Inflammatory cytokine receptor blockade in a rodent model of mild traumatic brain injury.

In rodent models of traumatic brain injury (TBI), both Interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNFα) levels increase early after injury to return later to basal levels. We have developed and characterized a rat mild fluid percussion model of TBI (mLFP injury) that results in righting reflex response times (RRRTs) that are less than those characteristic of moderate to severe LFP injury and yet increase IL-1α/ß and TNFα levels. Here we report that blockade of IL-1α/ß and TNFα binding to IL-1R and TNFR1, respectively, reduced neuropathology in parietal cortex, hippocampus, and thalamus and improved outcome. IL-1ß binding to the type I IL-1 receptor (IL-1R1) can be blocked by a recombinant form of the endogenous IL-1R antagonist IL-1Ra (Kineret). TNFα binding to the TNF receptor (TNFR) can be blocked by the recombinant fusion protein etanercept, made up of a TNFR2 peptide fused to an Fc portion of human IgG1. There was no benefit from the combined blockades compared with individual blockades or after repeated treatments for 11 days after injury compared with one treatment at 1 hr after injury, when measured at 6 hr or 18 days, based on changes in neuropathology. There was also no further enhancement of blockade benefits after 18 days. Given that both Kineret and etanercept given singly or in combination showed similar beneficial effects and that TNFα also has a gliotransmitter role regulating AMPA receptor traffic, thus confounding effects of a TNFα blockade, we chose to focus on a single treatment with Kineret.

A rodent model of mild traumatic brain blast injury.

One of the criteria defining mild traumatic brain injury (mTBI) in humans is a loss of consciousness lasting for less than 30 min. mTBI can result in long-term impairment of cognition and behavior. In rats, the length of time it takes a rat to right itself after injury is considered to be an analog for human return to consciousness. This study characterized a rat mild brain blast injury (mBBI) model defined by a righting response reflex time (RRRT) of more than 4 min but less than 10 min. Assessments of motor coordination relying on beam-balance and foot-fault assays and reference memory showed significant impairment in animals exposed to mBBI. This study's hypothesis is that there are inflammatory outcomes to mTBI over time that cause its deleterious effects. For example, mBBI significantly increased brain levels of interleukin (IL)-1ß and tumor necrosis factor-α (TNFα) protein. There were significant inflammatory responses in the cortex, hippocampus, thalamus, and amygdala 6 hr after mBBI, as evidenced by increased levels of the inflammatory markers associated with activation of microglia and macrophages, ionized calcium binding adaptor 1 (IBA1), impairment of the blood-brain barrier, and significant neuronal losses. There were significant increases in phosphorylated Tau (p-Tau) levels, a putative precursor to the development of neuroencephalopathy, as early as 6 hr after mBBI in the cortex and the hippocampus but not in the thalamus or the amygdala. There was an apparent correlation between RRRTs and p-Tau protein levels but not IBA1. These results suggest potential therapies for mild blast injuries via blockade of the IL-1ß and TNFα receptors.

Resolvin E1 protects the rat heart against reperfusion injury.

The purpose of the present study was to assess whether resolvin E1 (RvE1), an anti-inflammatory mediator derived from eicosapentaenoic acid, would limit myocardial infarct size in the rat. The H9c2 cell line was used to assess whether RvE1 has direct protective effects on cardiomyocytes. In in vivo experiments, Male Sprague-Dawley rats underwent 30 min of ischemia/4 h of reperfusion. Before reperfusion, rats received intravenous RvE1 (0, 0.03, 0.1, or 0.3mg/kg). In in vitro experiments, H9c2 cells were incubated with RvE1 (0, 1, 10, 100, or 1000 nM). Cells were subjected to 18 h of incubation under normoxic conditions, 16 h of hypoxia, or 16 h of hypoxia and 2 h of reoxygenation. In vivo, RvE1 dose dependently reduced infarct size (30.7 +/- 1.7% of the area at risk in the control group and 29.1 +/- 1.6%, 14.7 +/- 1.3%, and 9.0 +/- 0.6% in the 0.03, 0.1, and 0.3 mg/kg groups, respectively, P < 0.001). In vitro, RvE1 increased viability and decreased apoptosis in a dose-dependent fashion in cells exposed to hypoxia or hypoxia/reoxygenation. A maximal effect was achieved at a concentration of 100 nM. RvE1 augmented phosphoinositide 3-kinase activity, attenuated caspase-3 activity, and augmented calcium-dependent nitric oxide synthase activity in cells exposed to hypoxia or hypoxia/reoxygenation. RvE1 increased Akt, ERK1/2, and endothelial nitric oxide synthase phosphorylation and attenuated the levels of activated caspase-3 and phosphorylated p38 levels. AG-1478, an EGF receptor tyrosine kinase inhibitor, blocked the protective effect of RvE1 both in vivo and in vitro and attenuated the RvE1-induced increase in Akt and ERK1/2 phosphorylation. In conclusion, RvE1, an anti-inflammatory mediator derived from eicosapentaenoic acid, has a direct protective effect on cardiomyocytes against ischemia-reperfusion injury and limits infarct size when administered intravenously before reperfusion.

Normobaric hyperoximia increases hypoxia-induced cerebral injury: DTI study in rats.

Perinatal hypoxia affects normal neurological development and can lead to motor, behavioral and cognitive deficits. A common acute treatment for perinatal hypoxia is oxygen resuscitation (hyperoximia), a controversial treatment. Magnetic resonance imaging (MRI), including diffusion tensor imaging (DTI), was performed in a P7 rat model of perinatal hypoxia to determine the effect of hyperoximia. These studies were performed on two groups of animals: 1) animals which were subjected to ischemia followed by hypoxia (HI), and 2) HI followed by hyperoximic treatment (HHI). Lesion volumes on high resolution MRI and DTI derived measures, fractional anisotropy (FA), mean diffusivity (MD), and axial and radial diffusivities (lambda(l) and lambda(t), respectively) were measured in vivo one day, one week, and three weeks after injury. Most significant differences in the MRI and DTI measures were found at three weeks after injury. Specifically, three weeks after HHI injury resulted in significantly larger hyperintense lesion volumes (95.26 +/- 50.42 mm(3)) compared to HI (22.25 +/- 17.62 mm(3)). The radial diffusivity lambda(t) of the genu of corpus callosum was significantly larger in HHI (681 +/- 330 x 10(-6) mm(2)/sec) than in HI (486 +/- 96 x 10(-6) mm(2)/sec). Over all, most significant differences in all the DTI metrics (FA, MD, lambda(t), lambda(l)) at all time points were found in the corpus callosum. Our results suggest that treatment of perinatal hypoxia with normobaric oxygen does not ameliorate, but exacerbates damage.

Circadian clock in culture: N-acetyltransferase activity of chick pineal glands oscillates in vitro.

N-Acetyltransferase activity was measured in organ-cultured chick pineal glands. A circadian rhythm of enzyme activity persisted in cultured glands for up to 4 days. The phase of the rhythm in vitro closely approximates its phase in vivo. These observations demonstrate that the pineal gland of chicks contains (or is) a self-sustained circadian oscillator.

Increased numbers of thoracic dorsal root axons in rats given antibodies to nerve growth factor.

Sensory axons were counted in untreated 1-month-old rats and in littermates that were injected with antibodies to nerve growth factor. There were 45 percent more unmyelinated and 17 percent more myelinated axons in dorsal roots of the fifth thoracic spinal segment in treated rats. This suggests that the number of sensory axons can be changed by postnatal inactivation of nerve growth factor.

Aquaporin 1 - a novel player in spinal cord injury.

The role of water channel aquaporin 1 (AQP-1) in uninjured or injured spinal cords is unknown. AQP-1 is weakly expressed in neurons and gray matter astrocytes, and more so in white matter astrocytes in uninjured spinal cords, a novel finding. As reported before, AQP-1 is also present in ependymal cells, but most abundantly in small diameter sensory fibers of the dorsal horn. Rat contusion spinal cord injury (SCI) induced persistent and significant four- to eightfold increases in AQP-1 levels at the site of injury (T10) persisting up to 11 months post-contusion, a novel finding. Delayed AQP-1 increases were also found in cervical and lumbar segments, suggesting the spreading of AQP-1 changes over time after SCI. Given that the antioxidant melatonin significantly decreased SCI-induced AQP-1 increases and that hypoxia inducible factor-1alpha was increased in acutely and chronically injured spinal cords, we propose that chronic hypoxia contributes to persistent AQP-1 increases after SCI. Interestingly; AQP-1 levels were not affected by long-lasting hypertonicity that significantly increased astrocytic AQP-4, suggesting that the primary role of AQP-1 is not regulating isotonicity in spinal cords. Based on our results we propose possible novel roles for AQP-1 in the injured spinal cords: (i) in neuronal and astrocytic swelling, as AQP-1 was increased in all surviving neurons and reactive astrocytes after SCI and (ii) in the development of the neuropathic pain after SCI. We have shown that decreased AQP-1 in melatonin-treated SCI rats correlated with decreased AQP-1 immunolabeling in the dorsal horns sensory afferents, and with significantly decreased mechanical allodynia, suggesting a possible link between AQP-1 and chronic neuropathic pain after SCI.

Early postnatal development of rat brain: in vivo diffusion tensor imaging.

Perinatal hypoxia is a major cause of neurodevelopmental deficits. Neuronal migration patterns are particularly sensitive to perinatal hypoxia/ischemia and are associated with the clinical deficits. The rat model of hypoxia/ischemia at P7 mimics that of perinatal injury in humans. Before assessing the effects of postnatal injury on brain development, it is essential to determine the normal developmental trajectories of various brain structures in individual animals. In vivo longitudinal diffusion tensor imaging (DTI) was performed from postnatal day 0 (P0) to P56 on Wistar rats. The DTI metrics, mean diffusivity (MD), fractional anisotropy (FA), axial (lambdal) and radial (lambdat) diffusivities, were determined for four gray matter and eight white matter structures. The FA of the cortical plate and the body of corpus callosum decreased significantly during the first 3 weeks after birth. The decrease in the cortical plate's FA value was associated mainly with an increase in lambdat. The initial decrease in FA of corpus callosum was associated with a significant decrease in lambdal. The FA of corpus callosum increased during the rest of the observational period, which was mainly associated with a decrease in lambdat. The FA of gray matter structures, hippocampus, caudate putamen, and cortical mantle did not show significant changes between P0 and P56. In contrast, the majority of white matter structures showed significant changes between P0 and P56. These temporal changes in the DTI metrics were related to the neuronal and axonal pruning and myelination that are known to occur in the developing brain.

Acute and chronic changes in aquaporin 4 expression after spinal cord injury.

The effect of spinal cord injury (SCI) on the expression levels and distribution of water channel aquaporin 4 (AQP4) has not been studied. We have found AQP4 in gray and white matter astrocytes in both uninjured and injured rat spinal cords. AQP4 was detected in astrocytic processes that were tightly surrounding neurons and blood vessels, but more robustly in glia limitans externa and interna, which were forming an interface between spinal cord parenchyma and cerebrospinal fluid (CSF). Such spatial distribution of AQP4 suggests a critical role that astrocytes expressing AQP4 play in the transport of water from blood/CSF to spinal cord parenchyma and vice versa. SCI induced biphasic changes in astrocytic AQP4 levels, including its early down-regulation and subsequent persistent up-regulation. However, changes in AQP4 expression did not correlate well with the onset and magnitude of astrocytic activation, when measured as changes in GFAP expression levels. It appears that reactive astrocytes began expressing increased levels of AQP4 after migrating to the wound area (thoracic region) two weeks after SCI, and AQP4 remained significantly elevated for months after SCI. We also showed that increased levels of AQP4 spread away from the lesion site to cervical and lumbar segments, but only in chronically injured spinal cords. Although overall AQP4 expression levels increased in chronically-injured spinal cords, AQP4 immunolabeling in astrocytic processes forming glia limitans externa was decreased, which may indicate impaired water transport through glia limitans externa. Finally, we also showed that SCI-induced changes in AQP4 protein levels correlate, both temporally and spatially, with persistent increases in water content in acutely and chronically injured spinal cords. Although correlative, this finding suggests a possible link between AQP4 and impaired water transport/edema/syringomyelia in contused spinal cords.

The regulation of amyloid precursor protein metabolism by cholinergic mechanisms and neurotrophin receptor signaling.

The increased expression and/or abnormal processing of the amyloid precursor protein (APP) is associated with the formation of amyloid plaques and cerebrovascular amyloid deposits, which are one of the major morphological hallmarks of Alzheimer's disease (AD). Among the processes regulating APP metabolism, the proteolytic cleavage of APP into amyloidogenic or nonamyloidogenic fragments is of special interest. The cleavage of the APP by the alpha-secretase within the beta-amyloid sequence generates nonamyloidogenic C-terminal APP fragments and soluble APPs alpha, which has neurotrophic and neuroprotective activities. Proteolytic processing of APP by beta-secretase, on the other hand, exposes the N-terminus of beta-amyloid, which is liberated after gamma-secretase cleavage at the variable amyloid C-terminus. The resulting 39-43 amino acid beta-amyloid may be neurotoxic and disrupt neuronal connectivity after its accumulation in senile plaques. In this review, we discuss evidence derived from in vitro experiments, suggesting that the stimulation of protein kinase C (PKC)-coupled M1/M3 muscarinic acetylcholine receptors increases the nonamyloidogenic, secretory pathway of APP processing. It has also been shown in animal models that under conditions of reduced M1/M3 muscarinic acetylcholine receptor stimulation the secretory pathway of APP processing is inhibited and that constitutive upregulation of M1/M3-associated PKC increases APP secretion. Thus, the cortical cholinergic hypoactivity characteristic of AD may inhibit the nonamyloidogenic APP processing pathway and lead to increased beta-amyloid generation. It has been shown in vitro that nerve growth factor (NGF)-associated signaling also influences the expression and catabolism of APP. Recent experiments with NGF-responsive cells revealed a specific role for the high-affinity NGF receptor, TrkA, in the increases in secretory APP processing and a role for the low-affinity neurotrophin receptor, p75NTR, in the transcriptional regulation of APP. Therefore, treatments with NGF could ameliorate cortical cholinergic dysfunction in AD. These findings may influence the design of therapeutic strategies aimed at stimulating cholinergic function and at increasing nonamyloidogenic APP processing without elevating APP expression.

Nerve growth factor modulates the activation of the hypothalamo-pituitary-adrenocortical axis during the stress response.

In the present study, we have investigated the functional relationship between the nerve growth factor protein (NGF) and the hypothalamus-pituitary-adrenocortical axis (HPAA). We have found that while iv injected NGF is able to stimulate the HPAA activity in rats, NGF is not able to stimulate the axis after a block of the hypothalamus produced by chlorpromazine-morphine-Nembutal treatment. Also, the stress activation of the HPAA is significantly reduced by pretreatment of the rats with anti-NGF immunoglobulin G. These results suggest that the stimulatory action of NGF on HPAA activity requires the release of ACTH secretagogues from the hypothalamus and that NGF may modulate the HPAA response to stress stimuli.

NG-nitro-L-arginine methyl ester modifies the input function measured by dynamic susceptibility contrast magnetic resonance imaging.

In rat brain dynamic susceptibility contrast magnetic resonance (MR) images, vessels visible on the same scan plane as the brain tissue were used to measure the characteristics of the input function of the MR contrast agent gadopentetate dimeglumine. MR images were acquired 30 and 60 minutes after intravenous injections of 3 mg/kg and 15 mg/kg NG-Nitro-L-arginine methyl ester (L-NAME) (n = 9). The time of arrival (TOA) and the mean transit time corrected for TOA of the input function were increased by 3 mg/kg or 15 mg/kg L-NAME. The area of the input function was increased by 15 mg/kg L-NAME. In two animals, similar modifications of the input function induced by 20 mg/kg L-NAME were reversed by infusion of sodium nitroprusside. In two other animals, MABP was increased by phenylephrine to a similar extent as in L-NAME experiments, but did not induce the same modifications of the input function, showing that the action of L-NAME on the input function was not simply caused by an effect on MABP. These results show that the input function can be significantly altered by manipulations widely used in cerebrovascular studies. These input function changes have important implications for calculation of cerebral blood flow.

Nerve growth factor and neuronal cell death.

The regulation of neuronal cell death by the neuronotrophic factor, nerve growth factor (NGF), has been described during neural development and following injury to the nervous system. Also, reduced NGF activity has been reported for the aged NGF-responsive neurons of the sympathetic nervous system and cholinergic regions of the central nervous system (CNS) in aged rodents and man. Although there is some knowledge of the molecular structure of the NGF and its receptor, less is known as to the mechanism of action of NGF. Here, a possible role for NGF in the regulation of oxidant--antioxidant balance is discussed as part of a molecular explanation for the known effects of NGF on neuronal survival during development, after injury, and in the aged CNS.

Induction of apoptosis in the CNS during development by the combination of hyperoxia and inhibition of glutathione synthesis.

Apoptosis in the central nervous system (in contrast to necrosis) is an endogenous cell suicide mechanism triggered in response to biological factors and genotoxic stimuli often resulting from oxidative stress. Excessive neural apoptosis may result in longterm brain dysfunction. A significant proportion of prematurely born infants are exposed to high oxygen and nutritional regimens deficient in antioxidant precursors. Such infants frequently display cognitive deficits when studied in later childhood. Studies in cell culture have characterized a close relationship between oxidative stress, glutathione availability and cell death. Here, we assessed this relationship in rat brain, as a model approximation of the situation that occurs in human infants. Two day old rats were exposed to an atmosphere of 95% oxygen and treated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor. Control groups consisted of rat-pups kept in air, air plus BSO, or oxygen alone. At the end of 5 days of treatment, brains were harvested, dissected and nerve growth factor protein (NGF), glutathione, and extent of apoptosis were measured. Hyperoxia induced a decrease in NGF protein while BSO induced a decrease in glutathione concentrations. Animals treated with both hyperoxia and BSO had a dramatic increase in the extent of brain apoptosis detected. We conclude from these studies that the brains of animals exposed to both oxidative stress and limited antioxidant protection are liable to pro-apoptotic changes. Increased cell death via apoptosis reflecting changes in neurotrophin and glutathione homeostasis may represent the mechanism responsible for the induction of the longterm cognitive deficits observed in some preterm infants.

Model for aging in the basal forebrain cholinergic system.

A key component of the cognitive deficits associated with aging is the loss of function of cholinergic neurons in the basal forebrain due to neuronal losses and decreased cholinergic function of spared neurons. A model to mimic one aspect of this phenomenon is to kill cholinergic neurons selectively in the basal forebrain via administration of the immunotoxin IgG-192-saporin. Here we discuss apoptotic regulators, such as nerve growth factor, in age-associated changes present in the cholinergic system and the role of the NF-kappaB signaling system in cellular commitment to apoptosis. We also examine the age-associated decline in intrinsic response mechanisms, which may account for the age-associated reduction in recovery from both acute and chronic insults to the central nervous system.

In vivo ANTI-NGF induces sprouting of sensory axons in dorsal roots.

Newborn rats were given subcutaneous injections of antibodies to mouse beta -NGF (ANTI-NGF) daily for 1 month. The number of neurons in T4-T6 dorsal root ganglia (DRG) and the numbers of myelinated and unmyelinated axons in the dorsal roots of the same segments were counted in the ANTI-NGF animals and in normal littermates. The ANTI-NGF rats had 38% fewer neurons in thoracic ganglia but 17% more myelinated and 40% more unmyelinated fibers than their untreated littermates. Dorsal root ganglion cells also have a larger average size in the ANTI-NGF animals, which we interpret as a disproportionate loss of small cells. These data are interpreted as showing that some dorsal root ganglion cells, principally small ones, die when endogenous NGF is inactivated, and that the remaining cells emit more processes than normal. Thus, removal of NGF has what appears to be a paradoxical effect, a reduction in dorsal root ganglion cell numbers but an increase in dorsal root axon numbers. The relation of myelin thickness to fiber diameter is also altered, with small fibers being more thinly myelinated in the ANTI-NGF group. Thus, Schwann cell-neuronal interactions are also affected by inactivation of NGF.

Participation of active oxygen species in 6-hydroxydopamine toxicity to a human neuroblastoma cell line.

Catalase, superoxide dismutase, and dimethylsulfoxide were tested for their ability to prevent the cytotoxic effect of 6-hydroxydopamine (6-OHDA) on the human neuroblastoma line SY5Y. Viability was measured at two time points after 6-OHDA treatment: at 3 hr by means of amino acid incorporation and at 24 hr by trypan blue dye exclusion. Survival of cells treated concomitantly with catalase (50 microgram/ml) and 6-OHDA was at least 90 per cent that of untreated controls. Cells receiving 6-OHDA alone showed less than 30 per cent survival relative to untreated controls. Superoxide dismutase (50 microgram/ml) temporarily protected cells from a high concentration of 60-OHDA. Dimethylsulfoxide treatment increased survival from the control level 24 hr after treatment with 6-OHDA. Two other cell lines (A1B1 human glial cells and CHO fibroblasts) had intermediate and high resistance to the drug, respectively, compared to the low resistance of SY5Y cells. CHO and SY5Y cells had similar responses to 6-OHDA and to H2O2 when tested at twice the molarity of 6-OHDA. Specific activities of three enzymes known to detoxify H2O2 or H2O2-generated organic hydroperoxides (catalase, glutathione S-transferase, and glutathione peroxidase) were compared in the three cell lines. Catalase activity was 2.5 times as high as in A1B1 and CHO cells as in SY5Y cells when expressed as units/mg protein and 7 times as high in units/culture dish. Other enzyme activities showed no correlation to 6-OHDA resistance.

Stimulation of nerve growth factor receptors in PC12 by acetyl-L-carnitine.

Acetyl-L-carnitine (ALCAR) prevents some deficits associated with aging in the central nervous system (CNS), such as the aged-related reduction of nerve growth factor (NGF) binding. The aim of this study was to ascertain whether ALCAR could affect the expression of an NGF receptor (p75NGFR). Treatment of PC12 cells with ALCAR increased equilibrium binding of 125I-NGF. ALCAR treatment also increased the amount of immunoprecipitable p75NGFR from PC12 cells. Lastly, the level of p75NGFR messenger RNA (mRNA) in PC12 was increased following ALCAR treatment. These results are in agreement with the hypothesis that there is a direct action of ALCAR on p75NGFR expression in aged rodent CNS.

Cationic lipid-mediated NGF gene transfection increases neurofilament phosphorylation.

We examined the effect of cationic lipid-mediated gene transfection of nerve growth factor (NGF) in primary septo-hippocampal cell cultures. Rat NGF cDNA was subcloned into a pUC19-based plasmid containing a CMV promoter. Two days after NGF gene transfection in primary cell cultures, ELISA confirmed increases in NGF protein secretion from transfected cells. To study the biological effect of cationic lipid-mediated NGF gene transfection, we analyzed the amount of neurofilament protein from NGF-transfected cell cultures. Western blot and immunohistochemical analyses detected significant increases in the phosphorylated form of neurofilament proteins in the cultures after cationic lipid-mediated NGF cDNA transfection. Cationic lipid-mediated NGF cDNA transfection did rot cause significant changes in the total amount of neurofilament protein. Our studies suggest that cationic lipid-mediated NGF gene transfection can increase neurofilament phosphorylation but not total neurofilament protein.

Rodent model of chronic central pain after spinal cord contusion injury and effects of gabapentin.

Spinal cord injury (SCI) often results in abnormal pain syndromes in patients. We present a recently developed SCI mammalian model of chronic central pain in which the spinal cord is contused at T8 using the NYU impactor device (10-g rod, 2.0-mm diameter, 12.5-mm drop height), an injury which is characterized behaviorally as moderate. Recovery of locomotor function was assessed with an open field test and scored using the open field test scale (BBB scale). Somatosensory tests of paw withdrawal responses accompanied by supraspinal responses to both mechanical punctate (von Frey hairs) and nonpunctate (4 mm diameter blunt probe) as well as thermal (radiant heat) peripheral stimuli were performed. Comparisons at the level of the individual animal between precontusion and postcontusion responses indicated significant increases in reactions to low threshold punctate mechanical stimuli, non-punctate stimuli and thermal stimuli (p < 0.05). To demonstrate the validity of this model as a central pain model, gabapentin, an agent used clinically for central pain, was given i.p. at 10 or 30 mg/kg. Gabapentin treatment significantly and reversibly changed the responses, consistent with the attenuation of the abnormal sensory behavior, and the attenuated responses lasted for the duration of the drug effect (up to 6 h). These results support the use of the spinal contusion model in the study of chronic central pain after SCI.