Edward A. Armstrong’s research while affiliated with University of Alberta and other places

Our research group previously found that broccoli sprouts possess neuroprotective effects during pregnancy. The active compound has been identified as sulforaphane (SFA), obtained from glucosinolate and glucoraphanin, which are also present in other crucifers, including kale. Sulforaphene (SFE), obtained from glucoraphenin in radish, also has numerous biological benefits, some of which supersede those of sulforaphane. It is likely that other components, such as phenolics, contribute to the biological activity of cruciferous vegetables. Notwithstanding their beneficial phytochemicals, crucifers are known to contain erucic acid, an antinutritional fatty acid. The aim of this research was to phytochemically examine broccoli, kale, and radish sprouts to determine good sources of SFA and SFE to inform future studies of the neuroprotective activity of cruciferous sprouts on the fetal brain, as well as product development. Three broccoli: Johnny’s Sprouting Broccoli (JSB), Gypsy F1 (GYP), and Mumm’s Sprouting Broccoli (MUM), one kale: Johnny’s Toscano Kale (JTK), and three radish cultivars: Black Spanish Round (BSR), Miyashige (MIY), and Nero Tunda (NT), were analyzed. We first quantified the glucosinolate, isothiocyanate, phenolics, and DPPH free radical scavenging activity (AOC) of one-day-old dark- and light-grown sprouts by HPLC. Radish cultivars generally had the highest glucosinolate and isothiocyanate contents, and kale had higher glucoraphanin and significantly higher sulforaphane content than the broccoli cultivars. Lighting conditions did not significantly affect the phytochemistry of the one-day-old sprouts. Based on phytochemistry and economic factors, JSB, JTK, and BSR were chosen for further sprouting for three, five, and seven days and subsequently analyzed. The three-day-old JTK and radish cultivars were identified to be the best sources of SFA and SFE, respectively, both yielding the highest levels of the respective compound while retaining high levels of phenolics and AOC and markedly lower erucic acid levels compared to one-day-old sprouts.

Background: The interruption of oxygen and blood supply to the newborn brain around the time of birth is a risk factor for hypoxic-ischemic encephalopathy and may lead to infant mortality or lifelong neurological impairments. Currently, therapeutic hypothermia, the cooling of the infant's head or entire body, is the only treatment to curb the extent of brain damage. New method: In this study, we designed a focal brain cooling device that circulates cooled water at a steady state temperature of 19 ± 1 °C through a coil of tubing fitted onto the neonatal rat's head. We tested its ability to selectively decrease brain temperature and offer neuroprotection in a neonatal rat model of hypoxic-ischemic brain injury. Results: Our method cooled the brain to 30-33 °C in conscious pups, while keeping the core body temperature approximately 3.2 °C warmer. Furthermore, the application of the cooling device to the neonatal rat model demonstrated a reduction in brain volume loss compared to pups maintained at normothermia and achieved a level of brain tissue protection the same as that of whole-body cooling. Comparison with existing methods: Prevailing methods of selective brain hypothermia are designed for adult animal models rather than for immature animals such as the rat as a conventional model of developmental brain pathology. Contrary to existing methods, our method of cooling does not require surgical manipulation or anaesthesia. Conclusion: Our simple, economical, and effective method of selective brain cooling is a useful tool for rodent studies in neonatal brain injury and adaptive therapeutic interventions.

Antioxidants and anti-inflammatory compounds are potential candidates to prevent age-related chronic diseases. Broccoli sprouts (BrSp) are a rich source of sulforaphane—a bioactive metabolite known for its antioxidant and anti-inflammatory properties. We tested the effect of chronic BrSp feeding on age-related decline in cardiometabolic health and lifespan in rats. Male and female Long-Evans rats were fed a control diet with or without dried BrSp (300 mg/kg body weight, 3 times per week) from 4 months of age until death. Body weight, body composition, blood pressure, heart function, and glucose and insulin tolerance were measured at 10, 16, 20, and 22 months of age. Behavioral traits were also examined at 18 months of age. BrSp feeding prolonged life span in females, whereas in males the positive effects on longevity were more pronounced in a subgroup of males (last 25% of survivors). Despite having modest effects on behavior, BrSp profoundly affected cardiometabolic parameters in a sex-dependent manner. BrSp-fed females had a lower body weight and visceral adiposity while BrSp-fed males exhibited improved glucose tolerance and reduced blood pressure when compared to their control counterparts. These findings highlight the sex-dependent benefits of BrSp on improving longevity and delaying cardiometabolic decline associated with aging in rats.

Background Neonatal hypoxic-ischemic encephalopathy arises from a reduction of oxygen and blood supply to the infant brain and can lead to severe brain damage and life-long disability. The damage is greatest at the irreversibly injured necrotic core, whereas the penumbra is the surrounding, potentially salvageable tissue populated with a mix of alive and dying cells. To date, there exists no method for targeting drugs to the brain damage. Methods and Major Results Bacteriophages are viruses that propagate in bacteria but are biocompatible in humans and also amenable to genetic and chemical modification in a manner distinctive from conventional therapeutic nanoparticles. Here, a library of M13 bacteriophage was administered into a rat model of hypoxic-ischemic encephalopathy, and unique bacteriophage clones were confirmed to localize in healthy brain tissue versus the core and penumbra zones of injury. Conclusions For the first time, there is a potential to directly deliver therapeutics to different regions of the neonatal brain injury. This article is protected by copyright. All rights reserved

Background Perinatal brain injury results in neurodevelopmental disabilities (neuroDDs) that include cerebral palsy, autism, attention deficit disorder, epilepsy, learning disabilities and others. Commonly, injury occurs when placental circulation, that is responsible for transporting nutrients and oxygen to the fetus, is compromised. Placental insufficiency (PI) is a reduced supply of blood and oxygen to the fetus and results in a hypoxic-ischemic (HI) environment. A significant HI state in-utero leads to perinatal compromise, characterized by fetal growth restriction and brain injury. Given that over 80% of perinatal brain injuries that result in neuroDDs occur during gestation, prior to birth, preventive approaches are needed to reduce or eliminate the potential for injury and subsequent neuroDDs. Sulforaphane (SFA) derived from cruciferous vegetables such as broccoli sprouts (BrSps) is a phase-II enzyme inducer that acts via cytoplasmic Nrf2 to enhance the production of anti-oxidants in the brain through the glutathione pathway. We have previously shown a profound in vivo neuro-protective effect of BrSps/SFA as a dietary supplement in pregnant rat models of both PI and fetal inflammation. Strong evidence also points to a role for SFA as treatment for various cancers. Paradoxically, then SFA has the ability to enhance cell survival, and with conditions of cancer, enhance cell death. Given our findings of the benefit of SFA/Broccoli Sprouts as a dietary supplement during pregnancy, with improvement to the fetus, it is important to determine the beneficial and toxic dosing range of SFA. We therefore explored, in vitro, the dosing range of SFA for neuronal and glial protection and toxicity in normal and oxygen/glucose deprived (OGD) cell cultures. Methods OGD simulates, in vitro, the condition experienced by the fetal brain due to PI. We developed a cell culture model of primary cortical neuronal, astrocyte and combined brain cell co-cultures from newborn rodent brains. The cultures were exposed to an OGD environment for various durations of time to determine the LD50 (duration of OGD required for 50% cell death). Using the LD50 as the time point, we evaluated the efficacy of varying doses of SFA for neuroprotective and neurotoxicity effects. Control cultures were exposed to normal media without OGD, and cytotoxicity of varying doses of SFA was also evaluated. Immunofluorescence (IF) and Western blot analysis of cell specific markers were used for culture characterization, and quantification of LD50. Efficacy and toxicity effect of SFA was assessed by IF/high content microscopy and by AlamarBlue viability assay, respectively. Results We determined the LD50 to be 2 hours for neurons, 8 hours for astrocytes, and 10 hours for co-cultures. The protective effect of SFA was noticeable at 2.5 μM and 5 μM for neurons, although it was not significant. There was a significant protective effect of SFA at 2.5 μM (p<0.05) for astrocytes and co-cultures. Significant toxicity ranges were also confirmed in OGD cultures as ≥ 100 μM (p<0.05) for astrocytes, ≥ 50 μM (p<0.01) for co-cultures, but not toxic in neurons; and toxic in control cultures as ≥ 100 μM (p<0.01) for neurons, and ≥ 50 μM (p<0.01) for astrocytes and co-cultures. One Way ANOVA and Dunnett’s Multiple Comparison Test were used for statistical analysis. Conclusions Our results indicate that cell death shows a trend to reduction in neuronal and astrocyte cultures, and is significantly reduced in co-cultures treated with low doses of SFA exposed to OGD. Doses of SFA that were 10 times higher were toxic, not only under conditions of OGD, but in normal control cultures as well. The findings suggest that: 1. SFA shows promise as a preventative agent for fetal ischemic brain injury, and 2. Because the fetus is a rapidly growing organism with profound cell multiplication, dosing parameters must be established to insure safety within efficacious ranges. This study will influence the development of innovative therapies for the prevention of childhood neuroDD.

Introduction: Perinatal arterial ischemic stroke (PAIS) underlies approximately 10% of infantile spasms (IS). We aim to identify patterns of brain injury in ischemic stroke that may predispose infants to infantile spasms. Methods: Sixty-four perinatal arterial ischemic stroke patients were identified meeting the following inclusion criteria: term birth, magnetic resonance imaging (MRI) showing ischemic stroke or encephalomalacia in an arterial distribution, and follow-up records. Patients who developed infantile spasms (PAIS-IS) were analyzed descriptively for ischemic stroke injury patterns and were compared to a seizure-free control group (PAIS-only). Stroke injury was scored using the modified pediatric ASPECTS (modASPECTS). Results: The PAIS-IS (n = 9) group had significantly higher modASPECTS than the PAIS-only (n = 16) group (P = .002, Mann-Whitney). A greater proportion of PAIS-IS patients had injury to deep cerebral structures (67%) than PAIS-only (25%). Conclusion: Infarct size was significantly associated with infantile spasms development. Results support theories implicating deep cerebral structures in infantile spasms pathogenesis. This may help identify perinatal arterial ischemic stroke patients at risk of infantile spasms, facilitating more timely diagnosis.

Glucoraphanin is a glucosinolate prominently found in the cruciferous vegetables, broccoli, and broccoli sprouts. Following plant tissue damage, glucoraphanin combines with the enzyme myrosinase to form the isothiocyanate, sulforaphane. Sulforaphane is a natural health product with properties that have paradoxical effects. On the one hand it has been shown to be profoundly protective of cell death as a neuroprotective agent for neurodegenerative diseases, and on the other hand, it has been shown to be proapoptotic as an anticancer agent. Our laboratory has found broccoli sprouts and sulforaphane to be “protective” of the fetal brain in preclinical rodent models of placental insufficiency, fetal inflammation, and perinatal stroke. However, research intended to provide preventive strategies to the fetus, via the pregnant mother is sparse and difficult to undertake. This is especially true of conventional pharmaceuticals, likely related to the fear of litigation. This review sought to investigate the different mechanisms of action of sulforaphane when used as an antiapoptotic agent, versus as a proapoptotic agent in a cancer setting. Literature in maternal health and sulforaphane showed a pattern of low doses of sulforaphane acting as a phase II enzyme inducer and promotes antioxidant enzymes to reduce oxidative stress, thereby preventing cell death. Reviewing literature of sulforaphane as an anticancer agent, including our own studies in cell culture, shows that higher doses of sulforaphane act as a proapoptotic agents and possible HDAC inhibitors, leading to the death of a host of different types of cancer cells.

Stressors during the fetal and postnatal period affect the growth and developmental trajectories of offspring, causing lasting effects on physiologic regulatory systems. Here, we tested whether reduced uterine artery blood flow in late pregnancy would alter body composition in the offspring, and whether feeding offspring a western diet (WD) would aggravate these programming effects. Pregnant rats underwent bilateral uterine artery ligation (BUAL) or sham surgery on gestational day (GD)18 (term = GD22). At weaning, offspring from each group received either a normal diet (ND) or a WD. BUAL surgery increased fetal loss and caused offspring growth restriction, albeit body weights were no longer different at weaning, suggesting postnatal catch-up growth. BUAL did not affect body weight gain, fat accumulation, or plasma lipid profile in adult male offspring. In contrast, while ND-fed females from BUAL group were smaller and leaner than their sham-littermates, WD consumption resulted in excess weight gain, fat accumulation, and visceral adiposity. Moreover, WD increased plasma triglycerides and cholesterol in the BUAL-treated female offspring without any effect on sham littermates. These results demonstrate that reduced uterine artery blood flow during late pregnancy in rodents can impact body composition in the offspring in a sex-dependent manner, and these effects may be exacerbated by postnatal chronic WD consumption.

Inflammation plays a critical role in the development of hypoxia–ischemia (HI) induced newborn brain damage. A localized, sustained delivery of dexamethasone (Dex) through an intracerebral injection could reduce the inflammatory response in the injured perinatal brain while avoiding unnecessary side effects. Herein, investigated using anionic sulfobutyl ether β‐cyclodextrin (SBE‐β‐CD) to load Dex in the (RADA)4 nanofiber networks as a means of reducing the inflammatory response to HI injury is investigated. The ionic interaction between SBE‐β‐CD and (RADA)4 dramatically affects nanofiber formation and the stability of the nanoscaffold is highly dependent on the SBE‐β‐CD/(RADA)4 ratio. It is observed that the Dex release rate is affected by the concentration of SBE‐β‐CD and (RADA)4 peptide. A higher concentration of SBE‐β‐CD or (RADA)4 results in a higher drug encapsulation efficiency and slower release rate of Dex. This phenomenon may be related to the structure of fiber bundles. Animal studies show that nanoscaffold loaded with Dex inhibits both microglia activation and glial scar formation compared to controls (Dex alone or nanoscaffold alone) within 2 days of injury. It is thought that this is a step toward building a multifaceted nanoscaffold that can be used to treat HI events in perinates.

Perinatal arterial ischemic stroke (AIS) occurs in 1 in 4000 live births and is the most common stroke in children. Consequences include seizures and a spectrum of cognitive and motor disability. Infantile spasms (IS) is an epileptic encephalopathy of infancy with an incidence of 2 in 10,000 live births. Approximately 5% of IS is caused by perinatal AIS. Patterns of ischemic injury that may predispose infants to IS and predict treatment response have not yet been identified. This retrospective case series of infants with AIS and IS provides detailed descriptions of ischemic distribution, seizure presentation, treatment and outcomes. Inclusion criteria were: term birth, ischemic stroke or encephalomalacia in an arterial distribution identified or presumed to have occurred in the perinatal period, a diagnosis of infantile spasms. Patients with a watershed pattern of injury were excluded. The modified pediatric ASPECTS was used to qualify and quantify the type and distribution of stroke. Areas of injury were identified on MRI and scored from T2/Flair or DWI sequences. Eleven patients with AIS and IS were identified. Of nine who fit inclusion criteria, all had MCA territory involvement with 6/9 having basal ganglia injury. Five had ischemia identified retrospectively after developing IS and four presented as neonates. The highest ASPECTS (bilateral deep MCA) was associated with the worst outcome in motor function and epilepsy control. However, the second-highest ASPECTS (unilateral MCA, bilateral ACA) had mild motor deficits and no seizure recurrence after IS resolution. The three lowest ASPECTS (<10) involved unilateral cortical MCA strokes sparing the deep structures, and also had the best motor outcome and seizure control. The co-occurrence of perinatal AIS and IS often results in significant neurologic disability. Although there was no defined pattern of regional homogeneity, there was a trend towards basal ganglia injury with progression of epilepsy after IS. This study highlights that size of ischemic injury may be less important than location as a predictor of motor outcome and seizure intractability. Future research will focus on identifying areas of injury that may confer increased risk of IS compared to stroke patients who remain seizure-free.