Erythropoietin Ameliorates Neonatal Hypoxia-Ischemia-Induced Neurobehavioral Deficits, Neuroinflammation, and Hippocampal Injury in the Juvenile Rat.

The hematopoietic growth factor erythropoietin (EPO) has been shown to be neuroprotective against hypoxia-ischemia (HI) in Postnatal Day 7 (P7)-P10 or adult animal models. The current study was aimed to determine whether EPO also provides long-lasting neuroprotection against HI in P5 rats, which is relevant to immature human infants. Sprague-Dawley rats at P5 were subjected to right common carotid artery ligation followed by an exposure to 6% oxygen with balanced nitrogen for 1.5 h. Human recombinant EPO (rEPO, at a dose of 5 units/g) was administered intraperitoneally one hour before or immediately after insult, followed by additional injections at 24 and 48 h post-insult. The control rats were injected with normal saline following HI. Neurobehavioral tests were performed on P8 and P20, and brain injury was examined on P21. HI insult significantly impaired neurobehavioral performance including sensorimotor, locomotor activity and cognitive ability on the P8 and P20 rats. HI insult also resulted in brain inflammation (as indicated by microglia activation) and neuronal death (as indicated by Jade B positive staining) in the white matter, striatum, cortex, and hippocampal areas of the P21 rat. Both pre- and post-treatment with rEPO significantly improved neurobehavioral performance and protected against the HI-induced neuronal death, microglia activation (OX42+) as well as loss of mature oligodendrocytes (APC-CC1+) and hippocampal neurons (Nissl+). The long-lasting protective effects of rEPO in the neonatal rat HI model suggest that to exert neurotrophic activity in the brain might be an effective approach for therapeutic treatment of neonatal brain injury induced by hypoxia-ischemia.

Lipoprotein-associated phospholipase A2 and carotid intima-media thickness in individuals classified as low-risk according to Framingham.

BACKGROUND: The Framingham risk score (FRS) has long been used as a global tool to estimate coronary heart disease (CHD) risk, but data has shown that subclinical CHD may exist in those classified as low risk by FRS, and as a result, there is potential for misclassification. Lipoprotein-associated phospholipase A2 (Lp-PLA2) and carotid intima-media thickness (CIMT) are two emerging risk markers that are predictive of future CHD events. PURPOSE: To examine Lp-PLA2 and CIMT values in low risk individuals, and to explore the relationship between Lp-PLA2 and CIMT. METHODS: A total of 229 men and women (age =53±7 years) underwent body composition analysis, objective physical activity measurement, fasting blood draw to determine standard lipid values and Lp-PLA2 mass, and CIMT measurement through ultrasound. RESULTS: For all subjects, mean CIMT was 0.61±0.1 mm, mean Lp-PLA2 mass was 197±45 ng/dL. A total of 19.5% and 34.6% of women and 4.6% and 73.8% of men were considered at elevated risk for CHD by CIMT (>75(th) percentile for age) and Lp-PLA2 mass (>200 ng/dL) standards, respectively. Both CIMT and Lp-PLA2 mass were significant independent predictors of each other, whereas traditional risk markers (lipids, glucose) were not. CONCLUSIONS: Results suggest that in those classified as low risk by FRS, evidence of increased CHD risk may exist through the use of newer risk markers like CIMT and Lp-PLA2. These emerging markers may aid in the earlier detection and intervention of subclinical CHD.

Neuron-oligodendrocyte myelination co-culture derived from embryonic rat spinal cord and cerebral cortex.

An in vitro myelination model derived from rat central nervous system (CNS) remains to be established. Here, we describe a simple and reproducible myelination culture method using dissociated neuron-oligodendrocyte (OL) co-cultures from either the embryonic day 16 (E16) rat spinal cord or cerebral cortex. The dissociated cells are plated directly on poly-L-lysine-coated cover slips and maintained in a modified myelination medium that supports both OL and neuron differentiation. The spinal cord derived OL progenitor cells develop quickly into myelin basic protein (MBP)+ mature OLs and start to myelinate axons around 17 days in vitro (DIV17). Myelination reaches its peak around six weeks (DIV40) and the typical nodes of Ranvier are revealed by paranodal proteins Caspr and juxaparanodal protein Kv1.2 immunoreactivity. Electron microscopy (EM) shows typical myelination cytoarchitecture and synaptic organization. In contrast, the cortical-derived co-culture requires triiodothyronine (T3) in the culture medium for myelination. Finally, either hypomyelination and/or demyelination can be induced by exposing proinflammatory cytokines or demyelinating agents to the co-culture, suggesting the feasibility of this modified in vitro myelination model for myelin-deficit investigation.

Dexamethasone and betamethasone protect against lipopolysaccharide-induced brain damage in neonatal rats.

INTRODUCTION: The aim of this study was to test whether dexamethasone (Dex) and betamethasone (Beta), two of the most commonly used corticosteroids, protect against lipopolysaccharide (LPS)-induced white matter damage and neurobehavioral dysfunction. METHODS: LPS or sterile saline was injected into the brain white matter of rat pups at postnatal day 5 (P5), and Dex or Beta was given intraperitoneally to the rat pups 1 h before the LPS microinjection. Brain inflammatory response, brain damage, and myelination were examined at P6, P8, and P14. Neurobehavioral tests were performed from P3 through P22. RESULTS: Our results demonstrate that Dex and Beta markedly diminish the LPS-induced brain inflammatory response, restore myelin basic protein (MBP) expression, and alleviate lateral ventricle dilation. Both corticosteroids demonstrate significant protection against most LPS-induced behavioral deficits, including those in rearing, vibrissa-elicited forelimb-placing, beam walking, learning, and elevated plus-maze test. Of note, only Beta improved the locomotion and stereotype dysfunction. In contrast to their beneficial effects, neither drug prevented LPS-induced delay in body weight gain from P6 through P21. DISCUSSION: Our study suggests that if their adverse effects are minimized, corticosteroids may be the potential candidate drugs to prevent brain damage in premature infants.

Intracerebral lipopolysaccharide induces neuroinflammatory change and augmented brain injury in growth-restricted neonatal rats.

INTRODUCTION: Intrauterine growth restriction (IUGR) alters fetal development and is associated with neurodevelopmental abnormalities. We hypothesized that growth restriction from reduced intrauterine perfusion would predispose neonatal rats to subsequent inflammatory brain injury. METHODS: In this study, IUGR was achieved by induced placental insufficiency in pregnant rats at 14 days of gestation. IUGR offspring and sham-operated control pups were subsequently injected with intracerebral lipopolysaccharide (LPS) as a model of periventricular leukomalacia (PVL). RESULTS: LPS similarly elevates proinflammatory cytokines in the brains of both IUGR and control rat pups. However, the chemokines cytokine-induced neutrophil chemoattractant-1 (CINC-1) and macrophage chemoattractant protein-1 (MCP-1), as well as microglia activation, were significantly higher in LPS-treated IUGR rat pups as compared with LPS-treated controls. In addition to the unique brain inflammatory response, IUGR rat pups demonstrated increased brain damage with an increased number of apoptotic cells, larger lateral ventricular size, and more severe impairment of myelination. DISCUSSION: This study provides evidence that placental insufficiency may sensitize the innate immune system in the immature brain and reveals a possible link between brain inflammation and injury.

Neonatal exposure to lipopolysaccharide enhances vulnerability of nigrostriatal dopaminergic neurons to rotenone neurotoxicity in later life.

Brain inflammation in early life has been proposed to play important roles in the development of neurodegenerative disorders in adult life. To test this hypothesis, we used a neonatal rat model of lipopolysaccharide (LPS) exposure (1000 EU/g body weight, intracerebral injection on P5) to produce brain inflammation. By P70, when LPS-induced behavioral deficits were spontaneously recovered, animals were challenged with rotenone, a commonly used pesticide, through subcutaneous mini-pump infusion at a dose of 1.25 mg/kg per day for 14 days. This rotenone treatment regimen ordinarily does not produce toxic effects on behaviors in normal adult rats. Our results show that neonatal LPS exposure enhanced the vulnerability of nigrostriatal dopaminergic neurons to rotenone neurotoxicity in later life. Rotenone treatment resulted in motor neurobehavioral impairments in rats with the neonatal LPS exposure, but not in those without the neonatal LPS exposure. Rotenone induced losses of tyrosine hydroxylase immunoreactive neurons in the substantia nigra and decreased mitochondrial complex I activity in the striatum of rats with neonatal LPS exposure, but not in those without this exposure. Neonatal LPS exposure with later exposure to rotenone decreased retrogradely labeled nigrostriatal dopaminergic projecting neurons. The current study suggests that perinatal brain inflammation may enhance adult susceptibility to the development of neurodegenerative disorders triggered later on by environmental toxins at an ordinarily non-toxic or sub-toxic dose. Our model may be useful for studying mechanisms involved in the pathogenesis of nonfamilial Parkinson's disease and the development of potential therapeutic treatments.

Neonatal exposure to lipopolysaccharide enhances methamphetamine-induced reinstated behavioral sensitization in adult rats.

Our previous studies have shown that neonatal exposure to lipopolysaccharide (LPS) resulted in long-lasting dopaminergic injury and enhanced methamphetamine (METH)-induced increase of locomotion in the adult male rat. To further investigate the effect of neonatal LPS exposure-induced dopaminergic injury, we used our neonatal rat model of LPS exposure (1mg/kg, intracerebral injection in postnatal day 5, P5, rats) to examine the METH sensitization as an indicator of drug addiction in the adult rats. On P70, animals began a treatment schedule of 5 daily subcutaneous (s.c.) administration of METH (0.5mg/kg) or saline (P70-P74) to induce behavioral sensitization. Ninety-six hours after the 5th treatment with METH or saline (P78), animals received a single dose of 0.5mg/kg METH (s.c.) or saline. Neonatal LPS exposure enhanced the level of development of behavioral sensitization including distance traveled, rearing events and stereotypy to METH administration in both male and female rats. Neonatal LPS exposure also enhanced the reinstated behavioral sensitization in both male and female rats after the administration had ceased for 96h. However, neonatal LPS exposure induced alteration in the reinstated behaviors sensitization of distance traveled and rearing events to METH administration appears to be greater in male than in female rats. These results indicate that neonatal brain LPS exposure produces a persistent lesion in the dopaminergic system, as indicated by enhanced METH-induced locomotor and stereotyped behavioral sensitization in later life. These findings show that early-life brain inflammation may enhance susceptibility to the development of drug addiction in later life.

Whole body hypothermia broadens the therapeutic window of intranasally administered IGF-1 in a neonatal rat model of cerebral hypoxia-ischemia.

To investigate whether whole body hypothermia after neonatal cerebral hypoxia-ischemia (HI) could broaden the therapeutic window of intranasal treatment of IGF-1 (iN-IGF-1), postnatal day 7 rat pups were subjected to right common carotid artery ligation, followed by 8% oxygen inhalation for 2h. After HI, one group of pups were returned to their dams and kept at room temperature (24.5±0.2°C). A second group of pups were subjected to whole body hypothermia in a cool environment (21.5±0.3°C) for 2 or 4h before being returned to their dams. Two doses of 50 µg recombinant human IGF-1 were administered intranasally at a 1h interval starting at 0, 2 or 4h after hypothermia. Hypothermia decreased the rectal temperature of pups by 4.5°C as compared to those kept at room temperature. While hypothermia or iN-IGF-1 administered 2h after HI alone did not provide neuroprotection, the combined treatment of hypothermia with iN-IGF-1 significantly protected the neonatal rat brain from HI injury. Hypothermia treatment extended the therapeutic window of IGF-1 to 6h after HI. The extended IGF-1 therapeutic window by hypothermia was associated with decreases in infiltration of polymorphonuclear leukocytes and activation of microglia/macrophages and with attenuation of NF-κB activation in the ipsilateral hemisphere following HI.

Interleukin-1 receptor antagonist ameliorates neonatal lipopolysaccharide-induced long-lasting hyperalgesia in the adult rats.

An increasing amount of data show that central inflammation contributes to many debilitating diseases and produces spontaneous pain and hyperalgesia (an increased sensitivity to painful stimuli), and these processes may be associated with the production of proinflammatory cytokines by activated microglia. In the present study, we demonstrate that neonatal intracerebral injection of lipopolysaccharide (LPS) (1mg/kg) in postnatal day 5 (P5) rats produced hyperalgesia that lasted into adulthood as indicated by decreased latency in the tail-flick test. Neonatal LPS administration resulted in a long-lasting increase in the number of activated microglial in the P70 rat brain. The effects of interleukin-1beta (IL-1ß) and IL-1 receptor antagonists on hyperalgesia were determined to examine the possible role of inflammatory cytokines in LPS-induced hyperalgesia. Our data show that neonatal intracerebral injection of IL-1ß (1 µg/kg) produced a hyperalgesic tendency similar to that induced by LPS. Neonatal administration of an IL-1 receptor antagonist (0.1mg/kg) significantly attenuated long-lasting hyperalgesia induced by LPS and reduced the number of activated microglia in the adult rat brain. These data reveal that neonatal intracerebral LPS exposure results in long-lasting hyperalgesia and an elevated number of activated microglia in later life. This effect is similar to that induced by IL-1ß and can be prevented by an IL-1 receptor antagonist. The present study suggests that an IL-1 receptor antagonist effectively attenuates or blocks long-lasting hyperalgesia and microglia activation produced by LPS exposure in the neonatal period of rats.

Dopaminergic neuronal injury in the adult rat brain following neonatal exposure to lipopolysaccharide and the silent neurotoxicity.

Our previous studies have shown that neonatal exposure to lipopolysaccharide (LPS) resulted in motor dysfunction and dopaminergic neuronal injury in the juvenile rat brain. To further examine whether neonatal LPS exposure has persisting effects in adult rats, motor behaviors were examined from postnatal day 7 (P7) to P70 and brain injury was determined in P70 rats following an intracerebral injection of LPS (1 mg/kg) in P5 Sprague-Dawley male rats. Although neonatal LPS exposure resulted in hyperactivity in locomotion and stereotyped tasks, and other disturbances of motor behaviors, the impaired motor functions were spontaneously recovered by P70. On the other hand, neonatal LPS-induced injury to the dopaminergic system such as the loss of dendrites and reduced tyrosine hydroxylase immunoreactivity in the substantia nigra persisted in P70 rats. Neonatal LPS exposure also resulted in sustained inflammatory responses in the P70 rat brain, as indicated by an increased number of activated microglia and elevation of interleukin-1ß and interleukin-6 content in the rat brain. In addition, when challenged with methamphetamine (METH, 0.5 mg/kg) subcutaneously, rats with neonatal LPS exposure had significantly increased responses in METH-induced locomotion and stereotypy behaviors as compared to those without LPS exposure. These results indicate that although neonatal LPS-induced neurobehavioral impairment is spontaneously recoverable, the LPS exposure-induced persistent injury to the dopaminergic system and the chronic inflammation may represent the existence of silent neurotoxicity. Our data further suggest that the compromised dendritic mitochondrial function might contribute, at least partially, to the silent neurotoxicity.

IGF-1 can either protect against or increase LPS-induced damage in the developing rat brain.

Periventricular leukomalacia (PVL) is a major form of brain damage in premature infants. This study was to test whether IGF-1 can prevent PVL-like brain damage induced by lipopolysaccharide (LPS) in the neonatal rat. Intraventricular delivery of LPS resulted in an acute brain inflammatory response, i.e., rapid recruitment of polymorphonuclear leukocytes (PMNs), activation of microglia and astrocytes, and induction of IL-1beta (IL1beta) expression. Brain inflammation was associated with the loss of O4+ preoligodendrocytes (preOLs), a decrease of myelin basic protein (MBP) in the white matter and an increase of pyknotic cells in the cortex. IGF-1 at a low dose significantly prevented LPS-induced deleterious effects without alteration of IL-1beta expression and microglia/astrocytes activation. On the other hand, the low dose of IGF-1 enhanced LPS-induced PMNs recruitment and blood-brain barrier (BBB) permeability, and caused intracerebral hemorrhage. At higher doses, co-application of IGF-1 with LPS resulted in a high mortality rate. Brains from the surviving rats showed massive PMN infiltration and intracerebral hemorrhage. However, these adverse effects were not found in rats treated with IGF-1 alone. This study provides the alarming evidence that in an acute inflammatory condition, IGF-1 may have severe, harmful effects on the developing brain.

Hypoxic preconditioning provides neuroprotection and increases vascular endothelial growth factor A, preserves the phosphorylation of Akt-Ser-473 and diminishes the increase in caspase-3 activity in neonatal rat hypoxic-ischemic model.

Vascular endothelial growth factor A (VEGF) likely plays a role in the hypoxic preconditioning (PC) induced tolerance to subsequent hypoxic-ischemic (HI) injury to the brain. However, limited data is available concerning VEGF in the developing brain after HI following PC. Neuroprotection by VEGF involves activation of Akt which inhibits apoptotic processes that contribute significantly to the brain injury in neonatal HI. We evaluated whether PC provides neuroprotection and affects VEGF, Akt and caspase-3 following HI in the developing rat brain. Newborn rats (6 days) were subjected to normoxia (21% O(2)) or PC (8% O(2)) for 3h followed by 24h of reoxygenation. The rats then had the right carotid artery permanently ligated followed by 140 min of hypoxia (8% O(2)) (HI or PC+HI). Brains from rats at the corresponding age without any exposure to PC or HI were examined for comparison (Sham). PC significantly reduced brain damage as measured by weight loss of the right hemisphere at 22 days after HI and by gross and microscopic morphology. PC amplified and prolonged the induction of mRNA of VEGF splice variants measured by real-time RT-PCR and enhanced the increase in VEGF protein measured by ELISA in brain following HI. PC preserved the phosphorylation of Akt-Ser-473 and diminished the increase in caspase-3 activity in brain following HI. We conclude that PC provides neuroprotection and augments and preserves the increase in VEGF following HI in the newborn rat brain which may play an important role in neuroprotection.

Interleukin-1beta-induced brain injury in the neonatal rat can be ameliorated by alpha-phenyl-n-tert-butyl-nitrone.

To examine the possible role of inflammatory cytokines in mediating perinatal brain injury, we investigated effects of intracerebral injection of interleukin-1beta (IL-1beta) on brain injury in the neonatal rat and the mechanisms involved. Intracerebral administration of IL-1beta (1 microg/kg) resulted in acute brain injury, as indicated by enlargement of ventricles bilaterally, apoptotic death of oligodendrocytes (OLs) and loss of OL immunoreactivity in the neonatal rat brain. IL-1beta also induced axonal and neuronal injury in the cerebral cortex as indicated by elevated expression of beta-amyloid precursor protein, short beaded axons and dendrites, and loss of tyrosine hydroxylase-positive neurons in the substantia nigra and the ventral tegmental areas. Administration of alpha-phenyl-n-tert-butyl-nitrone (PBN, 100 mg/kg i.p.) immediately after the IL-1beta injection protected the brain from IL-1beta-induced injury. Protection of PBN was linked with the attenuated oxidative stress induced by IL-1beta, as indicated by decreased elevation of 8-isoprostane content and by the reduced number of 4-hydroxynonenal or malondialdehyde or nitrotyrosine-positive cells following IL-1beta exposure. PBN also attenuated IL-1beta-stimulated inflammatory responses as indicated by the reduced activation of microglia. The finding that IL-1beta induced perinatal brain injury was very similar to that induced by lipopolysaccharide (LPS), as we previously reported and that PBN was capable to attenuate the injury induced by either LPS or IL-1beta suggests that IL-1beta may play a critical role in mediating brain injury associated with perinatal infection/inflammation.

Intranasal administration of IGF-1 attenuates hypoxic-ischemic brain injury in neonatal rats.

To determine whether intranasal administration (iN) of recombinant human insulin-like growth factor-1 (rhIGF-1) provides neuroprotection to the neonatal rat brain following cerebral hypoxia-ischemia (HI), two doses of rhIGF-1 (50 microg at a 1 h interval) were infused into the right naris of postnatal day 7 (P7) rat pups with or without a prior HI insult (right common carotid artery ligation, followed by an exposure to 8% oxygen for 2 h). Our result showed that rhIGF-1 administered via iN was successfully delivered into the brain 30 min after the second dose. In the following studies rhIGF-1 was administered to P7 rat pups at 0, 1 or 2 h after HI at the dose described above. Pups in the control group received cerebral HI and vehicle treatment. Pups that underwent sham operation and vehicle treatment served as the sham group. Brain pathological changes were evaluated 2 and 15 days after HI. Our results showed that rhIGF-1 treatment up to 1 h after cerebral HI effectively reduced brain injury as compared to that in the vehicle-treated rats. Moreover, rhIGF-1 treatment improved neurobehavioral performance (tested on P5-P21) in juvenile rats subjected to HI. Our results further showed that rhIGF-1 inhibited apoptotic cell death, possibly through activating the Akt signal transduction pathway. rhIGF-1 enhanced proliferation of neuronal and oligodendroglial progenitors after cerebral HI as well. These data suggest that iN administration of IGF-1 has the potential to be used for clinical treatment.

alpha-Phenyl-n-tert-butyl-nitrone attenuates lipopolysaccharide-induced brain injury and improves neurological reflexes and early sensorimotor behavioral performance in juvenile rats.

Our previous study showed that treatment with alpha-phenyl-n-tert-butyl-nitrone (PBN) after exposure to lipopolysaccharide (LPS) reduced LPS-induced white matter injury in the neonatal rat brain. The object of the current study was to further examine whether PBN has long-lasting protective effects and ameliorates LPS-induced neurological dysfunction. Intracerebral (i.c.) injection of LPS (1 mg/kg) was performed in postnatal day (P) 5 Sprague Dawley rat pups and PBN (100 mg/kg) or saline was administered intraperitoneally 5 min after LPS injection. The control rats were injected (i.c.) with sterile saline. Neurobehavioral tests were carried out from P3 to P21, and brain injury was examined after these tests. LPS exposure resulted in severe brain damage, including enlargement of ventricles bilaterally, loss of mature oligodendrocytes, impaired myelination as indicated by the decrease in myelin basic protein immunostaining, and alterations in dendritic processes in the cortical gray matter of the parietal cortex. Electron microscopic examination showed that LPS exposure caused impaired myelination as indicated by the disintegrated myelin sheaths in the juvenile rat brain. LPS administration also significantly affected neurobehavioral functions such as performance in righting reflex, wire hanging maneuver, cliff avoidance, negative geotaxis, vibrissa-elicited forelimb-placing test, beam walking, and gait test. Treatment with PBN, a free radical scavenger and antioxidant, provided protection against LPS-induced brain injury and associated neurological dysfunction in juvenile rats, suggesting that antioxidation might be an effective approach for therapeutic treatment of neonatal brain injury induced by infection/inflammation.

Neuroprotective effects of vascular endothelial growth factor following hypoxic ischemic brain injury in neonatal rats.

Vascular Endothelial Growth Factor (VEGF) protects the brain against ischemic injury in adult animals. We evaluated whether VEGF has neuroprotective effects against hypoxic-ischemic (HI) brain injury in newborn rats. Seven-day-old rat pups had the right carotid artery permanently ligated followed by 140 min of hypoxia (8% oxygen). VEGF (5, 10, 20, or 40 ng) or vehicle was administered intracerebroventricularly 5 min after reoxygenation following HI. Brain damage was evaluated by weight loss of the right hemisphere at 22 d after HI and by gross and microscopic morphology. Body weight, rectal temperature, and mortality were not significantly different in the VEGF and vehicle treated groups. VEGF treatment increased brain VEGF levels at 15 min after injection. VEGF (10 and 20 ng) significantly reduced brain weight loss (p < 0.05) and gross brain injury (p < 0.05); however, treatment with 5 or 40 ng did not. VEGF (10 ng) also decreased brain damage assessed by histologic scoring. VEGF increased phosphorylation of protein kinase B (Akt) and extracellular-signal regulated kinase 1/2 (ERK1/2) in the cortex (p < 0.05). These results suggest that VEGF has neuroprotective effects in the neonatal rat HI model that may be related to activation of the Akt/ERK signaling pathway.

Neuroprotective effects of sodium orthovanadate after hypoxic-ischemic brain injury in neonatal rats.

Sodium orthovanadate (SOV), a competitive inhibitor of protein tyrosine phosphatases, is neuroprotective in adult animals following an ischemic event. The present study evaluated whether SOV might be protective in a rat pup hypoxic-ischemic (HI) model. Seven-day-old rat pups had the right carotid artery permanently ligated followed by 140 min of hypoxia (8% oxygen). SOV 1.15, 2.3, 4.6, 9.2 or 18.4 mg/kg and vehicle were administered by i.p. injection at 5 min after reoxygenation. Brain damage was evaluated by weight loss of the right hemisphere at 22 days after hypoxia and by gross and microscopic morphology. SOV lowered blood glucose at doses of 1.15, 2.3 and 4.6 mg/kg and induced toxic effects at 9.2mg/kg. The doses of 2.3 and 4.6 mg/kg of SOV significantly reduced brain weight loss (p<0.05), but treatment with 1.15 or 9.2mg/kg did not. SOV 4.6 mg/kg also improved the histopathologic score and diminished the HI induced reduction of Akt and ERK-1/2 phosphorylation in the cortex (p<0.05) and increased the density of BrdU-positive cells in the subventricular zone (p<0.01). In conclusion, SOV has neuroprotective effects in the neonatal rat HI model partially mediated by activating Akt and ERK-1/2 pathways.

Alpha-phenyl-n-tert-butyl-nitrone ameliorates hippocampal injury and improves learning and memory in juvenile rats following neonatal exposure to lipopolysaccharide.

Neonatal exposure to infectious agents may result in long-term neurological disability, and is particularly associated with the subsequent development of motor and cognitive disturbances. Our previous studies have shown that treatment with alpha-phenyl-n-tert-butyl-nitrone (PBN) following exposure to lipopolysaccharide (LPS) reduces LPS-induced brain injury in the neonatal rat. To examine whether PBN has long-lasting protective effects and ameliorates LPS-induced motor and cognitive dysfunction, PBN (100 mg/kg) was administered intraperitoneally 5 min after an LPS (1 mg/kg) intracerebral injection in postnatal day 5 (P5) Sprague-Dawley rat pups. Neurobehavioral tests were carried out from P3 to P21, and brain injury was examined at 24 h and 16 days after LPS injection. Neonatal LPS exposure resulted in hyperactivity from P13 to P17 in the open field task as compared with the control rat. Neurobehavioral deficits that were still observable at P21 included dysfunction in the beam-walking and pole tests, learning and memory deficits in the passive avoidance task, and less anxiety-like response in the elevated plus-maze task. These behavioral findings were matched by LPS-induced axonal injury in the CA1 region of the middle dorsal hippocampus (HP), reduction in the size of the HP and the number of neurons in the CA1 region of the middle dorsal HP, and loss of tyrosine hydroxylase immunoreactivity in neurons in the substantia nigra and ventral tegmental areas. Treatment with PBN provided long-lasting protection against the LPS-induced axonal injury and neuronal loss, and improved the associated neurological dysfunctions in juvenile rats.

alpha-Phenyl-n-tert-butyl-nitrone reduces lipopolysaccharide-induced white matter injury in the neonatal rat brain.

Lipopolysaccharide (LPS)-induced white matter injury in the neonatal rat brain is at least partially associated with oxidative stress. alpha-Phenyl-n-tert-butyl-nitrone (PBN) (100 mg/kg) significantly attenuated LPS (1 mg/kg)-induced brain injury, as indicated by the reduction in bilateral ventricular enlargement, apoptotic cell death of oligodendrocytes (OLs), and the loss of OL immunoreactivity in the neonatal rat brain. Protection of PBN was linked with the attenuated oxidative stress induced by LPS, as indicated by the decreased elevation of 8-isoprostane content and by the reduced number of 4-hydroxynonenal or malondialdehyde positive OLs following LPS exposure. Interestingly, while LPS exposure elevated, rather than depleted, levels of the reduced glutathione (GSH) and the GSH/GSSG (oxidized form) ratio, LPS exposure significantly suppressed glutathione peroxidase activity in the rat brain. PBN attenuated LPS-induced alterations in glutathione homeostasis in the rat brain. Additionally, the inflammatory responses were also reduced in the PBN-treated brain, as indicated by the decreased number of activated microglia following LPS exposure and by the consequently decreased elevation of interleukin1-beta and tumor necrosis factor-alpha contents in the rat brain. The overall results suggest that antioxidant PBN, more than a straightforward free radical scavenger, may also involve anti-inflammatory and anti-apoptotic properties in protection of the neonatal rat brain from LPS-induced injury.

IGF-1 protects oligodendrocyte progenitors against TNFalpha-induced damage by activation of PI3K/Akt and interruption of the mitochondrial apoptotic pathway.

Proinflammatory cytokine-mediated injury to oligodendrocyte progenitor cells (OPCs) has been proposed as a cause of periventricular leukomalacia (PVL), the most common brain injury found in preterm infants. Preventing death of OPCs is a potential strategy to prevent or treat PVL. In the current study, we utilized an in vitro cell culture system to investigate the effect of insulin-like growth factor-1 (IGF-1) on tumor necrosis factor-alpha (TNFalpha)-induced OPC injury and the possible mechanisms involved. OPCs were isolated from neonatal rat optic nerves and cultured in chemically defined medium (CDM) supplemented with platelet-derived growth factor and basic fibroblast growth factor. Exposure to TNFalpha resulted in death of OPCs. IGF-1 protected OPCs from TNFalpha cytotoxicity in a dose-dependent manner as measured by the XTT and TUNEL assays. IGF-1 activates both the PI3K/Akt and the extracellular signal-regulated kinase (ERK) pathway. However, IGF-1-enhanced cell survival signals were mediated by the PI3K/Akt, but not by the ERK pathway, as evidenced by the observation that IGF-1-enhanced cell survival was partially abrogated by Akti, the Akt inhibitor, or wortmannin, the PI3K inhibitor, but not by PD98,059, the MAPK kinase/ERK kinase inhibitor. The downstream events of IGF-1-triggered survival signals included phosphorylation of BAD, blockade of TNFalpha-induced translocation of Bax from the cytosol to the mitochondrial membrane, and suppression of caspase-9 and caspase-3 activation. These observations indicate that the protection of OPCs by IGF-1 is mediated, at least partially, by interruption of the mitochondrial apoptotic pathway via activation of PI3K/Akt.