In vivo and in vitro aspects of imidazoline-2(1/2)sites within the central nervous system

The in vivo and in vitro characteristics of the I2 binding site were probed using the technique of drug discrimination and receptor autoradiography. Data presented in this thesis indicates the I2 ligand 2-BFI generates a cue in drug discrimination. Further studies indicated agmatine, a proposed endogenous imidazoline ligand, and a number of imidazoline and imidazole analogues of 2-BFI substitute significantly for 2-BFI. In addition to specific I2 ligands the administration of NRl's (noradrenaline reuptake inhibitors), the sympathomimetic d-amphetamine, the α1-adrenoceptor agonist methoxamine, but not the β1 agonist dobutamine or the β2 agonist salbutamol, gave rise to significant levels of substitution for the 2-BFI cue. The administration of the α1-adrenoceptor antagonist WB4101, prior to 2- BFI itself significantly reduced levels of 2-BFI appropriate responding. Administration of the reversible MAO-A inhibitors moclobemide and Ro41-1049, but not the reversible MAO-B inhibitors lazabemide and Ro16-6491, gave rise to potent dose dependent levels of substitution for the 2-BFI cue. Further studies indicated the administration of a number of β-carbolines and the structurally related indole alkaloid ibogaine also gave rise to dose dependent significant levels of substitution. Due to the relationship of indole alkaloids to serotonin the 5-HT releaser fenfluramine and a number of SSRI's (selective serotonin reuptake inhibitor) were also administered and these compounds gave rise to significant partial (20-80% responses to the 2-BFI lever) levels of substitution. The autoradiographical studies reported here indicate [3H]2-BFI labels I2 sites within the rat arcuate nucleus, area postrema, pineal gland, interpeduncular nucleus and subfornical organ. Subsequent experiments confirmed that the drug discrimination dosing schedule significantly increases levels of [3H]2-BFI 12 binding within two of these nuclei. However, levels of [3H]2-BFI specific binding were significantly reduced within four of these nuclei after chronic treatment with the irreversible MAO inhibitors deprenyl and tranylcypromine but not pargyline, which only reduced levels significantly in two. Further autoradiographical studies indicated that the distribution of [3H]2-BFI within the C57/B mouse compares favourably to that within the rat. Comparison of these levels of binding to those from transgenic mice who over-express MAO-B indicates two possibly distinct populations of [3H]2-BFI 12 sites exist in mouse brain. The data presented here indicates the 2-BFI cue is associated with the selective activation of α1-adrenoceptors and possibly 5-HT receptors. 2-BFI trained rats recognise reversible MAO-A but not MAO-B inhibitors. However, data within this thesis indicates the autoradiographical distribution of I2 sites bears a closer resemblance to that of MAO-B not MAO-A and further studies using transgenic mice that over-express MAO-B suggests a non-MAO-B I2 site exists in mouse brain.

Behavioural correlates of ocular accomodation and the autonomic nervous system

The binding issue of th is thesis was the examination of workload, induced by relinotopic and spatiotopic stimuli, on both the ocu lomotor and cardiovascular systems together with investigating the covariation between the two systems - the 'eye-heart' link. Further, the influence of refractive error on ocular accommodation and cardiovascular function was assessed. A clinical evaluation was undertaken to assess the newly available open-view infrared Shin-Nippon NVision-K 5001 optometer, its benefit being the capability to measure through pupils = 2.3 mm. Measurements of refractive error taken with the NVision-K were found to be both accurate (Difference in Mean Spherical Equivalent: 0.14 ± 0.35 D; p = 0.67) and repeatable when compared to non-cycloplegic subjective refraction. Due to technical difficulties, however, the NVision-K could not be used for the purpose of the thesis, as such, measures of accommodation were taken using the continuously recording Shin-Nippon SRW-5000 openview infrared optometer, coupled with a piezo-electric finger pulse transducer to measure pulse. Heart rate variability (HRV) was spectrally analysed to determine the systemic sympathetic and parasympathetic components of the autonomic nervous system (ANS). A large sample (n = 60), cross-sectional study showed late-onset myopes (LOMs) display less accurate responses when compared to other refractive groups at high accommodative demand levels (3 .0 0 and 4.0D). Tonic accommodation (TA) was highest in the hypermetropes, fo llowed by emmetropes and early-onset myopes while the LOM subjects demonstrated statistically significant lower levels of TA. The root-meansquare (RMS) value of the accommodative response was shown to amplify with increased levels of accommodative demand. Changes in refractive error only became significant between groups at higher demand levels (3.0 D and 4.0 D) with the LOMs showing the largest magnification in oscilIations. Examination of the stimulus-response cross-over point with the unit ratio line and TA showed a correlation between the two (r = 0.45, p = 0.001), where TA is approximately twice the dioptric value of the stimulus-response cross-over point. Investigation of the relationship between ocular accommodation and systemic ANS function demonstrated covariation between the systems. Subjects with a faster heart rate (lower heart period) tended to have a higher TA value (r = -0.27, p < 0.05). Further, an increase in accommodative demand accompanies a faster heart rate. The influence of refractive error on the cardiovascular response to changes in accommodative demand, however, was equivocal. Examination of the microfluctuations ofacconunodation demonstrated a correlation between the temporal frequency location of the accommodative high Frequency component (HFC) and the arterial pulse frequency. The correlation was present at a range of accommodative demands from 0.0 D to 4.0 D and in all four refractive groups, suggesting that the HFC was augmented by physiological factors. Examination of the effect of visual cognition on ocular accommodation and the ANS confirmed that increasing levels of cognition affect the accommodative mechanism. The accommodative response shifted away from the subject at both near and far. This shift in accommodative response accompanied a decay in the systemic parasympathetic innervation to the heart. Differences between refractive groups also existed with LOMs showing less accurate responses compared to emmetropes. This disparity, however, appeared to be augmented by the systemic sympathetic nervous system. The investigations discussed explored Ihe role of oculomotor and cardiovascular fu nction in workload enviromnents, providing evidence for a behavioural link between the cardiovascular and oculomotor systems.

Specific lipidome signatures in central nervous system from methionine-restricted mice

Membrane lipid composition is an important correlate of the rate of aging of animals. Dietary methionine restriction (MetR) increases lifespan in rodents. The underlying mechanisms have not been elucidated but could include changes in tissue lipidomes. In this work, we demonstrate that 80% MetR in mice induces marked changes in the brain, spinal cord, and liver lipidomes. Further, at least 50% of the lipids changed are common in the brain and spinal cord but not in the liver, suggesting a nervous system-specific lipidomic profile of MetR. The differentially expressed lipids includes (a) specific phospholipid species, which could reflect adaptive membrane responses, (b) sphingolipids, which could lead to changes in ceramide signaling pathways, and (c) the physiologically redox-relevant ubiquinone 9, indicating adaptations in phase II antioxidant response metabolism. In addition, specific oxidation products derived from cholesterol, phosphatidylcholine, and phosphatidylethanolamine were significantly decreased in the brain, spinal cord, and liver from MetR mice. These results demonstrate the importance of adaptive responses of membrane lipids leading to increased stress resistance as a major mechanistic contributor to the lowered rate of aging in MetR mice. © 2013 American Chemical Society.

Psychological traits influence autonomic nervous system recovery following esophageal intubation in health and functional chest pain

Background: Esophageal intubation is a widely utilized technique for a diverse array of physiological studies, activating a complex physiological response mediated, in part, by the autonomic nervous system (ANS). In order to determine the optimal time period after intubation when physiological observations should be recorded, it is important to know the duration of, and factors that influence, this ANS response, in both health and disease. Methods: Fifty healthy subjects (27 males, median age 31.9 years, range 20-53 years) and 20 patients with Rome III defined functional chest pain (nine male, median age of 38.7 years, range 28-59 years) had personality traits and anxiety measured. Subjects had heart rate (HR), blood pressure (BP), sympathetic (cardiac sympathetic index, CSI), and parasympathetic nervous system (cardiac vagal tone, CVT) parameters measured at baseline and in response to per nasum intubation with an esophageal catheter. CSI/CVT recovery was measured following esophageal intubation. Key Results: In all subjects, esophageal intubation caused an elevation in HR, BP, CSI, and skin conductance response (SCR; all p < 0.0001) but concomitant CVT and cardiac sensitivity to the baroreflex (CSB) withdrawal (all p < 0.04). Multiple linear regression analysis demonstrated that longer CVT recovery times were independently associated with higher neuroticism (p < 0.001). Patients had prolonged CSI and CVT recovery times in comparison to healthy subjects (112.5 s vs 46.5 s, p = 0.0001 and 549 s vs 223.5 s, p = 0.0001, respectively). Conclusions & Inferences: Esophageal intubation activates a flight/flight ANS response. Future studies should allow for at least 10 min of recovery time. Consideration should be given to psychological traits and disease status as these can influence recovery. The psychological trait of neuroticism retards autonomic recovery following esophageal intubation in health and functional chest pain. © 2013 John Wiley & Sons Ltd.

Autonomic nervous system, circadian rhythms, and primary open-angle glaucoma

The etiology of primary open-angle glaucoma (POAG) remains the subject of continuing investigation. Despite the many known risk factors and mechanism of damage, the principal treatment objectives in POAG still consist of reduction of intraocular pressure, which although straightforward in many cases, often leaves the clinician with the question of how far to pursue a sufficiently low pressure to prevent further damage. Other risk factors such as hemodynamic insufficiency due to vascular dysregulation and abnormal blood pressure are often overlooked in the day-to-day practice; their harmful effects for glaucoma are, it seems, more potent at night while the patient sleeps and when clinical investigation is most difficult. Although the status of autonomic nervous system is an important determinant of the systemic hemodynamic parameters, this issue is usually ignored by the clinician in the process of glaucoma diagnosis. Consequently, there is a lack of alternative therapies tailored to address associated systemic risk factors for POAG on a case and chronological basis; this approach could be more effective in preventing the progression and visual loss in selected glaucoma cases. © 2004 Elsevier Inc. All rights reserved.

Development of a physiologically-based pharmacokinetic model of the rat central nervous system

Central nervous system (CNS) drug disposition is dictated by a drug’s physicochemical properties and its ability to permeate physiological barriers. The blood–brain barrier (BBB), blood-cerebrospinal fluid barrier and centrally located drug transporter proteins influence drug disposition within the central nervous system. Attainment of adequate brain-to-plasma and cerebrospinal fluid-to-plasma partitioning is important in determining the efficacy of centrally acting therapeutics. We have developed a physiologically-based pharmacokinetic model of the rat CNS which incorporates brain interstitial fluid (ISF), choroidal epithelial and total cerebrospinal fluid (CSF) compartments and accurately predicts CNS pharmacokinetics. The model yielded reasonable predictions of unbound brain-to-plasma partition ratio (Kpuu,brain) and CSF:plasma ratio (CSF:Plasmau) using a series of in vitro permeability and unbound fraction parameters. When using in vitro permeability data obtained from L-mdr1a cells to estimate rat in vivo permeability, the model successfully predicted, to within 4-fold, Kpuu,brain and CSF:Plasmau for 81.5% of compounds simulated. The model presented allows for simultaneous simulation and analysis of both brain biophase and CSF to accurately predict CNS pharmacokinetics from preclinical drug parameters routinely available during discovery and development pathways.

Phytochemical-loaded mesoporous silica nanoparticles for nose-to-brain olfactory drug delivery

Central nervous system (CNS) drug delivery is often hampered due to the insidious nature of the blood-brain barrier (BBB). Nose-to-brain delivery via olfactory pathways have become a target of attention for drug delivery due to bypassing of the BBB. The antioxidant properties of phytochemicals make them promising as CNS active agents but possess poor water solubility and limited BBB penetration. The primary aim of this study was the development of mesoporous silica nanoparticles (MSNs) loaded with the poorly water-soluble phytochemicals curcumin and chrysin which could be utilised for nose-to-brain delivery. We formulated spherical MSNP using a templating approach resulting in ∼220nm particles with a high surface porosity. Curcumin and chrysin were successfully loaded into MSNP and confirmed through Fourier transformation infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and HPLC approaches with a loading of 11-14% for curcumin and chrysin. Release was pH dependant with curcumin demonstrating increased chemical stability at a lower pH (5.5) with a release of 53.2%±2.2% over 24h and 9.4±0.6% for chrysin. MSNP were demonstrated to be non-toxic to olfactory neuroblastoma cells OBGF400, with chrysin (100μM) demonstrating a decrease in cell viability to 58.2±8.5% and curcumin an IC50 of 33±0.18μM. Furthermore confocal microscopy demonstrated nanoparticles of <500nm were able to accumulate within cells with FITC-loaded MSNP showing membrane localised and cytoplasmic accumulation following a 2h incubation. MSNP are useful carriers for poorly soluble phytochemicals and provide a novel vehicle to target and deliver drugs into the CNS and bypass the BBB through olfactory drug delivery.

Development of a post-mitotic, neuronal-astrocytic cell system, for the prediction of acute neurotoxicity in humans

The human NT2.D1 cell line was differentiated to form both a 1:2 co-culture of post-mitotic NT2 neuronal and NT2 astrocytic (NT2.N/A) cells and a pure NT2.N culture. The respective sensitivities to several test chemicals of the NT2.N/A, the NT2.N, and the NT2.D1 cells were evaluated and compared with the CCF-STTG1 astrocytoma cell line, using a combination of basal cytotoxicity and biochemical endpoints. Using the MTT assay, the basal cytotoxicity data estimated the comparative toxicities of the test chemicals (chronic neurotoxin 2,5-hexanedione, cytotoxins 2,3- and 3,4-hexanedione and acute neurotoxins tributyltin- and trimethyltin- chloride) and also provided the non-cytotoxic concentration-range for each compound. Biochemical endpoints examined over the non-cytotoxic range included assays for ATP levels, oxidative status (H2O2 and GSH levels) and caspase-3 levels as an indicator of apoptosis. although the endpoints did not demonstrate the known neurotoxicants to be consistently more toxic to the cell systems with the greatest number of neuronal properties, the NT2 astrocytes appeared to contribute positively to NT2 neuronal health following exposure to all the test chemicals. The NT2.N/A co-culture generally maintained superior ATP and GSH levels and reduced H2O2 levels in comparison with the NT2.N mono-culture. In addition, the pure NT2.N culture showed a significantly lower level of caspase-3 activation compared with the co-culture, suggesting NT2 astrocytes may be important in modulating the mode of cell death following toxic insult. Overall, these studies provide evidence that an in vitro integrated population of post-mitotic human neurons and astrocytes may offer significant relevance to the human in vivo heterogeneous nervous system, when initially screening compounds for acute neurotoxic potential.

Differentiating human NT2/D1 neurospheres as a versatile in vitro 3D model system for developmental neurotoxicity testing

Developmental neurotoxicity is a major issue in human health and may have lasting neurological implications. In this preliminary study we exposed differentiating Ntera2/clone D1 (NT2/D1) cell neurospheres to known human teratogens classed as non-embryotoxic (acrylamide), weakly embryotoxic (lithium, valproic acid) and strongly embryotoxic (hydroxyurea) as listed by European Centre for the Validation of Alternative Methods (ECVAM) and examined endpoints of cell viability and neuronal protein marker expression specific to the central nervous system, to identify developmental neurotoxins. Following induction of neuronal differentiation, valproic acid had the most significant effect on neurogenesis, in terms of reduced viability and decreased neuronal markers. Lithium had least effect on viability and did not significantly alter the expression of neuronal markers. Hydroxyurea significantly reduced cell viability but did not affect neuronal protein marker expression. Acrylamide reduced neurosphere viability but did not affect neuronal protein marker expression. Overall, this NT2/D1 -based neurosphere model of neurogenesis, may provide the basis for a model of developmental neurotoxicity in vitro.

Improving brain drug targeting through exploitation of the nose-to-brain route:a physiological and pharmacokinetic perspective

With an ageing population and increasing prevalence of central-nervous system (CNS) disorders new approaches are required to sustain the development and successful delivery of therapeutics into the brain and CNS. CNS drug delivery is challenging due to the impermeable nature of the brain microvascular endothelial cells that form the blood-brain barrier (BBB) and which prevent the entry of a wide range of therapeutics into the brain. This review examines the role intranasal delivery may play in achieving direct brain delivery, for small molecular weight drugs, macromolecular therapeutics and cell-based therapeutics, by exploitation of the olfactory and trigeminal nerve pathways. This approach is thought to deliver drugs into the brain and CNS through bypassing the BBB. Details of the mechanism of transfer of administrated therapeutics, the pathways that lead to brain deposition, with a specific focus on therapeutic pharmacokinetics, and examples of successful CNS delivery will be explored. © 2014 Bentham Science Publishers.