Intranasal insulin attenuates hypoxia-ischemia-induced short-term sensorimotor behavioral disturbances, neuronal apoptosis, and brain damage in neonatal rats.

There is a significant need for additional therapy to improve outcomes for newborns with acute Hypoxic-ischemic (HI) encephalopathy (HIE). New evidence suggests that insulin could be neuroprotective. This study aimed to investigate whether intranasal insulin attenuates HI-induced brain damage and neurobehavioral dysfunction in neonatal rats. Postnatal day 10 (P10), Sprague-Dawley rat pups were randomly divided into Sham + Vehicle, Sham + Insulin, HI + Vehicle, and HI + Insulin groups with equal male-to-female ratios. Pups either had HI by permanent ligation of the right common carotid artery followed by 90 min of hypoxia (8% O2) or sham surgery followed by room air exposure. Immediately after HI or Sham, pups were given fluorescence-tagged insulin (Alex-546-insulin)/vehicle, human insulin (25 µg), or vehicle in each nare under anesthesia. Shortly after administration, widespread Alex-546-insulin-binding cells were detected in the brain, primarily co-localized with neuronal nuclei-positive neurons on double-immunostaining. In the hippocampus, phospho-Akt was activated in a subset of Alex-546-insulin double-labeled cells, suggesting activation of the Akt/PI3K pathway in these neurons. Intranasal insulin (InInsulin) reduced HI-induced sensorimotor behavioral disturbances at P11. InInsulin prevented HI-induced increased Fluoro-Jade C+ degenerated neurons, cleaved caspase 3+ neurons, and volume loss in the ipsilateral brain at P11. There was no sex-specific response to HI or insulin. The findings confirm that intranasal insulin provides neuroprotection against HI brain injury in P10 rats associated with activation of intracellular cell survival signaling. If further pre-clinical research shows long-term benefits, intranasal insulin has the potential to be a promising non-invasive therapy to improve outcomes for newborns with HIE.

Maternal immune activation alters fetal and neonatal microglia phenotype and disrupts neurogenesis in mice.

BACKGROUND: Activation of microglia, increase in cortical neuron density, and reduction in GABAergic interneurons are some of the key findings in postmortem autism spectrum disorders (ASD) subjects. The aim of this study was to investigate how maternal immune activation (MIA) programs microglial phenotypes and abnormal neurogenesis in offspring mice. METHODS: MIA was induced by injection of lipopolysaccharide (LPS, i.p.) to pregnant mice at embryonic (E) day 12.5. Microglial phenotypes and neurogenesis were investigated between E15.5 to postnatal (P) day 21 by immunohistochemistry, flow cytometry, and cytokine array. RESULTS: MIA led to a robust increase in fetal and neonatal microglia in neurogenic regions. Homeostatic E15.5 and P4 microglia are heterogeneous, consisting of M1 (CD86+/CD206-) and mixed M1/M2 (CD86+/CD206+)-like subpopulations. MIA significantly reduced M1 but increased mixed M1/M2 microglia, which was associated with upregulation of numerous cytokines with pleotropic property. MIA resulted in a robust increase in Ki67+/Nestin+ and Tbr2+ neural progenitor cells in the subventricular zone (SVZ) of newborn mice. At juvenile stage, a male-specific reduction of Parvalbumin+ but increase in Reelin+ interneurons in the medial prefrontal cortex was found in MIA offspring mice. CONCLUSIONS: MIA programs microglia towards a pleotropic phenotype that may drive excessive neurogenesis in ASD patients. IMPACT: Maternal immune activation (MIA) alters microglial phenotypes in the brain of fetal and neonatal mouse offspring. MIA leads to excessive proliferation and overproduction of neural progenitors in the subventricular zone (SVZ). MIA reduces parvalbumin+ while increases Reelin+ interneurons in the prefrontal cortex. Our study sheds light on neurobiological mechanisms of abnormal neurogenesis in certain neurodevelopmental disorders, such as autism spectrum disorder (ASD).

Azithromycin Protects Oligodendrocyte Progenitor Cells against Lipopolysaccharide-Activated Microglia-Induced Damage.

Oligodendrocyte progenitor cells (OPC) are the primary cellular targets of brain white matter injury (WMI) in very low-birth weight (VLBW) infants. Microglia plays a significant role in inflammation-induced WMI. Our previous study showed that lipopolysaccharide (LPS)-induced OPC damage is mediated by activated microglia in vitro. We hypothesized that azithromycin (AZ) could protect OPCs against LPS-induced cytotoxicity by blocking microglial activation. Highly enriched primary rat microglia and OPCs were treated with LPS. There were 4 groups: control, LPS + Veh, AZ, and LPS + AZ. Microglia conditioned medium (MCM) was used to determine inflammatory cytokines by enzyme-linked immunosorbent assay or subsequent treatment of OPCs. We found that AZ significantly suppressed TNF-α, IL-1ß, and IL-6 in LPS+Veh-treated-microglial MCM and blocked microglial nuclear factor-κB p65 nuclear translocation. AZ prevented LPS-MCM-induced OPC death and improved OPC survival as measured by activated caspase-3 immunostaining and XTT assay, respectively. AZ ameliorated LPS-MCM-induced differentiation arrest and myelin basic protein deficit in oligodendrocytes. Our data suggest that AZ is a potent inhibitor for microglia activation and may hold the therapeutic potential for WMI in VLBW infants.

Intrauterine growth restriction and neonatal hypoxic ischemic brain injury causes sex-specific long-term neurobehavioral abnormalities in rats.

There is a lack of knowledge of factors preventing an adequate response to moderate hypothermia after hypoxic ischemic (HI) brain injury. We hypothesized that growth restriction from reduced intrauterine perfusion would predispose neonatal rats to have a worse outcome with HI brain injury. IUGR was induced by placental insufficiency in dams at 14 days of gestation. HI was induced at postnatal day (P) 10 by permanent right carotid artery ligation followed by 90 min of hypoxia (8% oxygen). Tests for early brain injury and neurobehavioral outcomes were subsequently done. All statistical analysis was done using Two-way ANOVA; post hoc Holm-Sidak test. HI in control and IUGR groups decreased the success rate of the contralateral vibrissa-elicited forelimb test, increased response latency in movement initiation test and increased the time to finish elevated beam walk test at P40 and P60. IUGR augmented HI-induced abnormality in vibrissa-elicited forelimb test at P40 but showed higher success rate when compared to HI only group at P60. IUGR's negative effect on HI-induced changes on the elevated beam walk test was sex-specific and exaggerated in P60 males. Increased TUNEL positive cells in the cortex were noted at 72 h after in HI in control but not in IUGR groups. In conclusion, the consequences of IUGR on subsequent neonatal HI varied based on age, sex and outcomes examined, and overall, male sex and IUGR had worse effects on the long-term neurobehavioral outcomes following HI.

Rapid transport of insulin to the brain following intranasal administration in rats.

We previously reported that intranasal insulin protects substantia nigra dopaminergic neurons against 6-hydroxydopamine neurotoxicity in rats. This study aimed to assess insulin pharmacokinetics in the rat brain following intranasal application. Recombinant human insulin (rh-Ins) or phosphate buffer solution was administered to both nostrils of rats. Animals were sacrificed at 15 minutes, 1, 2, and 6 hours to determine insulin levels in different brain regions by an ultrasensitive, human-specific enzyme-linked immunosorbent assay kit. For fluorescence tracing study, rats were administered with intranasal florescence-tagged insulin (Alex546-Ins), and brains were fixed at 10 and 30 minutes to prepare sagittal sections. rh-Ins was detected in all brain regions examined except the cerebral cortex. The highest levels were detected in the brainstem, followed by the cerebellum, substantia nigra/ventral tegmental area, olfactory bulb, striatum, hippocampus, and thalamus/hypothalamus. Insulin levels reached a peak at 15 minutes and then declined gradually overtime, but remained significantly higher than baseline levels at 6 hours in most regions. Consistently, widespread Alex546-Ins-binding cells were detected in the brain at 10 and 30 minutes, with the olfactory bulb and brainstem showing the highest while the cerebral cortex showing lowest fluorescence signals. Double-immunostaining showed that Alex546-Ins-bindings were primarily co-localized with neuronal nuclei-positive neurons. In the subtantia nigra, phospho-Akt was found to be activated in a subset of Alex546-Ins and tyrosine hydroxylase double-labeled cells, suggesting activation of the Akt/PI3K pathway in these dopaminergic neurons. Data from this study suggest that intranasal insulin could effectively reach deep brain structures including the nigrostriatal pathways, where it binds to dopaminergic neurons and activates intracellular cell survival signaling. This study was approved by the Institutional Animal Care Committee at the University of Mississippi Medical Center (protocol 1333A) on June 29, 2015.

Neuroprotective Effects of Intranasal IGF-1 against Neonatal Lipopolysaccharide-Induced Neurobehavioral Deficits and Neuronal Inflammation in the Substantia Nigra and Locus Coeruleus of Juvenile Rats.

Neonatal lipopolysaccharide (LPS) exposure-induced brain inflammation resulted in motor dysfunction and brain dopaminergic neuronal injury, and increased the risks of neurodegenerative disorders in adult rats. Our previous studies showed that intranasal administration of insulin-like growth factor-1 (IGF-1) protects against LPS-induced white matter injury in the developing rat brain. To further examine whether IGF-1 protects against LPS-induced brain neuronal injury and neurobehavioral dysfunction, recombinant human IGF-1 (rhIGF-1) at a dose of 50 µg/pup was administered intranasally 1 h following intracerebral injection of LPS (1 mg/kg) in postnatal day 5 (P5) Sprague-Dawley rat pups. Neurobehavioral tests were carried out from P7 to P21, and brain neuronal injury was examined at P21. Our results showed that LPS exposure resulted in disturbances of motor behaviors in juvenile rats. Moreover, LPS exposure caused injury to central catecholaminergic neurons, as indicated by a reduction of tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra (SN), ventral tegmental area (VTA) and olfactory bulb (OB), and brain noradrenergic neurons, as indicated by a reduction of TH immunoreactivity in the locus coeruleus (LC) of the P21 rat brain. The LPS-induced reduction of TH+ cells was observed at a greater degree in the SN and LC of the P21 rat brain. Intranasal rhIGF-1 treatment attenuated LPS-induced central catecholaminergic neuronal injury and motor behavioral disturbances, including locomotion, beam walking test and gait analysis. Intranasal rhIGF-1 administration also attenuated LPS-induced elevation of IL-1ß levels and numbers of activated microglia, and cyclooxygenase-2+ cells, which were double labeled with TH+ cells in the SN, VTA, OB and LC of the P21 rat brain. These results suggest that IGF-1 may provide protection against neonatal LPS exposure-induced central catecholaminergic neuronal injury and motor behavioral disturbances, and that the protective effects are associated with the inhibition of microglia activation and the reduction of neuronal oxidative stress by the suppression of the neuronal cyclooxygenase-2 expression.

Early Postnatal Lipopolysaccharide Exposure Leads to Enhanced Neurogenesis and Impaired Communicative Functions in Rats.

Perinatal infection is a well-identified risk factor for a number of neurodevelopmental disorders, including brain white matter injury (WMI) and Autism Spectrum Disorders (ASD). The underlying mechanisms by which early life inflammatory events cause aberrant neural, cytoarchitectural, and network organization, remain elusive. This study is aimed to investigate how systemic lipopolysaccharide (LPS)-induced neuroinflammation affects microglia phenotypes and early neural developmental events in rats. We show here that LPS exposure at early postnatal day 3 leads to a robust microglia activation which is characterized with mixed microglial proinflammatory (M1) and anti-inflammatory (M2) phenotypes. More specifically, we found that microglial M1 markers iNOS and MHC-II were induced at relatively low levels in a regionally restricted manner, whereas M2 markers CD206 and TGFß were strongly upregulated in a sub-set of activated microglia in multiple white and gray matter structures. This unique microglial response was associated with a marked decrease in naturally occurring apoptosis, but an increase in cell proliferation in the subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus. LPS exposure also leads to a significant increase in oligodendrocyte lineage population without causing discernible hypermyelination. Moreover, LPS-exposed rats exhibited significant impairments in communicative and cognitive functions. These findings suggest a possible role of M2-like microglial activation in abnormal neural development that may underlie ASD-like behavioral impairments.

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.

Corticosteroid responses following hypoxic preconditioning provide neuroprotection against subsequent hypoxic-ischemic brain injury in the newborn rats.

Limited research has evaluated the corticosteroids (CS) response in hypoxic preconditioning (PC) induced neuroprotection against subsequent hypoxic-ischemic (HI) brain injury in newborns. To measure, CS response to hypoxic PC, at postnatal day 6 (P6), rat pups were randomly divided into sham, NoPC (exposure to 21% O2) and PC (exposure to 8% O2 for 3h) groups. In a separate experiment, at P6, rat pups were randomly divided into three groups (sham, NoPC+HI, PC+HI). Rat pups in NoPC+HI and PC+HI groups, respectively had normoxic or hypoxic exposure for 3h at P6 and then had the right carotid artery permanently ligated followed by 140 min of hypoxia at P7 (HI). Plasma CS levels were measured at 0.5, 1, 3, 6 and 12h after hypoxic PC and hypoxic PC followed by HI. To investigate whether CS response to hypoxic PC provides neuroprotection against HI, at P6, rat pups were randomly divided into five groups. Fifteen minutes prior to PC or normoxic exposure, rat pups in DMSO+PC+HI and DMSO+NoPC+HI groups received DMSO while in RU486+PC+HI and RU486+NoPC+HI groups received RU486 (glucocorticoid receptor blocker, 60 mg/kg) s.c., respectively. Afterwards, rat pups were exposed to normoxia (DMSO+NoPC+HI, RU486+NoPC+HI) or hypoxia (DMSO+PC+HI, RU486+PC+HI) for 3h and then HI 24h later (P7). Rat pups at the corresponding age without any exposure to PC or HI or RU486/DMSO were used as sham. We found that hypoxic PC caused CS surge as well as augmented CS surge and preserved the glucocorticoid feedback regulation after HI. Hypoxic PC reduced HI induced early and delayed brain damage. RU486 partially but significantly inhibited hypoxic PC induced neuroprotection.

Interleukin-1 receptor antagonist reduces neonatal lipopolysaccharide-induced long-lasting neurobehavioral deficits and dopaminergic neuronal injury in adult rats.

Our previous study showed that a single lipopolysaccharide (LPS) treatment to neonatal rats could induce a long-lasting neuroinflammatory response and dopaminergic system injury late in life. This is evidenced by a sustained activation of microglia and elevated interleukin-1ß (IL-1ß) levels, as well as reduced tyrosine hydroxylase (TH) expression in the substantia nigra (SN) of P70 rat brain. The object of the current study was to test whether co-administration of IL-1 receptor antagonist (IL-1ra) protects against LPS-induced neurological dysfunction later in life. LPS (1 mg/kg) with or without IL-1ra (0.1 mg/kg), or sterile saline was injected intracerebrally into postnatal day 5 (P5) Sprague-Dawley male rat pups. Motor behavioral tests were carried out from P7 to P70 with subsequent examination of brain injury. Our results showed that neonatal administration of IL-1ra significantly attenuated LPS-induced motor behavioral deficits, loss of TH immunoreactive neurons, as well as microglia activation in the SN of P70 rats. These data suggest that IL-1ß may play a pivotal role in mediating a chronic neuroinflammation status by a single LPS exposure in early postnatal life, and blockading IL-1ß might be a novel approach to protect the dopaminergic system against perinatal infection/inflammation exposure.

Dexamethasone but not the equivalent doses of hydrocortisone induces neurotoxicity in neonatal rat brain.

BACKGROUND: The use of dexamethasone (Dex) in premature infants to treat or prevent chronic lung disease adversely affects neurodevelopment. Recent clinical studies suggest that hydrocortisone (HC) is a safer alternative to Dex. We compared the effects of Dex and HC on neurotoxicity in newborn rats. METHODS: Rat pups of a neurodevelopmental stage equivalent to premature human neonates were administered Dex or HC either as a single dose on postnatal day (PD) 6, repeated doses on PD 4 to 6 or tapering doses at PD 3 to 6 by i.p. injection. Brain weight, caspase-3 activity, and apoptotic cells were measured at PD 7; learning capability, memory, and motor function were measured at juvenile age. RESULTS: Dex decreased both body and brain weight gain, while HC did not. Tapering and repeated doses of Dex increased caspase-3 activity, cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells but HC, except at high doses, did not. Dex impaired learning and memory capability at juvenile age, while the rats exposed to HC showed normal cognitive behavior. CONCLUSION: HC is probably safer to use than Dex in the immediate postnatal period in neonatal rats. Cautious extrapolation of these findings to human premature infants is required.

IL-1 receptor antagonist attenuates neonatal lipopolysaccharide-induced long-lasting learning impairment and hippocampal injury in adult rats.

We have previously reported that neonatal lipopolysaccharide (LPS) exposure resulted in an increase in interleukin-1ß (IL-1ß) content, injury to the hippocampus, and cognitive deficits in juvenile male and female rats, as well as female adult rats. The present study aimed to determine whether an anti-inflammatory cytokine, interleukin-1 receptor antagonist (IL-1ra), protects against the neonatal LPS exposure-induced inflammatory responses, hippocampal injury, and long-lasting learning deficits in adult rats. LPS (1 mg/kg) or LPS plus IL-1ra (0.1 mg/kg) was injected intracerebrally to Sprague-Dawley male rat pups at postnatal day 5 (P5). Neurobehavioral tests were carried out on P21, P49, and P70, while neuropathological studies were conducted on P71. Our results showed that neonatal LPS exposure resulted in learning deficits in rats at both developmental and adult ages, as demonstrated by a significantly impaired performance in the passive avoidance task (P21, P49, and P70), reduced hippocampal volume, and reduced number of Nissl+ cells in the CA1 region of the middle dorsal hippocampus of P71 rat brain. Those neuropathological and neurobehavioral alterations by LPS exposure were associated with a sustained inflammatory response in the P71 rat hippocampus, indicated by increased number of activated microglia as well as elevated levels of IL-1ß. Neonatal administration of IL-1ra significantly attenuated LPS-induced long-lasting learning deficits, hippocampal injury, and sustained inflammatory responses in P71 rats. Our study demonstrates that neonatal LPS exposure leads to a persistent injury to the hippocampus, resulting in long-lasting learning disabilities related to chronic inflammation in rats, and these effects can be attenuated with an IL-1 receptor antagonist.

Exposure to serotonin adversely affects oligodendrocyte development and myelination in vitro.

Serotonin (5-hydroxytryptamine, 5-HT) has been implicated to play critical roles in early neural development. Recent reports have suggested that perinatal exposure to selective serotonin reuptake inhibitors (SSRIs) resulted in cortical network miswiring, abnormal social behavior, callosal myelin malformation, as well as oligodendrocyte (OL) pathology in rats. To gain further insight into the cellular and molecular mechanisms underlying SSRIs-induced OL and myelin abnormalities, we investigated the effect of 5-HT exposure on OL development, cell death, and myelination in cell culture models. First, we showed that 5-HT receptor 1A and 2A subtypes were expressed in OL lineages, using immunocytochemistry, Western blot, as well as intracellular Ca(2+) measurement. We then assessed the effect of serotonin exposure on the lineage development, expression of myelin proteins, cell death, and myelination, in purified OL and neuron-OL myelination cultures. For pure OL cultures, our results showed that 5-HT exposure led to disturbance of OL development, as indicated by aberrant process outgrowth and reduced myelin proteins expression. At higher doses, such exposure triggered a development-dependent cell death, as immature OLs exhibited increasing susceptibility to 5-HT treatment compared to OL progenitor cells (OPC). We showed further that 5-HT-induced immature OL death was mediated at least partially via 5-HT2A receptor, since cell death could be mimicked by 5-HT2A receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride, (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride, but atten-uated by pre-treatment with 5-HT2A receptor antagonist ritanserin. Utilizing a neuron-OL myelination co-culture model, our data showed that 5-HT exposure significantly reduced the number of myelinated internodes. In contrast to cell injury observed in pure OL cultures, 5-HT exposure did not lead to OL death or reduced OL density in neuron-OL co-cultures. However, abnormal patterns of contactin-associated protein (Caspr) clustering were observed at the sites of Node of Ranvier, suggesting that 5-HT exposure may affect other axon-derived factors for myelination. In summary, this is the first study to demonstrate that manipulation of serotonin levels affects OL development and myelination, which may contribute to altered neural connectivity noted in SSRIs-treated animals. The current in vitro study demonstrated that exposure to high level of serotonin (5-HT) led to aberrant oligodendrocyte (OL) development, cell injury, and myelination deficit. We propose that elevated extracellular serotonin levels in the fetal brain, such as upon the use of selective serotonin reuptake inhibitors (SSRIs) during pregnancy, may adversely affect OL development and/or myelination, thus contributing to altered neural connectivity seen in Autism Spectrum Disorders. OPC = oligodendrocyte progenitor cell.

Dexamethasone-induced neuroprotection in hypoxic-ischemic brain injury in newborn rats is partly mediated via Akt activation.

Prior treatment with dexamethasone (Dex) provides neuroprotection against hypoxia ischemia (HI) in newborn rats. Recent studies have shown that the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway plays an important role in the neuroprotection. The objective of this study is to evaluate the role of the PI3K/Akt pathway in the Dex-induced neuroprotection against subsequent HI brain injury. Seven-day-old rat pups had the right carotid artery permanently ligated followed by 160min of hypoxia (8% oxygen). Rat pups received i.p. injection of either saline or Dex (0.25mg/kg) at 24 and 4h before HI exposure. To quantify the effects of a PI3K/Akt inhibitor, wortmannin (1µl of 1µg/µl) or vehicle was injected intracerebroventricularly in the right hemisphere on postnatal day 6 at 30min prior to the first dose of Dex or saline treatment. Dex pretreatment significantly reduced the brain injury following HI which was quantified by the decrease in cleaved caspase-3 protein as well as cleaved caspase-3 and TUNEL positive cells at 24h and percent loss of ipsilateral hemisphere weight at 22d after HI, while wortmannin partially reversed these effects. We conclude that Dex provides robust neuroprotection against subsequent HI in newborn rats in part via activation of PI3/Akt pathway.

Strategies for myelin regeneration: lessons learned from development.

Myelin regeneration is indispensably important for patients suffering from several central nervous system (CNS) disorders such as multiple sclerosis (MS) and spinal cord injury (SCI), because it is not only essential for restoring neurophysiology, but also protects denuded axons for secondary degeneration. Understanding the cellular and molecular mechanisms underlying remyelination is critical for the development of remyelination-specific therapeutic approaches. As remyelination shares certain common mechanisms with developmental myelination, knowledge from study of developmental myelination contributes greatly to emerging myelin regeneration therapies, best evidenced as the recently developed human anti-Nogo receptor interacting protein-1 (LINGO-1) monoclonal antibodies to treat MS patients in clinical trials.

Dexamethasone induces apoptosis of progenitor cells in the subventricular zone and dentate gyrus of developing rat brain.

The use of dexamethasone in premature infants to prevent and/or treat bronchopulmonary dysplasia adversely affects neurocognitive development and is associated with cerebral palsy. The underlying mechanisms of these effects are multifactorial and likely include apoptosis. The objective of this study was to confirm whether dexamethasone causes apoptosis in different regions of the developing rat brain. On postnatal day 2, pups in each litter were randomly divided into the dexamethasone-treated (n = 91) or vehicle-treated (n = 92) groups. Rat pups in the dexamethasone group received tapering doses of dexamethasone on postnatal days 3-6 (0.5, 0.25, 0.125, and 0.06 mg/kg/day, respectively). Dexamethasone treatment significantly decreased the gain of body and brain weight and increased brain caspase-3 activity, DNA fragments, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and cleaved caspse-3-positive cells at 24 hr after treatment. Dexamethasone increased cleaved caspse-3-positive cells in the cortex, thalamus, hippocampus, cerebellum, dentate gyrus, and subventricular zone. Double-immunofluorescence studies show that progenitor cells in the subventricular zone and dentate gyrus preferentially undergo apoptosis following dexamethasone exposure. These results indicate that dexamethasone-induced apoptosis in immature cells in developing brain is one of the mechanisms of its neurodegenerative effects in newborn rats.

Celecoxib reduces brain dopaminergic neuronaldysfunction, and improves sensorimotor behavioral performance in neonatal rats exposed to systemic lipopolysaccharide.

BACKGROUND: Cyclooxygenase-2 (COX-2) is induced in inflammatory cells in response to cytokines and pro-inflammatory molecules, suggesting that COX-2 has a role in the inflammatory process. The objective of the current study was to examine whether celecoxib, a selective COX-2 inhibitor, could ameliorate lipopolysaccharide (LPS)-induced brain inflammation, dopaminergic neuronal dysfunction and sensorimotor behavioral impairments. METHODS: Intraperitoneal (i.p.) injection of LPS (2 mg/kg) was performed in rat pups on postnatal Day 5 (P5), and celecoxib (20 mg/kg) or vehicle was administered (i.p.) five minutes after LPS injection. Sensorimotor behavioral tests were carried out 24 h after LPS exposure, and brain injury was examined on P6. RESULTS: Our results showed that LPS exposure resulted in impairment in sensorimotor behavioral performance and injury to brain dopaminergic neurons, as indicated by loss of tyrosine hydroxylase (TH) immunoreactivity, as well as decreases in mitochondria activity in the rat brain. LPS exposure also led to increases in the expression of α-synuclein and dopamine transporter proteins and enhanced [3H]dopamine uptake. Treatment with celecoxib significantly reduced LPS-induced sensorimotor behavioral disturbances and dopaminergic neuronal dysfunction. Celecoxib administration significantly attenuated LPS-induced increases in the numbers of activated microglia and astrocytes and in the concentration of IL-1ß in the neonatal rat brain. The protective effect of celecoxib was also associated with an attenuation of LPS-induced COX-2+ cells, which were double labeled with TH + (dopaminergic neuron) or glial fibrillary acidic protein (GFAP) + (astrocyte) cells. CONCLUSION: Systemic LPS administration induced brain inflammatory responses in neonatal rats; these inflammatory responses included induction of COX-2 expression in TH neurons and astrocytes. Application of the COX-2 inhibitor celecoxib after LPS treatment attenuated the inflammatory response and improved LPS-induced impairment, both biochemically and behaviorally.

Neonatal exposure to lipopolysaccharide enhances accumulation of α-synuclein aggregation and dopamine transporter protein expression in the substantia nigra in responses to rotenone challenge in later life.

Brain inflammation in early life may enhance adult susceptibility to develop neurodegenerative disorders triggered by environmental toxins. Our previous studies show that perinatal lipopolysaccharide (LPS) exposure enhances adult susceptibility to rotenone-induced injury to the dopaminergic system in the substantia nigra (SN) of the adult rat brain. To further investigate the enhanced adult susceptibility by neonatal LPS exposure to rotenone neurotoxicity, we used our neonatal rat model of LPS exposure (1mg/kg, intracerebral injection in postnatal day 5, P5, neonatal rats) to examine the protein levels of α-synuclein and dopamine transporters (DAT) in the adult rat. By P70, rats from the saline- or LPS-exposed group were challenged with rotenone, a commonly used pesticide, through subcutaneous mini-pump infusion at a dose of 1.25mg/kg/day for 14 days. The accumulation of α-synuclein aggregation and increment of DAT protein content were found in the SN of LPS-exposed rats. Neonatal LPS exposure enhanced rotenone-stimulated accumulation of α-synuclein aggregation and increment in DAT protein expression in the cytoplasmic compartment of the SN, and in the synaptosomal compartment of the striatum of adult rats. Rotenone treatment also resulted in reduction of [(3)H]dopamine uptake and mitochondrial complex I activity in the striatum of rats with neonatal LPS exposure, but not in those without this exposure. The current study suggests possible roles of α-synuclein aggregate and DAT distribution in the cytoplasm and synaptosome triggered by environmental toxins in later life in the development of neurodegenerative disorders. Our model may be useful in studying mechanisms involved in the pathogenesis of nonfamilial Parkinson's disease and for developing potential therapeutic treatments for this disease.

Dexamethasone decreases insulin-like growth factor-I and -II via a glucocorticoid receptor dependent mechanism in developing rat brain.

OBJECTIVES: Dexamethasone (Dex) causes neurodegeneration in developing brain. Insulin-like growth factor-I (IGF-I) and -II (IGF-II) are potent neurotrophic and differentiation factors and play key roles in the regulation of growth and development of CNS. Current project evaluated the effects of Dex on IGF-I and -II in developing rat brains. MATERIAL AND METHODS: Sprague-Dawley rat pups in each litter were divided into vehicle (n=230) or Dex-treated (n=234) groups. Rat pups in the Dex group received one of the 3 different regimens of i.p. Dex: tapering doses (DexTD) on postnatal days (PD) 3 to PD 6 or repeated doses on PD 4 to PD 6 or single dose on PD 6. To quantify the glucocorticoid receptor (GR) blockade effect, rat pups in the DexTD group on PD 3 and 5 received vehicle or RU486 (GR blocker, 60 mg/kg) s.c., twenty minutes prior to Dex treatment. RESULTS: Dex decreased the gain of body and brain weight while RU486 inhibited these effects. RU486 also prevented the DexTD-induced increase in caspase-3 activity and reduction in IGF-I and -II proteins. Compared to the vehicle, the expression of mRNA of IGF-I and -II decreased at 24 h after DexTD treatment, while RU486 prevented this decrease on IGF-II but not IGF-I. CONCLUSIONS: Our findings indicate that Dex via GR decreases IGF-I and -II and causes neurodegeneration in the neonatal rat brain.

Differential roles of astrocyte and microglia in supporting oligodendrocyte development and myelination in vitro.

Oligodendrocyte (OL) development relies on many extracellular cues, most of which are secreted cytokines from neighboring neural cells. Although it is generally accepted that both astrocytes and microglia are beneficial for OL development, there is a lack of understanding regarding whether astrocytes and microglia play similar or distinct roles. The current study examined the effects of astrocytes and microglia on OL developmental phenotypes including cell survival, proliferation, differentiation, and myelination in vitro. Our data reveal that, although both astrocytes- and microglia-conditioned medium (ACDM and MCDM, respectively) protect OL progenitor cells (OPCs) against growth factor withdrawal-induced apoptosis, ACDM is significantly more effective than MCDM in supporting long-term OL survival. In contrast, MCDM preferentially promotes OL differentiation and myelination. These differential effects of ACDM and MCDM on OL development are highlighted by distinct pattern of cytokine/growth factors in the conditioned medium, which correlates with differentially activated intracellular signaling pathways in OPCs upon exposure to the conditioned medium.