Shuying Lin

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.

Minocycline alleviates hypoxic–ischemic injury to developing oligodendrocytes in the neonatal rat brain

The role of minocycline in preventing white matter injury, in particular the injury to developing oligodendrocytes was examined in a neonatal rat model of hypoxia-ischemia. Hypoxia-ischemia was achieved through bilateral carotid artery occlusion followed by exposure to hypoxia (8% oxygen) for 15 min in postnatal day 4 Sprague-Dawley rats. A sham operation was performed in control rats. Minocycline (45 mg/kg) or normal phosphate-buffered saline was administered intraperitoneally 12 h before and immediately after bilateral carotid artery occlusion+hypoxia and then every 24 h for 3 days. Nissl staining revealed pyknotic cells in the white matter area of the rat brain 1 and 5 days after hypoxia-ischemia. Hypoxia-ischemia insult also resulted in apoptotic oligodendrocyte cell death, loss of O4+ and O1+ oligodendrocyte immunoreactivity, and hypomyelination as indicated by decreased myelin basic protein immunostaining and by loss of mature oligodendrocytes in the rat brain. Minocycline significantly attenuated hypoxia-ischemia-induced brain injury. The protective effect of minocycline was associated with suppression of hypoxia-ischemia-induced microglial activation as indicated by the decreased number of activated microglia, which were also interleukin-1beta and inducible nitric oxide synthase expressing cells. The protective effect of minocycline was also linked with reduction in hypoxia-ischemia-induced oxidative and nitrosative stress as indicated by 4-hydroxynonenal and nitrotyrosine positive oligodendrocytes, respectively. The reduction in hypoxia-ischemia-induced oxidative stress was also evidenced by the decreases in the content of 8-isoprostane in the minocycline-treated hypoxia-ischemia rat brain as compared with that in the vehicle-treated hypoxia-ischemia rat brain. The overall results suggest that reduction in microglial activation may protect developing oligodendrocytes in the neonatal brain from hypoxia-ischemia injury.

Minocycline attenuates lipopolysaccharide-induced white matter injury in the neonatal rat brain

Our previous studies have shown that intracerebral administration of endotoxin, lipopolysaccharide (LPS), induces selective white matter injury and hypomyelination in the neonatal rat brain and that the LPS-induced brain injury is associated with activation of microglia. To test the hypothesis that inhibition of microglial activation may protect against LPS-induced white matter injury, we examined roles of minocycline, a putative suppressor of microglial activation, on LPS-induced brain injury in the neonatal rat. A stereotactic intracerebral injection of LPS (1 mg/kg) was performed in postnatal day 5 Sprague-Dawley rats and control rats were injected with sterile saline. Minocycline (45 mg/kg) was administered intraperitoneally 12 h before and immediately after LPS injection and then every 24 h for 3 days. Inflammatory responses, activation of microglia and brain injury were examined 1 and 3 days after LPS injection. LPS injection resulted in brain injury in selective brain areas, including bilateral ventricular enlargement, cell death at the sub- and periventricular areas, loss of O4+ and O1+ oligodendrocyte (OL) immunoreactivity and hypomyelination, as indicated by decreased myelin basic protein immunostaining, in the neonatal rat brain. Minocycline administration significantly attenuated LPS-induced brain injury in these rat brains. The protective effect of minocycline was associated with suppressed microglial activation as indicated by the decreased number of activated microglial cells following LPS stimulation and with consequently decreased elevation of interleukin 1beta and tumor necrosis factor-alpha concentrations induced by LPS and a reduced number of inducible nitric oxide synthase expressing cells. Protection of minocycline was also linked with the reduction in LPS-induced oxidative stress, as indicated by 4-hydroxynonenal positive OLs. The overall results suggest that reduction in microglial activation may protect the neonatal brain from LPS-induced white matter injury and inhibition of microglial activation might be an effective approach for the therapeutic treatment of infection-induced white matter injury.

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.

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.

Brain injury induced by intracerebral injection of interleukin-1beta and tumor necrosis factor-alpha in the neonatal rat.

To examine the possible role of inflammatory cytokines in mediating neonatal brain injury, we investigated effects of intra-cerebral injection of IL-1beta (IL-1β) or tumor necrosis factor-alpha (TNFα) on brain injury in the neonatal rat. A stereotaxic intracerebral injection of IL-1βor TNFα(10 ng per pup) was performed in postnatal day 5 (P5) SD rats. Although no necrosis of neurons was found, increased astrogliosis, as indicated by GFAP positive staining was observed 24 and 72 h following the injection of IL-1βor TNFα. IL-1βinduced apoptotic cell death in the rat brain 24 h after the injection, as indicated by increases in positive TUNEL staining and caspase-3 activity, and apoptotic cell death was partially blocked by systemic administration of NBQX, an antagonist of the AMPA glutamate receptor. IL-1βalso significantly reduced the number of developing oligodendrocytes (OLs) 24 h after the injection and this impairment was not prevented by NBQX. On the contrary, TNFα induced a much smaller increase in the number of TUNEL positive cells and did not reduce the number of developing OLs. By P8, myelin basic protein (MBP) was clearly detected in the control rat brain, while MBP positive staining was very weak, if any, in the IL-1βtreated rat brain. MBP expression in the TNFα treated rat brain was less affected. The overall results indicate that IL-1β may directly cause injuries to developing OLs and impair myelination in the neonatal rat brain and TNFα may have different roles in mediating brain injury.

α-Phenyl-n-tert-butyl-nitrone attenuates hypoxic–ischemic white matter injury in the neonatal rat brain ☆

White matter of the neonatal brain is highly sensitive to hypoxic–ischemic insult. The susceptibility of premature oligodendrocytes (OLs) to free radicals (FRs) produced during hypoxia–ischemia (HI) has been proposed as one of the mechanisms involved. To test this hypothesis, and to further investigate if the FR scavenger α-phenyl-N-tert-butyl-nitrone (PBN) attenuates hypoxic–ischemic white matter damage (WMD), postnatal day 4 (P4) SD rats were subjected to bilateral common carotid artery ligation (BCAL), followed by 8% oxygen exposure for 20 min. Pathological changes were evaluated on P6 and P9, 2 and 5 days after the HI insult. HI caused severe WMD including rarefaction, necrosis and cavity formation in the corpus callosum, external and internal capsule areas. OL injury was evidenced by degeneration of O4 positive OLs on P6. Disrupted myelination was verified by decreased immunostaining of myelin basic protein (MBP) on P9. Axonal injury was demonstrated by increased amyloid precursor protein (APP) immunostaining on both P6 and P9. Two lipid peroxidation end products, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), showed a one-fold elevation within 1–24 h following HI. 4-HNE immunostaining was found to specifically localize in the white matter area. Furthermore, pyknotic O4+ OLs were double-labeled with 4-HNE. These findings suggest that FRs are involved in the pathogenesis of neonatal WMD. PBN (100 mg/kg, i.p.) treatment alleviated the pathological changes of WMD following HI. It improved the survival of O4 positive OLs, attenuated hypomyelination and reduced axonal damage. PBN treatment also decreased the brain concentration of MDA/4-HNE and positive 4-HNE staining in the white matter area. These findings indicate that in the current WMD model, PBN protects both OLs and axons, the two main components in the white matter, from neonatal HI insult. FR scavenging appears to be the primary mechanism underlying its neuroprotective effect.

IGF-1 protects oligodendrocyte progenitor cells and improves neurological functions following cerebral hypoxia–ischemia in the neonatal rat

To investigate if insulin-like growth factor-1 (IGF-1) provides neuroprotection to oligodendrocyte progenitor cells (OPCs) following cerebral hypoxia-ischemia, a previously developed neonatal rat model of white matter damage was used in this study. Postnatal day 4 (P4) SD rat pups were subjected to bilateral common carotid artery ligation, followed by exposure to 8% oxygen for 10 min. IGF-1 (0.5 microg) or vehicle was injected into the left ventricle after artery ligation and before the hypoxic exposure. Cerebral hypoxia-ischemia caused death of O4+ late OPCs in the P5 rat brain and impaired myelination in the P9 and P21 rat brain. Caspase-3 activation was involved in the death of OPCs. Moreover, cerebral hypoxia-ischemia impaired neurobehavioral performance in juvenile rats. IGF-1 treatment attenuated damages to OPCs and improved neurological functions after cerebral hypoxia-ischemia. It reduced death of O4+ OPCs by 39% on P5 and enhanced myelination on P9 and P21. Bromodeoxyuridine uptake assay showed that cerebral hypoxia-ischemia inhibited proliferation of stem/progenitor cells in the subventricular zone and NG2+ early OPCs in the white matter area. IGF-1 treatment increased cell proliferation in the subventricular zone by 31% 1 day following hypoxic-ischemic insult. Proliferation of early and late OPCs in the IGF-1-treated group was 1.5- and 2.4-fold of that in the vehicle-treated group, respectively. In conclusion, IGF-1 provided potent neuroprotection to OPCs and improved neurological functions following cerebral hypoxia-ischemia in the neonatal rat. The neuroprotection of IGF-1 was associated with its antiapoptotic and mitogenic effects.