Edward A. Armstrong

Effect of caffeine and morphine on the developing pre-mature brain

Apnea of pre-maturity is common, occurring in 85% of infants born less than 34 week gestation. Oral caffeine is the most frequent form of therapy, often in conjunction with the use of intubation and intermittent ventilation. Morphine is used to reduce the pain believed to be associated with the latter. Little information is available on the effects of caffeine, morphine or their combination, on the developing brain. We determined the effect of caffeine and morphine alone and in combination of cell death on the developing brain of the rat. Cell death, measured by Fluoro-jade B and activated caspase-3, was significantly increased at 12 and 24 hour post-caffeine injection (P < 0.05) in the cortex, caudate, nucleus accumbens, hypothalamus, hippocampus and superior colliculus. No alterations were seen following morphine injection alone. However, in the thalamus, the combination of caffeine and morphine did increase cell death to a significantly greater extent than caffeine alone. Further research is required to determine the long-term pathologic and functional effects of caffeine and the combination of caffeine and morphine on the developing immature brain.

Housing environment and sex affect behavioral recovery from ischemic brain damage

In previous work from our laboratory, we have developed a model of stroke that allows the comparison of stroke injury across age groups (10-day old, 63-day old, 180-day old rats). In this current study, we incorporated environmental enrichment to determine whether this form of rehabilitation alters behavioral recovery, and whether age and/or sex interacts with enrichment. Our results indicated that ischemic male rats that were housed in standard cages (shoebox housed) performed the poorest on two tasks of motor ability, and that this performance was related to the size of the lesion. With the exception of lesion size, in which 10-day old enriched rats had significantly smaller lesions than either the enriched 63-day old rats or the 180-day old rats, we observed no significant effects of age on behavioral recovery. Thus, our results are consistent with the observation that male rats may be more vulnerable to stroke and that they may differentially benefit from enrichment.

Enriched environment and the effect of age on ischemic brain damage.

Stroke affects all age groups from the newborn to the elderly. Previous work from our laboratory has shown that despite a greater susceptibility to brain damage, the immature brain recovers more rapidly and to a greater extent than does the more mature nervous system. In the current study, we examined the influence of environmental enrichment on the effects of age on the brain damaging effects of stroke. Rats aged 10, 63, and 180 days received ischemic insults following stereotactic intra-cerebral injection of endothelin-1, and resulting in injury to the right middle cerebral artery territory. Rats were then housed in either environmentally enriched cages, or standard cages for 60 days, after which they were sacrificed, and brain volumes determined for the extent of neurologic injury. Rats receiving the insult at 10 days of age showed a reduction of pathologic injury when housed in the enriched cages compared to standard. Conversely, rats receiving the insult at 180 days and housed environmentally enriched cages actually showed an increased volume of brain damage compared to controls. Our findings clearly indicate the dramatic influence of age on the extent of stroke and the influence of rehabilitative therapies. Behavioral correlation to morphologic alterations is required. Attempts at therapeutic interventions clearly need to be age-specific.

The Effect of Postischemic Hypothermia on Apoptotic Cell Death in the Neonatal Rat Brain

Background and Objective: Hypothermia is the most effective neuroprotective therapy against ischemic injury in the developing brain. However, the mechanism of hypothermic neuroprotection is not well understood. We sought to investigate whether hypothermia mediates neuroprotection by modulating ischemia-induced apoptosis. Methods: Seven-day-old rat pups were randomly assigned to either control or hypoxia-ischemia (HI) groups. In the HI group, the internal carotid artery was ligated and cut. This was followed by transient hypoxia at 8% oxygen for 90 min. In the control rats, the internal carotid was isolated but not ligated. Immediately after the hypoxic episode, pups in the HI group were either placed in water baths maintained at 28°C for 24 h (core temperatures at 31°C) or they remained in a normothermic environment. Animals were sacrificed at 24, 48 and 72 h and 1 week after the HI insult. Brain sections were processed for immunohistochemistry and Western blots. Results: Caspase 3 expression was significantly higher in the core compared with the peri-infarct area at all time points in normothermic rats. Hypothermia reduced caspase 3 expression in the core but had little effect in the peri-infarct area. Hypothermia reduced apoptosis-inducing factor translocation to the nucleus in the core and peri-infarct area. Concurrently, X-linked inhibitor of apoptosis (XIAP) expression was significantly potentiated in the hypothermic-ischemic core but not in the peri-infarct area. Conclusion: Hypothermic modulation of caspase-dependent apoptosis may be mediated by upregulating XIAP. However, the effect of hypothermia on caspase-independent apoptosis may be mediated by XIAP-independent mechanisms. Importantly, these effects are mediated in both the core and the penumbral regions of ischemic lesion.

Treatment of the Term Newborn With Brain Injury: Simplicity As the Mother of Invention

Neonatal brain injury remains a common cause of developmental disability, despite tremendously enhanced obstetrical and neonatal care. The timing of brain injury occurs throughout gestation, labor, and delivery, providing an evolving form of brain injury and a moving target for therapeutic intervention. Nonetheless, markedly improved methods are available to identify those infants injured at birth, via clinical presentation with neonatal encephalopathy and neuroimaging techniques. Postischemic hypothermia has been shown to be of tremendous clinical promise in several completed and ongoing trials. As part of this approach to the treatment of the newborn, other parameters of physiologic homeostasis can and should be attended to, with strong animal and clinical evidence that their correction will have dramatic influence on the outcome of the newborn infant. This review addresses aspects of newborn care to which we can direct our attention currently, and which should result in a safe and efficacious improvement in the prognosis of the newborn with neonatal encephalopathy.

Mechanisms of neurodegeneration after severe hypoxic-ischemic injury in the neonatal rat brain.

PURPOSE Apoptosis is implicated in mild-moderate ischemic injury. Cell death pathways in the severely ischemic brain are not characterized. We sought to determine the role of apoptosis in the severely ischemic immature brain. METHODS Seven-day old rats were randomly assigned to mild-moderate or severe cerebral hypoxia-ischemia (HI) group. After ligating the right common carotid artery, animals were subjected to hypoxia for 90min in the mild-moderate HI or 180min in the severe HI. The core and peri-infarct area were measured in H&E stained brain sections using NIS Elements software. Brain sections were processed for caspase-3, AIF and RIP3 immuno-staining. Number of positive cells were counted and compared between the two groups. RESULTS The core constituted a significantly higher proportion of the ischemic lesion in the severely compared to the moderately injured brain (P<0.04) up to 7 days post-injury. Apoptotic cell death was significantly higher (P<0.05) in the core than the peri-infarct of the severe HI brain. In the peri-infarct area of severe HI, AIF-induced cell death increased over time and caspase-3 and AIF equally mediated neuronal death. Necroptosis was significantly higher (P=0.02) in the peri-infarct of the severe HI lesion compared to the moderate HI lesion. In males, but not in females, apoptosis was higher in moderate compared to severe HI. CONCLUSIONS Caspase-independent cell death plays an important role in severe ischemic injury. Injury severity, timing of intervention post-injury and sex of the animal are important determinants in designing neuroprotective intervention for the severely ischemic immature brain.

Broccoli sprout supplementation during pregnancy prevents brain injury in the newborn rat following placental insufficiency

Chronic placental insufficiency and subsequent intrauterine growth restriction (IUGR) increase the risk of hypoxic-ischemic encephalopathy in the newborn by 40 fold. The latter, in turn, increases the risk of cerebral palsy and developmental disabilities. This study seeks to determine the effectiveness of broccoli sprouts (BrSp), a rich source of the isothiocyanate sulforaphane, as a neuroprotectant in a rat model of chronic placental insufficiency and IUGR. Placental insufficiency and IUGR was induced by bilateral uterine artery ligation (BUAL) on day E20 of gestation. Dams were fed standard chow or chow supplemented with 200mg of dried BrSp from E15 - postnatal day 14 (PD14). Controls received Sham surgery and the same dietary regime. Pups underwent neurologic reflex testing and open field testing, following which they were euthanized and their brains frozen for neuropathologic assessment. Compared to Sham, IUGR pups were delayed in attaining early reflexes and performed worse in the open field, both of which were significantly improved by maternal supplementation of BrSp (p<0.05). Neuropathology revealed diminished white matter, ventricular dilation, astrogliosis and reduction in hippocampal neurons in IUGR animals compared to Sham, whereas broccoli sprout supplementation improved outcome in all histological assessments (p<0.05). Maternal dietary supplementation with BrSp prevented the detrimental neurocognitive and neuropathologic effects of chronic intrauterine ischemia. These findings suggest a novel approach for prevention of cerebral palsy and/or developmental disabilities associated with placental insufficiency.

Pioglitazone attenuates hepatic inflammation and fibrosis in phosphatidylethanolamine N-methyltransferase-deficient mice.

Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt(-/-) mice are protected against high-fat diet (HFD)-induced obesity and insulin resistance; however, these mice develop nonalcoholic fatty liver disease (NAFLD). We hypothesized that peroxisomal proliferator-activated receptor-γ (PPARγ) activation by pioglitazone might stimulate adipocyte proliferation, thereby directing lipids from the liver toward white adipose tissue. Pioglitazone might also act directly on PPARγ in the liver to improve NAFLD. Pemt(+/+) and Pemt(-/-) mice were fed a HFD with or without pioglitazone (20 mg·kg(-1)·day(-1)) for 10 wk. Pemt(-/-) mice were protected from HFD-induced obesity but developed NAFLD. Treatment with pioglitazone caused an increase in body weight gain in Pemt(-/-) mice that was mainly due to increased adiposity. Moreover, pioglitazone improved NAFLD in Pemt(-/-) mice, as indicated by a 35% reduction in liver weight and a 57% decrease in plasma alanine transaminase levels. Livers from HFD-fed Pemt(-/-) mice were steatotic, inflamed, and fibrotic. Hepatic steatosis was still evident in pioglitazone-treated Pemt(-/-) mice; however, treatment with pioglitazone reduced hepatic fibrosis, as evidenced by reduced Sirius red staining and lowered mRNA levels of collagen type Iα1 (Col1a1), tissue inhibitor of metalloproteinases 1 (Timp1), α-smooth muscle actin (Acta2), and transforming growth factor-β (Tgf-β). Similarly, oxidative stress and inflammation were reduced in livers from Pemt(-/-) mice upon treatment with pioglitazone. Together, these data show that activation of PPARγ in HFD-fed Pemt(-/-) mice improved liver function, while these mice were still protected against diet-induced obesity and insulin resistance.

Consumption of broccoli sprouts during late gestation and lactation confers protection against developmental delay induced by maternal inflammation

BACKGROUND The presence of a fetal inflammatory response is linked to cerebral palsy. Unfortunately no preventive therapies are available. In this study, we determined whether dietary supplementation with broccoli sprouts (BrSp), a phase-II enzyme inducer, would be effective in preventing the behavioural and pathologic manifestations in a rodent model of inflammation during late pregnancy. METHODS Pregnant Long-Evans rats were administered i.p. Injections of saline (100μl) or lipopolysaccharide (LPS, 200μg/kg), every 12h on embryonic day (E) 19 and 20. In the treatment groups, dams were supplemented with 200mg/day of dried BrSp from E14 until postnatal day 21. Pups underwent a series of neurodevelopmental reflex tests from postnatal day 3-21 followed by neuropathological analyses. RESULTS Pups born from the LPS group were significantly growth restricted (p<0.001) and delayed in hindlimb placing (p<0.05), cliff avoidance (p<0.05), and gait (p<0.001) compared to controls. In the open field behaviour analyses, LPS pups had an increase in grooming behaviour (p<0.05) and a decreased amount of time spent in the center of the box compared to controls. Dietary supplementation with BrSp to offspring exposed to LPS had increased birth weights (p<0.001), were no longer delayed in acquiring hindlimb placing, cliff avoidance, gait, and posture, and groomed less compared to LPS alone pups (p<0.01). Histological analyses revealed that LPS pups had reduced myelin basic protein compared to controls. CONCLUSIONS Our data suggest that BrSp dietary supplementation during pregnancy may be effective in preventing growth restriction and neurodevelopmental delays.

Evidence for Therapeutic Intervention in the Prevention of Cerebral Palsy: Hope from Animal Model Research

Knowledge translation, as defined by the Canadian Institute of Health Research, is defined as the exchange, synthesis, and ethically sound application of knowledge--within a complex system of interactions among researchers and users--to accelerate the capture of the benefits of research through improved health, more effective services and products, and a strengthened healthcare system. The requirement for this to occur lies in the ability to continue to determine mechanistic actions at the molecular level, to understand how they fit at the in vitro and in vivo levels, and for disease states, to determine their safety, efficacy, and long-term potential at the preclinical animal model level. In this regard, particularly as it relates to long-term disabilities such as cerebral palsy that begin in utero, but only express their full effect in adulthood, animal models must be used to understand and rapidly evaluate mechanisms of injury and therapeutic interventions. In this review, we hope to provide the reader with a background of animal data upon which therapeutic interventions for the prevention and treatment of cerebral palsy, benefit this community, and increasingly do so in the future.