Protective Effect of Vitreous against Hemoglobin Neurotoxicity

Abstract

Hemorrhage into the brain parenchyma or subarachnoid space is associated with edema and vascular injury that is likely mediated at least in part by the toxicity of hemoglobin. In contrast, extravascular blood appears to be less neurotoxic when localized to the retina or adjacent vitreous, the gel filling the posterior segment of the eye. In this study, the hypothesis that vitreous protects neurons from hemoglobin toxicity was investigated in a primary cortical cell culture model. Consistent with prior observations, hemoglobin exposure for 24 hours resulted in death of most neurons without injury to co-cultured glia. Neuronal loss was reduced in a concentration-dependent fashion by bovine vitreous, with complete protection produced by 3% vitreous solutions. This effect was associated with a reduction in malondialdehyde but an increase in cell iron. At low vitreous concentrations, its ascorbate content was sufficient to account for most neuroprotection, as equivalent concentrations of ascorbate alone had a similar effect. However, other vitreous antioxidants provided significant protection when applied at concentrations present in undiluted vitreous, and prevented all neuronal loss when combined in the absence of ascorbate. These results indicate that vitreous is an antioxidant cocktail that robustly protects neurons from hemoglobin toxicity, and may contribute to the relative resistance of retinal neurons to hemorrhagic injury.

Introduction

Hemoglobin (Hb) is a pro-oxidant protein that is released into CNS tissue in millimolar concentrations after spontaneous or traumatic hemorrhage. Its location within erythrocytes provides an effective barrier that prevents its toxicity within the circulation, and also in the initial hours after parenchymal or subarachnoid hemorrhage. However, subsequent erythrophagocytosis by microglia and infiltrating macrophages is apparently insufficient to prevent significant local Hb release and breakdown. At one week after experimental subarachnoid hemorrhage, heme concentrations within the hematoma are two orders of magnitude above those required to kill cultured neurons [1], the cell population most vulnerable to Hb and iron [2, 3]. In vivo, parenchymal injection of autologous blood or Hb produces a delayed iron-dependent injury that is attenuated in rodent and pig models by the ferric chelator deferoxamine [4–7].

In contrast to its toxicity in the brain parenchyma and subarachnoid space, the deleterious effects of extravascular blood appear to be mitigated in the eye. Retinal neurons and photoreceptors sustain relatively little injury after hemorrhage localized to the retina or extending into the vitreous [8], the hyaluronan-based gel in the posterior segment of the eye. Accordingly, management is primarily conservative, limited to postural changes to promote erythrocyte settling or observation alone, and usually results in a satisfactory outcome [9, 10]. While this phenomenon may merely indicate that retinal cells are selectively resistant to heme or iron-mediated injury, two observations suggest otherwise. First, photoreceptor degeneration is observed when hemorrhage is localized to the subretinal space rather than the retina or vitreous, and can be reduced by deferoxamine [11–13]. Second, the vulnerability of cultured retinal neurons to iron resembles that of other central neurons [3, 14, 15].

An alternative hypothesis is that vitreous is inherently protective against Hb, and reduces the vulnerability of adjacent cells to its oxidative toxicity. If that is so, then elucidation of its protective mechanisms may have implications beyond ocular hemorrhage, and may provide information relevant to the design of safe and effective therapies for hemorrhagic stroke and trauma. As an initial step towards this end, we investigated the effect of bovine vitreous in a characterized model of Hb neurotoxicity.

Materials and Methods

Materials.

Bovine vitreous was purchased from InVision Bioresources, Seattle, Washington, USA. It was frozen after harvesting and shipped on dry ice. For use in experiments, it was quickly thawed, homogenized while ice-cold, and sterile-filtered. Aliquots were then stored at −80°C until used.

Human Hb A was obtained as a gift from Hemosol, Inc, Etobicoke, Ontario, Canada.

Apotransferrin was purchased from Millipore-Sigma, Burlington, MA, USA and apoferritin was purchased from Sigma-Aldrich, St Louis, MO, USA.

Hyaluronic acid was purchased from R&D Systems, Minneapolis, MN, USA.

Culture media (MEM and DMEM) were purchased from Gibco-Thermo Fisher Scientific, Gaithersburg, MD, USA; serum was purchased from GE Healthcare Hyclone, Logan, UT, USA. Other experimental reagents were purchased from Sigma-Aldrich unless otherwise indicated.

Primary cell cultures.

Mouse breeding, rearing and culture preparation followed protocols approved by the Thomas Jefferson University Institutional Animal Care and Use Committee, and were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). Cultures containing neurons and glia (> 90% GFAP+ astrocytes) were prepared from the pooled cortices of fetal C57BL/6 × 129/Sv mice that were collected at gestational age 14–16 days, as previously described [16]. After trypsin digestion, tissue was dissociated to a cell suspension by trituration and diluted in medium containing 5% fetal bovine serum, 5% equine serum, 2 mM glutamine and MEM (Gibco 11430) or DMEM (Gibco 11960). Two-thirds of the medium was replaced with fresh medium containing 10% equine serum and lacking fetal bovine serum on days 5 and 9, and then daily beginning on day 11. Cultures were used for experiments on days 12–17.

Hemoglobin toxicity experiments.

All Hb exposures were conducted in serum-free MEM containing 10 mM glucose (MEM10). On the evening preceding the experiment, the serum content was reduced by feeding cells with MEM10 as described above. Remaining serum was washed out (>1000-fold dilution) immediately prior to adding Hb alone or with diluted bovine vitreous and other study drugs. Vitreous was diluted with MEM10 only. Cultures were then incubated at 37°C in a 5% CO2 atmosphere until completion of the experiment. Cultures were randomly assigned to treatment groups using a random number generator (www.random.com)

Injury assessment.

Personnel assessing cell injury were blinded to experimental conditions. Multiple prior studies (e.g. [17]) have demonstrated that micromolar Hb concentrations are not toxic to glial cells in this model. After sampling medium (25 μl) for LDH release assay, cultures were washed with MEM10 and incubated with 13 μg/ml propidium iodide (PI) at 37°C for 5 minutes. After dye washout, fluorescence intensity was measured (iVision-Mac, Biovision Technologies Inc, Exton, PA, USA) following an established protocol [18]. Medium LDH activity was then assessed using a microplate assay previously described in detail [19]. LDH activity and PI fluorescence intensity due to Hb toxicity were calculated by subtracting the low mean value in control cultures not treated with Hb from all experimental values. Fluorescence intensity and LDH activity values were normalized to the mean values of sister cultures treated with 300 μM N-methyl-D-aspartate (NMDA), which kills all neurons without injuring glial cells, in order control for variability of neuronal density in different culture platings.

Cell nonheme iron assay.

After washing cultures with saline, cell nonheme iron content was determined with the ferrozine assay, following the protocol of Pountney et al. [20, 21].

Lipid oxidation assay.

After protein precipitation with 4.5% trichoroacetic acid, lipid oxidation was quantified by malondialdehyde assay, using a modification of the method of Ohkawa et al. [22] that has previously been described in detail [23].

Ascorbic acid assay.

The OxiSelectTM FRASC (Ferric Reducing/Antioxidant Ascorbic Acid chemistry) assay kit (Cell Biolabs Inc., San Diego, CA. Cat. No. STA-860) was used, following the manufacturer’s instructions.

Statistical analysis.

Data were analyzed with GraphPad Prism 4 software. Differences between treatment groups were assessed using one way ANOVA and the Bonferroni multiple comparisons test.

Results

Vitreous protects against Hb toxicity.

Consistent with prior observations [17], treatment with 10 μM Hb for 24 hours resulted in degeneration of most phase-bright cells with the typical appearance of neurons in this model, without injury to the background glial monolayer (Fig. 1 A-D ). This morphological change was associated with increased LDH activity in the culture medium and increased culture fluorescence after PI staining (Fig. 1 E, F). Both cell injury markers were significantly attenuated in a concentration-dependent fashion by bovine vitreous. At a 3% vitreous concentration, values were similar to those in sham-treated control cultures.

Figure 1.

Vitreous protects cortical neurons against hemoglobin (Hb) toxicity. A-D) Phase contrast photomicrographs of cultures after 24 hour exposure to: A) sham media exchange; phase-bright neuronal cell bodies overlie a confluent glial monolayer; B) Hb 10 μM; most neuronal cell bodies have degenerated; C,D) Hb 10 μM plus 0.3% and 3% bovine vitreous dilutions, respectively; concentration-dependent protection is apparent. E, F) Bars represent mean percentage cell death (±SEM), as measured by LDH release (E) and propidium iodide fluorescence assays (PI, F), in cultures treated for 24 hours with Hb 10 μM alone or with indicated bovine vitreous (V) dilutions. The low mean LDH or background fluorescence values in sham cultures subjected to medium exchange only were subtracted from each value to determine the signal specific for Hb neurotoxicity. *P < 0.05, **P < 0.01, ***P < 0.001 v. Hb alone conditions, Bonferroni multiple comparisons test, n = 8–9 cultures/condition).

Vitreous decreases cell malondialdehyde but increases iron.

Hb is an iron-dependent oxidative neurotoxin that increases the concentration of the lipid oxidation product malondialdehyde in this and other models [24, 25]. Consistent with an antioxidant effect, 1–3% vitreous dilutions significantly decreased culture malondialdehyde (Fig. 2A). In order to test the hypothesis that this observation was mediated by mitigation of the increase in cell iron produced by Hb, cultures treated with Hb for 24 hours were lysed and nonheme iron was quantified. Contrary to hypothesis, the 2.8-fold increase in cell iron produced by Hb was further increased by concomitant vitreous treatment (Fig. 2B).

Figure 2.

Vitreous reduces cell malondialdehyde (MDA) but increases iron after hemoglobin exposure. Mean malondialdehyde (A, 10 cultures/condition) and iron (B, 8–10 cultures/condition) after treatment for 24 hours with Hb 10 μM alone or with indicated bovine vitreous dilutions. *P < 0.05, **P < 0.01, ***P < 0.001 v. Hb alone conditions, ###P < 0.001 v. sham, Bonferroni multiple comparisons test.

Ascorbate contributes to the antioxidant effect of vitreous.

Increased cell iron uptake with a concomitant decrease in malondialdehyde is a characteristic signature of ascorbate in neural cell cultures [26, 27]. The ascorbate concentration of the bovine vitreous used in these experiments was 451±16 μM. Ascorbate concentrations replicating those present in 0.3–3% vitreous provided concentration-dependent protection against Hb neurotoxicity similar to that produced by vitreous, and also reduced culture malondialdehyde (Fig. 3A, B). Addition of ascorbate oxidase to vitreous at an activity sufficient to completely oxidize it to dehydroascorbate at the dilutions used decreased its efficacy, but only by about 30% as measured by LDH release assay (Fig. 3C).

Figure 3.

Ascorbate contributes to the antioxidant effect of vitreous. A) Mean LDH release (n = 16–24 cultures/condition) in cultures treated for 24 hours with Hb 10 μM alone or with indicated ascorbate (ASC) concentrations, which are equivalent to the ascorbate concentrations in 0.3%, 1% and 3% bovine vitreous dilutions. B) Mean malondialdehyde (MDA, 12/condition) in cultures treated as in A. C) Mean LDH release in cultures treated with Hb 10 μM alone, with 3% vitreous (V), or with 3% vitreous that had been pretreated with 30 units/ml ascorbate oxidase (n = 1012/condition). ***P < 0.05, ***P < 0.001 v. Hb alone condition, Bonferroni multiple comparisons test.

Effect of other vitreous components on Hb neurotoxicity.

Prior studies have demonstrated that vitreous contains low concentrations of ferritin and selenium and higher concentrations of transferrin and hyaluronan [28, 29]. All of these components have antioxidant effects that may protect cells from hemoglobin. At concentrations approximating those present in a 3% vitreous, none reduced neuronal death in the absence of ascorbate (Fig. 4A). However, at concentrations similar to those in undiluted vitreous, iron-poor transferrin and selenium (applied as sodium selenite) provided significant protection (Fig. 4B). The combination of iron-poor transferrin, ferritin, selenium and hyaluronan provided near-complete neuroprotection in the absence of ascorbate.

Figure 4.

Effect of other vitreous antioxidants on Hb neurotoxicity. A) Percentage LDH release in cultures (12–17/condition) treated with Hb 10 μM alone or with hyaluronon (HA, 16.65 μg/ml), selenium (Se, 0.0031 μM), iron poor ferritin (F, 0.585 ng/ml), iron-poor transferrin (T, 2.634 μg/ml), combined hyaluronan, selenium, ferritin, and transferrin (+C), or ascorbate (2.38 μg/ml), approximating concentrations present in a 3% vitreous solution. B) Cultures (7–15/condition) treated with Hb as in A, but with concentrations approximating those in undiluted vitreous (555 μg/ml hyaluronon, 0.1035 μM selenium, 19.5 ng/ml ferritin, 87.8 μg/ml transferrin, 79.48 ug/ml ascorbate. ***P < 0.001 v. Hb alone condition, Bonferroni multiple comparisons test.

All data are available on Mendeley Data (http://dx.doi.Org/10.17632/yyk22tn93y.1).

Discussion

This study provides novel evidence that vitreous robustly protects neurons from Hb neurotoxicity. At a concentration 3% of that in vivo, bovine vitreous completely prevented the widespread neuronal death produced by sustained exposure to 10 μM Hb. At this vitreous dilution, a primary protectant was its ascorbate, and the result was replicated by treating cells with the same concentrations of ascorbate in culture medium alone. However, at concentrations found in undiluted vitreous, other antioxidants were also effective and prevented most neuronal loss when combined in a hydrogel resembling ascorbate-free vitreous.

Regulation of iron trafficking and metabolism by ascorbate may contribute to its benefit after Hb exposure [30], and likely accounts for the increase in cell iron produced by vitreous in the present study. Extracellular ascorbate increases neural cell iron levels at least in part by reducing nontransferrin-bound iron to its ferrous form, which facilitates uptake via divalent metal transporter-1 (DMT1) [26, 27]. This pathway appears to be particularly relevant to astrocytes, which upregulate DMT1 in response to inflammatory or toxic stimuli [31, 32]. Astrocytes are better-equipped for iron detoxification than neurons due to their potent antioxidant defenses, including the ability to rapidly increase intracellular ferritin expression in response to iron exposure or oxidative stress [33, 34]. Each ferritin heteropolymer can deposit up to 4000 iron atoms in its mineral core, where it is generally unavailable to participate in free radical reactions [35]. In the presence of ascorbate, astrocytes serve as high-capacity iron sinks, thereby enhancing tolerance for the localized iron loading that accompanies hemorrhagic CNS injuries [36]. Of note, the ascorbate concentration in the vitreous used in these experiments, while similar to those previously reported in bovine samples, is only about one-quarter of that in human vitreous [37]. Protection provided by the ascorbate component of human vitreous may therefore be even more prominent than that observed in the current series of experiments.

Ascorbate oxidase pretreatment attenuated the efficacy of vitreous in this model, consistent with the contribution of ascorbate to neuroprotection. However, the effect was incomplete, with significant benefit persisting despite complete oxidation of ascorbate in the culture medium. Two factors likely contribute to this observation. First, some dehydroascorbate enters cells via glucose transporters, where it may be reduced to ascorbate by glutathione-dependent dehydroascorbate reductase [30]. Second, other vitreous components may have an additive effect when ascorbate is near-depletion. Prior studies have quantified the levels of three antioxidants in vitreous that may be directly relevant to Hb neurotoxicity [28, 29]. Transferrin and ferritin bind ferric iron with high affinity and protect neurons from Hb [19, 25]. Selenium is incorporated into selenoproteins, is critical for the activity of glutathione peroxidases and thioredoxin reductases [38], and attenuates Hb-mediated lipid peroxidation in vivo [39]. The low concentrations of these antioxidants that approximate those present in a 3% vitreous dilution were ineffective in the absence of ascorbate, but physiologic concentrations of selenium and transferrin provided moderate protection alone. A hyaluronon hydrogel containing these vitreous components but lacking ascorbate prevented all Hb-mediated neuronal loss. These results indicate that endogenous vitreous is an antioxidant cocktail that provides multiple lines of defense against the iron-mediated neurotoxicity of Hb.

The slow course of Hb toxicity after CNS hemorrhage suggests that it may be a feasible therapeutic target [4, 40], and identification of mitigating pharmacotherapies has been an active area of preclinical research. A major focus of clinical trials to date has been iron chelation with deferoxamine, a compound in clinical use for decades to treat acute iron poisoning and transfusion-related iron overload. However, a multicenter Phase 2 deferoxamine trial was halted due to pulmonary toxicity, an adverse effect of this compound that was first recognized over two decades ago [41]. The primary challenge of systemic chelator therapy for CNS hemorrhage is delivery of sufficient iron binding capacity to perihematomal tissue while avoiding off-target toxicity, which is likely caused by iron sequestration and metalloenzyme inhibition [42, 43]. The present results suggest an alternative and more physiologic approach. Lifetime exposure to vitreous has no reported toxicity to any human or animal cell population in vivo. Bovine vitreous dilutions robustly protected neurons from Hb neurotoxicity in this cell culture model without producing concomitant iron deprivation. It remains to be determined if local application of this natural hydrogel is beneficial in hemorrhagic stroke models.

Highlights.

• Bovine vitreous protected cultured neurons from the oxidative toxicity of hemoglobin.

• Complete neuroprotection was provided by a 3% vitreous solution.

• Ascorbate accounted for most protection in diluted vitreous.

• Transferrin and selenium content may contribute in undiluted vitreous.

• Vitreous may protect adjacent neurons from hemorrhagic injury.

Abbreviations:

Hb

hemoglobin

LDH

lactate dehydrogenase

PI

propidium iodide