. The Po ₂ levels were significantly increased after ischemia. The Po ₂ levels during reperfusion were enhanced by d-allose. Data represent the mean ± SEM.

Figure 5.

 

Effect of d-allose on the release of H₂O₂. The brown color indicates the release of H₂O₂. Ischemia induction was for 45 minutes. Color photographs were taken before ischemia induction and at 30, 45, 60, and 75 minutes after starting ischemia induction. (A) There was no brown staining in a control flat-mounted retina. Rats were killed 30 minutes after the instillation of saline (B) or d-allose (C). With d-allose treatment, the retina became brown 60 minutes after ischemia and was lighter stained than the retina without d-allose treatment at every time point. Bar, 500 μm.

Table 1.

 

Distribution of d-Allose in Vitreous and Blood

Vitreous	46.4 ± 5.2	(8.4 ± 1.0)
Blood	42.8 ± 3.0	(7.7 ± 0.5)

  d-[1-¹⁴C]Allose (555 Bq/μL) was intraperitoneally injected, and radioactivity in vitreous and blood were measured at 30 minutes after injection. Data are mean Bq/μL ± SEM (% of distribution) of four preparations.