Non-specific protection from respiratory tract infections in cattle generated by intranasal administration of an innate immune stimulant.

Alternatives to antibiotics for prevention of respiratory tract infections in cattle are urgently needed given the increasing public and regulatory pressure to reduce overall antibiotic usage. Activation of local innate immune defenses in the upper respiratory tract is one strategy to induce non-specific protection against infection with the diverse array of viral and bacterial pathogens associated with bovine respiratory disease complex (BRDC), while avoiding the use of antibiotics. Our prior studies in rodent models demonstrated that intranasal administration of liposome-TLR complexes (LTC) as a non-specific immune stimulant generated high levels of protection against lethal bacterial and viral pathogens. Therefore, we conducted studies to assess LTC induction of local immune responses and protective immunity to BRDC in cattle. In vitro, LTC were shown to activate peripheral blood mononuclear cells in cattle, which was associated with secretion of INFγ and IL-6. Macrophage activation with LTC triggered intracellular killing of Mannheimia hemolytica and several other bacterial pathogens. In studies in cattle, intranasal administration of LTC demonstrated dose-dependent activation of local innate immune responses in the nasopharynx, including recruitment of monocytes and prolonged upregulation (at least 2 weeks) of innate immune cytokine gene expression by nasopharyngeal mucosal cells. In a BRDC challenge study, intranasal administration of LTC prior to pathogen exposure resulted in significant reduction in both clinical signs of infection and disease-associated euthanasia rates. These findings indicate that intranasal administration of a non-specific innate immune stimulant can be an effective method of rapidly generating generalized protection from mixed viral and bacterial respiratory tract infections in cattle.

Lipopolysaccharide-induced functional and structural injury of the mitochondria in the nigrostriatal pathway.

Accumulating evidence suggests that chronic inflammation plays a role in the progressive dopaminergic neurodegeneration that occurs in Parkinson's disease. It has been hypothesized that inflammation mediates neuronal damage via exacerbation of a vicious cycle of oxidative stress and mitochondrial dysfunction. The bacterial endotoxin, lipopolysaccharide (LPS), induces microglial activation and inflammation driven dopaminergic neurodegeneration. In order to test the hypothesis that LPS-induced inflammatory response might damage mitochondrial structure and function leading to nigral dopaminergic neuron loss, we injected LPS or saline into the striatum of rats. Here, we found that intrastriatal LPS induced deficit in mitochondrial respiration, damage to mitochondrial cristae, mitochondrial oxidation and nitration. Finally, we found significant loss of dopaminergic neurons in the substantia nigra one week after LPS injection. This study indicates that LPS-induced dopaminergic neurodegeneration might be exerted by mitochondrial injury.

Traumatic brain injury and trichloroethylene exposure interact and produce functional, histological, and mitochondrial deficits.

Mitochondria play a pivotal role in the development of pathology associated with Parkinson's disease (PD), traumatic brain injury (TBI), and following exposure to the environmental toxin trichloroethylene (TCE). Evidence from humans indicates that both TBI and TCE can play a role in the development of PD and that each of these insults result in significant mitochondrial dysfunction. In the current studies we hypothesized that exposure to both TCE and TBI would result in increased pathology associated with PD. To test this hypothesis, 16 week old male Fischer 344 rats were administered TCE for either one or two weeks by oral gavage. Following exposure to TCE, rats were subjected to either a sham, mild (1.0mm), or moderate (2.0mm) controlled cortical impact TBI. Given the strong connection between mitochondrial function and PD, TBI, and TCE, tissue from the striatum and substantia nigra were analyzed 6h after the TBI. Neither TCE exposure, TBI, nor the combination of the two insults resulted in mitochondrial deficits at 6h post-TBI in the substantia nigra. Unlike the substantia nigra, the striatum exhibited significant mitochondrial dysfunction. Exposure to TCE alone for two weeks resulted in approximately a 75% reduction in mitochondrial function (p<0.05) in the striatum whereas TBI alone resulted in approximately a 30% reduction in striatal mitochondrial function. Following 1 week exposure to TCE followed by TBI, there was a significant reduction (50%) in mitochondrial function (p<0.05) which required the presence of both insults. Beginning 12 days after the injury significant motor impairment was observed with Rotarod testing. Animals exposed to TCE and a moderate TBI exhibited performance which was approximately 50% of controls (p<0.01). Cylinder testing revealed that at 30 days post-injury animals exposed to TCE and a moderate TBI also had about a 34% reduction in the usage of the contralateral fore paw and this impairment was significantly worse than both control animals and animals exposed to TCE and a mild TBI (p<0.05). At 30 days post-injury there was a 13-17% reduction in the number of tyrosine hydroxylase (TH) positive neurons in the substantia nigra (p<0.05), which was the result of protein loss and not cell death. Loss of TH positive neurons did not result in changes in striatal TH fiber density or levels of the dopamine transporter or type-2 dopamine receptor. Additionally, exposure to TCE prior to the TBI did not increase the loss of cortical tissue, indicating regional specificity for TCE induced deficits. These studies provide further evidence for the connection between TCE, TBI, and PD and lend support to the concept that PD develops from a multifactorial injury scenario.

Glial cell line-derived neurotrophic factor protects midbrain dopaminergic neurons against lipopolysaccharide neurotoxicity.

Aberrant microglia activation causes dopaminergic neuronal loss and nitric oxide produced by microglia plays a critical role in dopaminergic neuronal degeneration. However, no study has determined if GDNF protects dopaminergic neurons via inhibiting nitric oxide generation in Parkinson's disease animal model. We report that GDNF not only reduces lipopolysaccharide-induced degeneration of dopaminergic neurons, suppresses microglia activation and nitric oxide generation, but also reverses the inhibition of phosphoinositide 3-kinase (PI3K) in dopaminergic neurons and microglia. It suggests that the neuroprotective effect of GDNF on dopaminergic neurons may be related to its suppression of microglia activation-mediated nitric oxide via releasing the inhibition of PI3K in both neurons and microglia.

Trichloroethylene induces dopaminergic neurodegeneration in Fisher 344 rats.

Trichloroethylene, a chlorinated solvent widely used as a degreasing agent, is a common environmental contaminant. Emerging evidence suggests that chronic exposure to trichloroethylene may contribute to the development of Parkinson's disease. The purpose of this study was to determine if selective loss of nigrostriatal dopaminergic neurons could be reproduced by systemic exposure of adult Fisher 344 rats to trichloroethylene. In our experiments, oral administration of trichloroethylene induced a significant loss of dopaminergic neurons in the substantia nigra pars compacta in a dose-dependent manner, whereas the number of both cholinergic and GABAergic neurons were not decreased in the striatum. There was a robust decline in striatal levels of 3, 4-dihydroxyphenylacetic acid without a significant depletion of striatal dopamine. Rats treated with trichloroethylene showed defects in rotarod behavior test. We also found a significantly reduced mitochondrial complex I activity with elevated oxidative stress markers and activated microglia in the nigral area. In addition, we observed intracellular alpha-synuclein accumulation in the dorsal motor nucleus of the vagus nerve, with some in nigral neurons, but little in neurons of cerebral cortex. Overall, our animal model exhibits some important features of Parkinsonism, and further supports that trichloroethylene may be an environmental risk factors for Parkinson's disease.

Striatal neuroinflammation promotes Parkinsonism in rats.

BACKGROUND: Sporadic Parkinson's disease (PD) is a progressive neurodegenerative disorder with unknown cause, but it has been suggested that neuroinflammation may play a role in pathogenesis of the disease. Neuroinflammatory component in process of PD neurodegeneration was proposed by postmortem, epidemiological and animal model studies. However, it remains unclear how neuroinflammatory factors contribute to dopaminergic neuronal death in PD. FINDINGS: In this study, we analyzed the relationship among inducible nitric oxide synthase (iNOS)-derived NO, mitochondrial dysfunction and dopaminergic neurodegeneration to examine the possibility that microglial neuroinflammation may induce dopaminergic neuronal loss in the substantia nigra. Unilateral injection of lipopolysaccharide (LPS) into the striatum of rat was followed by immunocytochemical, histological, neurochemical and biochemical analyses. In addition, behavioral assessments including cylinder test and amphetamine-induced rotational behavior test were employed to validate ipsilateral damage to the dopamine nigrostriatal pathway. LPS injection caused progressive degeneration of the dopamine nigrostriatal system, which was accompanied by motor impairments including asymmetric usage of forelimbs and amphetamine-induced turning behavior in animals. Interestingly, some of the remaining nigral dopaminergic neurons had intracytoplasmic accumulation of alpha-synuclein and ubiquitin. Furthermore, defect in the mitochondrial respiratory chain, and extensive S-nitrosylation/nitration of mitochondrial complex I were detected prior to the dopaminergic neuronal loss. The mitochondrial injury was prevented by treatment with L-N(6)-(l-iminoethyl)-lysine, an iNOS inhibitor, suggesting that iNOS-derived NO is associated with the mitochondrial impairment. CONCLUSIONS: These results implicate neuroinflammation-induced S-nitrosylation/nitration of mitochondrial complex I in mitochondrial malfunction and subsequent degeneration of the nigral dopamine neurons.

Intrastriatal lipopolysaccharide injection induces parkinsonism in C57/B6 mice.

A role for inflammation has been hypothesized in the etiology and progression of Parkinson's disease (PD). In this study, we generated, characterized, and validated the first progressive PD-related mouse model (C57/B6) with intrastriatal injection of lipopolysaccharide (LPS). We showed progressive and specific dopaminergic neurodegeneration in the substantia nigra, which is accompanied by striatal dopamine depletion and progressive behavioral impairment, which was alleviated by the use of the PD drug L-Dopa. We focused on the role of nitric oxide (NO) in inflammation-promoted cell death and suggest that the expression of the inducible NO synthase plays a role in the progressive loss of dopaminergic neurons but not the initial loss induced by LPS. With this model, future research can be performed in gene knockout mice to study other potential mechanisms of inflammation-induced neurodegeneration. In addition, this model can be used to screen therapeutics for PD at a more clinically relevant time (i.e., after LPS injection but before manifestation of PD-related behavioral impairment), because most PD drugs are screened in animal models in which inhibitors are given predisease induction. Thus, this novel PD-related model should be further characterized and strongly considered as a tool for future drug studies.

Phenidone protects the nigral dopaminergic neurons from LPS-induced neurotoxicity.

Anti-inflammatory drugs such as ibuprofen appear to prevent the development of Parkinson's disease (PD); however, long-term use has undesirable side-effects. A new strategy for anti-inflammatory drug therapy is using a dual inhibitor of COX and lipooxygenase (LOX). Here, we compared the dopaminergic neuroprotective property of phenidone (a dual COX and LOX inhibitor) with COX or LOX inhibitors including SC-560 (a COX-1 inhibitor), aspirin (a COX-1/2 inhibitor), meloxicam (a preferential COX-2 inhibitor), caffeic acid (a 5-LOX inhibitor), and esculetin (a 5, 12-LOX inhibitor) in our lipopolysaccharide (LPS)-induced PD animal model. Our results show that COX-2 and 5-LOX play a major role in LPS-induced dopaminergic neurotoxicity, as meloxicam and phenidone attenuated LPS-induced oxidative stress and meloxicam, phenidone, and caffeic acid attenuated dopaminergic neurodegeneration, while SC-560, aspirin, and esculetin did not. In addition, phenidone was superior in attenuating LPS-induced dopaminergic neurodegeneration and microglia activation, probably as a result of dual inhibition of COX-2 and LOX. Therefore, dual inhibition of COX and LOX with phenidone represents a promising new candidate for anti-inflammatory drug therapy, and may provide a novel therapeutic approach for inflammation-related neurodegenerative diseases including PD.

Microsomal epoxide hydrolase deletion enhances tyrosine hydroxylase phosphorylation in mice after MPTP treatment.

Parkinson's disease (PD) is the most prevalent neurodegenerative movement disorder. Epidemiological studies have suggested most cases of PD are linked to environmental risk factors. Microsomal epoxide hydrolase (mEH) is a conserved enzyme that catalyzes hydrolysis of a large number of epoxide intermediates such as drugs and epoxides of environmental toxins. We hypothesize that changes in mEH are involved in the pathogenesis of PD by modulating the vulnerability of dopaminergic neurons to environmental stress. Herein we reported that acute treatment with the neurotoxin MPTP (1-methyl-4-phemyl-1,2,3,6-tetrahydropyridine) markedly increased the mEH immunoreactivity in the nigrostriatal system of C57BL/6 mice. Next, mEH knockout (KO) mice were used, and we found that tyrosine hydroxylase (TH)-positive cell loss was significantly lower in the substantia nigra of mEH KO mice compared with wild-type (WT) mice after MPTP treatment. The mean dopamine turnover ratios were significantly increased in MPTP-treated mEH KO mice compared with WT. In addition, TH is the rate-limiting enzyme for dopamine biosynthesis, and its activity is mainly regulated by TH phosphorylation at Ser-31 (pSer31) and Ser-40 (pSer40). Double immunofluorescence showed that both pSer31 and pSer40 are completely colocalized in total TH-positive cells. However, immunoblotting confirmed that there was a significantly higher level of pSer31 in mEH-KO mice when compared with WT mice after MPTP, and no marked differences among TH and its phosphorylation levels occurred after saline injection. These data suggested that mEH deficiency facilitates TH phosphorylation in the nigrostriatal dopamine system, which may be associated with an increased resistance of dopaminergic neurons to environmental toxins.

Protective properties afforded by pioglitazone against intrastriatal LPS in Sprague-Dawley rats.

We created an inflammation-induced Parkinson's disease model, where microglia activation leads to oxidative stress, mitochondrial dysfunction, and dopaminergic neurodegeneration in the substantia nigra. Pioglitazone, an agonist of peroxisome proliferator activated receptor-gamma (PPAR-gamma), can prevent these deficits and protect dopaminergic neurons. To continue exploring the effects of pioglitazone in this model we focused on the expression of PPAR-gamma, uncoupling protein 2 (UCP2), and mitoNEET. We report that intrastriatal lipopolysaccharide (LPS) increases striatal PPAR-gamma, UCP2, and mitoNEET expression, and pioglitazone attenuates these LPS-induced changes.

Pioglitazone inhibition of lipopolysaccharide-induced nitric oxide synthase is associated with altered activity of p38 MAP kinase and PI3K/Akt.

BACKGROUND: Previous studies have suggested that peroxisome proliferator activated receptor-gamma (PPAR-gamma)-mediated neuroprotection involves inhibition of microglial activation and decreased expression and activity of inducible nitric oxide synthase (iNOS); however, the underlying molecular mechanisms have not yet been well established. In the present study we explored: (1) the effect of the PPAR-gamma agonist pioglitazone on lipopolysaccharide (LPS)-induced iNOS activity and nitric oxide (NO) generation by microglia; (2) the differential role of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun NH(2)-terminal kinase (JNK), and phosphoinositide 3-kinase (PI3K) on LPS-induced NO generation; and (3) the regulation of p38 MAPK, JNK, and PI3K by pioglitazone. METHODS: Mesencephalic neuron-microglia mixed cultures, and microglia-enriched cultures were treated with pioglitazone and/or LPS. The protein levels of iNOS, p38 MAPK, JNK, PPAR-gamma, PI3K, and protein kinase B (Akt) were measured by western blot. Different specific inhibitors of iNOS, p38MAPK, JNK, PI3K, and Akt were used in our experiment, and NO generation was measured using a nitrite oxide assay kit. Tyrosine hydroxylase (TH)-positive neurons were counted in mesencephalic neuron-microglia mixed cultures. RESULTS: Our results showed that pioglitazone inhibits LPS-induced iNOS expression and NO generation, and inhibition of iNOS is sufficient to protect dopaminergic neurons against LPS insult. In addition, inhibition of p38 MAPK, but not JNK, prevented LPS-induced NO generation. Further, and of interest, pioglitazone inhibited LPS-induced phosphorylation of p38 MAPK. Wortmannin, a specific PI3K inhibitor, enhanced p38 MAPK phosphorylation upon LPS stimulation of microglia. Elevations of phosphorylated PPAR-gamma, PI3K, and Akt levels were observed with pioglitazone treatment, and inhibition of PI3K activity enhanced LPS-induced NO production. Furthermore, wortmannin prevented the inhibitory effect of pioglitazone on the LPS-induced NO increase. CONCLUSION: We demonstrate that pioglitazone protects dopaminergic neurons against LPS insult at least via inhibiting iNOS expression and NO generation, which is potentially mediated via inhibition of p38 MAPK activity. In addition, the PI3K pathway actively participates in the negative regulation of LPS-induced NO production. Our findings suggest that PPAR-gamma activation may involve differential regulation of p38 MAPK and of the PI3K/Akt pathway in the regulation of the inflammatory process.

Inflammation and age-related iron accumulation in F344 rats.

Evidence suggests chronic inflammation and iron accumulation may play a role in the pathogenesis of Parkinson's disease (PD) as inflammation and iron levels increase with age and appear in the disease pathology. It is hypothesized that an aggravated inflammatory response and iron accumulation, as a function of age, increase oxidative stress and participate in the pathogenesis of PD. Intracranial injection of the bacterial endotoxin, lipopolysaccharide (LPS), has been shown to induce microglia activation, oxidative stress, mitochondrial impairment, iron accumulation, and dopaminergic neurodegeneration within the substantia nigra. We tested the hypothesis that injection of LPS into the striatum would increase iron accumulation in the substantia nigra of aged rats compared to young ones. Our results showed that four weeks post injection, LPS significantly increased microglia activation, lipid peroxidation, ferritin expression, and total nigral iron content in aged rats. In addition, LPS significantly altered the turnover ratio of homovanillic acid to dopamine. Thus, an age-related increase in iron as well as susceptibility to inflammation may play an important role in PD-related neurodegeneration, as free radicals produced from the inflammatory response can become more toxic through increased ferrous iron catalyzed Fenton chemistry. This may enhance oxidative stress, exacerbate microglia activation, and drive the progression of PD.

Trichloroethylene: Parkinsonism and complex 1 mitochondrial neurotoxicity.

OBJECTIVE: To analyze a cluster of 30 industrial coworkers with Parkinson's disease and parkinsonism subjected to long-term (8-33 years) chronic exposure to trichloroethylene. METHODS: Neurological evaluations were conducted on the 30 coworkers, including a general physical and neurological examination and the Unified Parkinson's Disease Rating Scale. In addition, fine motor speed was quantified and an occupational history survey was administered. Next, animal studies were conducted to determine whether trichloroethylene exposure is neurotoxic to the nigrostriatal dopamine system that degenerates in Parkinson's disease. The experiments specifically analyzed complex 1 mitochondrial neurotoxicity because this is a mechanism of action of other known environmental dopaminergic neurotoxins. RESULTS: The three workers with workstations adjacent to the trichloroethylene source and subjected to chronic inhalation and dermal exposure from handling trichloroethylene-soaked metal parts had Parkinson's disease. Coworkers more distant from the trichloroethylene source, receiving chronic respiratory exposure, displayed many features of parkinsonism, including significant motor slowing. Neurotoxic actions of trichloroethylene were demonstrated in accompanying animal studies showing that oral administration of trichloroethylene for 6 weeks instigated selective complex 1 mitochondrial impairment in the midbrain with concomitant striatonigral fiber degeneration and loss of dopamine neurons. INTERPRETATION: Trichloroethylene, used extensively in industry and the military and a common environmental contaminant, joins other mitochondrial neurotoxins, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and some pesticides, as a risk factor for parkinsonism.

Fenbendazole treatment may influence lipopolysaccharide effects in rat brain.

In evaluating discrepant results between experiments in our laboratory, we collected data that challenge the notion that anthelminthic drugs like FBZ do not alter inflammatory responses. We found that FBZ significantly modulates inflammation in F344 rats intrastriatally injected with LPS. FBZ treatment of LPS-injected rats significantly increased weight loss, microglial activation, and dopamine loss; in addition, FBZ attenuated the LPS-induced loss of astrocytes. Therefore, FBZ treatment altered the effects of LPS injection. Caution should be used in interpreting data collected from rats treated with LPS and FBZ.

Inflammation induces mitochondrial dysfunction and dopaminergic neurodegeneration in the nigrostriatal system.

Evidence suggests that chronic inflammation, mitochondrial dysfunction, and oxidative stress play significant and perhaps synergistic roles in Parkinson's disease (PD), where the primary pathology is significant loss of the dopaminergic neurons in the substantia nigra. The use of anti-inflammatory drugs for PD treatment has been proposed, and inhibition of cyclo-oxygenase-2 (COX-2) or activation of peroxisome proliferator-activated receptor gamma (PPAR-gamma) yields neuroprotection in MPTP-induced PD. Lipopolysaccharide (LPS) induces inflammation-driven dopaminergic neurodegeneration. We tested the hypothesis that celecoxib (Celebrex, COX-2 inhibitor) or pioglitazone (Actos, PPAR-gamma agonist) will reduce the LPS-induced inflammatory response, spare mitochondrial bioenergetics, and improve nigral dopaminergic neuronal survival. Rats were treated with vehicle, celecoxib, or pioglitazone and were intrastriatally injected with LPS. Inflammation, mitochondrial dysfunction, oxidative stress, decreased dopamine, and nigral dopaminergic neuronal loss were observed post-LPS. Celecoxib and pioglitazone provided neuroprotective properties by decreasing inflammation and restoring mitochondrial function. Pioglitazone also attenuated oxidative stress and partially restored striatal dopamine as well as demonstrated dopaminergic neuroprotection and reduced nigral microglial activation. In summary, intrastriatal LPS served as a model for inflammation-induced dopaminergic neurodegeneration, anti-inflammatory drugs provided protective properties, and pioglitazone or celecoxib may have therapeutic potential for the treatment of neuro-inflammation and PD.

Cyclooxygenase-2 mediates microglial activation and secondary dopaminergic cell death in the mouse MPTP model of Parkinson's disease.

BACKGROUND: Accumulating evidence suggests that inflammation plays an important role in the progression of Parkinson's disease (PD). Among many inflammatory factors found in the PD brain, cyclooxygenase (COX), specifically the inducible isoform, COX-2, is believed to be a critical enzyme in the inflammatory response. Induction of COX-2 is also found in an experimental model of PD produced by administration of 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). METHOD: COX-2-deficient mice or C57BL/6 mice were treated with MPTP to investigate the effects of COX-2 deficiency or by using various doses of valdecoxib, a specific COX-2 inhibitor, which induces inhibition of COX-2 on dopaminergic neuronal toxicity and locomotor activity impairment. Immunohistochemistry, stereological cell counts, immunoblotting, an automated spontaneous locomotor activity recorder and rotarod behavioral testing apparatus were used to assess microglial activation, cell loss, and behavioral impairments. RESULTS: MPTP reduced tyrosine hydroxylase (TH)-positive cell counts in the substantia nigra pars compacta (SNpc); total distance traveled, vertical activity, and coordination on a rotarod; and increased microglia activation. Valdecoxib alleviated the microglial activation, the loss of TH-positive cells and the decrease in open field and vertical activity. COX-2 deficiency attenuated MPTP-induced microglial activation, degeneration of TH-positive cells, and loss of coordination. CONCLUSION: These results indicate that reducing COX-2 activity can mitigate the secondary and progressive loss of dopaminergic neurons as well as the motor deficits induced by MPTP, possibly by suppression of microglial activation in the SNpc.

Competition modeling of American football: observational data and implications for high school, collegiate, and professional player conditioning.

In an effort to increase the transfer of training to sport performance, sport-specific training programs should be developed. Competition modeling has been proposed as a method for developing metabolic conditioning programs that mimic competition environments. This process involves both a qualitative and quantitative evaluation of competitive conditions of a sport. The purpose of this observational research was to construct a competition model of American football for 3 different levels: high school, collegiate, and professional. Observations of 30 football games at different levels were conducted and modeled with respect to length of play, length of recovery between plays, plays per series, and stoppages per series. The resultant data demonstrated that differences in these variables exist between levels of play. High school plays lasted, on average, 5.6 +/- 2.0 seconds and were slightly longer than college (+0.47 seconds) and professional (+0.44 seconds) plays. The average time for recovery between plays was longest in National Football League (NFL) games and shortest in high school. On average, the work to recovery ratio was most strenuous in high school (1:5.5), college (1:6.1), and NFL (1:6.2), respectively. Differences in the identified competitive conditions, although slight, do exist among high school, collegiate, and professional football. In order to design specific metabolic training programs for American football, coaches should consider the identified models. Exercise to rest ratios and volume of work performed in a training session should be designed to ensure that players are preparing specifically for identified game conditions.