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. Author manuscript; available in PMC: 2021 Dec 10.
Published in final edited form as: Dev Neurosci. 2020 Dec 10;42(2-4):124–134. doi: 10.1159/000511888

Sulforaphane Protects Piglet Brains from Neonatal Hypoxic-Ischemic Injury

Bing Wang a, Ewa Kulikowicz a, Jennifer K Lee a, Raymond C Koehler a, Zeng-Jin Yang a
PMCID: PMC7864873  NIHMSID: NIHMS1639349  PMID: 33302269

Abstract

The striatal, primary sensorimotor cortical, and thalamic neurons are highly vulnerable to hypoxia-ischemia (HI) in term newborns. In a piglet model of HI that exhibits similar selective regional vulnerability, we tested the hypothesis that early treatment with sulforaphane, an activator of the Nrf2 transcription factor, protects vulnerable neurons from HI injury. Anesthetized 3–7 days old piglets were subjected to 45 min of hypoxia and 7 min of airway occlusion. At 15 min after resuscitation, the piglets received intravenous vehicle or sulforaphane. At 4 days of recovery, the density of viable neurons in the putamen of vehicle-treated piglets was 31 ± 34% (±SD) that of sham-operated controls. Treatment with sulforaphane significantly increased viability to 77 ± 31%. In the sensorimotor cortex, neuronal viability was also increased from 59 ± 35% in the vehicle-treated group to 89 ± 15% in the sulforaphane-treated animals. Treatment with sulforaphane increased the nuclear Nrf2 and γ-glutamylcysteine synthetase expression at 6 h of recovery in these regions. We conclude that systemic administration of sulforaphane at 15 min after HI can induce the translocation of Nrf2 to the nucleus, increase expression of an enzyme involved in glutathione synthesis, and salvage neurons in the highly vulnerable putamen and sensorimotor cortex in a large-animal model of HI. Therefore, targeting Nrf2 activation soon after recovery from HI is a feasible approach for neuroprotection in the newborn brain.

Keywords: Hypoxic-ischemic encephalopathy, pig, Neuroprotection, Sulforaphane, Nrf2

Introduction

Neonatal hypoxic-ischemic encephalopathy (HIE) causes significant infant mortality and morbidity. Oxidative stress is a major contributor to neuronal damage after hypoxia-ischemia (HI) in immature brains [1–3]. Various markers of oxidative stress are prominent in HI brains, including decreased glutathione, increased formation of protein carbonyl groups, increased nitration of proteins and nucleic acids, and increased hydroxylation of nucleic acids [1, 4–6]. Antioxidant drugs such as allopurinol [7], deferoxamine [8], edaravone [9], and sulforaphane [10] reduce oxidative stress and improve outcomes in postnatal rodents after HI. Thus, early treatment with antioxidant reagents may serve as a potential therapeutic strategy against neonatal HIE in humans.

Sulforaphane is an isothiocyanate compound that is found in cruciferous vegetables such as broccoli. Systemic sulforaphane administration protects brains in various experimental models, such as focal cerebral ischemia [11], intracerebral hemorrhage [12], subarachnoid hemorrhage [13], controlled cortical impact brain injury [14–16], and contusive spinal cord injury [17]. Sulforaphane is not a direct antioxidant. Instead, it induces Phase II antioxidant enzymes via activation of nuclear factor erythroid 2-related factor 2 (Nrf2) [18, 19]. Nrf2 then translocates into the nucleus and binds to the antioxidant response element (ARE) on hundreds of genes to produce a broad set of antioxidant and cell defense proteins that increase survival and suppress inflammation to various forms of cell stress [20–23]. Although pretreatment with sulforaphane has shown benefit in postnatal day 7 rats exposed to HI [10], it is unclear whether post-HI treatment will be effective because damage to the endoplasmic reticulum and Golgi apparatus after reoxygenation [1] could impair synthesis of Phase II antioxidant proteins.

Rodent HIE models are advantageous because genetic modifications can be made that permit modulation of specific molecular pathways. However, larger animals, including pigs, are more similar to humans in terms of behavior and sensorimotor integration [24]. In addition, large-animal models exhibit neuroanatomical advantages over rodent HIE models. The larger gyrencephalic piglet brain permits study of selective cortical areas and neuronal vulnerability, regional white matter injury, and connectivity [25]. The Stroke Therapy Academic Industry Roundtable (STAIR) guidelines agree that positive results from small-animal drug studies should be confirmed in a higher species before clinical evaluation in humans [26]. Here, we used a model of hypoxia followed by asphyxia in newborn piglets to study the effects of sulforaphane in HI brains. This piglet injury model [27] causes regionally selective brain damage very similar to the pattern of injury found in human newborns who have experienced HI [25]. The most vulnerable regions include the striatum, primary sensorimotor cortex, and somatosensory thalamic nuclei (ventral posterior nucleus) [28]. Therefore, we evaluated the efficacy of sulforaphane for ameliorating HI damage in vulnerable regions of piglet brains and tested whether it induces Nrf2 nuclear translocation and upregulates glutathione synthetic enzymes and its related detoxifying metabolic pathways.

Material and Methods

A total of 53 male piglets (1.7 to 2.5 kg, 3–7 days old) were used in this study. Piglets were not studied at 0–2 days of age because some piglets have gastrointestinal injury after HI that affects outcome with this global model. In contrast to rodents, brain maturation in swine occurs over a period of at least 6 months [29].

Hypoxia-asphyxia Model

Hypoxia-asphyxia was induced according to the methods described previously [30]. In brief, piglets were anesthetized via nose cone with 5% isoflurane in a 50%/50% nitrous oxide/oxygen mixture, orally intubated, and maintained under anesthesia with 2% isoflurane in a 70%/30% nitrous oxide/oxygen mixture during aseptic surgery for catheterization of the femoral artery and vein. Piglets received an intravenous injection of 5% dextrose and 0.45% sodium chloride solution (4 mL/kg/h), fentanyl (10 μg/kg, bolus), and vecuronium (0.2 mg/kg, bolus). Inspired O2 was decreased to 10.0 ± 0.2% for 45 min (hypoxia). Then, piglets were ventilated with room air for 5 min (required for cardiac resuscitation from ensuing asphyxia) before their endotracheal tubes were occluded for 7 min to produce asystolic or bradycardic cardiac arrest, hypotension, and loss of arterial pulse. Piglets were resuscitated by mechanical ventilation with 50% O2, manual chest compressions, and, if necessary, intravenous injection of epinephrine until the return of spontaneous circulation. Piglets that did not exhibit the return of spontaneous circulation within 3 min were excluded from studies. After resuscitation, inspired O2 was gradually reduced to 30% to maintain arterial O2 saturation greater than 95%. Sham-operated animals received only catheterization but no hypoxia or asphyxia. Arterial blood gases, pH, glucose concentration, arterial blood pressure, and rectal temperature were monitored until piglets regained consciousness. Sodium bicarbonate was administered to correct metabolic acidosis when necessary. In some groups, neuropathology was evaluated at 4 days of recovery. Considering that ischemic cytopathology occurs primarily after 6 h of recovery [28], biochemical measurements were made at 6 h to avoid the possible effect of neuronal loss on molecular changes. Piglets that survived the severe HI insult were kept on mechanical ventilation with a 70%/30% nitrous oxide/oxygen mixture and received continuous intravenous infusion of fentanyl (10 μg/kg/h) and vecuronium (0.2 mg/kg/h). At 6 h, some were deeply anesthetized by intravenous injection with 50 mg/kg pentobarbital and 6.4 mg/kg phenytoin and perfused transcardially with cold phosphate-buffered saline before their brain tissue was collected.

Sulforaphane Treatment

At 15 min of recovery or at an equivalent time in sham-operated piglets, 10 mg/kg sulforaphane (LKT Labs, St Paul, MN) or vehicle (0.5% DMSO in normal saline) was infused intravenously. Treatment group assignment was predetermined by a randomization schedule, and the experimenter was blind to treatment group.

Western Blotting

Expression levels of Nrf2, glutathione synthesis enzyme γ-glutamylcysteine synthetase (γ-GCS), glutathione peroxidase (GPx, an antioxidant enzyme that effectively reduces H2O2 and lipid peroxides), and glutathione-S-transferase (GST, a detoxification enzyme) were measured by Western blot analysis of total lysates from putamen and sensorimotor cortical tissues harvested at 6 h of recovery. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the total protein loading control. For nuclear Nrf2 measurement, we obtained nuclear-enriched fractions of putamen and sensorimotor cortex with a sucrose gradient technique as previously described [31]. Histone H3 was used as the nuclear protein loading control. Samples were separated by 4% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were probed with the following primary antibodies: mouse anti-γ-GCS monoclonal antibody (sc-55586; Santa Cruz Biotech, Dallas, TX), rabbit anti-GPx (ABN63; MilliporeSigma, Burlington, MA), rabbit anti-GST (ABN116; MilliporeSigma), rabbit anti-Nrf2 antibody (PA5–27882, ThermoFisher, Carlsbad, CA), rabbit anti-GAPDH antibody (ABS16, MilliporeSigma), and rabbit anti-Histone H3 antibody (GTX122148, GeneTex, Irvine CA).

Histologic Assessment

At 4 days of recovery, piglets were injected intraperitoneally with 50 mg/kg pentobarbital and 6.4 mg/kg phenytoin. When they were deeply anesthetized, they were perfused transcardially with cold phosphate-buffered saline followed by ice-cold 4% paraformaldehyde. After overnight post-fixation, the brain was removed from the skull, bisected mid-sagittally, and cut into a 20-mm coronal slab between −2 and +18 mm from bregma to include basal ganglia, somatosensory cortex, and thalamus. The entire slab was then cut into four 5-mm slices and embedded in paraffin. Profile counting was performed on 10-μm sections stained with hematoxylin and eosin under oil immersion at 1000× power by an investigator who was blinded to treatment. On each of four equally spaced slides from four blocks covering the entire length of the putamen, the number of viable neurons, showing as round or oval cell bodies with a thin rim of cytoplasm, an open nucleus, and evident nucleolus, was counted in seven nonoverlapping fields in the putamen and caudate nucleus, and in nine nonoverlapping fields in para-sagittal cortical gyrus. On the most caudal block, the number of viable neurons was counted in seven nonoverlapping fields in the ventral posterior thalamus. For each brain region, the data are expressed as a percent of the mean value in the sham group.

Statistical Analysis

All values are expressed as means ± SD. Two-way analysis of variance (ANOVA) was used for comparing HI groups with regard to blood gas, mean arterial pressure, and rectal temperature. One-way ANOVA followed by the Holm-Sidak post hoc procedure was used for comparing viable neuronal counts among groups after we ensured that the data passed the Shapiro-Wilk normality test. For Western blot analysis with each treatment group balanced on each of eight independent gels, a one-way analysis of variance was performed with the optical density normalized by the naïve pig sample on that gel. Because the overall optical density can differ among gels and be correlated among lanes on the same gel, each gel was considered as a within-subject factor. If the F-value was significant, comparisons between groups were made with the Student-Newman-Keuls post hoc test. p<0.05 was considered statistically different.

Results

Arterial PO2 decreased to 35 ± 4 mmHg (mean ± SD, HI + vehicle group) or 28 ± 5 mmHg (HI + sulforaphane group) during the 45 min of ventilation with 10% O2, increased to 112 ± 31 mmHg (HI + vehicle group) or 123 ± 28 mmHg (HI + sulforaphane group) during the 5 min of ventilation with room air, and then decreased to 17 ± 6 mmHg (HI + vehicle group) or 15 ± 5 mmHg (HI + sulforaphane group) by 6 min of asphyxia (Fig. 1A). Arterial PO2 was purposely kept above the normal level during the early recovery period to counter the acidemia-produced decrease in O2-hemoglobin affinity for and thereby ensure that oxyhemoglobin saturation was in the 95–99% range. Arterial PO2 did not differ significantly between HI piglets treated with vehicle and those treated with sulforaphane. Arterial PCO2 remained unchanged during hypoxia (Fig. 1B), and arterial pH decreased modestly to around 7.30 at 42 min of hypoxia (Fig. 1C). However, by 6 min of asphyxia, arterial PCO2 had increased to 120 ± 14 mmHg (HI + vehicle group) or 107 ± 11 mmHg (HI + sulforaphane group), and arterial pH had decreased profoundly to 6.82 ± 0.09 (HI + vehicle group) or 6.86 ± 0.08 (HI + sulforaphane group). With the administration of sodium bicarbonate and rapid normalization of arterial PCO2 after resuscitation, arterial pH had mostly recovered by 15–30 min and remained stable near a value of 7.35–7.40. No significant difference in arterial PCO2 or pH was observed between vehicle-treated and sulforaphane-treated HI piglets. Similarly, we did not see a significant difference in arterial glucose concentration between the two HI groups during hypoxia, asphyxia, or early recovery (Fig. 1D).

Fig. 1.

Fig. 1.

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Arterial PO2 (A), PCO2 (B), pH (C), and glucose concentration (D) at baseline, during 45 min of hypoxia (Hyp, inspired O2 of 10%), at 5 min of room air (RA) ventilation, at 6 min of the 7-min asphyxia period, and during the first 3 h after return of spontaneous circulation (ROSC) in HI groups treated with vehicle (Veh) or sulforaphane (SFN) and at corresponding time points in sham-operated piglets. Breaks in the x-axis allow for changes in the time scale to better illustrate the changes that occur during hypoxia, asphyxia, and the recovery period. An increase in PO2 at 5 min of recovery is related to a brief rise in inspired O2 to 50% before returning to 30%. Values are means ± SD. The two HI groups did not differ significantly at any time point.

The mean arterial blood pressure increased slightly during hypoxia and decreased markedly during the last 3–4 min of asphyxia (Fig. 2A). After resuscitation, arterial pressure briefly increased above baseline levels. Hence, the level of hypoxia and hypotension during asphyxia and early recovery were well matched between the two HI groups. Moreover, the dose of epinephrine administered (3.4 ± 2.0 mg in the vehicle group and 3.0 ± 3.0 mg in the sulforaphane group) and the duration of cardiopulmonary resuscitation required to restore spontaneous circulation (37 ± 13 s in the vehicle group and 45 ± 34 s in the sulforaphane group) were well matched between the two HI groups. Mean arterial blood pressure did not differ significantly between the groups. Rectal temperature was maintained at the near-normal piglet temperature of 38.5 ± 1.0°C during HI and early recovery in all groups (Fig. 2B).

Fig. 2.

Fig. 2.

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The mean arterial blood pressure (MAP, A) and rectal temperature (Temp, B) at baseline, during 45 min of hypoxia (Hyp, inspired O2 of 10%), at 5 min of room air (RA) ventilation, during the 7-min asphyxia period, and during the 3 h after return of spontaneous circulation (ROSC) in HI groups treated with vehicle (Veh) or sulforaphane (SFN) and at corresponding time points in sham-operated piglets. Breaks in the x-axis allow for changes in the time scale to better illustrate the changes that occur during hypoxia, asphyxia, and the recovery period. Values are means ± SD. The two HI groups did not differ significantly at any time point.

Sulforaphane-treated and vehicle-treated piglets in the sham-operated groups had similar neuronal counts in caudate, putamen, sensorimotor cortex, and thalamus. Therefore, we combined all sham-operated piglets into one sham group for histologic analysis. Neuronal profile counts were quantified on day 4 of recovery in 7 sham piglets, 7 HI-vehicle piglets, and 7 HI-sulforaphane piglets. Caudate, putamen, sensorimotor cortex, and ventral posterior thalamus in sham-operated brains exhibited typical anatomical architecture with normal neuronal morphology: a round or oval cell body, thin rim of cytoplasm, and open nucleus with visible chromatin strands and a nucleolus (Fig. 3). HI led to extensive neuronal damage in caudate, putamen, sensorimotor cortex, and ventral posterior thalamic nucleus at 4 days of recovery. The putamen was the most severely damaged region. The ischemic neurons exhibited a shrunken and acutely angular cell body with eosinophilic cytoplasm consisting of microvacuoles, a hematoxylin-stained pyknotic, bony nucleus, and no nucleolus. The density of viable neurons in the HI-vehicle group was significantly less than that in the sham group in the putamen (p < 0.001), caudate (p = 0.015), sensorimotor cortex (p = 0.008), and ventral posterior thalamus (p = 0.004). As a percent of the sham group values, the remaining viable neurons were 31 ± 34% (mean ± SD) in the putamen, 68 ± 26% in the caudate, 59 ± 35% in the sensorimotor cortex, and 59 ± 22% in the ventral posterior thalamus (Fig. 4). With sulforaphane treatment, more viable neurons survived, although some neurons retained ischemic morphology. Compared to that of the HI-vehicle group, the number of viable neurons was significantly increased in the putamen and sensorimotor cortex of the HI-sulforaphane group. The density of viable neurons was 77 ± 31% in the putamen (p = 0.014 vs. vehicle group) and 89 ± 15% in the sensorimotor cortex (p = 0.036 vs. vehicle group). In addition, the viable neurons in the caudate and the ventral posterior thalamus of sulforaphane-treated animals were 84 ± 16% and 78 ± 21% of sham controls. respectlvely. These values were not significantly different from those of HI vehicle group or sham controls.

Fig. 3.

Fig. 3.

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Representative images from H&E-stained sections of piglet caudate, putamen, sensorimotor cortex, and ventral posterior thalamus 4 days after sham surgery or hypoxic-ischemic (HI) injury. In time-matched sham controls treated with vehicle (0.5% DMSO in normal saline) or sulforaphane, neurons had a normal round shape, noncondensed nucleus, and discrete nucleolus. The neuropil appeared smooth and uniform. In HI piglets treated with vehicle, most of the neurons underwent ischemic neurodegeneration, and the surrounding neuropil became pale and vacuolated. In HI piglets treated with sulforaphane, some of the neurons remained undamaged. Arrows indicate normal neurons, and arrowheads indicate ischemic neurons. The Insert showed the representative image of ischemic neurons. Scale bar = 50 μm. Scale bar in insert = 10 μm.

Fig. 4.

Fig. 4.

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Quantification of viable neurons in putamen, caudate, sensorimotor cortex, and ventral posterior thalamus after sham operation (n = 7) or hypoxia-ischemia (HI) in piglets treated with vehicle (n = 7) or sulforaphane (n = 7). The data are shown as mean ± SD. * p < 0.05 versus sham; † p < 0.05 versus HI-vehicle.

Treatment of sham-operated piglets with sulforaphane (n = 8) did not significantly alter the expression of total γ-GCS, GPx, GST, or Nrf2 in the putamen (Fig. 5) or sensorimotor cortical tissue (Fig. 6) compared with that in vehicle-treated sham animals (n = 8). The level of Nrf2 in the nuclear fraction was also unchanged at 6 h after treatment. HI (n = 8) significantly reduced total Nrf2 expression in sensorimotor cortex at 6 h of recovery but did not alter the levels of total γ-GCS, GPx, GST, or nuclear Nrf2 in the putamen or sensorimotor cortical tissue. Compared to those of vehicle-treated animals, treatment with sulforaphane after HI (n = 8) significantly increased the nuclear Nrf2 and total γ-GCS levels in the putamen and sensorimotor cortical tissue after HI.

Fig. 5.

Fig. 5.

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Effect of sulforaphane (SFN) treatment after hypoxia-ischemia (HI) on γ-GCS, GPx, GST, total Nrf2, and nuclear Nrf2 in the putamen at 6 h of recovery (n = 8 per group). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the total protein loading control. Histone H3 was used as the nuclear protein loading control. Optical density (OD) data (mean ± SD) were normalized to the naive value (N). * p<0.05 versus sham-vehicle; # p<0.05 versus HI-vehicle.

Fig. 6.

Fig. 6.

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Effect of sulforaphane (SFN) treatment after hypoxia-ischemia (HI) on γ-GCS, GPx, GST, total Nrf2, and nuclear Nrf2 in the sensorimotor cortex at 6 h of recovery (n = 8 per group). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the total protein loading control. Histone H3 was used as the nuclear protein loading control. Optical density (OD) data (mean ± SD) were normalized to the naive value (N). * p<0.05 versus sham vehicle; # p<0.05 versus HI-vehicle.

Discussion

Our results are consistent with previous work [28] showing that HI leads to ischemic neuronal injury in selectively vulnerable regions, including the caudate nucleus, putamen, primary sensorimotor cortex, and ventral posterior thalamus, of neonatal piglet brains. At 4 days of recovery, piglets that were treated with post-HI sulforaphane exhibited neuronal protection in the putamen and sensorimotor cortex. Nuclear Nrf2 levels were increased in putamen and sensorimotor cortical tissues of sulforaphane-treated piglets at 6 h of recovery, but total Nrf2 expression in tissue lysates was unchanged.

HI leads to a cascade of toxic events in newborn brains: energy failure, excessive glutamate release, activation of N-methyl-D-aspartic acid receptors, calcium influx, and massive nitric oxide production [32]. These changes further result in mitochondrial dysfunction with superoxide leakage and formation of other reactive oxygen species, such as hydrogen peroxide [33]. It is now believed that oxidative stress is a significant contributor to the pathogenesis of HI complications through increased oxidative/nitrosative injury [2]. Oxidants can then damage all biologic molecules, including DNA, RNA, lipids, proteins, carbohydrates, and antioxidants [34]. Thus, the brains of newborns with HI exhibit glutathione reduction and increased formation of protein carbonyl groups, nitration of proteins and nucleic acids, and hydroxylation of nucleic acids [1, 4–6]. Our previous work showed that systemic administration of antioxidant EUK134 or edaravone at 30 min of recovery reduces oxidative stress and salvages the highly vulnerable neurons of newborn HI piglet brain [35].

Nrf2 belongs to the essential leucine zipper transcription factor family [36]. It is widely expressed in cellular cytoplasm and predominately bound to the Kelch-like ECH-associating protein 1 (Keap1), a negative Nrf2 regulator that normally sequesters Nrf2 with cytoskeletal actin and facilitates Nrf2 degradation through ubiquinitation [37]. When it dissociates from Keap1, the Nrf2 translocates into the nucleus and binds to the ARE in the promoter region of hundreds of antioxidant and detoxifying enzymes, thus upregulating their expression and activating endogenous glutathione-based, thioredoxin-based, and other antioxidant defense systems [38, 39]. Nrf2 knockout mice subjected to ischemia produce more reactive oxygen species, exhibit worse neurologic deficits, and develop more extensive infarction than their wild-type counterparts [40, 41, 12]. Therefore, Nrf2 coordinately regulates key antioxidant components and precisely controls the antioxidant defense system at multiple levels against brain ischemia.

Sulforaphane is one of the most extensively studied Nrf2 activators [42]. It interacts directly with sulfhydryl residues on Keap1 to disrupt Nrf2 binding. Moreover, sulforaphane may work through Akt, mitogen-activated protein kinase, and protein kinase C to regulate the phosphorylation state of Nrf2, alter the integrity and stability of Nrf2, and promote Nrf2 release from Keap1 [43–46]. Systemic sulforaphane administration can increase expression of γ-GCS, GPx, GST, NAD(P)H: quinone 1 oxidoreductase, and heme oxygenase 1 in brain tissues, neurons, astrocytes, and brain microvessels [11, 15, 13]. Protective effects of sulforaphane are lost in Nrf2 deficient mice [12] and cells [47], thereby indicating that protection requires Nrf2. By targeting multiple antioxidant pathways, sulforaphane is highly effective at reducing oxidative stress markers, such as 3-nitrotyrosine and 4-hydroxynonenal, and the lipid peroxidation marker malondialdehyde in rodent models of intracerebral hemorrhage and neonatal HI [12, 10]. Here, we found that sulforaphane increased nuclear Nrf2 in the putamen and sensorimotor cortex at 6 h after HI. Nuclear translocation is essential for Nrf2 activation [48]. Therefore, it is not surprising that we observed upregulation of γ-GCS, an enzyme involved in the rate-limiting step of glutathione synthesis. In addition to directly protecting neurons from ischemic injury [49], sulforaphane might target glial cells and brain microvessels that are affected by HI. It activates the astrocytic Nrf2 pathway and protects astrocytes in in vitro ischemia model [50]. It also stimulates Nrf2-driven genes and preserves blood-brain barrier integrity after brain injury [15]. Effects of sulforaphane on non-neuronal cells may support survival of neurons after ischemia.

On the other hand, two additional Phase II antioxidant enzymes, GPx and GST, which represent some of the downstream effectors of Nrf2 signaling, did not increase significantly. These enzymes are involved in catalyzing the transfer of glutathione to its substrates. Previous studies have shown that mitochondrial GPx protein levels and activities are reduced at 20 h of recovery in HI rat pups [6], and Nrf2 activation induces GST at 72 h after ischemia and GPx at 24 h of recovery [51, 52]. In this context, sulforaphane treatment may affect the levels of GPx and GST beyond the 6-h recovery time point. We selected 6 h for our study because, in this model, neuronal cell death, as assessed histologically, occurs primarily between 6 and 24 h in the striatum and between 24 and 48 h in the sensorimotor cortex [1]. Furthermore, some disruption of endoplasmic reticulum and the Golgi apparatus is already apparent by 3–6 h in the striatum and may limit the synthesis of Phase II antioxidant proteins beyond that time [53]. In addition, we did not observe an increase in overall Nrf2 expression. Oxidative products affect the conformation of Keap1, disrupt the association between Keap1 and Nrf2, and enhance Nrf2 expression [38, 54]. Some work suggests the induction of Nrf2 in ischemic brains [55]. However, Nrf2 mRNA can be reduced in ischemic brains [56]. Other reports indicate that whole tissue Nrf2 expression is unchanged or even reduced in ischemic brains [57, 58]. Here, we found that HI did not alter total Nrf2 expression in the putamen and even reduced total Nrf2 level in sensorimotor cortical tissue.

Sulforaphane may exert effects independent of Nrf2 activation. For example, it has recently been shown to produce rapid dilation of piglet pial arterioles by a mechanism involving the generation of hydrogen sulfide [59]. However, we believe that the neuroprotection in our model is unlikely related to vasodilation because cerebral blood flow quickly returns to normal levels after resuscitation [60]. Moreover, sulforaphane can lead to the deacetylation of histones in cancer cells [61]. That effect, however, may generally be required for the induction of Phase II antioxidant enzymes.

The injury was less severe in the caudate nucleus than in the putamen both in our present and previous work [35]. Sulforaphane treatment after HI did not produce significant neuroprotection in the caudate nucleus (increase in viable neurons from 68% to 84%, p = 0.23). The lack of statistical significance may be related to the presence of fewer neurons that can be rescued. In contrast to the rapid neurodegeneration that occurs in the striatum, neurodegeneration in the thalamus is much more delayed in this model [25]. The apoptotic morphology is often seen in thalamic sensory nuclei between 2 and 4 days [62]. Sulforaphane treatment also produced a trend toward neuroprotection in ventral posterior thalamus (increase in viable neurons from 59% to 77%, p = 0.103). It has been shown that sulforaphane plasma concentration reaches a peak at ~2 h, with a subsequent circulating half-life of ~2 h [20, 63]. The concentration of sulforaphane in brain is approximately 10% of that in splanchnic organs and 25% of the plasma concentration [64]. We injected sulforaphane intravenously only once at 15 min of recovery. Multiple sulforaphane injections or a much larger sample size may be needed to show significant protection of sulforaphane in caudate and thalamic neurons of HI piglet brains.

In summary, sulforaphane administered intravenously to piglets at 15 min after HI increased nuclear Nrf2 expression and resulted in partial neuroprotection in the highly vulnerable putamen and primary sensorimotor cortex. These results from a large-animal model support the early use of antioxidant treatment for neonatal HIE.

Acknowledgements

We thank Claire Levine for assistance with manuscript preparation.

Funding Sources

This work was supported by a grant from the National Institutes of Health (R01 NS060703 to RCK) and by an American Heart Association Grant-in-Aid (to JKL).

Footnotes

Statement of Ethics

All animal experiment protocols were approved by the Animal Care and Use Committee of the Johns Hopkins University (SW19M296) and were performed in accordance with the National Institutes of Health Guidelines and the ARRIVE guidelines (http://www.nc3rs.org.uk/arrive-guidelines).

Conflict of Interest Statement

The authors declare no conflicts of interest.

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