Cranial manipulation affects cholinergic pathway gene expression in aged rats.

CONTEXT: Age-dependent dementia is a devastating disorder afflicting a growing older population. Although pharmacological agents improve symptoms of dementia, age-related comorbidities combined with adverse effects often outweigh their clinical benefits. Therefore, nonpharmacological therapies are being investigated as an alternative. In a previous pilot study, aged rats demonstrated improved spatial memory after osteopathic cranial manipulative medicine (OCMM) treatment. OBJECTIVES: In this continuation of the pilot study, we examine the effect of OCMM on gene expression to elicit possible explanations for the improvement in spatial memory. METHODS: OCMM was performed on six of 12 elderly rats every day for 7 days. Rats were then euthanized to obtain the brain tissue, from which RNA samples were extracted. RNA from three treated and three controls were of sufficient quality for sequencing. These samples were sequenced utilizing next-generation sequencing from Illumina NextSeq. The Cufflinks software suite was utilized to assemble transcriptomes and quantify the RNA expression level for each sample. RESULTS: Transcriptome analysis revealed that OCMM significantly affected the expression of 36 genes in the neuronal pathway (false discovery rate [FDR] <0.004). The top five neuronal genes with the largest-fold change were part of the cholinergic neurotransmission mechanism, which is known to affect cognitive function. In addition, 39.9% of 426 significant differentially expressed (SDE) genes (FDR<0.004) have been previously implicated in neurological disorders. Overall, changes in SDE genes combined with their role in central nervous system signaling pathways suggest a connection to previously reported OCMM-induced behavioral and biochemical changes in aged rats. CONCLUSIONS: Results from this pilot study provide sufficient evidence to support a more extensive study with a larger sample size. Further investigation in this direction will provide a better understanding of the molecular mechanisms of OCMM and its potential in clinical applications. With clinical validation, OCMM could represent a much-needed low-risk adjunct treatment for age-related dementia including Alzheimer's disease.

A glutamate concentration-biased allosteric modulator potentiates NMDA-induced ion influx in neurons.

Precisely controlled synaptic glutamate concentration is essential for the normal function of the N-methyl D-aspartate (NMDA) receptors. Atypical fluctuations in synaptic glutamate homeostasis lead to aberrant NMDA receptor activity that results in the pathogenesis of neurological and psychiatric disorders. Therefore, glutamate concentration-dependent NMDA receptor modulators would be clinically useful agents with fewer on-target adverse effects. In the present study, we have characterized a novel compound (CNS4) that potentiates NMDA receptor currents based on glutamate concentration. This compound alters glutamate potency and exhibits no voltage-dependent effect. Patch-clamp electrophysiology recordings confirmed agonist concentration-dependent changes in maximum inducible currents. Dynamic Ca2+ and Na+ imaging assays using rat brain cortical, striatal and cerebellar neurons revealed CNS4 potentiated ion influx through native NMDA receptor activity. Overall, CNS4 is novel in chemical structure, mechanism of action and agonist concentration-biased allosteric modulatory effect. This compound or its future analogs will serve as useful candidates to develop drug-like compounds for the treatment of treatment-resistant schizophrenia and major depression disorders associated with hypoglutamatergic neurotransmission.

Intracellular potassium depletion enhances apoptosis induced by staurosporine in cultured trigeminal satellite glial cells.

PURPOSE: Satellite glial cells (SGC) surrounding neurons in sensory ganglia can buffer extracellular potassium, regulating the excitability of injured neurons and possibly influencing a shift from acute to neuropathic pain. SGC apoptosis may be a key component in this process. This work evaluated induction or enhancement of apoptosis in cultured trigeminal SGC following changes in intracellular potassium [K]ic. MATERIALS AND METHODS: We developed SGC primary cultures from rat trigeminal ganglia (TG). Purity of our cultures was confirmed using immunofluorescence and western blot analysis for the presence of the specific marker of SGC, glutamine synthetase (GS). SGC [K]ic was depleted using hypo-osmotic shock and 4 mM bumetanide plus 10 mM ouabain. [K]ic was measured using the K+ fluorescent indicator potassium benzofuran isophthalate (PBFI-AM). RESULTS: SGC tested positive for GS and hypo-osmotic shock induced a significant decrease in [K]ic at every evaluated time. Cells were then incubated for 5 h with either 2 mM staurosporine (STS) or 20 ng/ml of TNF-α and evaluated for early apoptosis and late apoptosis/necrosis by flow cytometry using annexin V and propidium iodide. A significant increase in early apoptosis, from 16 to 38%, was detected in SGC with depleted [K]ic after incubation with STS. In contrast, TNF-α did not increase early apoptosis in normal or [K]ic depleted SGC. CONCLUSION: Hypo-osmotic shock induced a decrease in intracellular potassium in cultured trigeminal SGC and this enhanced apoptosis induced by STS that is associated with the mitochondrial pathway. These results suggest that K+ dysregulation may underlie apoptosis in trigeminal SGC.

Mechanoceutics Alters Alzheimer's Disease Phenotypes in Transgenic Rats: A Pilot Study.

Current advancements in neurovascular biology relates a mechanoceutics treatment, known as cranial osteopathic manipulation (COM), Alzheimer's disease (AD). COM could be used as an evidence-based treatment strategy to improve the symptoms of AD if molecular mechanisms, which currently remain unclear, are elucidated. In the present pilot study, using transgenic rats, we have identified COM mediated changes in behavioral and biochemical parameters associated with AD phenotypes. We expect these changes may have functional implications that might account for improved clinical outcomes of COM treatment. Further investigations on COM will be helpful to establish an adjunct treatment for AD.

Effect of Osteopathic Cranial Manipulative Medicine on an Aged Rat Model of Alzheimer Disease.

CONTEXT: In the aging brain, reduction in the pulsation of cerebral vasculature and fluid circulation causes impairment in the fluid exchange between different compartments and lays a foundation for the neuroinflammation that results in Alzheimer disease (AD). The knowledge that lymphatic vessels in the central nervous system play a role in the clearance of brain-derived metabolic waste products opens an unprecedented capability to increase the clearance of macromolecules such as amyloid ß proteins. However, currently there is no pharmacologic mechanism available to increase fluid circulation in the aging brain. OBJECTIVE: To demonstrate the influence of an osteopathic cranial manipulative medicine (OCMM) technique, specifically, compression of the fourth ventricle, on spatial memory and changes in substrates associated with mechanisms of metabolic waste clearance in the central nervous system using the naturally aged rat model of AD. RESULTS: Significant improvement was found in spatial memory in 6 rats after 7 days of OCMM sessions. Live animal positron emission tomographic imaging and immunoassays revealed that OCMM reduced amyloid ß levels, activated astrocytes, and improved neurotransmission in the aged rat brains. CONCLUSION: These findings demonstrate the molecular mechanism of OCMM in aged rats. This study and further investigations will help physicians promote OCMM as an evidence-based adjunctive treatment for patients with AD.

Ligand binding domain interface: A tipping point for pharmacological agents binding with GluN1/2A subunit containing NMDA receptors.

N-methyl D-aspartate (NMDA) receptors play a crucial role in normal brain function, pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDA receptor contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that evolve from six different genes including four GluN2 (A-D) and two GluN3 (A-B) subunits. Since NMDA receptors confer varied physiological properties and spatiotemporal distributions in the brain, pharmacological agents that target NMDA receptors with specific GluN2 subunits have significant potential for therapeutic applications. In the present work, by using electrophysiology techniques, we have studied the role of ligand binding domain (LBD) interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, channel blockers and an allosteric modulator. Remarkably, point mutations at the distal end (site-II&III) of GluN1 LBD interface increased memantine potency up to sevenfold when co-expressed with wild type GluN2A receptors but exhibit no effect on Mg2+ activity. Conversely, mutations at the proximal end (site-I) of the LBD interface did not affect the memantine but altered Zn2+ and Mg2+ potency towards opposite directions. These results indicate that GluN1/2A LBD interface interactions play a key role in determining channel function. Further, subtle changes in LBD interaction can be readily translated to the downstream extracellular vestibule of channel pore to adopt a conformation that may affect memantine, Zn2+ and Mg2+ binding. Further studies on NMDA receptor LBD to transmembrane domain signal propagation mechanisms will help develop GluN2 subunit selective biomolecules that can be used for the treatment of neurological and psychiatric disorders.

Clinical risk factors for acute ischaemic and haemorrhagic stroke in patients with infective endocarditis.

BACKGROUND: Stroke as a complication of infective endocarditis portends a poor prognosis, yet risk factors for stroke subtypes have not been well defined. AIM: To identify risk factors associated with ischaemic and haemorrhagic strokes. METHODS: A retrospective patient chart review was performed at a single US academic centre to identify risk factors and imaging for patients who were 18 years or older with infectious endocarditis (IE) and stroke diagnoses. Differences in patient characteristics by stroke status were assessed using univariate analysis, χ2 or student's t-test as well as logistic regression models for multivariable analyses and correlation matrices to identify possible collinearity between variables and to obtain odds ratios (OR) and their 95% confidence intervals. RESULTS: A final sample of 1157 participants was used for this analysis. The total number of non-surgical strokes was 178, with a prevalence of 15.4% (78% ischaemic, 10% parenchymal haemorrhages, 8% subarachnoid haemorrhages and 4% mixed ischaemic/haemorrhagic). Multivariate risk factors for ischaemic stroke included prior stroke (OR 2.0, 1.3-3.1), Staphylococcus infection (OR 2.0, 1.3-3.0), mitral vegetations (OR 2.2, 1.4-3.3) and valvular abscess (OR 2.7, 1.7-4.3). Risk factors for haemorrhagic stroke included fungal infection (OR 6.4, 1.2-34.0), male gender (OR 3.5, 1.4-8.3) and rheumatic heart disease (OR 3.3, 1.1-10.4). CONCLUSION: Among patients with IE, there exist characteristics that relate differentially to ischaemic and haemorrhagic stroke risk.

Positive Modulatory Interactions of NMDA Receptor GluN1/2B Ligand Binding Domains Attenuate Antagonists Activity.

N-methyl D-aspartate receptors (NMDAR) play crucial role in normal brain function and pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDAR contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that include six different gene products; four GluN2 (A-D) and two GluN3 (A-B) subunits. The heterogeneity of subunit combination facilities the distinct function of NMDARs. All GluN subunits contain an extracellular N-terminal Domain (NTD) and ligand binding domain (LBD), transmembrane domain (TMD) and an intracellular C-terminal domain (CTD). Interaction between the GluN1 and co-assembling GluN2/3 subunits through the LBD has been proven crucial for defining receptor deactivation mechanisms that are unique for each combination of NMDAR. Modulating the LBD interactions has great therapeutic potential. In the present work, by amino acid point mutations and electrophysiology techniques, we have studied the role of LBD interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, and allosteric modulators. The results reveal that agonists (glycine and glutamate) potency was altered based on mutant amino acid sidechain chemistry and/or mutation site. Most antagonists inhibited mutant receptors with higher potency; interestingly, clinically used NMDAR channel blocker memantine was about three-fold more potent on mutated receptors (N521A, N521D, and K531A) than wild type receptors. These results provide novel insights on the clinical pharmacology of memantine, which is used for the treatment of mild to moderate Alzheimer's disease. In addition, these findings demonstrate the central role of LBD interactions that can be exploited to develop novel NMDAR based therapeutics.

Occupational health outcomes for workers in the agriculture, forestry and fishing sector: implications for immigrant workers in the southeastern US.

BACKGROUND: Workers in the Agriculture, Forestry, and Fisheries (AgFF) sector experience exposures directly related to the work itself, as well as the physical environment in which the work occurs. Health outcomes vary from immediate to delayed, and from acute to chronic. METHODS: We reviewed existing literature on the health outcomes of work in the AgFF sector and identified areas where further research is needed to understand the impact of these exposures on immigrant Latino workers in the southeastern US. RESULTS: Outcomes related to specific body systems (e.g., musculoskeletal, respiratory) as well as particular exposure sources (e.g., pesticides, noise) were reviewed. The most extensive evidence exists for agriculture, with a particular focus on chemical exposures. Little research in the southeastern US has examined health outcomes of exposures of immigrant workers in forestry or fisheries. CONCLUSION: As the AgFF labor force includes a growing number of Latino immigrants, more research is needed to characterize a broad range of exposures and health outcomes experienced by this population, particularly in forestry and fisheries.

Congenital infection of mice with Toxoplasma gondii induces minimal change in behavior and no change in neurotransmitter concentrations.

We examined the effect of maternal Toxoplasma gondii infection on behavior and the neurotransmitter concentrations of congenitally infected CD-1 mice at 4 and 8 wk of age when latent tissue cysts would be present in their brains. Because of sex-associated behavioral changes that develop during aging, infected female mice were compared with control females and infected male mice were compared with control males. Only the short memory behavior (distance between goal box and first hole investigated) of male mice congenitally infected with T. gondii was significantly different (P < 0.05) from that of uninfected control males at both 4 and 8 wk by using the Barnes maze test. The other parameters examined in the latter test, i.e., functional observational battery tests, virtual cliff, visual placement, and activity tests, were not significantly different (P > 0.05) at 4 and 8 wk. Concentrations of neurotransmitters and their metabolites (dopamine; 3,4-dihydroxyphenylacetic acid; homovanillic acid; norepinephrine; epinephrine; 3-methoxy-4-hydroxyphenylglycol; serotonin; and 5-hydroxyindoleacetic acid) in the frontal cortex and striatum were not different (P > 0.05) between infected and control mice at 8 wk of age. The exact mechanism for the observed effect on short-term memory in male mice is not known, and further investigation may help elucidate the molecular mechanisms associated with the proposed link between behavioral changes and T. gondii infection in animals. We were not able, however, to confirm the widely held belief that changes in neurotransmitters result from chronic T. gondii infection of the brain.

Examination of concurrent exposure to repeated stress and chlorpyrifos on cholinergic, glutamatergic, and monoamine neurotransmitter systems in rat forebrain regions.

Repeated stress has been reported to cause reversible impairment in the central nervous system (CNS). It was proposed that alterations in glutamatergic, cholinergic, and monoamine neurotransmitter systems after exposure to stress are initial CNS events contributing to this impairment and that exacerbation could occur with concurrent exposure to cholinesterase inhibitors. Effects of concurrent exposure to repeated stress and chlorpyrifos on activities of acetylcholinesterase (AChE), carboxylesterase, and choline acetyltransferase (ChAT); concentrations of excitatory amino acids, monoamines, and their metabolites; and maximum binding densities (B(max)) and equilibrium dissociation rate constants (K(d)) of glutamatergic N-methyl-d-aspartate (NMDA) and total muscarinic cholinergic receptors were studied in the blood, hippocampus, cerebral cortex, or hypothalamus of adult Long-Evans rats. Stress treatments extended over 28 days included (1) control rats handled 5 days/week; (2) rats restrained 1 h/day for 5 days/week; (3) rats swum 30 min for 1 day/week; or (4) rats restrained 4 days/week and swum for 1 day/week. On day 24, each stress treatment group was randomly divided and injected either with corn oil or chlorpyrifos, 160 mg/kg subcutaneously (sc) (60% of the maximum tolerated dose), 4 h after restraint. Blood and brain tisssues were collected on day 28. Rats restrained and swum had a statistical trend toward increasing concentrations of glutamate in the hippocampus when compared to rats only swum (p = .064). Chlorpyrifos administration decreased restraint-induced elevated aspartate in the hippocampus, and decreased B(max) of total muscarinic receptors in the cerebral cortex. In addition, chlorpyrifos decreased B(max) and K(d) of total muscarinic receptors in the cerebral cortex of swum rats. Results demonstrated that chlorpyrifos inhibited AChE activity in blood, cerebral cortex, and hippocampus, but stress did not affect AChE activity. Carboxylesterase activity was inhibited by chlorpyrifos and by repeated restraint with swim. Swim stress decreased concentrations of norepinephrine in the hippocampus and hypothalamus, and increased concentrations of dopamine and its metabolite, DOPAC, in the hypothalamus. Both stress and chlorpyrifos altered serotonin concentrations, and the interactions of repeated stress and chlorpyrifos on serotonin approached significance in the hippocampus (p = .06) and hypothalamus (p = .08). Therefore, stress models were demonstrated to alter glutamatergic and monoamine responses, whereas chlorpyrifos alone had effects on cholinergic and monoamine systems in the rat CNS. However, the interactions between stress and chlorpyrifos significant at p < 0.05 were restricted to attenuation of elevated aspartate in the hippocampus of restrained with swim rats and decreased K(d) of acetylcholine receptors in the cerebral cortex of swum rats and restrained rats.

Basal Ganglia accumulation and motor assessment following manganese chloride exposure in the C57BL/6 mouse.

Equivocal clinical evidence for involvement of manganese in development of Parkinson's disease necessitates experimental studies on this issue. The aged, 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine-treated C57BL/6 mouse is one of the most common models for Parkinson's disease. However, there is little information on brain bioaccumulation of manganese, and little or no information on clinical/behavioral manifestations of manganese neurotoxicity, in this strain. Male C57BL/6 retired breeder mice were given a single subcutaneous injection of either 0, 50, or 100 mg/kg of MnCl(2) (single-dose regimen) or three injections of either of these doses over 7 days (multiple-dose regimen). Behavioral assessment was performed 24 h after final injection, followed by sacrifice, and body weight was recorded each day. There was a 105% increase in striatal manganese concentration 1 day after a single 100 mg/kg injection, and 421% and 647% increases, respectively, 1 day after multiple doses of 50 or 100 mg/kg of MnCl(2). One day after a single injection, there were respective 30.9% and 38.9% decreases in horizontal movement (grid crossing) for the 50 and 100 mg/kg doses and a 43.2% decrease for the multiple dose of 100 mg/kg. There was no significant main effect of dose level on rearing, swimming, grip strength, or grip fatigue. Unlike previous work with the C57BL/6 strain using smaller intraperitoneal doses, this study established dosing regimens that produced significant increases in basal ganglia manganese concentration reminiscent of brain increases in the CD-1 mouse following subcutaneous doses close to our lowest. A decrease in locomotor behavior, significant but not severe in this study, has been reported following manganese exposure in other mouse strains. These data, particularly the significant increase in basal ganglia manganese concentration, provide guidance for designing studies of the potential role of manganese in Parkinson's disease using the most common animal model for the disorder.

Immunohistochemical changes in the mouse striatum induced by the pyrethroid insecticide permethrin.

Epidemiological studies have linked insecticide exposure and Parkinson's disease. In addition, some insecticides produce damage or physiological disruption within the dopaminergic nigrostriatal pathway of non-humans. This study employed immunohistochemical analysis in striatum of the C57BL/6 mouse to clarify tissue changes suggested by previous pharmacological studies of the pyrethroid insecticide permethrin. Dopamine transporter, tyrosine hydroxylase, and glial fibrillary acidic protein immunoreactivities were examined in caudate-putamen to distinguish changes in amount of dopamine transporter immunoreactive protein from degeneration or other damage to dopaminergic neuropil. Weight-matched pairs of pesticide-treated and vehicle-control mice were dosed and sacrificed on the same days. Permethrin at 0.8, 1.5 and 3.0 mg/kg were the low doses and at 200 mg/kg the high dose. Brains from matched pairs of mice were processed on the same slides using the avidin-biotin technique. Four fields were morphometrically located in each of the serial sections of caudate-putamen, digitally photographed, and immunopositive image pixels were counted and compared between members of matched pairs of permethrin-treated and vehicle-control mice. For low doses, only 3.0 mg/kg produced a significant decrease in dopamine transporter immunostaining. The high dose of permethrin did not produce a significant change in dopamine transporter or tyrosine hydroxylase immunostaining, but resulted in a significant increase in glial fibrillary acidic protein immunostaining. These data suggest that a low dose of permethrin can reduce the amount of dopamine transporter immunoreactive protein in the caudate-putamen. They also suggest that previously reported reductions in dopamine uptake of striatal synaptosomes of high-dose mice may be due to nondegenerative tissue damage within this region as opposed to reductions of dopamine transporter protein or death of nigrostriatal terminals. These data provide further evidence that insecticides can affect the primary neurodegenerative substrate of Parkinson's disease.

Corticosterone in drinking water: altered kinetics of a single oral dose of corticosterone and concentrations of plasma sodium, albumin, globulin, and total protein.

Effects of chronic exposure to corticosterone in drinking water on corticosterone kinetics, blood chemistry, and concentrations of catecholamines in parts of brain were studied in Long-Evans rats. Rats were randomly grouped into 3 x 2 treatments (n=4), with three treatments of drinking water (tap water, or 2.5% ethanol, or 400 microg/mL of corticosterone in 2.5% ethanol) for 28 days and two treatments of gavage with a single dose of either corn oil or corticosterone 20 mg/kg on day 28. Blood samples were collected at 0, 15, 30, 60, 120, 240, 480, and 720 min after dosing to determine plasma corticosterone concentrations. Blood samples were collected for clinical pathology on day 42. Hippocampus, cerebral cortex, caudate-putamen, and pons were examined to determine concentrations of catecholamines and activities of esterases. Concentrations of plasma corticosterone before gavage of the corticosterone-drinking rats (47.61 +/- 1.13 ng/mL) were lower than the water (418.47 +/- 1.13 ng/mL) or the ethanol rats (383.71 +/- 1.13 ng/mL, P < 0.0001). Plasma corticosterone rose to peak concentrations by 15 min after gavage in all three groups of drinking rats. Corticosterone-drinking rats had concentrations of plasma corticosterone that returned to basal levels slower than water- and ethanol-drinking rats. Plasma sodium and chloride concentrations were lower in the corticosterone-drinking rats than the water-drinking rats (P < 0.01). Plasma albumin, globulin, and total protein were highest in the corticosterone-drinking rats when compared to the other groups of drinking rats (P < 0.001, P < 0.05, and P < 0.001, respectively). Corticosterone in drinking water did not affect activities of brain neurotoxic esterase, carboxylesterase, acetylcholinesterase, or concentrations of monoamines and their metabolites. A single oral dose of corticosterone reduced neurotoxic esterase activity in the cerebral cortex (P < 0.05) and increased norepinephrine concentrations in the hippocampus (P < 0.05).