- Email: [email protected]
Ischemic preconditioning of the rat retina: Protective role of ferritin
Available online at www.sciencedirect.com
Free Radical Biology & Medicine 44 (2008) 1286 – 1294 www.elsevier.com/locate/freeradbiomed
Original Contribution
Ischemic preconditioning of the rat retina: Protective role of ferritin Alexey Obolensky a,b,1 , Eduard Berenshtein b,1 , Abraham M. Konijn c , Eyal Banin a,1 , Mordechai Chevion b,⁎,1 b
a Department of Ophthalmology, The Hebrew University–Hadassah Schools of Medicine and Dental Medicine, Jerusalem 91120, Israel Department of Cellular Biochemistry and Human Genetics, The Hebrew University–Hadassah Schools of Medicine and Dental Medicine, Jerusalem 91120, Israel c Department of Human Nutrition and Metabolism, The Hebrew University–Hadassah Schools of Medicine and Dental Medicine, Jerusalem 91120, Israel
Received 30 September 2007; revised 29 October 2007; accepted 30 October 2007 Available online 21 November 2007
Abstract Ischemic preconditioning (IPC) of the retina, accomplished by ischemia of short duration, is highly effective in preventing subsequent severe injury caused by iron-dependent free radical burst after prolonged ischemia. To investigate the mechanistic basis for IPC rescue, we examined changes in the levels of the retinal redox-active and labile iron pool, ferritin, and ferritin-bound iron. Prolonged ischemia severely impaired retinal function, with total loss of the full-field electroretinographic response. IPC provided marked protection against such injury. Histological examination revealed that ischemia-associated structural damage and loss of cells in the outer and inner nuclear layers were largely prevented by IPC. Ferritin levels decreased after prolonged ischemia but remained close to normal when the ischemic episode was preceded by IPC. The protective effect of IPC on retinal function and ferritin was blocked by a zinc–desferrioxamine complex known to interfere with iron signaling. The results suggest a mechanism whereby IPC activates an iron signaling pathway leading to a marked increase in ferritin levels, which mediates resistance to prolonged ischemia. © 2007 Elsevier Inc. All rights reserved. Keywords: Iron; Ferritin; Free radicals; Retina; Preconditioning
Subjecting a tissue to prolonged ischemia causes detrimental effects, often leading to cell injury and death via necrosis and apoptosis [1,2]. A number of ocular pathologies, such as occlusions of the central retinal artery and vein, are associated with acute retinal ischemia and rapid visual loss [3,4]. In other conditions which develop gradually (e.g., diabetic retinopathy and, possibly, glaucoma) chronic retinal ischemia is a major pathogenic factor [5–8]. Ischemic challenges also contribute to the pathogenesis of less prevalent conditions, such as sickle cell retinopathy, radiation retinopathy, and retinopathy of prematurity [9–12]. Ischemia in the eye often leads to severe visual impairment, due either to irreversible loss of neuronal tissue or to abnormal ocular neovascularization. Currently, intense research is focused on the mechanisms of ischemia/reperfusion (I/R) injury, including a search for novel and more effective therapies [13].
⁎ Corresponding author. Fax: +972 2 641 5848. E-mail address: [email protected] (M. Chevion). 1 These authors contributed equally to this article. 0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.10.060
Ischemic injury is at least partially mediated by the formation of reactive oxygen-derived species (ROS), including free radicals, which burst with the resumption of blood flow (reperfusion). Under normal conditions, a balance between the continuous generation of low levels of ROS and adequate levels of relevant antioxidants is maintained. However, in the presence of redoxactive and labile (chelatable) iron, low-reactive oxygen species, such as the superoxide radical anion (O2·−), convert into highly reactive radical species, such as the hydroxyl radical (HO·) [14]. Thus, redox-active iron can augment ROS-induced cell damage caused by reperfusion injury and other pathological conditions of the eye [15–19]. Normally, the cell tightly controls the level of labile iron by binding most of it within ferritin—the major iron storage protein [20]. After I/R, iron may be released from ferritin and becomes involved in redox cycling, thus challenging the cellular antioxidant defense mechanisms [15,16,21]. Characterizing the iron which catalyzes redox reactions, including injury of cellular components, is complicated and not straightforward. Various terms have been used by various investigators and include “redox-active iron,” “labile iron,” “chelatable
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
iron,” and “free iron.” Although the first term best describes the iron activity, the others are also extensively used. It is noteworthy that due to the low solubility of iron, in particular in the ferric state, the term “free iron” is misleading and should be avoided. Ischemic preconditioning (IPC) is a well-established procedure to protect tissues against prolonged ischemia. It consists of a brief noninjurious ischemic episode (or repeated episodes) applied before the prolonged ischemia and confers remarkable resistance against I/R injury in various organs including the heart [22,23], liver [24,25], kidney [26], and brain [27]. Recently, IPC was also shown to protect the rat retina against a subsequent prolonged ischemic insult [28]. The protective effect was activated hours after the IPC procedure had been applied, and remained in effect for 72 h, similar to other neuronal tissues [29]. A variety of factors have been suggested to play roles in retinal IPC, including adenosine and adenosine receptors, de novo protein synthesis [28,30], opening of mitochondrial KATP channels [31–33], expression of heme oxygenase-1 and HIF-1α [34], reduction of apoptosis-related expression of genes, elevation of VEGF-A levels, alterations in protein phosphorylation [1], attenuation of ischemia-associated hypoperfusion [35], inhibition of leukocyte rolling and accumulation [36], and induction of the heat shock protein HSP27 [37] associated with inhibition of caspase-3 activation. IPC caused up-regulation of bFGF, GFAP, and Bcl-2 and also “cross-protected” photoreceptors against light-induced injury [38]. Despite this growing body of data and knowledge, evidence on the iron-related mechanisms of IPC in the retina is still lacking. Given the important role of iron-mediated mechanisms in I/R injury [39], we studied the major components of iron homeostasis (labile iron, ferritin, and ferritin-bound iron) in relation to I/R and IPC in the rat retina. Materials and methods Animals and anesthesia One hundred six Sprague–Dawley rats weighing 225–250 g were maintained on a 12:12 h light:dark cycle, with unlimited food and water. For induction of ischemia and for electro-
1287
retinographic (ERG) recordings, the rats were anesthetized by intraperitoneal injections of ketamine HCl (100 mg/kg; Ketalar, Parke Davis, UK) in combination with the relaxing agent xylazine (5 mg/kg; VMD, Arendonk, Belgium). Subsequent intramuscular injections of the same mixture were used to extend anesthesia when required. All the experiments were approved by the Institutional Animal Care and Use Committee of the Hebrew University of Jerusalem, conforming to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996). Induction of retinal ischemia Retinal ischemia was induced as described by Roth et al. [28]. A 3-O silk suture loop was passed behind one eye, around the optic nerve and associated blood vessels. Suture ends were then passed through a small piece of polyethylene tubing. Ocular ischemia was induced by pulling the sutures tight to occlude blood flow in the main retinal blood vessels. Cessation of retinal blood flow was verified by indirect ophthalmoscopy of the fundus. Occlusion of retinal blood vessels was maintained for 7 min to induce IPC and for 1 h to cause the prolonged ischemic insult, as previously established by Roth et al. [28]. The fellow eye was left as an untreated control in all cases. Experimental groups and protocol Animals were divided into five experimental groups (Table 1). In group I (control), no ischemia was induced. Group II rats (I/R) were subjected to 1 h ocular ischemia followed by 24 h reperfusion. In group III (IPC alone) eyes were subjected to 7 min ischemia followed by 64 ± 2 h reperfusion, without any additional intervention. Group IV rats (IPC + I/R) were exposed to the same IPC procedure as group III and 40 ± 2 h later challenged with 1 h prolonged ischemia followed by 24 h reperfusion. Subgroups of animals from groups III and IV (IIIa and IVa) were intravenously (iv) injected with an aqueous solution of the zinc–desferrioxamine complex (Zn/DFO; at a dose of either 0.5 or 2.5 mg/kg
Table 1 Experimental groups and outline of the protocols used
Electroretinographic recordings were performed 24 h after prolonged ischemia, and retinal samples were collected for biochemical analyses immediately afterward. Tissue for histological assessment was collected 1 week after the ischemic episode. a In groups IIIa and IVa, Zn/DFO was injected iv 15 min before IPC. b The ischemic preconditioning (IPC) procedure consisted of a short period (7 min) of retinal ischemia induced at 40 ± 2 h before the prolonged (1-h-long) ischemic insult.
1288
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
body wt), given 15 min before IPC. In order to examine the possible effects of the complex itself, group V (Zn/DFO alone) animals were injected with 0.5 mg/kg Zn/DFO followed by 64 ± 2 h of perfusion with no additional intervention. Electroretinography ERG recordings were performed 24 h after the prolonged ischemic insult (or 64 ± 2 h after IPC induction). Full-field ERGs were recorded in anesthetized rats after overnight dark adaptation using a Ganzfeld dome and a computerized system (UTAS-E 3000; LKC Instruments, Gaithersburg, MD, USA). Pupils were dilated with 1% tropicamide and 2.5% phenylephrine (Fisher Pharmaceuticals, Tel-Aviv, Israel). Local anesthetic drops (benoxinate HCl, 0.4%; Fisher Pharmaceuticals) were administered before insertion of rat unipolar corneal contact lens electrodes (Medical Workshop, Groningen, The Netherlands), which served as the active lead. A reference electrode was placed on the tongue and a needle ground electrode was placed subcutaneously on the animal's head. Two series of responses were recorded in the dark-adapted state: rod responses to dim blue flashes and mixed cone–rod responses to a series of white flashes of increasing intensities (0.05–48 cd · s/m2). All ERG responses were filtered at 0.3 to 500 Hz, and signal averaging was applied.
and blocked with 0.5% gelatin and 0.1% sodium azide. The sample was then introduced and rabbit anti-H rat ferritin was added. After 3 h of incubation, plates were treated with the third antibody. Chlorophenol red–β-D-galactopyranoside (Roche Diagnostics, Indianapolis, IN, USA) at a concentration of 0.35 mg/ml was used as the substrate. The plates were washed and assayed, using a microplate reader (MR 5000; Dynatech Laboratories) with a 570 nm test filter and a 630 nm reference filter [41]. Iron content within a ferritin molecule Ferritin iron was measured as described previously [41]. Briefly, lysates of retinal tissue (pools of samples from each group) were incubated at 70°C for 10 min, cooled on ice, and centrifuged at 14,000 rpm for 20 min, and the supernatant was collected. A sample containing 3–4 μg of ferritin was incubated with the rabbit anti-rat ferritin antibody (mixture of anti-H and anti-L (50/50)) in order to precipitate the ferritin. After 72 h the precipitate was separated by centrifugation, washed, and dried. Concentrated nitric acid (100 μl) was added and the sample incubated at 37°C for 30 min. The total amount of iron atoms was determined by Zeeman atomic absorption spectroscopy. The average number of iron atoms per molecule of ferritin was then calculated.
Tissue preparation for biochemical assays
Assay of the labile iron pool (LIP)
Lysis buffer (1% deionized Triton X-100 and 0.1% sodium azide in 50 mM Tris–HCl, pH 7.5) was incubated with Chelex100 at room temperature for a minimum of 24 h to get rid of traces of iron. Phenylmethylsulfonyl fluoride, a protease inhibitor, was added to the lysis buffer just before use. Immediately after ERG recording, eyes were enucleated, and retinas were gently dissected out, placed in the lysis buffer, homogenized mechanically, and sonicated for 30 s. Samples were then incubated on ice for 30 min (vortexed every 5–6 min). After centrifugation at 3000 rpm for 15 min, the supernatant was collected and used for analysis.
Fifty-microliter aliquots of the supernatant (from the homogenized sample) was transferred to clear-bottom, 96-well plates (Maxisorp; Nunc), in quadruplicate. Fluorescent transferrin (Fl-T) was prepared by conjugating 5-(4,6-dichlorotriazinyl) aminofluorescein (Molecular Probes, Eugene, OR, USA) to apo-transferrin (Apo-Tf), as previously described [42,43]. Fl-T (50 μl, 1 μM) was added to 2 wells, whereas 50 μl of a mixture containing Fl-T (1 μM) and Apo-Tf (100 μM) was added to the other 2 wells of the quadruplicate (for inhibition of fluorescence quenching). The fluorescence was measured after 20 min (using a BMG Galaxy Fluostar microplate reader; BMG Lab Instruments, Offenburg, Germany) with a 485/538 nm excitation/emission filter pair. Calibration curves were obtained by spiking Hepes-buffered saline with ferric iron in the form of Fe/ nitrilotriacetate (1/7 mol/mol) to give final concentrations of 0.015 to 0.33 μM.
Measurement of total protein Total protein content was estimated using the bicinchoninic acid assay kit (Pierce, Rockford, IL, USA) in 96-well plates (TC Microwell 96F SI W/LID Nunclon D; Nunc, Roskilde, Denmark). The results were read using a microplate reader (MR 5000; Dynatech Laboratories, Chantilly, VA, USA) equipped with a 570 nm filter. Measurement of ferritin Levels of ferritin were determined by ELISA with 96-well microplates, using three antibodies: (i) goat anti-L rat ferritin and (ii) rabbit anti-H rat ferritin, both prepared by Professor A. M. Konijn [40]; (iii) goat anti-rabbit IgG (from Jackson ImmunoResearch Laboratories, West Grove, PA, USA), which was conjugated to β-galactosidase (Roche Applied Science, Mannheim, Germany). The plates were precoated with goat anti-L rat ferritin
Retinal histology Eyes of two or three animals from each experimental group were collected for histological examination 1 week after ERG recording. After enucleation, the eyes were fixed in Davidson fixative and the cornea, iris, and lens were removed. Eyecups were embedded in paraffin, cut into 4-μm-thick sections, and stained with hematoxylin and eosin. Statistical analysis Comparison of biochemical parameters was performed using repeated one-way ANOVA followed by post hoc test for multiple
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
comparisons. Differences were considered statistically significant when p ≤ 0.05. All results are presented as means ± SEM. Results Prolonged ischemia had profound deleterious effects on retinal function (Fig. 1) and structure (Fig. 2). Twenty-four hours after the prolonged ischemic insult (1 h), ERG recordings revealed a total loss of response to flashes of white light in dark-
1289
adapted animals, even when high-intensity stimuli were used (Fig. 1C). The nonischemic fellow eye showed normal responses (Fig. 1A). A single episode of IPC (7 min duration) applied 40 ± 2 h before the prolonged ischemia provided marked protection against this severe injury (Fig. 1B). In these retinas, the ERG amplitude was only marginally reduced compared to the fellow (nonischemic) eyes. At high-intensity stimuli, the mean ERG amplitudes were approximately 80% of the control for the a-wave component and 80–90% for the b-wave component (Fig. 1D
Fig. 1. Retinal function as measured by full-field ERG in eyes subjected to prolonged unilateral ischemia and reperfusion with and without ischemic preconditioning. ERGs were recorded in vivo 24 h after the prolonged ischemic insult (of 1 h) and 64 ± 2 h after a single episode of short ischemia (IPC; 7 min ischemia). Waveforms in (A–C) show ERG responses to white flashes of increasing intensity under dark-adapted conditions. (A) Control, nonischemic eye. (B) Eye subjected to IPC 40 ± 2 h before the prolonged ischemic insult. (C) Prolonged ischemia alone. (D) ERG a-wave amplitudes and (E) b-wave amplitudes in IPC + I/R eyes versus eyes exposed to I/R alone. (F) Log b-wave amplitude versus log stimulus intensity plots of eyes subjected to IPC+ I/R versus control (no ischemia) eyes. (Control eyes, squares; IPC + ischemia, diamonds; ischemia alone, triangles; n = 5–7 animals in each experimental group.)
1290
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
Fig. 2. Histology of retinas after IPC ± I/R showing the protective effects of IPC. Samples were collected 1 week after prolonged ischemia and stained with hematoxylin and eosin. (A) Control, nonischemic retina. (B) Retina subjected to prolonged (1 h) ischemia (I/R). (C) Retina subjected to IPC 40 ± 2 h before I/R. The large circular structure in the GCL is a patent retinal vessel. (D) Retina subjected to IPC alone. (GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segments of photoreceptors. Original magnification ×40 with digital zoom.)
and E). The log b-wave amplitude–intensity relations in eyes after IPC followed by I/R slightly differed from control nonischemic fellow eyes at low intensities, but converged at higher intensities (Fig. 1F). Thus, IPC provided marked and nearcomplete protection against the loss of retinal function associated with subsequent prolonged ischemia. IPC alone (without subsequent prolonged ischemia but with 64 h of subsequent reperfusion) did not affect the ERG response compared to controls (results not shown). These electrophysiological results were corroborated by histological examination (Fig. 2). One week after prolonged ischemia, a decrease in the thickness of the retina was observed (Fig. 2B). The number of rows of photoreceptor nuclei in the outer nuclear layer (ONL) decreased from 11–12 in controls (Fig. 2A) to 5–6 in I/R retinas. A reduction in the density of nuclei in the inner nuclear layer (INL) and ganglion cell layer was also evident. Furthermore, many nuclei contained condensed chromatin (Fig. 2B), indicative of cell injury. Cellular infiltration into the photoreceptor outer segment layer was also observed in I/R retinas. In contrast, a marked protective effect was evident in eyes subjected to IPC before the prolonged ischemia with both the ONL and the INL highly preserved (Fig. 2C). No changes in retinal histology were observed in eyes subjected to IPC alone, and their structure was identical to that of control eyes (Fig. 2D). The LIP was evaluated in retinal tissues after the same ischemia protocols. As shown in Fig. 3, prolonged ischemia followed by reperfusion induced a massive release of redoxactive and labile iron, noticeable even after 24 h of reperfusion; this amount of labile iron exceeded the control level by approximately threefold ( p = 0.0012). IPC, applied at 40 ± 2 h before prolonged ischemia, moderated this increase in LIP levels. It was still approximately twice higher than in control retinas ( p = 0.006), but significantly lower than that in the I/R-alone group ( p = 0.040). Similar changes in LIP levels were observed in retinas subjected to IPC alone ( p = 0.006, compared to nonischemic control). These pronounced changes in the retinal LIP after IPC alone raised the possibility that iron release not only correlated with but mediated IPC-induced protection. To examine this hypoth-
esis, we attempted to scavenge the labile iron liberated during/ after IPC and tested whether this intervention affected the protective effect. This was achieved by introducing the selective and cell-permeable iron chelator Zn/DFO shortly before the IPC phase. The Zn/DFO complex acts via the so-called “push-andpull” mechanism: it binds iron into a low-reactive complex and eventually pulls it out from the cell by substituting iron for zinc [44]. Indeed, Zn/DFO (0.5 mg/kg) administered iv 15 min before IPC effectively prevented the rise in labile iron caused by IPC
Fig. 3. Labile iron pool in retinas subjected to IPC ± I/R. The experimental groups are as described in Table 1 and included (i) control (naive) retinas (group I, n = 6 eyes); (ii) retinas subjected to prolonged (1 h) ischemia followed by 24 h of reperfusion (group II, n = 6); (iii) retinas subjected to IPC alone (single ischemic episode of 7 min) followed by 64 ± 2 h of reperfusion (group III, n = 7); (iv) retinas subjected to IPC followed by 40 ± 2 h reperfusion, then prolonged 1 h ischemia, and finally an additional 24 h of reperfusion (group IV, n = 12); (v) retinas of animals treated with Zn/DFO complex followed by 64 ± 2 h of perfusion (group V, n = 5); (vi) retinas subjected to IPC alone followed by 64 ± 2 h of reperfusion, and Zn/DFO complex was applied 15 min before IPC (group IIIa, n = 6); and (vii) retinas subjected to IPC followed by 40 ± 2 h reperfusion, then prolonged ischemia, and an additional 24 h of reperfusion, and Zn/DFO complex was applied 15 min before IPC (group IVa, n = 7). Each bar represents mean labile iron in μg/mg of total protein (±SEM). Zn/DFO complex was administrated iv at a concentration of 0.5 mg/kg of animal body weight. ⁎p b 0.05 from groups control, IPC + I/R, Zn/DFO alone, and Zn/DFO + IPC; † p b 0.05 from groups control and Zn/DFO + IPC.
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
Fig. 4. Low levels of the Zn/DFO complex reverse the protective effect of IPC. ERG responses to white flashes of increasing intensity in the dark-adapted state were recorded 24 h after exposing the retina to prolonged ischemia, with IPC applied at 40 ± 2 h earlier. Zn/DFO was administered intravenously 15 min before IPC. White bars, no Zn/DFO (protective effect of IPC evident). Black bars, 0.5 mg/kg Zn/DFO (the height of each of the black bars is near zero; the protective effect of IPC abolished). Hatched bars, 2.5 mg/kg Zn/DFO (emergence of partial protective effect). For each stimulus intensity, and between every two groups (of the three experimental groups), p b 0.05.
1291
enging at the reperfusion phase [45]. This mechanism may be similar to IPC in the heart, where iron mobilization and redistribution have been proposed to activate a signaling cascade underlying the protective effect of IPC [41]. In the heart, an additional major iron-related event associated with IPC is increased expression of ferritin [41]. Therefore, we examined ferritin levels in retinas subjected to ischemia and reperfusion with and without IPC (Fig. 5). One hour (prolonged) ischemia alone led to an insignificant decrease in retinal ferritin by about 20%, compared to controls. When IPC was applied before I/R, this decrease not only was abolished but turned into a marked increase in retinal ferritin level compared to control ( p = 0.031). Likewise, retinas of animals subjected to IPC alone showed a similar increase in ferritin ( p = 0.032 vs. control), indicating that IPC itself triggered this event.
alone and brought the LIP level to that of controls (Fig. 3). Then, when Zn/DFO + IPC was followed by prolonged ischemia (Zn/DFO + IPC + I/R, group IVa) the level of LIP was quite high and close to that observed in I/R alone (Fig. 3). Notably, in these eyes, the protective effect of IPC against I/R injury was abolished and retinal function (as quantified by ERG recordings) was severely impaired, similar to I/R alone (Fig. 4). These observations suggest that labile iron mobilized during and after the IPC phase plays a causative role in IPC protection. When Zn/DFO was given at a high concentration (2.5 mg/kg), the effect of IPC was also largely abolished (Fig. 4). However, at this large dose, residual amounts of complex are apparently still present and are able to provide partial protection by iron scav-
Fig. 5. Ferritin levels in retinas subjected to IPC ± I/R. The experimental groups are as described in Table 1 and for Fig. 3 and included (i) control (naive) retinas (group I, n = 20 eyes); (ii) I/R retinas (group II, n = 6); (iii) IPC alone (group III, n = 7); (iv) IPC + I/R (group IV, n = 12); (v) Zn/DFO alone (group V, n = 6); (vi) Zn/DFO + IPC (group IIIa, n = 6); and (vii) Zn/DFO + IPC + I/R (group IVa, n = 6). Each bar represents mean ferritin levels (±SEM). Zn/DFO complex was administrated iv at a concentration of 0.5 mg/kg of animal body weight. ⁎p b 0.05 for the specific group, compared to the following groups: control, I/R, Zn/ DFO alone, Zn/DFO + IPC, and Zn/DFO + IPC + I/R.
Fig. 6. (A) Iron content per ferritin molecule in retinas subjected to IPC ± I/R (mean ± SEM). Due to the small weight of each retina sample, and the requirement for several micrograms of ferritin for an adequate immunoprecipitation reaction, a minimum of six retina samples were pooled together and a minimum of two pools were analyzed for each group, in duplicate, monitoring the amounts of iron and ferritin. The experimental groups are as described in Table 1 and for Fig. 3 and included (i) control (naive) retinas (group I); (ii) I/R retinas (group II); (iii) IPC alone (group III); (iv) IPC + I/R (group IV); (v) Zn/ DFO alone (group V); (vi) Zn/DFO + IPC (group IIIa); and (vii) Zn/DFO + IPC + I/R (group IVa). Each bar represents the iron content per molecule of ferritin. Zn/ DFO complex was administrated iv at a concentration of 0.5 mg/kg of animal body weight. ⁎p b 0.05 of the specific group compared to the following groups: IPC only and IPC + I/R; †p b 0.05 of the specific group (I/R), compared to all other groups. (B) The relative total amounts of ferritin-bound iron (per milligram of retinal protein) of the experimental groups: control (set to 100%), ischemia and reperfusion (I/R), IPC only, Zn/DFO alone, Zn/DFO + IPC, and Zn/DFO + IPC + I/R. The values were calculated and normalized by multiplying the concentration of ferritin in the retina (ng-Ft/mg protein) by the amount of iron in a ferritin molecule.
1292
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
Administration of Zn/DFO 15 min before the IPC procedure prevented the rise in ferritin seen after IPC alone and partially prevented the increase in ferritin caused by IPC + I/R. The chelator did not influence ferritin levels when applied to control animals (Fig. 5). For more detailed analysis of iron–ferritin relationships we calculated the iron content within a ferritin molecule (Fig. 6A). In control retinas, each ferritin molecule contained an average of 2457 iron atoms. After prolonged ischemia, the average iron content within a ferritin molecule dramatically increased and ferritin became oversaturated by iron (4764 atoms/molecule). IPC not only prevented such ferritin overload, but even led to a decrease in iron content within ferritin molecules in comparison with control. The same effect was shown by IPC alone. Administration of the Zn/DFO complex 15 min before IPC with or without subsequent prolonged ischemia did not affect ferritin iron content (Fig. 6A). It is important that not only the LIP but also the total iron content changed in the I/R and Zn/DFO + IPC + I/R groups, thus pointing to the proposed mechanism of IPC protection (Fig. 6B). Discussion Prolonged ischemia causes severe functional and structural injury to the retina. IPC, when applied 40 ± 2 h before the prolonged ischemic insult, provided nearly complete protection against this I/R-induced damage, as evident from functional and histological assessments. These findings are in accord with data previously published by others [1,28,33]. The present study suggests new components of the molecular response that underlie functional protection of the retina by IPC. First, IPC induced a significant increase in cellular ferritin levels. We, therefore, surmise that the increased levels and availability of ferritin induced by retinal IPC serve to scavenge the redoxactive and labile iron released by I/R, thus reducing ironmediated injury. Second, the reduction in ferritin content observed in retinas subjected to prolonged ischemia alone could be related to increased protein catabolism in ischemic tissues and subsequent depletion of ferritin. As a result, the labile iron released during I/R remains unbound and adds to the LIP. The elevated levels of labile iron could initiate the formation of aggressive free radicals and aggravate tissue damage upon reperfusion [46,47]. In similarity to the heart, ferritin accumulation observed in the retina after IPC could be due to IPC-induced mobilization and redistribution of relatively small amounts of iron that bind to the iron regulatory proteins (IRPs), which act to reduce the tight inhibitory control of ferritin translation. Under normal iron balance, the two IRPs block translation of the ferritin message. After IPC, these proteins are, probably, converted to their nonfunctional forms, allowing initiation of translation [20]. In our previous studies on isolated rat hearts it has been proposed that the minute amounts of labile iron released by the IPC procedure serve as a signaling species that triggers de novo synthesis of apo-ferritin. This enables the heart to scavenge much larger and deleterious amounts of labile iron released after a subsequent prolonged ischemic insult and contributes to IPCinduced cardiac protection [41].
In analogy to the heart, we suggest that similar iron release and similar iron-mediated processes also occur after IPC and ischemic insults in the retina, albeit with different windows of effect temporally. In the heart, the protective effect of IPC is rapid (within minutes), whereas in the retina the conditioning and protective effects become manifest only after hours or days. However, despite these temporal differences, the similarities between the heart and the retina are apparent and may imply a common mechanism underlying IPC protection. Mobilization of iron depends on its valency state: it is ferrous rather than ferric iron that dissociates from ferritin [20]. It is therefore likely that the iron release from ferritin and iron accumulation in LIP occur during prolonged ischemia which is associated with oxygen deficiency and prevalence of reductive conditions [39]. Then, after reperfusion and restoration of oxygen supply, these high levels of labile iron may markedly accelerate oxidative reactions and free radical formation. Retinal cells are extremely sensitive to oxidative damage [48]. It is well known that enhanced ROS production and oxidative stress occur after ischemia and reperfusion. ROS can oxidize a wide spectrum of biological molecules including lipids, DNA, and proteins [49–56], which underlie morphological and functional injuries to the cell. The fact that a small amount of Zn/DFO administered shortly before IPC abolishes IPC protection (Fig. 4) supports the proposed mechanism: the complex enters retinal cells [57,58], binds the equivalent amount of chelatable iron just liberated by IPC, and thus prevents this iron from binding to the IRPs and from inducing the protective ferritin accumulation. This stimulating result raises the question of whether an ironcontaining molecule able to trigger an IPC-like protective response can be designed. Such a compound is expected to be protective against prolonged ischemia, without the need to subject the eye to pretreatment by IPC. If proven successful, this approach could provide more support for the proposed ironbased mechanism of IPC and be relevant for future clinical therapeutic applications. In summary, we suggest that iron plays a dual role in both I/R injury and IPC protection. Thus, excess iron mediates prolonged ischemia-induced injury by catalyzing the formation of ROS upon reperfusion. On the other hand, minute amounts of labile iron released after IPC play a signaling role and activate the regulatory process leading to protection [41]. From this study, a major protective mechanism is the accumulation of ferritin that protects the tissue against the excessive iron released by subsequent prolonged ischemia, thus reducing the degree of ischemia–reperfusion injury. We also hypothesize that the proposed mechanism of IPC-induced protection is common to different organs. A deeper understanding of these pathways and how they are activated should inspire novel approaches to preventative therapy of ischemic injury. Acknowledgments M.C. is the incumbent of the Dr. William Ganz Chair of Heart Studies at the Hebrew University of Jerusalem. This study was supported by grants from the Israel Science Foundation
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
(585/02 and 316/05), the Pepka and Dr. Moshe Bergman Memorial Fund at the Hebrew University of Jerusalem, and the Yedidut Research Fund. The contribution of Dr. Leonid Grinberg in editing the manuscript is acknowledged.
[19]
[20]
References [1] Zhang, C.; Rosenbaum, D. M.; Shaikh, A. R.; Li, Q.; Rosenbaum, P. S.; Pelham, D. J., et al. Ischemic preconditioning attenuates apoptotic cell death in the rat retina. Invest. Ophthalmol. Visual Sci. 43:3059–3066; 2002. [2] Joo, C. K.; Choi, J. S.; Ko, H. W.; Park, K. Y.; Sohn, S.; Chun, M. H., et al. Necrosis and apoptosis after retinal ischemia: involvement of NMDAmediated excitotoxicity and p53. Invest. Ophthalmol. Visual Sci. 40:713–720; 1999. [3] Iida, T.; Spaide, R. F.; Kantor, J. Retinal and choroidal arterial occlusion in Wegener's granulomatosis. Am. J. Ophthalmol. 133:151–152; 2002. [4] Hayreh, S. S. Prevalent misconceptions about acute retinal vascular occlusive disorders. Prog. Retin. Eye Res. 24:493–519; 2005. [5] Aiello, L. P. The potential role of PKC beta in diabetic retinopathy and macular edema. Surv. Ophthalmol. 47 (Suppl. 2):S263–S269; 2002. [6] Butler, J. M.; Guthrie, S. M.; Koc, M.; Afzal, A.; Caballero, S.; Brooks, H. L., et al. SDF-1 is both necessary and sufficient to promote proliferative retinopathy. J. Clin. Invest. 115:86–93; 2005. [7] Madl, J. E.; McIlnay, T. R.; Powell, C. C.; Gionfriddo, J. R. Depletion of taurine and glutamate from damaged photoreceptors in the retinas of dogs with primary glaucoma. Am. J. Vet. Res. 66:791–799; 2005. [8] Mondal, L. K.; Baidya, K. P.; Bhattacharya, B.; Chatterjee, P. R.; Bhaduri, G. The efficacy of topical administration of brimonidine to reduce ischaemia in the very early stage of diabetic retinopathy in good controlled type-2 diabetes mellitus. J. Indian Med. Assoc. 102:724–725 (729); 2004. [9] Kaja, S.; Yang, S. H.; Wei, J.; Fujitani, K.; Liu, R.; Brun-Zinkernagel, A. M., et al. Estrogen protects the inner retina from apoptosis and ischemia-induced loss of Vesl-1L/Homer 1c immunoreactive synaptic connections. Invest. Ophthalmol. Vis. Sci. 44:3155–3162; 2003. [10] Peachey, N. S.; Charles, H. C.; Lee, C. M.; Fishman, G. A.; Cunha-Vaz, J. G.; Smith, R. T. Electroretinographic findings in sickle cell retinopathy. Arch. Ophthalmol. 105:934–938; 1987. [11] Keshet, E. More weapons in the arsenal against ischemic retinopathy. J. Clin. Invest. 107:945–946; 2001. [12] Suganami, E.; Takagi, H.; Ohashi, H.; Suzuma, K.; Suzuma, I.; Oh, H., et al. Leptin stimulates ischemia-induced retinal neovascularization: possible role of vascular endothelial growth factor expressed in retinal endothelial cells. Diabetes 53:2443–2448; 2004. [13] Osborne, N. N.; Casson, R. J.; Wood, J. P.; Chidlow, G.; Graham, M.; Melena, J. Retinal ischemia: mechanisms of damage and potential therapeutic strategies. Prog. Retin. Eye Res. 23:91–147; 2004. [14] Gutteridge, J. M.; Rowley, D. A.; Halliwell, B. Superoxide-dependent formation of hydroxyl radicals in the presence of iron salts: detection of ‘free’ iron in biological systems by using bleomycin-dependent degradation of DNA. Biochem. J. 199:263–265; 1981. [15] Dunaief, J. L.; Richa, C.; Franks, E. P.; Schultze, R. L.; Aleman, T. S.; Schenck, J. F., et al. Macular degeneration in a patient with aceruloplasminemia, a disease associated with retinal iron overload. Ophthalmology 112:1062–1065; 2005. [16] Hahn, P.; Milam, A. H.; Dunaief, J. L. Maculas affected by age-related macular degeneration contain increased chelatable iron in the retinal pigment epithelium and Bruch's membrane. Arch. Ophthalmol. 121:1099–1105; 2003. [17] McGahan, M. C.; Harned, J.; Mukunnemkeril, M.; Goralska, M.; Fleisher, L.; Ferrell, J. B. Iron alters glutamate secretion by regulating cytosolic aconitase activity. Am. J. Physiol. Cell. Physiol. 288:C1117–C1124; 2005. [18] Harned, J.; Grimes, A. M.; McGahan, M. C. The effect of UVB irradiation on ferritin subunit synthesis, ferritin assembly and Fe metabolism in
[21]
[22]
[23]
[24]
[25] [26] [27]
[28]
[29] [30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
1293
cultured canine lens epithelial cells. Photochem. Photobiol. 77:440–445; 2003. Goralska, M.; Holley, B.; McGahan, M. C. The effects of Tempol on ferritin synthesis and Fe metabolism in lens epithelial cells. Biochim. Biophys. Acta 1497:51–60; 2000. Ponka, P. Cellular iron metabolism. Kidney Inter., Suppl. 69:S2–S11; 1999. Hahn, P.; Qian, Y.; Dentchev, T.; Chen, L.; Beard, J.; Harris, Z. L., et al. Disruption of ceruloplasmin and hephaestin in mice causes retinal iron overload and retinal degeneration with features of age-related macular degeneration. Proc. Natl. Acad. Sci. U. S. A. 101:13850–13855; 2004. Sommerschild, H. T.; Kirkeboen, K. A. Preconditioning—endogenous defence mechanisms of the heart. Acta Anaesthesiol. Scand. 46:123–137; 2002. Nakano, A.; Cohen, M. V.; Downey, J. M. Ischemic preconditioning: from basic mechanisms to clinical applications. Pharmacol. Ther. 86:263–275; 2000. Serafin, A.; Fernandez-Zabalegui, L.; Prats, N.; Wu, Z. Y.; Rosello-Catafau, J.; Peralta, C. Ischemic preconditioning: tolerance to hepatic ischemia– reperfusion injury. Histol. Histopathol. 19:281–289; 2004. Carini, R.; Albano, E. Recent insights on the mechanisms of liver preconditioning. Gastroenterology 125:1480–1491; 2003. Bonventre, J. V. Kidney ischemic preconditioning. Curr. Opin. Nephrol. Hypertens. 11:43–48; 2002. Schaller, B.; Graf, R. Cerebral ischemic preconditioning: an experimental phenomenon or a clinical important entity of stroke prevention? J. Neurol. 249:1503–1511; 2002. Roth, S.; Li, B.; Rosenbaum, P. S.; Gupta, H.; Goldstein, I. M.; Maxwell, K. M., et al. Preconditioning provides complete protection against retinal ischemic injury in rats. Invest. Ophthalmol. Vis. Sci. 39:777–785; 1998. Roth, S. Endogenous neuroprotection in the retina. Brain Res. Bull. 62: 461–466; 2004. Li, B.; Yang, C.; Rosenbaum, D. M.; Roth, S. Signal transduction mechanisms involved in ischemic preconditioning in the rat retina in vivo. Exp. Eye Res. 70:755–765; 2000. Sakamoto, K.; Kuwagata, M.; Nakahara, T.; Ishii, K. Late preconditioning in rat retina: involvement of adenosine and ATP-sensitive K(+) channel. Eur. J. Pharmacol. 418:89–93; 2001. Ettaiche, M.; Heurteaux, C.; Blondeau, N.; Borsotto, M.; Tinel, N.; Lazdunski, M. ATP-sensitive potassium channels (K(ATP)) in retina: a key role for delayed ischemic tolerance. Brain Res. 890:118–129; 2001. Roth, S.; Dreixler, J. C.; Shaikh, A. R.; Lee, K. H.; Bindokas, V. Mitochondrial potassium ATP channels and retinal ischemic preconditioning. Invest. Ophthalmol. Vis. Sci. 47:2114–2124; 2006. Zhu, Y.; Zhang, Y.; Ojwang, B. A.; Brantley Jr., M. A.; Gidday, J. M. Long-term tolerance to retinal ischemia by repetitive hypoxic preconditioning: role of HIF-1alpha and heme oxygenase-1. Invest. Ophthalmol. Vis. Sci. 48:1735–1743; 2007. Lin, J.; Roth, S. Ischemic preconditioning attenuates hypoperfusion after retinal ischemia in rats. Invest. Ophthalmol. Vis. Sci. 40:2925–2931; 1999. Nonaka, A.; Kiryu, J.; Tsujikawa, A.; Yamashiro, K.; Nishijima, K.; Miyamoto, K., et al. Inhibitory effect of ischemic preconditioning on leukocyte participation in retinal ischemia–reperfusion injury. Invest. Ophthalmol. Vis. Sci. 42:2380–2385; 2001. Li, Y.; Roth, S.; Laser, M.; Ma, J. X.; Crosson, C. E. Retinal preconditioning and the induction of heat-shock protein 27. Invest. Ophthalmol. Vis. Sci. 44:1299–1304; 2003. Casson, R. J.; Wood, J. P.; Melena, J.; Chidlow, G.; Osborne, N. N. The effect of ischemic preconditioning on light-induced photoreceptor injury. Invest. Ophthalmol. Vis. Sci. 44:1348–1354; 2003. Chevion, M.; Jiang, Y.; Har-El, R.; Berenshtein, E.; Uretzky, G.; Kitrossky, N. Copper and iron are mobilized following myocardial ischemia: possible predictive criteria for tissue injury. Proc. Natl. Acad. Sci. U. S. A. 90: 1102–1106; 1993. Konijn, A. M.; Tal, R.; Levy, R.; Matzner, Y. Isolation and fractionation of ferritin from human term placenta—a source for human isoferritins. Anal. Biochem. 144:423–428; 1985.
1294
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
[41] Berenshtein, E.; Vaisman, B.; Goldberg-Langerman, C.; Kitrossky, N.; Konijn, A. M.; Chevion, M. Roles of ferritin and iron in ischemic preconditioning of the heart. Mol. Cell. Biochem. 234–235:283–292; 2002. [42] Breuer, W.; Cabantchik, Z. I. A fluorescence-based one-step assay for serum non-transferrin-bound iron. Anal. Biochem. 299:194–202; 2001. [43] Esposito, B. P.; Breuer, W.; Sirankapracha, P.; Pootrakul, P.; Hershko, C.; Cabantchik, Z. I. Labile plasma iron in iron overload: redox activity and susceptibility to chelation. Blood 102:2670–2677; 2003. [44] Chevion, M. Protection against free radical-induced and transition metalmediated damage: the use of “pull” and “push” mechanisms. Free Radic. Res. Commun. 12–13 (Pt 2):691–696; 1991. [45] Ophir, A.; Berenshtein, E.; Kitrossky, N.; Berman, E. R.; Photiou, S.; Rothman, Z., et al. Hydroxyl radical generation in the cat retina during reperfusion following ischemia. Exp. Eye Res. 57:351–357; 1993. [46] Minotti, G. NADPH-and adriamycin-dependent microsomal release of iron and lipid peroxidation. Arch. Biochem. Biophys. 277:268–276; 1990. [47] Kurz, T.; Leake, A.; Von Zglinicki, T.; Brunk, U. T. Relocalized redoxactive lysosomal iron is an important mediator of oxidative-stress-induced DNA damage. Biochem. J. 378:1039–1045; 2004. [48] Rose, R. C.; Richer, S. P.; Bode, A. M. Ocular oxidants and antioxidant protection. Proc. Soc. Exp. Biol. Med. 217:397–407; 1998. [49] Aust, S. D. Iron redox reactions and lipid peroxidation. Basic Life Sci. 49:137–144; 1988. [50] Chevion, M. A site-specific mechanism for free radical induced biological
[51] [52]
[53]
[54] [55]
[56]
[57]
[58]
damage: the essential role of redox-active transition metals. Free Radic. Biol. Med. 5:27–37; 1988. Minotti, G. Metals and membrane lipid damage by oxy-radicals. Ann. N. Y. Acad. Sci. 551:34–44 (discussion 45-6); 1988. Ursini, F.; Maiorino, M.; Hochstein, P.; Ernster, L. Microsomal lipid peroxidation: mechanisms of initiation. The role of iron and iron chelators. Free Radic. Biol. Med. 6:31–36; 1989. Richter, C.; Park, J. W.; Ames, B. N. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc. Natl. Acad. Sci. U. S. A. 85:6465–6467; 1988. Imlay, J. A.; Linn, S. DNA damage and oxygen radical toxicity. Science 240:1302–1309; 1988. Stadtman, E. R. Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free Radic. Biol. Med. 9:315–325; 1990. Tatarkova, Z.; Aplan, P.; Matejovicova, M.; Lehotsky, J.; Dobrota, D.; Flameng, W. Effect of ischemia and reperfusion on protein oxidation in isolated rabbit hearts. Physiol. Res. 54:185–191; 2004. Ophir, A.; Berenshtein, E.; Kitrossky, N.; Averbukh, E. Protection of the transiently ischemic cat retina by zinc–desferrioxamine. Invest. Ophthalmol. Vis. Sci. 35:1212–1222; 1994. Banin, E.; Berenshtein, E.; Kitrossky, N.; Pe'er, J.; Chevion, M. Gallium– desferrioxamine protects the cat retina against injury after ischemia and reperfusion. Free Radic. Biol. Med. 28:315–323; 2000.
Free Radical Biology & Medicine 44 (2008) 1286 – 1294 www.elsevier.com/locate/freeradbiomed
Original Contribution
Ischemic preconditioning of the rat retina: Protective role of ferritin Alexey Obolensky a,b,1 , Eduard Berenshtein b,1 , Abraham M. Konijn c , Eyal Banin a,1 , Mordechai Chevion b,⁎,1 b
a Department of Ophthalmology, The Hebrew University–Hadassah Schools of Medicine and Dental Medicine, Jerusalem 91120, Israel Department of Cellular Biochemistry and Human Genetics, The Hebrew University–Hadassah Schools of Medicine and Dental Medicine, Jerusalem 91120, Israel c Department of Human Nutrition and Metabolism, The Hebrew University–Hadassah Schools of Medicine and Dental Medicine, Jerusalem 91120, Israel
Received 30 September 2007; revised 29 October 2007; accepted 30 October 2007 Available online 21 November 2007
Abstract Ischemic preconditioning (IPC) of the retina, accomplished by ischemia of short duration, is highly effective in preventing subsequent severe injury caused by iron-dependent free radical burst after prolonged ischemia. To investigate the mechanistic basis for IPC rescue, we examined changes in the levels of the retinal redox-active and labile iron pool, ferritin, and ferritin-bound iron. Prolonged ischemia severely impaired retinal function, with total loss of the full-field electroretinographic response. IPC provided marked protection against such injury. Histological examination revealed that ischemia-associated structural damage and loss of cells in the outer and inner nuclear layers were largely prevented by IPC. Ferritin levels decreased after prolonged ischemia but remained close to normal when the ischemic episode was preceded by IPC. The protective effect of IPC on retinal function and ferritin was blocked by a zinc–desferrioxamine complex known to interfere with iron signaling. The results suggest a mechanism whereby IPC activates an iron signaling pathway leading to a marked increase in ferritin levels, which mediates resistance to prolonged ischemia. © 2007 Elsevier Inc. All rights reserved. Keywords: Iron; Ferritin; Free radicals; Retina; Preconditioning
Subjecting a tissue to prolonged ischemia causes detrimental effects, often leading to cell injury and death via necrosis and apoptosis [1,2]. A number of ocular pathologies, such as occlusions of the central retinal artery and vein, are associated with acute retinal ischemia and rapid visual loss [3,4]. In other conditions which develop gradually (e.g., diabetic retinopathy and, possibly, glaucoma) chronic retinal ischemia is a major pathogenic factor [5–8]. Ischemic challenges also contribute to the pathogenesis of less prevalent conditions, such as sickle cell retinopathy, radiation retinopathy, and retinopathy of prematurity [9–12]. Ischemia in the eye often leads to severe visual impairment, due either to irreversible loss of neuronal tissue or to abnormal ocular neovascularization. Currently, intense research is focused on the mechanisms of ischemia/reperfusion (I/R) injury, including a search for novel and more effective therapies [13].
⁎ Corresponding author. Fax: +972 2 641 5848. E-mail address: [email protected] (M. Chevion). 1 These authors contributed equally to this article. 0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.10.060
Ischemic injury is at least partially mediated by the formation of reactive oxygen-derived species (ROS), including free radicals, which burst with the resumption of blood flow (reperfusion). Under normal conditions, a balance between the continuous generation of low levels of ROS and adequate levels of relevant antioxidants is maintained. However, in the presence of redoxactive and labile (chelatable) iron, low-reactive oxygen species, such as the superoxide radical anion (O2·−), convert into highly reactive radical species, such as the hydroxyl radical (HO·) [14]. Thus, redox-active iron can augment ROS-induced cell damage caused by reperfusion injury and other pathological conditions of the eye [15–19]. Normally, the cell tightly controls the level of labile iron by binding most of it within ferritin—the major iron storage protein [20]. After I/R, iron may be released from ferritin and becomes involved in redox cycling, thus challenging the cellular antioxidant defense mechanisms [15,16,21]. Characterizing the iron which catalyzes redox reactions, including injury of cellular components, is complicated and not straightforward. Various terms have been used by various investigators and include “redox-active iron,” “labile iron,” “chelatable
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
iron,” and “free iron.” Although the first term best describes the iron activity, the others are also extensively used. It is noteworthy that due to the low solubility of iron, in particular in the ferric state, the term “free iron” is misleading and should be avoided. Ischemic preconditioning (IPC) is a well-established procedure to protect tissues against prolonged ischemia. It consists of a brief noninjurious ischemic episode (or repeated episodes) applied before the prolonged ischemia and confers remarkable resistance against I/R injury in various organs including the heart [22,23], liver [24,25], kidney [26], and brain [27]. Recently, IPC was also shown to protect the rat retina against a subsequent prolonged ischemic insult [28]. The protective effect was activated hours after the IPC procedure had been applied, and remained in effect for 72 h, similar to other neuronal tissues [29]. A variety of factors have been suggested to play roles in retinal IPC, including adenosine and adenosine receptors, de novo protein synthesis [28,30], opening of mitochondrial KATP channels [31–33], expression of heme oxygenase-1 and HIF-1α [34], reduction of apoptosis-related expression of genes, elevation of VEGF-A levels, alterations in protein phosphorylation [1], attenuation of ischemia-associated hypoperfusion [35], inhibition of leukocyte rolling and accumulation [36], and induction of the heat shock protein HSP27 [37] associated with inhibition of caspase-3 activation. IPC caused up-regulation of bFGF, GFAP, and Bcl-2 and also “cross-protected” photoreceptors against light-induced injury [38]. Despite this growing body of data and knowledge, evidence on the iron-related mechanisms of IPC in the retina is still lacking. Given the important role of iron-mediated mechanisms in I/R injury [39], we studied the major components of iron homeostasis (labile iron, ferritin, and ferritin-bound iron) in relation to I/R and IPC in the rat retina. Materials and methods Animals and anesthesia One hundred six Sprague–Dawley rats weighing 225–250 g were maintained on a 12:12 h light:dark cycle, with unlimited food and water. For induction of ischemia and for electro-
1287
retinographic (ERG) recordings, the rats were anesthetized by intraperitoneal injections of ketamine HCl (100 mg/kg; Ketalar, Parke Davis, UK) in combination with the relaxing agent xylazine (5 mg/kg; VMD, Arendonk, Belgium). Subsequent intramuscular injections of the same mixture were used to extend anesthesia when required. All the experiments were approved by the Institutional Animal Care and Use Committee of the Hebrew University of Jerusalem, conforming to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996). Induction of retinal ischemia Retinal ischemia was induced as described by Roth et al. [28]. A 3-O silk suture loop was passed behind one eye, around the optic nerve and associated blood vessels. Suture ends were then passed through a small piece of polyethylene tubing. Ocular ischemia was induced by pulling the sutures tight to occlude blood flow in the main retinal blood vessels. Cessation of retinal blood flow was verified by indirect ophthalmoscopy of the fundus. Occlusion of retinal blood vessels was maintained for 7 min to induce IPC and for 1 h to cause the prolonged ischemic insult, as previously established by Roth et al. [28]. The fellow eye was left as an untreated control in all cases. Experimental groups and protocol Animals were divided into five experimental groups (Table 1). In group I (control), no ischemia was induced. Group II rats (I/R) were subjected to 1 h ocular ischemia followed by 24 h reperfusion. In group III (IPC alone) eyes were subjected to 7 min ischemia followed by 64 ± 2 h reperfusion, without any additional intervention. Group IV rats (IPC + I/R) were exposed to the same IPC procedure as group III and 40 ± 2 h later challenged with 1 h prolonged ischemia followed by 24 h reperfusion. Subgroups of animals from groups III and IV (IIIa and IVa) were intravenously (iv) injected with an aqueous solution of the zinc–desferrioxamine complex (Zn/DFO; at a dose of either 0.5 or 2.5 mg/kg
Table 1 Experimental groups and outline of the protocols used
Electroretinographic recordings were performed 24 h after prolonged ischemia, and retinal samples were collected for biochemical analyses immediately afterward. Tissue for histological assessment was collected 1 week after the ischemic episode. a In groups IIIa and IVa, Zn/DFO was injected iv 15 min before IPC. b The ischemic preconditioning (IPC) procedure consisted of a short period (7 min) of retinal ischemia induced at 40 ± 2 h before the prolonged (1-h-long) ischemic insult.
1288
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
body wt), given 15 min before IPC. In order to examine the possible effects of the complex itself, group V (Zn/DFO alone) animals were injected with 0.5 mg/kg Zn/DFO followed by 64 ± 2 h of perfusion with no additional intervention. Electroretinography ERG recordings were performed 24 h after the prolonged ischemic insult (or 64 ± 2 h after IPC induction). Full-field ERGs were recorded in anesthetized rats after overnight dark adaptation using a Ganzfeld dome and a computerized system (UTAS-E 3000; LKC Instruments, Gaithersburg, MD, USA). Pupils were dilated with 1% tropicamide and 2.5% phenylephrine (Fisher Pharmaceuticals, Tel-Aviv, Israel). Local anesthetic drops (benoxinate HCl, 0.4%; Fisher Pharmaceuticals) were administered before insertion of rat unipolar corneal contact lens electrodes (Medical Workshop, Groningen, The Netherlands), which served as the active lead. A reference electrode was placed on the tongue and a needle ground electrode was placed subcutaneously on the animal's head. Two series of responses were recorded in the dark-adapted state: rod responses to dim blue flashes and mixed cone–rod responses to a series of white flashes of increasing intensities (0.05–48 cd · s/m2). All ERG responses were filtered at 0.3 to 500 Hz, and signal averaging was applied.
and blocked with 0.5% gelatin and 0.1% sodium azide. The sample was then introduced and rabbit anti-H rat ferritin was added. After 3 h of incubation, plates were treated with the third antibody. Chlorophenol red–β-D-galactopyranoside (Roche Diagnostics, Indianapolis, IN, USA) at a concentration of 0.35 mg/ml was used as the substrate. The plates were washed and assayed, using a microplate reader (MR 5000; Dynatech Laboratories) with a 570 nm test filter and a 630 nm reference filter [41]. Iron content within a ferritin molecule Ferritin iron was measured as described previously [41]. Briefly, lysates of retinal tissue (pools of samples from each group) were incubated at 70°C for 10 min, cooled on ice, and centrifuged at 14,000 rpm for 20 min, and the supernatant was collected. A sample containing 3–4 μg of ferritin was incubated with the rabbit anti-rat ferritin antibody (mixture of anti-H and anti-L (50/50)) in order to precipitate the ferritin. After 72 h the precipitate was separated by centrifugation, washed, and dried. Concentrated nitric acid (100 μl) was added and the sample incubated at 37°C for 30 min. The total amount of iron atoms was determined by Zeeman atomic absorption spectroscopy. The average number of iron atoms per molecule of ferritin was then calculated.
Tissue preparation for biochemical assays
Assay of the labile iron pool (LIP)
Lysis buffer (1% deionized Triton X-100 and 0.1% sodium azide in 50 mM Tris–HCl, pH 7.5) was incubated with Chelex100 at room temperature for a minimum of 24 h to get rid of traces of iron. Phenylmethylsulfonyl fluoride, a protease inhibitor, was added to the lysis buffer just before use. Immediately after ERG recording, eyes were enucleated, and retinas were gently dissected out, placed in the lysis buffer, homogenized mechanically, and sonicated for 30 s. Samples were then incubated on ice for 30 min (vortexed every 5–6 min). After centrifugation at 3000 rpm for 15 min, the supernatant was collected and used for analysis.
Fifty-microliter aliquots of the supernatant (from the homogenized sample) was transferred to clear-bottom, 96-well plates (Maxisorp; Nunc), in quadruplicate. Fluorescent transferrin (Fl-T) was prepared by conjugating 5-(4,6-dichlorotriazinyl) aminofluorescein (Molecular Probes, Eugene, OR, USA) to apo-transferrin (Apo-Tf), as previously described [42,43]. Fl-T (50 μl, 1 μM) was added to 2 wells, whereas 50 μl of a mixture containing Fl-T (1 μM) and Apo-Tf (100 μM) was added to the other 2 wells of the quadruplicate (for inhibition of fluorescence quenching). The fluorescence was measured after 20 min (using a BMG Galaxy Fluostar microplate reader; BMG Lab Instruments, Offenburg, Germany) with a 485/538 nm excitation/emission filter pair. Calibration curves were obtained by spiking Hepes-buffered saline with ferric iron in the form of Fe/ nitrilotriacetate (1/7 mol/mol) to give final concentrations of 0.015 to 0.33 μM.
Measurement of total protein Total protein content was estimated using the bicinchoninic acid assay kit (Pierce, Rockford, IL, USA) in 96-well plates (TC Microwell 96F SI W/LID Nunclon D; Nunc, Roskilde, Denmark). The results were read using a microplate reader (MR 5000; Dynatech Laboratories, Chantilly, VA, USA) equipped with a 570 nm filter. Measurement of ferritin Levels of ferritin were determined by ELISA with 96-well microplates, using three antibodies: (i) goat anti-L rat ferritin and (ii) rabbit anti-H rat ferritin, both prepared by Professor A. M. Konijn [40]; (iii) goat anti-rabbit IgG (from Jackson ImmunoResearch Laboratories, West Grove, PA, USA), which was conjugated to β-galactosidase (Roche Applied Science, Mannheim, Germany). The plates were precoated with goat anti-L rat ferritin
Retinal histology Eyes of two or three animals from each experimental group were collected for histological examination 1 week after ERG recording. After enucleation, the eyes were fixed in Davidson fixative and the cornea, iris, and lens were removed. Eyecups were embedded in paraffin, cut into 4-μm-thick sections, and stained with hematoxylin and eosin. Statistical analysis Comparison of biochemical parameters was performed using repeated one-way ANOVA followed by post hoc test for multiple
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
comparisons. Differences were considered statistically significant when p ≤ 0.05. All results are presented as means ± SEM. Results Prolonged ischemia had profound deleterious effects on retinal function (Fig. 1) and structure (Fig. 2). Twenty-four hours after the prolonged ischemic insult (1 h), ERG recordings revealed a total loss of response to flashes of white light in dark-
1289
adapted animals, even when high-intensity stimuli were used (Fig. 1C). The nonischemic fellow eye showed normal responses (Fig. 1A). A single episode of IPC (7 min duration) applied 40 ± 2 h before the prolonged ischemia provided marked protection against this severe injury (Fig. 1B). In these retinas, the ERG amplitude was only marginally reduced compared to the fellow (nonischemic) eyes. At high-intensity stimuli, the mean ERG amplitudes were approximately 80% of the control for the a-wave component and 80–90% for the b-wave component (Fig. 1D
Fig. 1. Retinal function as measured by full-field ERG in eyes subjected to prolonged unilateral ischemia and reperfusion with and without ischemic preconditioning. ERGs were recorded in vivo 24 h after the prolonged ischemic insult (of 1 h) and 64 ± 2 h after a single episode of short ischemia (IPC; 7 min ischemia). Waveforms in (A–C) show ERG responses to white flashes of increasing intensity under dark-adapted conditions. (A) Control, nonischemic eye. (B) Eye subjected to IPC 40 ± 2 h before the prolonged ischemic insult. (C) Prolonged ischemia alone. (D) ERG a-wave amplitudes and (E) b-wave amplitudes in IPC + I/R eyes versus eyes exposed to I/R alone. (F) Log b-wave amplitude versus log stimulus intensity plots of eyes subjected to IPC+ I/R versus control (no ischemia) eyes. (Control eyes, squares; IPC + ischemia, diamonds; ischemia alone, triangles; n = 5–7 animals in each experimental group.)
1290
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
Fig. 2. Histology of retinas after IPC ± I/R showing the protective effects of IPC. Samples were collected 1 week after prolonged ischemia and stained with hematoxylin and eosin. (A) Control, nonischemic retina. (B) Retina subjected to prolonged (1 h) ischemia (I/R). (C) Retina subjected to IPC 40 ± 2 h before I/R. The large circular structure in the GCL is a patent retinal vessel. (D) Retina subjected to IPC alone. (GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segments of photoreceptors. Original magnification ×40 with digital zoom.)
and E). The log b-wave amplitude–intensity relations in eyes after IPC followed by I/R slightly differed from control nonischemic fellow eyes at low intensities, but converged at higher intensities (Fig. 1F). Thus, IPC provided marked and nearcomplete protection against the loss of retinal function associated with subsequent prolonged ischemia. IPC alone (without subsequent prolonged ischemia but with 64 h of subsequent reperfusion) did not affect the ERG response compared to controls (results not shown). These electrophysiological results were corroborated by histological examination (Fig. 2). One week after prolonged ischemia, a decrease in the thickness of the retina was observed (Fig. 2B). The number of rows of photoreceptor nuclei in the outer nuclear layer (ONL) decreased from 11–12 in controls (Fig. 2A) to 5–6 in I/R retinas. A reduction in the density of nuclei in the inner nuclear layer (INL) and ganglion cell layer was also evident. Furthermore, many nuclei contained condensed chromatin (Fig. 2B), indicative of cell injury. Cellular infiltration into the photoreceptor outer segment layer was also observed in I/R retinas. In contrast, a marked protective effect was evident in eyes subjected to IPC before the prolonged ischemia with both the ONL and the INL highly preserved (Fig. 2C). No changes in retinal histology were observed in eyes subjected to IPC alone, and their structure was identical to that of control eyes (Fig. 2D). The LIP was evaluated in retinal tissues after the same ischemia protocols. As shown in Fig. 3, prolonged ischemia followed by reperfusion induced a massive release of redoxactive and labile iron, noticeable even after 24 h of reperfusion; this amount of labile iron exceeded the control level by approximately threefold ( p = 0.0012). IPC, applied at 40 ± 2 h before prolonged ischemia, moderated this increase in LIP levels. It was still approximately twice higher than in control retinas ( p = 0.006), but significantly lower than that in the I/R-alone group ( p = 0.040). Similar changes in LIP levels were observed in retinas subjected to IPC alone ( p = 0.006, compared to nonischemic control). These pronounced changes in the retinal LIP after IPC alone raised the possibility that iron release not only correlated with but mediated IPC-induced protection. To examine this hypoth-
esis, we attempted to scavenge the labile iron liberated during/ after IPC and tested whether this intervention affected the protective effect. This was achieved by introducing the selective and cell-permeable iron chelator Zn/DFO shortly before the IPC phase. The Zn/DFO complex acts via the so-called “push-andpull” mechanism: it binds iron into a low-reactive complex and eventually pulls it out from the cell by substituting iron for zinc [44]. Indeed, Zn/DFO (0.5 mg/kg) administered iv 15 min before IPC effectively prevented the rise in labile iron caused by IPC
Fig. 3. Labile iron pool in retinas subjected to IPC ± I/R. The experimental groups are as described in Table 1 and included (i) control (naive) retinas (group I, n = 6 eyes); (ii) retinas subjected to prolonged (1 h) ischemia followed by 24 h of reperfusion (group II, n = 6); (iii) retinas subjected to IPC alone (single ischemic episode of 7 min) followed by 64 ± 2 h of reperfusion (group III, n = 7); (iv) retinas subjected to IPC followed by 40 ± 2 h reperfusion, then prolonged 1 h ischemia, and finally an additional 24 h of reperfusion (group IV, n = 12); (v) retinas of animals treated with Zn/DFO complex followed by 64 ± 2 h of perfusion (group V, n = 5); (vi) retinas subjected to IPC alone followed by 64 ± 2 h of reperfusion, and Zn/DFO complex was applied 15 min before IPC (group IIIa, n = 6); and (vii) retinas subjected to IPC followed by 40 ± 2 h reperfusion, then prolonged ischemia, and an additional 24 h of reperfusion, and Zn/DFO complex was applied 15 min before IPC (group IVa, n = 7). Each bar represents mean labile iron in μg/mg of total protein (±SEM). Zn/DFO complex was administrated iv at a concentration of 0.5 mg/kg of animal body weight. ⁎p b 0.05 from groups control, IPC + I/R, Zn/DFO alone, and Zn/DFO + IPC; † p b 0.05 from groups control and Zn/DFO + IPC.
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
Fig. 4. Low levels of the Zn/DFO complex reverse the protective effect of IPC. ERG responses to white flashes of increasing intensity in the dark-adapted state were recorded 24 h after exposing the retina to prolonged ischemia, with IPC applied at 40 ± 2 h earlier. Zn/DFO was administered intravenously 15 min before IPC. White bars, no Zn/DFO (protective effect of IPC evident). Black bars, 0.5 mg/kg Zn/DFO (the height of each of the black bars is near zero; the protective effect of IPC abolished). Hatched bars, 2.5 mg/kg Zn/DFO (emergence of partial protective effect). For each stimulus intensity, and between every two groups (of the three experimental groups), p b 0.05.
1291
enging at the reperfusion phase [45]. This mechanism may be similar to IPC in the heart, where iron mobilization and redistribution have been proposed to activate a signaling cascade underlying the protective effect of IPC [41]. In the heart, an additional major iron-related event associated with IPC is increased expression of ferritin [41]. Therefore, we examined ferritin levels in retinas subjected to ischemia and reperfusion with and without IPC (Fig. 5). One hour (prolonged) ischemia alone led to an insignificant decrease in retinal ferritin by about 20%, compared to controls. When IPC was applied before I/R, this decrease not only was abolished but turned into a marked increase in retinal ferritin level compared to control ( p = 0.031). Likewise, retinas of animals subjected to IPC alone showed a similar increase in ferritin ( p = 0.032 vs. control), indicating that IPC itself triggered this event.
alone and brought the LIP level to that of controls (Fig. 3). Then, when Zn/DFO + IPC was followed by prolonged ischemia (Zn/DFO + IPC + I/R, group IVa) the level of LIP was quite high and close to that observed in I/R alone (Fig. 3). Notably, in these eyes, the protective effect of IPC against I/R injury was abolished and retinal function (as quantified by ERG recordings) was severely impaired, similar to I/R alone (Fig. 4). These observations suggest that labile iron mobilized during and after the IPC phase plays a causative role in IPC protection. When Zn/DFO was given at a high concentration (2.5 mg/kg), the effect of IPC was also largely abolished (Fig. 4). However, at this large dose, residual amounts of complex are apparently still present and are able to provide partial protection by iron scav-
Fig. 5. Ferritin levels in retinas subjected to IPC ± I/R. The experimental groups are as described in Table 1 and for Fig. 3 and included (i) control (naive) retinas (group I, n = 20 eyes); (ii) I/R retinas (group II, n = 6); (iii) IPC alone (group III, n = 7); (iv) IPC + I/R (group IV, n = 12); (v) Zn/DFO alone (group V, n = 6); (vi) Zn/DFO + IPC (group IIIa, n = 6); and (vii) Zn/DFO + IPC + I/R (group IVa, n = 6). Each bar represents mean ferritin levels (±SEM). Zn/DFO complex was administrated iv at a concentration of 0.5 mg/kg of animal body weight. ⁎p b 0.05 for the specific group, compared to the following groups: control, I/R, Zn/ DFO alone, Zn/DFO + IPC, and Zn/DFO + IPC + I/R.
Fig. 6. (A) Iron content per ferritin molecule in retinas subjected to IPC ± I/R (mean ± SEM). Due to the small weight of each retina sample, and the requirement for several micrograms of ferritin for an adequate immunoprecipitation reaction, a minimum of six retina samples were pooled together and a minimum of two pools were analyzed for each group, in duplicate, monitoring the amounts of iron and ferritin. The experimental groups are as described in Table 1 and for Fig. 3 and included (i) control (naive) retinas (group I); (ii) I/R retinas (group II); (iii) IPC alone (group III); (iv) IPC + I/R (group IV); (v) Zn/ DFO alone (group V); (vi) Zn/DFO + IPC (group IIIa); and (vii) Zn/DFO + IPC + I/R (group IVa). Each bar represents the iron content per molecule of ferritin. Zn/ DFO complex was administrated iv at a concentration of 0.5 mg/kg of animal body weight. ⁎p b 0.05 of the specific group compared to the following groups: IPC only and IPC + I/R; †p b 0.05 of the specific group (I/R), compared to all other groups. (B) The relative total amounts of ferritin-bound iron (per milligram of retinal protein) of the experimental groups: control (set to 100%), ischemia and reperfusion (I/R), IPC only, Zn/DFO alone, Zn/DFO + IPC, and Zn/DFO + IPC + I/R. The values were calculated and normalized by multiplying the concentration of ferritin in the retina (ng-Ft/mg protein) by the amount of iron in a ferritin molecule.
1292
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
Administration of Zn/DFO 15 min before the IPC procedure prevented the rise in ferritin seen after IPC alone and partially prevented the increase in ferritin caused by IPC + I/R. The chelator did not influence ferritin levels when applied to control animals (Fig. 5). For more detailed analysis of iron–ferritin relationships we calculated the iron content within a ferritin molecule (Fig. 6A). In control retinas, each ferritin molecule contained an average of 2457 iron atoms. After prolonged ischemia, the average iron content within a ferritin molecule dramatically increased and ferritin became oversaturated by iron (4764 atoms/molecule). IPC not only prevented such ferritin overload, but even led to a decrease in iron content within ferritin molecules in comparison with control. The same effect was shown by IPC alone. Administration of the Zn/DFO complex 15 min before IPC with or without subsequent prolonged ischemia did not affect ferritin iron content (Fig. 6A). It is important that not only the LIP but also the total iron content changed in the I/R and Zn/DFO + IPC + I/R groups, thus pointing to the proposed mechanism of IPC protection (Fig. 6B). Discussion Prolonged ischemia causes severe functional and structural injury to the retina. IPC, when applied 40 ± 2 h before the prolonged ischemic insult, provided nearly complete protection against this I/R-induced damage, as evident from functional and histological assessments. These findings are in accord with data previously published by others [1,28,33]. The present study suggests new components of the molecular response that underlie functional protection of the retina by IPC. First, IPC induced a significant increase in cellular ferritin levels. We, therefore, surmise that the increased levels and availability of ferritin induced by retinal IPC serve to scavenge the redoxactive and labile iron released by I/R, thus reducing ironmediated injury. Second, the reduction in ferritin content observed in retinas subjected to prolonged ischemia alone could be related to increased protein catabolism in ischemic tissues and subsequent depletion of ferritin. As a result, the labile iron released during I/R remains unbound and adds to the LIP. The elevated levels of labile iron could initiate the formation of aggressive free radicals and aggravate tissue damage upon reperfusion [46,47]. In similarity to the heart, ferritin accumulation observed in the retina after IPC could be due to IPC-induced mobilization and redistribution of relatively small amounts of iron that bind to the iron regulatory proteins (IRPs), which act to reduce the tight inhibitory control of ferritin translation. Under normal iron balance, the two IRPs block translation of the ferritin message. After IPC, these proteins are, probably, converted to their nonfunctional forms, allowing initiation of translation [20]. In our previous studies on isolated rat hearts it has been proposed that the minute amounts of labile iron released by the IPC procedure serve as a signaling species that triggers de novo synthesis of apo-ferritin. This enables the heart to scavenge much larger and deleterious amounts of labile iron released after a subsequent prolonged ischemic insult and contributes to IPCinduced cardiac protection [41].
In analogy to the heart, we suggest that similar iron release and similar iron-mediated processes also occur after IPC and ischemic insults in the retina, albeit with different windows of effect temporally. In the heart, the protective effect of IPC is rapid (within minutes), whereas in the retina the conditioning and protective effects become manifest only after hours or days. However, despite these temporal differences, the similarities between the heart and the retina are apparent and may imply a common mechanism underlying IPC protection. Mobilization of iron depends on its valency state: it is ferrous rather than ferric iron that dissociates from ferritin [20]. It is therefore likely that the iron release from ferritin and iron accumulation in LIP occur during prolonged ischemia which is associated with oxygen deficiency and prevalence of reductive conditions [39]. Then, after reperfusion and restoration of oxygen supply, these high levels of labile iron may markedly accelerate oxidative reactions and free radical formation. Retinal cells are extremely sensitive to oxidative damage [48]. It is well known that enhanced ROS production and oxidative stress occur after ischemia and reperfusion. ROS can oxidize a wide spectrum of biological molecules including lipids, DNA, and proteins [49–56], which underlie morphological and functional injuries to the cell. The fact that a small amount of Zn/DFO administered shortly before IPC abolishes IPC protection (Fig. 4) supports the proposed mechanism: the complex enters retinal cells [57,58], binds the equivalent amount of chelatable iron just liberated by IPC, and thus prevents this iron from binding to the IRPs and from inducing the protective ferritin accumulation. This stimulating result raises the question of whether an ironcontaining molecule able to trigger an IPC-like protective response can be designed. Such a compound is expected to be protective against prolonged ischemia, without the need to subject the eye to pretreatment by IPC. If proven successful, this approach could provide more support for the proposed ironbased mechanism of IPC and be relevant for future clinical therapeutic applications. In summary, we suggest that iron plays a dual role in both I/R injury and IPC protection. Thus, excess iron mediates prolonged ischemia-induced injury by catalyzing the formation of ROS upon reperfusion. On the other hand, minute amounts of labile iron released after IPC play a signaling role and activate the regulatory process leading to protection [41]. From this study, a major protective mechanism is the accumulation of ferritin that protects the tissue against the excessive iron released by subsequent prolonged ischemia, thus reducing the degree of ischemia–reperfusion injury. We also hypothesize that the proposed mechanism of IPC-induced protection is common to different organs. A deeper understanding of these pathways and how they are activated should inspire novel approaches to preventative therapy of ischemic injury. Acknowledgments M.C. is the incumbent of the Dr. William Ganz Chair of Heart Studies at the Hebrew University of Jerusalem. This study was supported by grants from the Israel Science Foundation
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
(585/02 and 316/05), the Pepka and Dr. Moshe Bergman Memorial Fund at the Hebrew University of Jerusalem, and the Yedidut Research Fund. The contribution of Dr. Leonid Grinberg in editing the manuscript is acknowledged.
[19]
[20]
References [1] Zhang, C.; Rosenbaum, D. M.; Shaikh, A. R.; Li, Q.; Rosenbaum, P. S.; Pelham, D. J., et al. Ischemic preconditioning attenuates apoptotic cell death in the rat retina. Invest. Ophthalmol. Visual Sci. 43:3059–3066; 2002. [2] Joo, C. K.; Choi, J. S.; Ko, H. W.; Park, K. Y.; Sohn, S.; Chun, M. H., et al. Necrosis and apoptosis after retinal ischemia: involvement of NMDAmediated excitotoxicity and p53. Invest. Ophthalmol. Visual Sci. 40:713–720; 1999. [3] Iida, T.; Spaide, R. F.; Kantor, J. Retinal and choroidal arterial occlusion in Wegener's granulomatosis. Am. J. Ophthalmol. 133:151–152; 2002. [4] Hayreh, S. S. Prevalent misconceptions about acute retinal vascular occlusive disorders. Prog. Retin. Eye Res. 24:493–519; 2005. [5] Aiello, L. P. The potential role of PKC beta in diabetic retinopathy and macular edema. Surv. Ophthalmol. 47 (Suppl. 2):S263–S269; 2002. [6] Butler, J. M.; Guthrie, S. M.; Koc, M.; Afzal, A.; Caballero, S.; Brooks, H. L., et al. SDF-1 is both necessary and sufficient to promote proliferative retinopathy. J. Clin. Invest. 115:86–93; 2005. [7] Madl, J. E.; McIlnay, T. R.; Powell, C. C.; Gionfriddo, J. R. Depletion of taurine and glutamate from damaged photoreceptors in the retinas of dogs with primary glaucoma. Am. J. Vet. Res. 66:791–799; 2005. [8] Mondal, L. K.; Baidya, K. P.; Bhattacharya, B.; Chatterjee, P. R.; Bhaduri, G. The efficacy of topical administration of brimonidine to reduce ischaemia in the very early stage of diabetic retinopathy in good controlled type-2 diabetes mellitus. J. Indian Med. Assoc. 102:724–725 (729); 2004. [9] Kaja, S.; Yang, S. H.; Wei, J.; Fujitani, K.; Liu, R.; Brun-Zinkernagel, A. M., et al. Estrogen protects the inner retina from apoptosis and ischemia-induced loss of Vesl-1L/Homer 1c immunoreactive synaptic connections. Invest. Ophthalmol. Vis. Sci. 44:3155–3162; 2003. [10] Peachey, N. S.; Charles, H. C.; Lee, C. M.; Fishman, G. A.; Cunha-Vaz, J. G.; Smith, R. T. Electroretinographic findings in sickle cell retinopathy. Arch. Ophthalmol. 105:934–938; 1987. [11] Keshet, E. More weapons in the arsenal against ischemic retinopathy. J. Clin. Invest. 107:945–946; 2001. [12] Suganami, E.; Takagi, H.; Ohashi, H.; Suzuma, K.; Suzuma, I.; Oh, H., et al. Leptin stimulates ischemia-induced retinal neovascularization: possible role of vascular endothelial growth factor expressed in retinal endothelial cells. Diabetes 53:2443–2448; 2004. [13] Osborne, N. N.; Casson, R. J.; Wood, J. P.; Chidlow, G.; Graham, M.; Melena, J. Retinal ischemia: mechanisms of damage and potential therapeutic strategies. Prog. Retin. Eye Res. 23:91–147; 2004. [14] Gutteridge, J. M.; Rowley, D. A.; Halliwell, B. Superoxide-dependent formation of hydroxyl radicals in the presence of iron salts: detection of ‘free’ iron in biological systems by using bleomycin-dependent degradation of DNA. Biochem. J. 199:263–265; 1981. [15] Dunaief, J. L.; Richa, C.; Franks, E. P.; Schultze, R. L.; Aleman, T. S.; Schenck, J. F., et al. Macular degeneration in a patient with aceruloplasminemia, a disease associated with retinal iron overload. Ophthalmology 112:1062–1065; 2005. [16] Hahn, P.; Milam, A. H.; Dunaief, J. L. Maculas affected by age-related macular degeneration contain increased chelatable iron in the retinal pigment epithelium and Bruch's membrane. Arch. Ophthalmol. 121:1099–1105; 2003. [17] McGahan, M. C.; Harned, J.; Mukunnemkeril, M.; Goralska, M.; Fleisher, L.; Ferrell, J. B. Iron alters glutamate secretion by regulating cytosolic aconitase activity. Am. J. Physiol. Cell. Physiol. 288:C1117–C1124; 2005. [18] Harned, J.; Grimes, A. M.; McGahan, M. C. The effect of UVB irradiation on ferritin subunit synthesis, ferritin assembly and Fe metabolism in
[21]
[22]
[23]
[24]
[25] [26] [27]
[28]
[29] [30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
1293
cultured canine lens epithelial cells. Photochem. Photobiol. 77:440–445; 2003. Goralska, M.; Holley, B.; McGahan, M. C. The effects of Tempol on ferritin synthesis and Fe metabolism in lens epithelial cells. Biochim. Biophys. Acta 1497:51–60; 2000. Ponka, P. Cellular iron metabolism. Kidney Inter., Suppl. 69:S2–S11; 1999. Hahn, P.; Qian, Y.; Dentchev, T.; Chen, L.; Beard, J.; Harris, Z. L., et al. Disruption of ceruloplasmin and hephaestin in mice causes retinal iron overload and retinal degeneration with features of age-related macular degeneration. Proc. Natl. Acad. Sci. U. S. A. 101:13850–13855; 2004. Sommerschild, H. T.; Kirkeboen, K. A. Preconditioning—endogenous defence mechanisms of the heart. Acta Anaesthesiol. Scand. 46:123–137; 2002. Nakano, A.; Cohen, M. V.; Downey, J. M. Ischemic preconditioning: from basic mechanisms to clinical applications. Pharmacol. Ther. 86:263–275; 2000. Serafin, A.; Fernandez-Zabalegui, L.; Prats, N.; Wu, Z. Y.; Rosello-Catafau, J.; Peralta, C. Ischemic preconditioning: tolerance to hepatic ischemia– reperfusion injury. Histol. Histopathol. 19:281–289; 2004. Carini, R.; Albano, E. Recent insights on the mechanisms of liver preconditioning. Gastroenterology 125:1480–1491; 2003. Bonventre, J. V. Kidney ischemic preconditioning. Curr. Opin. Nephrol. Hypertens. 11:43–48; 2002. Schaller, B.; Graf, R. Cerebral ischemic preconditioning: an experimental phenomenon or a clinical important entity of stroke prevention? J. Neurol. 249:1503–1511; 2002. Roth, S.; Li, B.; Rosenbaum, P. S.; Gupta, H.; Goldstein, I. M.; Maxwell, K. M., et al. Preconditioning provides complete protection against retinal ischemic injury in rats. Invest. Ophthalmol. Vis. Sci. 39:777–785; 1998. Roth, S. Endogenous neuroprotection in the retina. Brain Res. Bull. 62: 461–466; 2004. Li, B.; Yang, C.; Rosenbaum, D. M.; Roth, S. Signal transduction mechanisms involved in ischemic preconditioning in the rat retina in vivo. Exp. Eye Res. 70:755–765; 2000. Sakamoto, K.; Kuwagata, M.; Nakahara, T.; Ishii, K. Late preconditioning in rat retina: involvement of adenosine and ATP-sensitive K(+) channel. Eur. J. Pharmacol. 418:89–93; 2001. Ettaiche, M.; Heurteaux, C.; Blondeau, N.; Borsotto, M.; Tinel, N.; Lazdunski, M. ATP-sensitive potassium channels (K(ATP)) in retina: a key role for delayed ischemic tolerance. Brain Res. 890:118–129; 2001. Roth, S.; Dreixler, J. C.; Shaikh, A. R.; Lee, K. H.; Bindokas, V. Mitochondrial potassium ATP channels and retinal ischemic preconditioning. Invest. Ophthalmol. Vis. Sci. 47:2114–2124; 2006. Zhu, Y.; Zhang, Y.; Ojwang, B. A.; Brantley Jr., M. A.; Gidday, J. M. Long-term tolerance to retinal ischemia by repetitive hypoxic preconditioning: role of HIF-1alpha and heme oxygenase-1. Invest. Ophthalmol. Vis. Sci. 48:1735–1743; 2007. Lin, J.; Roth, S. Ischemic preconditioning attenuates hypoperfusion after retinal ischemia in rats. Invest. Ophthalmol. Vis. Sci. 40:2925–2931; 1999. Nonaka, A.; Kiryu, J.; Tsujikawa, A.; Yamashiro, K.; Nishijima, K.; Miyamoto, K., et al. Inhibitory effect of ischemic preconditioning on leukocyte participation in retinal ischemia–reperfusion injury. Invest. Ophthalmol. Vis. Sci. 42:2380–2385; 2001. Li, Y.; Roth, S.; Laser, M.; Ma, J. X.; Crosson, C. E. Retinal preconditioning and the induction of heat-shock protein 27. Invest. Ophthalmol. Vis. Sci. 44:1299–1304; 2003. Casson, R. J.; Wood, J. P.; Melena, J.; Chidlow, G.; Osborne, N. N. The effect of ischemic preconditioning on light-induced photoreceptor injury. Invest. Ophthalmol. Vis. Sci. 44:1348–1354; 2003. Chevion, M.; Jiang, Y.; Har-El, R.; Berenshtein, E.; Uretzky, G.; Kitrossky, N. Copper and iron are mobilized following myocardial ischemia: possible predictive criteria for tissue injury. Proc. Natl. Acad. Sci. U. S. A. 90: 1102–1106; 1993. Konijn, A. M.; Tal, R.; Levy, R.; Matzner, Y. Isolation and fractionation of ferritin from human term placenta—a source for human isoferritins. Anal. Biochem. 144:423–428; 1985.
1294
A. Obolensky et al. / Free Radical Biology & Medicine 44 (2008) 1286–1294
[41] Berenshtein, E.; Vaisman, B.; Goldberg-Langerman, C.; Kitrossky, N.; Konijn, A. M.; Chevion, M. Roles of ferritin and iron in ischemic preconditioning of the heart. Mol. Cell. Biochem. 234–235:283–292; 2002. [42] Breuer, W.; Cabantchik, Z. I. A fluorescence-based one-step assay for serum non-transferrin-bound iron. Anal. Biochem. 299:194–202; 2001. [43] Esposito, B. P.; Breuer, W.; Sirankapracha, P.; Pootrakul, P.; Hershko, C.; Cabantchik, Z. I. Labile plasma iron in iron overload: redox activity and susceptibility to chelation. Blood 102:2670–2677; 2003. [44] Chevion, M. Protection against free radical-induced and transition metalmediated damage: the use of “pull” and “push” mechanisms. Free Radic. Res. Commun. 12–13 (Pt 2):691–696; 1991. [45] Ophir, A.; Berenshtein, E.; Kitrossky, N.; Berman, E. R.; Photiou, S.; Rothman, Z., et al. Hydroxyl radical generation in the cat retina during reperfusion following ischemia. Exp. Eye Res. 57:351–357; 1993. [46] Minotti, G. NADPH-and adriamycin-dependent microsomal release of iron and lipid peroxidation. Arch. Biochem. Biophys. 277:268–276; 1990. [47] Kurz, T.; Leake, A.; Von Zglinicki, T.; Brunk, U. T. Relocalized redoxactive lysosomal iron is an important mediator of oxidative-stress-induced DNA damage. Biochem. J. 378:1039–1045; 2004. [48] Rose, R. C.; Richer, S. P.; Bode, A. M. Ocular oxidants and antioxidant protection. Proc. Soc. Exp. Biol. Med. 217:397–407; 1998. [49] Aust, S. D. Iron redox reactions and lipid peroxidation. Basic Life Sci. 49:137–144; 1988. [50] Chevion, M. A site-specific mechanism for free radical induced biological
[51] [52]
[53]
[54] [55]
[56]
[57]
[58]
damage: the essential role of redox-active transition metals. Free Radic. Biol. Med. 5:27–37; 1988. Minotti, G. Metals and membrane lipid damage by oxy-radicals. Ann. N. Y. Acad. Sci. 551:34–44 (discussion 45-6); 1988. Ursini, F.; Maiorino, M.; Hochstein, P.; Ernster, L. Microsomal lipid peroxidation: mechanisms of initiation. The role of iron and iron chelators. Free Radic. Biol. Med. 6:31–36; 1989. Richter, C.; Park, J. W.; Ames, B. N. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc. Natl. Acad. Sci. U. S. A. 85:6465–6467; 1988. Imlay, J. A.; Linn, S. DNA damage and oxygen radical toxicity. Science 240:1302–1309; 1988. Stadtman, E. R. Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free Radic. Biol. Med. 9:315–325; 1990. Tatarkova, Z.; Aplan, P.; Matejovicova, M.; Lehotsky, J.; Dobrota, D.; Flameng, W. Effect of ischemia and reperfusion on protein oxidation in isolated rabbit hearts. Physiol. Res. 54:185–191; 2004. Ophir, A.; Berenshtein, E.; Kitrossky, N.; Averbukh, E. Protection of the transiently ischemic cat retina by zinc–desferrioxamine. Invest. Ophthalmol. Vis. Sci. 35:1212–1222; 1994. Banin, E.; Berenshtein, E.; Kitrossky, N.; Pe'er, J.; Chevion, M. Gallium– desferrioxamine protects the cat retina against injury after ischemia and reperfusion. Free Radic. Biol. Med. 28:315–323; 2000.