Malin Gustavsson

PARP-1 gene disruption in mice preferentially protects males from perinatal brain injury

Poly(ADP‐ribose) polymerase‐1 is over‐activated in the adult brain in response to ischemia and contributes to neuronal death, but its role in perinatal brain injury remains uncertain. To address this issue, 7‐day‐old wild‐type (wt) and PARP‐1 gene deficient (parp+/– and parp–/–) Sv129/CD‐1 hybrid mice were subjected to unilateral hypoxia‐ischemia and histologic damage was assessed 10 days later by two evaluators. Poly(ADP‐ribose) polymerase‐1 knockout produced moderate but significant (p < 0.05) protection in the total group of animals, but analysis by sex revealed that males were strongly protected (p < 0.05) in contrast to females in which there was no significant effect. Separate experiments demonstrated that PARP‐1 was activated over 1–24 h in both females and males after the insult in neonatal wt mice and rats using immnocytochemistry and western blotting for poly(ADP‐ribose). Brain levels of NAD+ were also significantly reduced, but the decrease of NAD+ during the early post‐hypoxia‐ischemia (HI) phase was only seen in males. The results indicate that hypoxia‐ischemia activates Poly(ADP‐ribose) polymerase‐1 in the neonatal brain and that the sex of the animal strongly influences its role in the pathogenesis of brain injury.

IGF-I neuroprotection in the immature brain after hypoxia-ischemia, involvement of Akt and GSK3β?

Insulin‐like growth factor I (IGF‐I) is a neurotrophic factor that promotes neuronal growth, differentiation and survival. Neuroprotective effects of IGF‐I have previously been shown in adult and juvenile rat models of brain injury. We wanted to investigate the neuroprotective effect of IGF‐I after hypoxia‐ischemia (HI) in 7‐day‐old neonatal rats and the mechanisms of IGF‐I actions in vivo. We also wanted to study effects of HI and/or IGF‐I on the serine/threonine kinases Akt and glycogen synthase kinase 3β (GSK3β) in the phophatidylinositol‐3 kinase (PI3K) pathway. Immediately after HI, phosphorylated Akt (pAkt) and phosphorylated GSK3β (pGSK3β) immunoreactivity was lost in the ipsilateral and reduced in the contralateral hemisphere. After 45 min, pAkt levels were restored to control values, whereas pGSK3β remained low 4 h after HI. Administration of IGF‐I (50 µg i.c.v.) after HI resulted in a 40% reduction in brain damage (loss of microtubule‐associated protein) compared with vehicle‐treated animals. IGF‐I treatment without HI was shown to increase pAkt whereas pGSK3β decreased in the cytosol, but increased in the nuclear fraction. IGF‐I treatment after HI increased pAkt in the cytosol and pGSK3β in both the cytosol and the nuclear fraction in the ipsilateral hemisphere compared with vehicle‐treated rats, concomitant with a reduced caspase‐3‐ and caspase‐9‐like activity. In conclusion, IGF‐I induces activation of Akt during recovery after HI which, in combination with inactivation of GSK3β, may explain the attenuated activation of caspases and reduction of injury in the immature brain.

N-acetylcysteine reduces lipopolysaccharide-sensitized hypoxic-ischemic brain injury.

Maternal inflammation/infection alone or in combination with birth asphyxia increases the risk for perinatal brain injury. Free radicals are implicated as major mediators of inflammation and hypoxia‐ischemia (HI)–induced perinatal brain injury. This study evaluated the neuroprotective efficacy of a scavenging agent, N‐acetylcysteine (NAC), in a clinically relevant model.

Vascular response to hypoxic preconditioning in the immature brain

We hypothesized that hypoxic preconditioning (PC) modifies the microvasculature in the immature brain and thereby affects the cerebral blood flow (CBF) during a subsequent hypoxic—ischemic (HI) insult. On postnatal day 6 rats were exposed to hypoxia (36°C, 8.0% O2) or normoxia for 3 h. Unilateral HI (unilateral carotid ligation and 8% hypoxia) was induced 24 h later. Cortical CBF was measured with the 14C-iodoantipyrine technique (at the end of HI) or with laser Doppler flowmetry (Perimed PF5001) before and during HI. At 0, 2, 8, and 24 h cerebral cortex was sampled and analyzed with gene arrays (Affymetrix 230 2.0). l-nitroarginine or vehicle was administrated before hypoxic PC or 30 mins before HI followed by CBF measurement (laser Doppler) during subsequent HI. Twenty-four hours after PC animals were perfusion-fixed and brains immunolabeled for von Willebrand factor and vascular density was determined by stereological quantification. The decrease in CBF during HI was attenuated significantly in PC versus control animals (P < 0.01), as detected by both techniques. Several vascular genes (Angpt2, Adm, Apln, Vegf, Flt1, Kdr, Pdgfra, Agtrap, Adora2a, Ednra, serpine1, caveolin, Id1, Prrx1, Ero1l, Acvrl1, Egfl7, Nudt6, Angptl4, Anxa2, and NOS3) were upregulated and a few (Csrp2, Adora2b) were downregulated after PC. A significant increase in vascular density (P < 0.05) was seen after PC. Nitric oxide synthase inhibition did not affect CBF during HI after PC. In conclusion, hypoxic PC upregulates vascular genes, increases vascular density and attenuates the decrease of CBF during a subsequent HI, which could contribute to tolerance.

Hypoxic preconditioning confers long-term reduction of brain injury and improvement of neurological ability in immature rats.

Exposure to preconditioning (PC) hypoxia 24 h before a severe hypoxic-ischemic (HI) insult reduces development of injury in the immature brain. Several protective regimens have proved effective in the short-term but not in the long-term perspective. The aim of the present study, therefore, was to evaluate the PC effect on long-term morphologic and neurologic outcome in the developing brain. Six-day-old rats were subjected to hypoxia (36°C, 8.0% O2; PC/HI group) and sham controls to normoxia (36°C; HI group) for 3 h. Twenty-four hours later, all rats were exposed to cerebral HI produced by unilateral carotid artery occlusion combined with 1 h, 15 min of hypoxia (36°C, 7.7% O2). A cylinder test was used to evaluate forelimb asymmetry to determine sensorimotor function at 4, 6, and 8 wk of age. Spatial/cognitive ability was assessed by Morris water maze trials at 7 wk of recovery. Neuropathologic analysis was performed 8 wk after insult. Brain damage was reduced (p < 0.0001) in PC/HI (45.0 ± 11.1 mm3) in comparison with HI (159.3 ± 12.2 mm3) rats. A bias for using the ipsilateral forelimb in wall movements was observed in the cylinder test in HI compared with PC/HI rats at 4 (p < 0.001), 6 (p < 0.01), and 8 (p < 0.0001) wk of age. Results of the Morris water maze test revealed differences (p < 0.0001) in average path length between groups on the third and fourth day of trials. Hypoxic PC before HI reduced brain injury by 72% at 8 wk after the insult and provided long-term improvement of sensorimotor and spatial/cognitive functions.

Global Gene Expression in the Developing Rat Brain After Hypoxic Preconditioning: Involvement of Apoptotic Mechanisms?

Exposure to hypoxia before hypoxia-ischemia (HI) confers substantial protection referred to as preconditioning (PC). We hypothesized that PC induces critical changes of genes related to apoptotic cell death to render the brain more resistant. PC hypoxia (8% O2, 36°C, 3 h) was induced in rats on postnatal day (PND) 6, and the rats were killed at 0, 2, 8, and 24 h. Total RNA was extracted from cerebral cortex and analyzed using Affymetrix rat genome 230 2.0 array. PC induced significant changes in 906 genes at 0 h, 927 at 2 h, 389 at 8 h, and 114 at 24 h. Ontology analysis revealed significant alterations in genes involved in cell communication, signal transduction, transcription, phosphorylation, and transport. Genes involved in cell death/apoptosis as well as those related to brain development (cell differentiation, neurogenesis, organogenesis, blood vessel development) were overrepresented. A detailed analysis demonstrated that 77 significantly regulated genes were involved in apoptosis, specifically related to the Bcl-2 family, JNK pathway, trophic factor pathways, inositol triphosphate (PI3) kinase/Akt pathway, extrinsic or intrinsic pathway, or the p53 pathway. The study supports that the epidermal growth factor receptor family, mitogen-activated protein kinase phosphatases, and Bcl-2–related proteins and the PI3 kinase/Akt pathway may have roles in providing resistance in the developing central nervous system (CNS).

Delayed Peripheral Administration of a GPE Analogue Induces Astrogliosis and Angiogenesis and Reduces Inflammation and Brain Injury following Hypoxia-Ischemia in the Neonatal Rat

Glycine 2-methyl proline glutamate (G-2mPE) is a proline-modified analogue to the naturally existing N-terminal tripeptide glycine-proline-glutamate that is a cleaved product from insulin-like growth factor-1. G-2mPE is designed to be more enzymatically resistant than glycine-proline-glutamate and to increase its bioavailability. The current study has investigated the protective effects of G-2mPE following hypoxic-ischemic brain injury in the neonatal brain. On postnatal day 7, Wistar rats were exposed to hypoxia-ischemia (HI). HI was induced by unilateral ligation of the left carotid artery followed by hypoxia (7.7% O2, 36°C) for 60 min. The drug treatment started 2 h after the insult, and the pups were given either 1.2 mg/kg (bolus), 1.2 mg/ml once a day for 7 days, or vehicle. The degree of brain damage was determined histochemically by thionin/acid fuchsin staining. G-2mPE’s anti-inflammatory properties were investigated by IL-1β, IL-6, and IL-18 ELISA, and effects on apoptosis by caspase 3 activity. Vascularization was determined immunohistochemically by the total length of isolectin-positive blood vessels. Effect on astrocytosis was also determined in the hippocampus. Animals treated with multiple doses of G-2mPE demonstrated reduced overall brain injury 7 days after HI, particularly in the hippocampus and thalamus compared to vehicle-treated rats. The expression of IL-6 was decreased in G-2mPE-treated animals compared to vehicle-treated pups, and both the capillary length and astrogliosis were increased in the drug-treated animals. There was no effect on caspase 3 activity. This study indicates that peripheral administration of G-2mPE, starting 2 h after a hypoxic-ischemic insult, reduces the degree of brain injury in the immature rat brain. The normalization of IL-6 levels and the promotion of both neovascularization and reactive astrocytosis may be potential mechanisms that underlie its protective effects.

Supplementation with a mixture of complex lipids derived from milk to growing rats results in improvements in parameters related to growth and cognition

Alterations in nutritional factors during early development can exert long-term effects on growth, neural function, and associated behaviors. The lipid component of milk provides a critical nutritional source for generating both energy and essential nutrients for the growth of the newborn. The present study, therefore, investigated the hypothesis that nutritional supplementation with a complex milk lipid (CML) preparation, derived from the milk fat globule membrane rich in phospholipids and gangliosides from young rats, has beneficial effects on learning behavior and postnatal growth and development. Male Wistar rat offspring from normal pregnancies were treated from neonatal day 10 until postnatal day 80 with either vehicle or CML at a dose of 0.2% (low) and 1.0% (high) based on total food intake (n = 16 per group). Neonatal dosing was via daily oral gavage, while postweaning dosing was via gel supplementation to a standard chow diet. Animals underwent behavioral tasks related to spatial memory, learning, and cognitive function. Complex milk lipid supplementation significantly increased linear growth rate (P < .05), and the improved growth trajectory was not related to changes in body composition as quantified by dual-energy x-ray absorptiometry scanning or altered plasma lipid profiles. Moreover, this effect was not dose dependent and not attributable to the contribution to total energy intake of the CML composition. Supplementation of the CML to growing rats resulted in statistically significant improvements in parameters related to novelty recognition (P < .02) and spatial memory (P < .05) using standard behavioral techniques, but operant testing showed no significant differences between treatment groups. Supplementation with a CML containing gangliosides had positive growth and learning behavioral effects in young normal growing rats.

Maternal supplementation with a complex milk lipid mixture during pregnancy and lactation alters neonatal brain lipid composition but lacks effect on cognitive function in rats.

Complex milk lipids (CMLs) provide a critical nutritional source for generating both energy and essential nutrients for the growth of the newborn. The present study investigated nutritional supplementation with a CML containing gangliosides and phospholipids in pregnant and lactating rats on learning behavior and postnatal growth in male offspring. Wistar female rats were supplemented during pregnancy and lactation with either control or CML to provide gangliosides at a dose of 0.01% (low) and 0.05% (high) based on total food intake. The CML-supplemented dams showed no differences in comparison to controls regarding growth, food intake, and litter characteristics. There were significant differences in brain composition in male offspring at postnatal day 2 (P2) with higher concentrations of gangliosides (high dose, P < .05) and lower concentrations of phospholipids (low and high dose, P < .05) in the CML-supplemented groups. The distribution of individual ganglioside species was not significantly different between treatment groups. Brain weight at P2 was also significantly higher in the CML groups. Differences in the brain composition and weight were not significant by weaning (P21). As adults (P80), adiposity was reduced in the low CML-supplemented group compared to controls. No significant differences were detected between any of the treatment groups in any of the behavioral tasks (water maze, object recognition, and operant learning). These data suggest that maternal supplementation with a CML during pregnancy and lactation is safe and has a significant early impact on brain weight and ganglioside and phospholipid content in offspring but did not alter long-term behavioral function using standard behavioral techniques.