Behavioral and Physiological Alterations in Angus Steers Grazing Endophyte-Infected Toxic Fescue during Late Fall.

Fescue toxicosis is caused by grazing ergot alkaloid-producing endophyte (Epichloë coenophiala)-infected tall fescue (E+). Summer grazing of E+ leads to decreased productivity, associated impaired thermoregulation, and altered behavior. The goal of this study was to determine the role of E+ grazing-climate interaction on animal behavior and thermoregulation during late fall. Eighteen Angus steers were placed on nontoxic (NT), toxic (E+) and endophyte-free (E-) fescue pastures for 28 days. Physiological parameters, such as rectal temperature (RT), respiration rate (RR), ear and ankle surface temperature (ET, AT), and body weights, were measured. Skin surface temperature (SST) and animal activity were recorded continuously with temperature and behavioral activity sensors, respectively. Environmental conditions were collected using paddocks-placed data loggers. Across the trial, steers on E+ gained about 60% less weight than the other two groups. E+ steers also had higher RT than E- and NT, and lower SST than NT post-pasture placement. Importantly, animals grazing E+ spent more time lying, less time standing, and took more steps. These data suggest that late fall E+ grazing impairs core and surface temperature regulation and increases non-productive lying time, which may be partly responsible for the observed decreased weight gains.

Evaluation of delayed LNFPIII treatment initiation protocol on improving long-term behavioral and neuroinflammatory pathology in a mouse model of Gulf War Illness.

Chemical overexposures and war-related stress during the 1990-1991 Gulf War (GW) are implicated in the persisting pathological symptoms that many GW veterans continue to endure. These symptoms culminate into a disease known as Gulf War Illness (GWI) and affect about a third of the GW veteran population. Currently, comprehensive effective GWI treatment options are unavailable. Here, an established GWI mouse model was utilized to explore the (1) long-term behavioral and neuroinflammatory effects of deployment-related GWI chemicals exposure and (2) ability of the immunotherapeutic lacto-N-fucopentaose III (LNFPIII) to improve deficits when given months after the end of exposure. Male C57BL6/J mice (8-9 weeks old) were administered pyridostigmine bromide (PB) and DEET for 14 days along with corticosterone (CORT; latter 7 days) to emulate wartime stress. On day 15, a single injection of the nerve agent surrogate diisopropylfluorophosphate (DFP) was given. LNFPIII treatment began 7 months post GWI chemicals exposure and continued until study completion. A battery of behavioral tests for assessment of cognition/memory, mood, and motor function in rodents was performed beginning 8 months after exposure termination and was then followed by immunohistochemcal evaluation of neuroinflammation and neurogenesis. Within tests of motor function, prior GWI chemical exposure led to hyperactivity, impaired sensorimotor function, and altered gait. LNFPIII attenuated these motor-related deficits and improved overall grip strength. GWI mice also exhibited more anxiety-like behavior that was reduced by LNFPIII; this was test-specific. Short-term, but not long-term memory, was impaired by prior GWI exposure; LNFPIII improved this measure. In the brains of GWI mice, but not in mice treated with LNFPIII, glial activation was increased. Overall, it appears that months after exposure to GWI chemicals, behavioral deficits and neuroinflammation are present. Many of these deficits were attenuated by LNFPIII when treatment began long after GWI chemical exposure termination, highlighting its therapeutic potential for veterans with GWI.

Integrative interactomics applied to bovine fescue toxicosis.

Bovine fescue toxicosis (FT) is caused by grazing ergot alkaloid-producing endophyte (Epichloë coenophiala)-infected tall fescue. Endophyte's effects on the animal's microbiota and metabolism were investigated recently, but its effects in planta or on the plant-animal interactions have not been considered. We examined multi-compartment microbiota-metabolome perturbations using multi-'omics (16S and ITS2 sequencing, plus untargeted metabolomics) in Angus steers grazing non-toxic (Max-Q) or toxic (E+) tall fescue for 28 days and in E+ plants. E+ altered the plant/animal microbiota, decreasing most ruminal fungi, with mixed effects on rumen bacteria and fecal microbiota. Metabolic perturbations occurred in all matrices, with some plant-animal overlap (e.g., Vitamin B6 metabolism). Integrative interactomics revealed unique E+ network constituents. Only E+ had ruminal solids OTUs within the network and fecal fungal OTUs in E+ had unique taxa (e.g., Anaeromyces). Three E+-unique urinary metabolites that could be potential biomarkers of FT and targeted therapeutically were identified.

Sex differences in behavior, response to LPS, and glucose homeostasis in middle-aged mice.

Sex and age have distinct influences and roles in behavior and immune reactivity; yet, most studies use adult male rodents with little attention to middle age, a time associated with key physiological transitions in both sexes. Thus, this study investigated sex differences during middle age in behavior, immune response to lipopolysaccharide (LPS), and glucose regulation in C57BL/6 mice with GFP-tagged monocytes/microglia. Behaviorally, males performed better in tests of motor function (Open Field [OF], Grip Strength, Sticker Removal, Gait, and Pole tests) and displayed less depressive- and anxiety-like behaviors across multiple mood tests (OF, Elevated Zero Maze, Sucrose Preference, and Swim test). However, females performed better in tests of cognition (Barnes Maze and Novel Object Recognition). Following behavioral assessment, mice were given LPS to characterize sex-dependent inflammagen responses. Females displayed greater sickness behavior in the OF, higher levels of peripheral cytokines, and subtle neuroinflammation in the cortex, striatum, and hippocampus. A separate middle-aged cohort was used for glucose tolerance and insulin sensitivity testing. Both sexes had excessive blood glucose rebound after insulin challenge, but displayed differences following glucose administration, where males had higher baseline glucose and females remained hyperglycemic. This study suggests that during middle-age male mice have better emotional regulation and motor function, but not cognitive ability than females. Further, males are less sensitive than females to the acute effects of LPS peripherally and centrally, but both sexes showed sex-specific impairments in blood glucose regulation. Overall, it appears that middle age is an important transition point with multiple sex differences, some of which are unique to this stage of life.

Delayed treatment with the immunotherapeutic LNFPIII ameliorates multiple neurological deficits in a pesticide-nerve agent prophylactic mouse model of Gulf War Illness.

Residual effects of the 1990-1991 Gulf War (GW) still plague veterans 30 years later as Gulf War Illness (GWI). Thought to stem mostly from deployment-related chemical overexposures, GWI is a disease with multiple neurological symptoms with likely immunological underpinnings. Currently, GWI remains untreatable, and the long-term neurological disease manifestation is not characterized fully. The present study sought to expand and evaluate the long-term implications of prior GW chemicals exposure on neurological function 6-8 months post GWI-like symptomatology induction. Additionally, the beneficial effects of delayed treatment with the glycan immunotherapeutic lacto-N-fucopentaose III (LNFPIII) were evaluated. Male C57BL/6J mice underwent a 10-day combinational exposure (i.p.) to GW chemicals, the nerve agent prophylactic pyridostigmine bromide (PB) and the insecticide permethrin (PM; 0.7 and 200 mg/kg, respectively). Beginning 4 months after PB/PM exposure, a subset of the mice were treated twice a week until study completion with LNFPIII. Evaluation of cognition/memory, motor function, and mood was performed beginning 1 month after LNFPIII treatment initiation. Prior exposure to PB/PM produced multiple locomotor, neuromuscular, and sensorimotor deficits across several motor tests. Subtle anxiety-like behavior was also present in PB/PM mice in mood tests. Further, PB/PM-exposed mice learned at a slower rate, mostly during early phases of the learning and memory tests employed. LNFPIII treatment restored or improved many of these behaviors, particularly in motor and cognition/memory domains. Electrophysiology data collected from hippocampal slices 8 months post PB/PM exposure revealed modest aberrations in basal synaptic transmission and long-term potentiation in the dorsal or ventral hippocampus that were improved by LNFPIII treatment. Immunohistochemical analysis of tyrosine hydroxylase (TH), a dopaminergic marker, did not detect major PB/PM effects along the nigrostriatal pathway, but LNFPIII increased striatal TH. Additionally, neuroinflammatory cells were increased in PB/PM mice, an effect reduced by LNFPIII. Collectively, long-term neurobehavioral and neurobiological dysfunction associated with prior PB/PM exposure was characterized; delayed LNFPIII treatment provided multiple behavioral and biological beneficial effects in the context of GWI, highlighting its potential as a GWI therapeutic.

Lacto-N-fucopentaose-III ameliorates acute and persisting hippocampal synaptic plasticity and transmission deficits in a Gulf War Illness mouse model.

AIMS: The present study investigated if treatment with the immunotherapeutic, lacto-N-fucopentaose-III (LNFPIII), resulted in amelioration of acute and persisting deficits in synaptic plasticity and transmission as well as trophic factor expression along the hippocampal dorsoventral axis in a mouse model of Gulf War Illness (GWI). MAIN METHODS: Mice received either coadministered or delayed LNFPIII treatment throughout or following, respectively, exposure to a 15-day GWI induction paradigm. Subsets of animals were subsequently sacrificed 48 h, seven months, or 11 months post GWI-related (GWIR) exposure for hippocampal qPCR or in vitro electrophysiology experiments. KEY FINDINGS: Progressively worsened impairments in hippocampal synaptic plasticity, as well as a biphasic effect on hippocampal synaptic transmission, were detected in GWIR-exposed animals. Dorsoventral-specific impairments in hippocampal synaptic responses became more pronounced over time, particularly in the dorsal hippocampus. Notably, delayed LNFPIII treatment ameliorated GWI-related aberrations in hippocampal synaptic plasticity and transmission seven and 11 months post-exposure, an effect that was consistent with enhanced hippocampal trophic factor expression and absence of increased interleukin 6 (IL-6) in animals treated with LNFPIII. SIGNIFICANCE: Approximately a third of Gulf War Veterans have GWI; however, GWI therapeutics are presently limited to targeted and symptomatic treatments. As increasing evidence underscores the substantial role of persisting neuroimmune dysfunction in GWI, efficacious neuroactive immunotherapeutics hold substantial promise in yielding GWI remission. The findings in the present report indicate that LNFPIII may be an efficacious candidate for ameliorating persisting neurological abnormalities presented in GWI.

Lacto-N-fucopentaose-III (LNFPIII) ameliorates acute aberrations in hippocampal synaptic transmission in a Gulf War Illness animal model.

Approximately one-third of Persian Gulf War veterans are afflicted by Gulf War Illness (GWI), a chronic multisymptom condition that fundamentally presents with cognitive deficits (i.e., learning and memory impairments) and neuroimmune dysfunction (i.e., inflammation). Factors associated with GWI include overexposures to neurotoxic pesticides and nerve agent prophylactics such as permethrin (PM) and pyridostigmine bromide (PB), respectively. GWI-related neurological impairments associated with PB-PM overexposures have been recapitulated in animal models; however, there is a paucity of studies assessing PB-PM-related aberrations in hippocampal synaptic plasticity and transmission that may underlie behavioral impairments. Importantly, FDA-approved neuroactive treatments are currently unavailable for GWI. In the present study, we assessed the efficacy of an immunomodulatory therapeutic, lacto-N-fucopentaose-III (LNFPIII), on ameliorating acute effects of in vivo PB-PM exposure on synaptic plasticity and transmission as well as trophic factor/cytokine expression along the hippocampal dorsoventral axis. PB-PM exposure resulted in hippocampal synaptic transmission deficits 48 h post-exposure, a response that was ameliorated by LNFPIII coadministration, particularly in the dorsal hippocampus (dH). LNFPIII coadministration also enhanced synaptic transmission in the dH and the ventral hippocampus (vH). Notably, LNFPIII coadministration elevated long-term potentiation in the dH. Further, PB-PM exposure and LNFPIII coadministration uniquely altered key inflammatory cytokine and trophic factor production in the dH and the vH. Collectively, these findings demonstrate that PB-PM exposure impaired hippocampal synaptic responses 48 h post-exposure, impairments that differentially manifested along the dorsoventral axis. Importantly, LNFPIII ameliorated GWI-related electrophysiological deficits, a beneficial effect indicating the potential efficacy of LNFPIII for treating GWI.

Sex- and age-dependent alterations of splenic immune cell profile and NK cell phenotypes and function in C57BL/6J mice.

BACKGROUND: Physiological homeostasis decline, immunosenescence, and increased risk for multiple diseases, including neurodegeneration, are all hallmarks of ageing. Importantly, it is known that the ageing process is sex-biased. For example, there are sex differences in predisposition for multiple age-related diseases, including neurodegenerative and autoimmune diseases. However, sex differences in age-associated immune phenotypes are not clearly understood. RESULTS: Here, we examined the effects of age on immune cell phenotypes in both sexes of C57BL/6J mice with a particular focus on NK cells. We found female-specific spleen weight increases with age and concordant reduction in the number of splenocytes per gram of spleen weight compared to young females. To evaluate sex- and age-associated changes in splenic immune cell composition, we performed flow cytometry analysis. In male mice, we observed an age-associated reduction in the frequencies of monocytes and NK cells; female mice displayed a reduction in B cells, NK cells, and CD8 + T cells and increased frequency of monocytes and neutrophils with age. We then performed a whole blood stimulation assay and multiplex analyses of plasma cytokines and observed age- and sex-specific differences in immune cell reactivity and basal circulating cytokine concentrations. As we have previously illustrated a potential role of NK cells in Parkinson's disease, an age-related neurodegenerative disease, we further analyzed age-associated changes in NK cell phenotypes and function. There were distinct differences between the sexes in age-associated changes in the expression of NK cell receptors, IFN-γ production, and impairment of α-synuclein endocytosis. CONCLUSIONS: This study demonstrates sex- and age-specific alterations in splenic lymphocyte composition, circulating cytokine/chemokine profiles, and NK cell phenotype and effector functions. Our data provide evidence that age-related physiological perturbations differ between the sexes which may help elucidate sex differences in age-related diseases, including neurodegenerative diseases, particularly Parkinson's disease, where immune dysfunction is implicated in their etiology.

Assessing the Beneficial Effects of the Immunomodulatory Glycan LNFPIII on Gut Microbiota and Health in a Mouse Model of Gulf War Illness.

The microbiota's influence on host (patho) physiology has gained interest in the context of Gulf War Illness (GWI), a chronic disorder featuring dysregulation of the gut-brain-immune axis. This study examined short- and long-term effects of GWI-related chemicals on gut health and fecal microbiota and the potential benefits of Lacto-N-fucopentaose-III (LNFPIII) treatment in a GWI model. Male C57BL/6J mice were administered pyridostigmine bromide (PB; 0.7 mg/kg) and permethrin (PM; 200 mg/kg) for 10 days with concurrent LNFPIII treatment (35 µg/mouse) in a short-term study (12 days total) and delayed LNFPIII treatment (2×/week) beginning 4 months after 10 days of PB/PM exposure in a long-term study (9 months total). Fecal 16S rRNA sequencing was performed on all samples post-LNFPIII treatment to assess microbiota effects of GWI chemicals and acute/delayed LNFPIII administration. Although PB/PM did not affect species composition on a global scale, it affected specific taxa in both short- and long-term settings. PB/PM elicited more prominent long-term effects, notably, on the abundances of bacteria belonging to Lachnospiraceae and Ruminococcaceae families and the genus Allobaculum. LNFPIII improved a marker of gut health (i.e., decreased lipocalin-2) independent of GWI and, importantly, increased butyrate producers (e.g., Butyricoccus, Ruminococcous) in PB/PM-treated mice, indicating a positive selection pressure for these bacteria. Multiple operational taxonomic units correlated with aberrant behavior and lipocalin-2 in PB/PM samples; LNFPIII was modulatory. Overall, significant and lasting GWI effects occurred on specific microbiota and LNFPIII treatment was beneficial.

Structure and redox properties of the diheme electron carrier cytochrome c4 from Pseudomonas aeruginosa.

At low oxygen concentrations, respiration of Pseudomonas aeruginosa (Pa) and other bacteria relies on activity of cytochrome cbb3 oxidases. A diheme cytochrome c4 (cyt c4) donates electrons to Pa cbb3 oxidases to enable oxygen reduction and proton pumping by these enzymes. Given the importance of this redox pathway for bacterial pathogenesis, both cyt c4 and cbb3 oxidase are potential targets for new antibacterial strategies. The structural information about these two proteins, however, is scarce, and functional insights for Pa and other bacteria have been primarily drawn from analyses of the analogous system from Pseudomonas stutzeri (Ps). Herein, we describe characterization of structural and redox properties of cyt c4 from Pa. The crystal structure of Pa cyt c4 has revealed that this protein is organized in two monoheme domains. The interdomain interface is more hydrophobic in Pa cyt c4, and the protein surface does not show the dipolar distribution of charges found in Ps cyt c4. The reduction potentials of the two hemes are similar in Pa cyt c4 but differ by about 100 mV in Ps cyt c4. Analyses of structural models of these and other cyt c4 proteins suggest that multiple factors contribute to the potential difference of the two hemes in these proteins, including solvent accessibility of the heme group, the distribution of surface charges, and the nature of the interdomain interface. The distinct properties of cyt c4 proteins from closely-related Pa and Ps bacteria emphasize the importance of examining the cbb3/cyt c4 redox pathway in multiple species.

Cellular and behavioral effects of lipopolysaccharide treatment are dependent upon neurokinin-1 receptor activation.

BACKGROUND: Several psychiatric conditions are affected by neuroinflammation and neuroimmune activation. The transcription factor nuclear factor kappa light-chain-enhancer of activated B cells (NFkB) plays a major role in inflammation and innate immunity. The neurokinin-1 receptor (NK1R) is the primary endogenous target of the neuroactive peptide substance P, and some data suggests that NK1R stimulation may influence NFkB activity. Both NK1R and NFkB have been shown to play a functional role in complex behaviors including stress responsivity, depression, and addiction. In this study, we test whether NFkB activity in the brain (stimulated by lipopolysaccharide administration) is dependent upon the NK1R. METHODS: Adult male Wistar rats were treated systemically with the NK1R antagonist L822429 followed by administration of systemic lipopolysaccharide (LPS, a strong activator of NFkB). Hippocampal extracts were used to assess expression of proinflammatory cytokines and NFkB-DNA-binding potential. For behavioral studies, rats were trained to consume 1% (w/v) sucrose solution in a continuous access two-bottle choice model. After establishment of baseline, animals were treated with L822429 and LPS and sucrose preference was measured 12 h post-treatment. RESULTS: Systemic LPS treatment causes a significant increase in proinflammatory cytokine expression and NFkB-DNA-binding activity within the hippocampus. These increases are attenuated by systemic pretreatment with the NK1R antagonist L822429. Systemic LPS treatment also led to the development of anhedonic-like behavior, evidenced by decreased sucrose intake in the sucrose preference test. This behavior was significantly attenuated by systemic pretreatment with the NK1R antagonist L822429. CONCLUSIONS: Systemic LPS treatment induced significant increases in NFkB activity, evidenced by increased NFkB-DNA binding and by increased proinflammatory cytokine expression in the hippocampus. LPS also induced anhedonic-like behavior. Both the molecular and behavioral effects of LPS treatment were significantly attenuated by systemic NK1R antagonism, suggesting that NK1R stimulation lies upstream of NFkB activation following systemic LPS administration and is at least in part responsible for NFkB activation.

The effects of repeated nitroglycerin administrations in rats; modeling migraine-related endpoints and chronification.

BACKGROUND: Rodent models typically use a single nitroglycerin injection to induce migraine, yet migraine in clinical populations presents as recurrent episodes. Further, these models quantify behavioral endpoints that do not align with the clinical features of episodic migraine or migraine chronification and therefore may limit translational relevance. NEW METHOD: Rats received 5 nitroglycerin (10mg/kg/2ml), propylene glycol/ethanol vehicle, or saline injections every third day over 15days. Behavioral endpoints were assessed 110min post nitroglycerin administration and included time spent light/dark chambers for photophobia as well as activity, facial pain expressions, and tactile allodynia. RESULTS: Animals administered nitroglycerin displayed photophobia, decreased activity, and increased facial pain expression. Similar alterations in photophobia and activity were seen in the vehicle treated animals, but these tended to diminish by the 4th or 5th injection. The presentation of spontaneous tactile allodynia was observed in the nitroglycerin group by the 5th episode. COMPARISON WITH EXISTING METHODS: Most NTG migraine models entail a single NTG administration and quantification of evoked allodynia. This paradigm employs recurring NTG episodes and clinically-relevant measures of photophobia, hypoactivity and facial grimace endpoints as well as introduces a novel arena apparatus to quantify spontaneous allodynia. CONCLUSIONS: This repeated NTG procedure and endpoint measures aligns with the frequency and clinical presentation of episodic migraine and its chronification, respectively. Further, propylene glycol ethanol vehicle contributes to migraine endpoints.

The effects of Sceletium tortuosum (L.) N.E. Br. extract fraction in the chick anxiety-depression model.

ETHNOPHARMACOLOGICAL RELEVANCE: Sceletium tortuosum (L.) N.E. Br. has been reported to elevate mood, reduce anxiety and stress and alleviate pain. AIM OF STUDY: This study sought to examine the effects of an S. tortuosum alkaloid enriched fraction in the chick anxiety-depression model, a model that shows high predictive validity as a pharmacological screening assay. MATERIAL AND METHODS: Socially-raised male Silver Laced Wyandotte chicks (4-6 days old) were given IP vehicle, imipramine (10mg/kg), or S. tortuosum fraction (10, 20, 30mg/kg in Exp. 1 or 50, 75, 100mg/kg in Exp. 2) 15min prior to a 60min isolation test period in which distress vocalizations (DVoc) were continuously recorded. RESULTS: Vehicle chicks displayed high DVoc rates in the anxiety phase (first 3min). DVoc rates declined about 50% (i.e., behavioral despair) in the depression phase (30-60min). S. tortuosum fraction at 75 and 100mg/kg decreased DVoc rates during the anxiety phase indicative of an anxiolytic effect. Imipramine, but not S. tortuosum groups, increased DVoc rates in the depression phase indicative of an antidepressant effect. CONCLUSIONS: The findings suggest that an alkaloid enriched S. tortuosum fraction may benefit some forms of stress-related disorders.

Comparative effects of Mitragyna speciosa extract, mitragynine, and opioid agonists on thermal nociception in rats.

This study sought to compare the effects of Mitragyna speciosa (Korth.) Havil. extract, alkaloids fraction, and mitragynine, a µ-opioid receptor agonist, to that of morphine and oxycodone in a test of thermal nociception. In Experiment 1, male Sprague-Dawley rats were administered test articles intraperitoneally (IP) 30 min prior to testing to compare the effects of M. speciosa articles to opioid reference compounds on the hotplate assay. Test articles were vehicle, 10 mg/kg morphine, 3 mg/kg oxycodone, 300 mg/kg M. speciosa extract, 75 mg/kg M. speciosa alkaloids fraction, or 30 mg/kg mitragynine. To mirror consumer usage, Experiment 2 sought to determine whether M. speciosa articles retained their biological activity when given orally (PO). Test articles were vehicle, 6 mg/kg oxycodone, 300 mg/kg M. speciosa extract, or 100mg/kg mitragynine with hotplate tests conducted 30 and 60 min after administration. Mitragynine produced antinociceptive effects similar to the reference opioid agonists when administered IP and PO routes. These data suggest that M. speciosa extracts containing significant quantities of mitragynine may warrant consideration for further studies in primate self-administration models to yield insight into the abuse liability of this commercially available product.