Lu-Tai Tien

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-1β 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.

Neonatal lipopolysaccharide exposure induces long-lasting learning impairment, less anxiety-like response and hippocampal injury in adult rats

Infection during early neonatal period has been shown to cause lasting neurological disabilities and is associated with the subsequent impairment in development of learning and memory ability and anxiety-related behavior in adults. We have previously reported that neonatal lipopolysaccharide (LPS) exposure resulted in cognitive deficits in juvenile rats (P21); thus, the goal of the present study was to determine whether neonatal LPS exposure has long-lasting effects in adult rats. After an LPS (1mg/kg) intracerebral (i.c.) injection in postnatal day 5 (P5) Sprague-Dawley female rat pups, neurobehavioral tests were carried out on P21 and P22, P49 and P50 or P70 and P71 and brain injury was examined at 66days after LPS injection (P71). Our data indicate that neonatal LPS exposure resulted in learning deficits in the passive avoidance task, less anxiety-like (anxiolytic-like) responses in the elevated plus-maze task, reductions in the hippocampal volume and the number of neuron-specific nuclear protein (NeuN)+ cells, as well as axonal injury in the CA1 region of the middle dorsal hippocampus in P71 rats. Neonatal LPS exposure also resulted in sustained inflammatory responses in the P71 rat hippocampus, as indicated by an increased number of activated microglia and elevation of interleukin-1β content in the rat hippocampus. This study reveals that neonatal LPS exposure causes persistent injuries to the hippocampus and results in long-lasting learning disabilities, and these effects are related to the chronic inflammation in the rat hippocampus.

Neonatal Systemic Exposure to Lipopolysaccharide Enhances Susceptibility of Nigrostriatal Dopaminergic Neurons to Rotenone Neurotoxicity in Later Life

Brain inflammation via intracerebral injection with lipopolysaccharide (LPS) in early life has been shown to increase risks for the development of neurodegenerative disorders in adult rats. To determine if neonatal systemic LPS exposure has the same effects on enhancement of adult dopaminergic neuron susceptibility to rotenone neurotoxicity as centrally injected LPS does, LPS (2 μg/g body weight) was administered intraperitoneally into postnatal day 5 (P5) rats and when grown to P70, rats were challenged with rotenone, a commonly used pesticide, through subcutaneous minipump infusion at a dose of 1.25 mg/kg/day for 14 days. Systemically administered LPS can penetrate into the neonatal rat brain and cause acute and chronic brain inflammation, as evidenced by persistent increases in IL-1β levels, cyclooxygenase-2 expression and microglial activation in the substantia nigra (SN) of P70 rats. Neonatal LPS exposure resulted in suppression of tyrosine hydroxylase (TH) expression, but not actual death of dopaminergic neurons in the SN, as indicated by the reduced number of TH+ cells and unchanged total number of neurons (NeuN+) in the SN. Neonatal LPS exposure also caused motor function deficits, which were spontaneously recoverable by P70. A small dose of rotenone at P70 induced loss of dopaminergic neurons, as indicated by reduced numbers of both TH+ and NeuN+ cells in the SN, and Parkinsons disease (PD)-like motor impairment in P98 rats that had experienced neonatal LPS exposure, but not in those without the LPS exposure. These results indicate that although neonatal systemic LPS exposure may not necessarily lead to death of dopaminergic neurons in the SN, such an exposure could cause persistent functional alterations in the dopaminergic system and indirectly predispose the nigrostriatal system in the adult brain to be damaged by environmental toxins at an ordinarily nontoxic or subtoxic dose and develop PD-like pathological features and motor dysfunction.

α-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 α‐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.

Melatonin ameliorates brain injury induced by systemic lipopolysaccharide in neonatal rats

Our previous study showed that lipopolysaccharide (LPS)-induced brain injury in the neonatal rat is associated with nitrosative and oxidative stress. The present study was conducted to examine whether melatonin, an endogenous molecule with antioxidant properties, reduces systemic LPS-induced nitrosative and oxidative damage in the neonatal rat brain. Intraperitoneal (i.p.) injection of LPS (2mg/kg) was administered to Sprague-Dawley rat pups on postnatal day 5 (P5), and i.p. administration of melatonin (20mg/kg) or vehicle was performed 5min after LPS injection. Sensorimotor behavioral tests were performed 24h after LPS exposure, and brain injury was examined after these tests. The results show that systemic LPS exposure resulted in impaired sensorimotor behavioral performance, and acute brain injury, as indicated by the loss of oligodendrocyte immunoreactivity and a decrease in mitochondrial activity in the neonatal rat brain. Melatonin treatment significantly reduced LPS-induced neurobehavioral disturbances and brain damage in neonatal rats. The neuroprotective effect of melatonin was associated with attenuation of LPS-induced nitrosative and oxidative stress, as indicated by the decreased nitrotyrosine- and 4-hydroxynonenal-positive staining in the brain following melatonin and LPS exposure in neonatal rats. Further, melatonin significantly attenuated LPS-induced increases in the number of activated microglia in the neonatal rat brain. The protection provided by melatonin was also associated with a reduced number of inducible nitric oxide synthase (iNOS)+ cells, which were double-labeled with ED1 (microglia). Our results show that melatonin prevents the brain injury and neurobehavioral disturbances induced by systemic LPS exposure in neonatal rats, and its neuroprotective effects are associated with its impact on nitrosative and oxidative stress.

Celecoxib attenuates systemic lipopolysaccharide-induced brain inflammation and white matter injury in the neonatal rats.

Lipopolysaccharide (LPS)-induced white matter injury in the neonatal rat brain is associated with inflammatory processes. Cyclooxygenase-2 (COX-2) can be induced by inflammatory stimuli, such as cytokines and pro-inflammatory molecules, suggesting that COX-2 may be considered as the target for anti-inflammation. The objective of the present study was to examine whether celecoxib, a selective COX-2 inhibitor, can reduce systemic LPS-induced brain inflammation and brain damage. Intraperitoneal (i.p.) injection of LPS (2mg/kg) was performed in postnatal day 5 (P5) of Sprague-Dawley rat pups and celecoxib (20mg/kg) or vehicle was administered i.p. 5 min after LPS injection. The body weight and wire-hanging maneuver test was performed 24h after the LPS exposure, and brain injury was examined after these tests. Systemic LPS exposure resulted in an impairment of behavioral performance and acute brain injury, as indicated by apoptotic death of oligodendrocytes (OLs) and loss of OL immunoreactivity in the neonatal rat brain. Treatments with celecoxib significantly reduced systemic LPS-induced neurobehavioral disturbance and brain damage. Celecoxib administration significantly attenuated systemic LPS-induced increments in the number of activated microglia and astrocytes, concentrations of IL-1β and TNFα, and protein levels of phosphorylated-p38 MAPK in the neonatal rat brain. The protection of celecoxib was also associated with a reduction of systemic LPS-induced COX-2+ cells which were double labeled with GFAP+ (astrocyte) cells. The overall results suggest that celecoxib was capable of attenuating the brain injury and neurobehavioral disturbance induced by systemic LPS exposure, and the protective effects are associated with its anti-inflammatory properties.

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 Parkinsons disease and the development of potential therapeutic treatments.

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

BackgroundCyclooxygenase-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.MethodsIntraperitoneal (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.ResultsOur 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-1β 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.ConclusionSystemic 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.

Interleukin-1β-induced brain injury in the neonatal rat can be ameliorated by α-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.

INTERLEUKIN-1β-INDUCED BRAIN INJURY AND NEUROBEHAVIORAL DYSFUNCTIONS IN JUVENILE RATS CAN BE ATTENUATED BY α-PHENYL-N-TERT-BUTYL-NITRONE

Our previous study showed that perinatal exposure to interleukin-1beta (IL-1beta), an inflammatory cytokine, induces acute injury to developing white matter in the neonatal rat brain, and alpha-phenyl-n-tert-butyl-nitrone (PBN), a free radical scavenger and antioxidant, protects against IL-1beta-induced acute brain injury. The objective of the present study was to further examine whether perinatal exposure to IL-1beta resulted in persistent brain damage and neurological disabilities, and whether PBN offers lasting protection. Intracerebral injection of IL-1beta (1 microg/kg) was performed in postnatal day 5 (P5) Sprague-Dawley rat pups and PBN (100 mg/kg) or saline was administered intraperitoneally 5 min after IL-1beta injection. Perinatal IL-1beta exposure significantly affected neurobehavioral functions in juvenile rats. Although some neurobehavioral deficits such as performance in negative geotaxis, cliff avoidance, beam walking, and locomotion were spontaneously reversible, sustained deficits such as poor performance in the vibrissa-elicited forelimb-placing test, the pole test, the passive avoidance task, and the elevated plus-maze task were still observable at P21. Perinatal IL-1beta exposure resulted in persistent brain damage including enlargement of ventricles, loss of mature oligodendrocytes, impaired myelination as indicated by the decrease in myelin basic protein immunostaining, axonal and dendritic injury, and loss of hippocampal CA1 neurons and tyrosine hydroxylase positive neurons in the substantia nigra and ventral tegmental areas of the rat brain. Treatments with PBN provided lasting protection against the IL-1beta-induced brain injury and improved the associated neurological dysfunctions in juvenile rats, suggesting that prompt treatments for brain injury induced by perinatal infection/inflammation might have important long-term consequences.