Philip G. Rhodes

Cytokine Induction in Fetal Rat Brains and Brain Injury in Neonatal Rats after Maternal Lipopolysaccharide Administration

Induction of proinflammatory cytokines has been proposed to be a link between prenatal maternal intrauterine infection and neonatal brain damage. It is known that the endotoxin, lipopolysaccharide (LPS), released during bacterial infection crosses the placenta. Cytokine induction in the fetal rat brain after maternal administration of LPS was determined by reverse transcriptase-polymerase chain reaction method. LPS suspension in pyrogen-free saline was administered (i.p.) to pregnant rats at 18 d of gestation. The control group was treated with pyrogen-free saline. Expression of the proinflammatory cytokines, tumor necrosis factor-α and IL 1-β mRNA, in the fetal rat brain was increased in a dose-dependent manner at 1 h after LPS administration. The great increase in expression of IL-1β mRNA was only observed at 1 h after injection of LPS (4 mg/kg), whereas the increased expression of tumor necrosis factor-α was still detectable from 4 to 24 h after LPS administration. Brain injuries were examined by immunohistochemistry in 8-d-old rat pups born to the dams that were consecutively treated with LPS (500 μg/kg) or pyrogen-free saline on gestation d 18 and 19. No apparent necrotic tissue damage was found in either the LPS group or the control group. Myelin basic protein staining, as a marker of myelin, was clearly observed in the internal capsule and the fimbria hippocampus in the rat brain from the control group. Myelin basic protein staining was much less and weaker in the brains of the LPS-treated group. Glial fibrillary acidic protein–positive astrocytes were observed in both the control and the LPS-treated groups. The LPS-treated group appeared to have more glial fibrillary acidic protein–positive astrocytes in the hippocampal and the cortex areas of the brain than the control group. Immunoblotting data showed that glial fibrillary acidic protein content in the cortex or the hippocampus of the LPS-treated rat brain was higher than in the control group. OX-42–positive staining (a marker of the type 3 complement receptors) of microglial cells was greatly reduced in the 8-d-old rat brain after maternal LPS administration. However, histochemistry with tomato lectin showed that staining of both amoeboid and ramified microglial cells in the LPS-treated rat brain was similar to that in the control group. The overall results indicate that maternal LPS administration induces an increased expression of IL-1β and tumor necrosis factor-α mRNA in the fetal brain. Maternal LPS administration also increases glial fibrillary acidic protein–positive astrocytes, decreases myelin basic protein and alters immunoreactivity of microglia in the brain of offspring. Although results from the current study do not provide direct evidence linking LPS-induced cytokines with the abnormalities in the neonatal rat brain, our animal model may be appropriate for exploring the mechanisms involved in the effects of maternal infection on glial cells in the brains of offspring.

Disturbance of oligodendrocyte development, hypomyelination and white matter injury in the neonatal rat brain after intracerebral injection of lipopolysaccharide.

Increasing data provide support for the hypothesis that brain inflammation plays an important role in injury to developing white matter. In the present study, inflammatory responses in the neonatal rat brain were investigated following lipopolysaccharide (LPS) administration at postnatal day 5. LPS-induced brain injury was examined in brain sections 24 h, 3 and 9 days after LPS injection. White matter rarefaction was observed in 50% of the rat brains (three out of six) 24 h after LPS injection. Lateral ventricle enlargement was found in 100% (four out of four) and 89% (eight out of nine) of rat brains 3 and 9 days after LPS administration, respectively. White matter necrosis was found in three out of nine brains injected with LPS on P14. None of these injuries was observed in any control rat brains. No histological changes in gray matter were noted in the LPS-injected rat brain. Proinflammatory cytokines, tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) in the rat brain were greatly induced after LPS administration. Activated astrocytes and microglia/macrophages were found in the affected rat brains. Double-labeling showed that IL-1beta and iNOS expressing cells were microglia/macrophages. Injury to or delayed development of immature oligodendrocytes (OLs) was evident by decreased immunostaining for both O4 and O1 antibodies, markers for developing immature OLs, in the LPS-injected as compared to the control rat brain. LPS also resulted in hypomyelination, as indicated by reduced myelin basic protein (MBP) immunostaining in the P8 rat brain. Co-administration of IL-1 receptor antagonist (IL-1Ra) with LPS reduced brain injury by improving myelination and subsequent reduction of lateral ventricle enlargement. These results indicate that developing OLs may be the target cells for LPS-induced brain injury and inflammatory cytokines are possible mediators of LPS-induced brain injury.

Differential roles of tumor necrosis factor-α and interleukin-1 β in lipopolysaccharide-induced brain injury in the neonatal rat

Increasing data provide support for the hypothesis that inflammatory cytokines mediate inflammation-induced injury to developing white matter. In the present study, roles of tumor necrosis factor-alpha (TNFalpha) and interleukin-1 beta (IL-1beta) in mediating lipopolysaccharide (LPS)-induced brain injury were investigated by co-administration of LPS with IL-1 receptor antagonist (IL-1ra) or TNFalpha antibody in the 5-day-old rat brain. Intracerebral injection of LPS and other agents was performed in a stereotaxic apparatus at the location of 1.0 mm posterior and 1.0 mm lateral to the bregma, and 2.0 mm deep to the skull surface at the left hemisphere. Brain injury was examined in brain sections 3 and 11 days after LPS injection. LPS-induced inflammatory responses were evidenced by great increases in TNFalpha and IL-1beta concentrations in the neonatal rat brain 6 h after LPS injection. White matter rarefaction was observed in 71% (five out of seven) of the rat brains 3 days after LPS injection and bilateral ventricle dilation was found in 71% (five out of seven) of the P8 rat brains and in 100% of the P16 rat brains (four out of four). These alterations were not found in the control rat brains. No apparent histological changes in gray matter were observed in the LPS-injected rat brains. LPS injection also resulted in injuries to oligodendrocytes (OLs) and hypomyelination, as indicated by reduced immunostaining for O4 and myelin basic protein (MBP). Increased astrogliosis, as indicated by increased glial fibrillary acidic protein (GFAP) immunostaining, was also observed in the LPS-injected, but not the control rat brain. Co-administration of LPS with IL-1ra, but not with TNFalpha antibody, significantly attenuated LPS-induced white matter injury, as indicated by decreases in ventricle dilation, white matter rarefaction, GFAP positive staining and by improved O4 and MBP immunostaining. Co-administration of LPS with IL-1ra significantly reduced LPS-induced elevation of caspase-3 activity in the rat brain. While TNFalpha antibody had no effect on LPS-induced elevation of caspase-3 activity, co-administration of LPS with TNFalpha antibody partially, but significantly, decreased LPS-stimulated increase in IL-1beta in the neonatal rat brain. These data suggest that IL-1beta may play an important role in mediating LPS-induced brain injury and TNFalpha may have complicated, probably dual, effects in LPS-induced brain injury.

Long-Term Feeding of Formulas High in Linolenic Acid and Marine Oil to Very Low Birth Weight Infants: Phospholipid Fatty Acids

ABSTRACT: Red blood cell (RBC) phospholipids of infants fed human milk compared with formula have more arachidonic acid (AA) and docosahexanoic acid (DHA). The addition of low levels of marine oil to infant formula with 0.6 to 2.0% α-linolenic acid (LLA, 18:3n-3) prevented declines in DHA in formula-fed infants; however, the feeding trials were short (4 to 6 wk), LLA concentrations were low compared with current formulas (3.0 to 5.0% LLA), and the formulas were unstable. Trials with stable formulas were necessary to determine if dietary DHA could maintain phospholipid DHA after discharge from the hospital and, in fact, if it was necessary with higher intakes of LLA. The results of acute (4 wk) and extended (to 79 wk postconception) feeding of such formulas on RBC and plasma phospholipid AA and DHA are reported here. Control formulas were identical to commercially available formulas. Experimental formulas differed only in the addition of small amounts of marine oil. DHA in RBC and plasma phosphatidylethanolamine (PE) declined during four weeks of feeding but not if marine oil provided DHA (0.2% or 0.4%) and plasma phospholipid AA (g/100 g) decreased with time and marine oil feeding. Extended feeding with marine oil accounted for half the DHA in RBC and plasma phosphatidylethanolamine at equilibrium; however, RBC (g/100g) and plasma AA (g/100 g; mg/L plasma) decreased progressively until late infancy and were depressed further by marine oil. We conclude that 1) AA and DHA decline in RBC and plasma phospholipids of preterm infants when only their n-6 and n-3 fatty acid precursors are consumed; and 2) marine oil can maintain cord concentrations of RBC phosphatidylethanolamine DHA but further reduces AA.

Effect of fish oil supplementation on the n-3 fatty acid content of red blood cell membranes in preterm infants.

ABSTRACT.: Very low birth weight infants demonstrate significant reductions in red blood cell membrane docosahexaenoic acid (DHA, 22:6n-3) following delivery unless fed human milk. The purpose of the present study was to determine if a dietary source of DHA (MaxEPA, R. P. Scherer Corporation, Troy, MI) could prevent the decline in red blood cell phospholipid DHA in very low birth weight infants whose enteral feeding consisted of a preterm formula without DHA. Longitudinal data were obtained on membrane phospholipid DHA in both un supplemented and MaxEPA-supplemented infants by a combination of thin layer and gas chromatography. These infants (n = 39) ranged in age from 10 to 53 days at enrollment (0 time). At enrollment, phospholipid DHA and arachidonic acid (20:4n-6) were inversely correlated with age in days. During the study, mean red blood cell phospholipid DHA declined without supplementary DHA as determined by biweekly measurement, but infants supplemented with MaxEPA maintained the same weight percent of phospholipid (phosphatidylethanolanine, phosphatidylcholine, and phospharidylserine) DHA as at enrollment. The pattern of red blood cell phospholipid fatty acids in supplemented infants was similar to that reported for preterm infants fed human milk.

Minocycline reduces lipopolysaccharide-induced neurological dysfunction and brain injury in the neonatal rat.

Preferential brain white matter injury and hypomyelination induced by intracerebral administration of the endotoxin lipopolysaccharide (LPS) in the neonatal rat brain has been characterized as associated with the activation of microglia. To examine whether inhibition of microglial activation might provide protection against LPS‐induced brain injury and behavioral deficits, minocycline (45 mg/kg) was administered intraperitoneally 12 hr before and immediately after an LPS (1 mg/kg) intracerebral injection in postnatal day 5 (P5) Sprague‐Dawley rats and then every 24 hr for 3 days. Brain injury and myelination were examined on postnatal day 21 and the tests for neurobehavioral toxicity were carried out from P3 to P21. LPS administration resulted in severe white matter injury, enlarged ventricles, deficits in the hippocampus, loss of oligodendrocytes and tyrosine hydroxylase neurons, damage to axons and dendrites, and impaired myelination as indicated by the decrease in myelin basic protein immunostaining in the P21 rat brain. LPS administration also significantly affected physical development (body weight) and neurobehavioral performance, such as righting reflex, wire hanging maneuver, cliff avoidance, locomotor activity, gait analysis, and responses in the elevated plus‐maze and passive avoidance task. Treatment with minocycline significantly attenuated the LPS‐induced brain injury and improved neurobehavioral performance. The protective effect of minocycline was associated with its ability to attenuate LPS‐induced microglial activation. These results suggest that inhibition of microglial activation by minocycline may have long‐term protective effects in the neonatal brain on infection‐induced brain injury and associated neurologic dysfunction in the rat.

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.

Minocycline attenuates hypoxia–ischemia-induced neurological dysfunction and brain injury in the juvenile rat

To investigate whether minocycline provides long‐lasting protection against neonatal hypoxia–ischemia‐induced brain injury and neurobehavioral deficits, minocycline was administered intraperitoneally in postnatal day 4 Sprague–Dawley rats subjected to bilateral carotid artery occlusion followed by exposure to hypoxia (8% oxygen for 15 min). Brain injury and myelination were examined on postnatal day 21 (P21) and tests for neurobehavioral toxicity were performed from P3 to P21. Hypoxic–ischemic insults resulted in severe white matter injury, enlarged ventricles, deficits in the hippocampus, reduction in numbers of mature oligodendrocytes and tyrosine hydroxylase‐positive neurons, damage to axons and dendrites, and impaired myelination, as indicated by the decrease in myelin basic protein immunostaining in the P21 rat brain. Hypoxic–ischemic insult also significantly affected physical development (body weight gain and eye opening) and neurobehavioral performance, including sensorimotor and locomotor function, anxiety and cognitive ability in the P21 rat. Treatments with minocycline significantly attenuated the hypoxia–ischemia‐induced brain injury and improved neurobehavioral performance. The protection of minocycline was associated with its ability to reduce microglial activation. The present results show that minocycline has long‐lasting protective effects in the neonatal rat brain in terms of both hypoxia–ischemia‐induced brain injury and the associated neurological dysfunction.

IGF-1 protects oligodendrocyte progenitors against TNFα-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‐α (TNFα)‐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 TNFα resulted in death of OPCs. IGF‐1 protected OPCs from TNFα 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 PD98059, the MAPK kinase/ERK kinase inhibitor. The downstream events of IGF‐1‐triggered survival signals included phosphorylation of BAD, blockade of TNFα‐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.

Hypoxia-ischemia induced neurological dysfunction and brain injury in the neonatal rat

Bilateral carotid artery occlusion (BCAO) followed by exposure to a hypoxic condition (8% oxygen for 10 or 15 min) was performed in postnatal day 4 SD rats. Brain injury and myelination changes were examined on postnatal day 21 (P21) and tests for neurobehavioral toxicity were performed from P3 to P21. BCAO followed by 10 or 15 min hypoxic insult resulted in mild and severe, respectively, brain injury, reduction in mature oligodendrocytes and tyrosine hydroxylase positive neurons and impaired myelination as indicated by decreased myelin basic protein immunostaining in the P21 rat brain. Hypoxia-ischemia also affected physical development (body weight gain and eye opening) and neurobehavioral performance, such as righting reflex, wire hanging maneuver, cliff avoidance, locomotor activity, gait analysis, responses in the elevated plus-maze and passive avoidance. BCAO followed by 15 min of hypoxia caused more severely impaired neurobehavioral performance as compared with BCAO followed by 10 min of hypoxia in the rat. The overall results demonstrate that hypoxia-ischemia-induced brain injury not only persists, but also is linked with neurobehavioral deficits in juvenile rats. The present data also indicate that the degree of brain injury and the deficits of neurobehavioral performance in the rat are dependent on the hypoxic-ischemic condition, i.e., the exposure time to hypoxia.