Driving force to detect Alzheimer's disease biomarkers: application of a thioflavine T@Er-MOF ratiometric fluorescent sensor for smart detection of presenilin 1, amyloid ß-protein and acetylcholine.

Currently, the highly sensitive detection of Alzheimer's Disease (AD) biomarkers, namely presenilin 1, amyloid ß-protein (Aß), and acetylcholine (ACh), is vital to helping us prevent and diagnose AD. In this work, a novel metal-organic framework [Er(L)(DMF)1.27]n (Er-MOF) (H3L = terphenyl-3,4'',5-tricarboxylic acid) has been synthesized by solvothermal and ultrasonic methods. Further, through the post-synthesis assembly strategy, the fluorescent dye thioflavine T (ThT) has been introduced into Er-MOF to construct a dual-emission ThT@Er-MOF ratiometric fluorescent sensor. This is the first time that ThT@Er-MOF has been successfully applied in the highly sensitive detection of three main Alzheimer's disease biomarkers in the cerebrospinal fluid through three different low cost and facile detection strategies. Firstly, with the spilted DNA strategy, this is the first time that ThT@Er-MOF can be applied in the label-free detection of SSODN (part of the presenilin 1 gene). Secondly, for the detection of Aß, because ThT can be specifically combined with Aß and has an excellent characteristic fluorescence band, the dual-emission ThT@Er-MOF sensor can be selectively applied to detect Aß over the analog protein, which shows far more sensitivity than other Aß sensors. Thirdly, through the acetylcholine esterase (AchE) enzymatic cleavage and release strategy, ThT@Er-MOF enhances the detection of acetylcholine (ACh) with a low limit of detection (LOD) value (0.03226 nM). It should be noticed that the three different detection methods are low cost and facile. This study also provides the first example of utilizing laser scanning confocal microscopy (LSCM) to investigate the fluorescence resonance energy transfer (FRET) detection mechanism by ThT@Er-MOF in more detail. The location of FRET occurrence and FRET efficiency can also be investigated by LSCM, which can be helpful to understand the FRET detection process by these unique MOF-based hybrid materials.

In Situ Sensing of the Neurotransmitter Acetylcholine in a Dynamic Range of 1 nM to 1 mM.

The neurotransmitter acetylcholine (ACh) plays a key role in the pathophysiology of brain disorders such as Alzheimer's disease. Understanding the dynamics of ACh concentration changes and kinetics of ACh degradation in the living brain is crucial to unravel the pathophysiology of such diseases and the rational design of therapeutics. In this work, an electrochemical sensor capable of dynamic, label-free, selective, and in situ detection of ACh in a range of 1 nM to 1 mM (with temporal resolution of less than one second) was developed. The sensor was employed for the direct detection of ACh in artificial cerebrospinal fluid and rat brain homogenate, without any prior separation steps. A potentiometric receptor-doped ion-selective electrode (ISE) with selectivity for ACh was designed by taking advantage of the positive charge of ACh. The dynamic range, limit of detection (LOD), and the selectivity of the sensor were optimized stepwise by (i) screening of hydrophobic biomimetic calixarenes to identify receptors that strongly bind to ACh based on shape-selective multitopic recognition, (ii) doping of the ISE sensing membrane with an ACh-binding hydrophobic calixarene to enable selective detection of ACh in complex matrices, (iii) utilizing a hydrophilic calixarene in the inner filling solution of the ISE to buffer the concentration of ACh and, thereby, lower the LOD of the sensor, and (iv) introducing a surface treatment step prior to the measurement by placing the sensor for ∼1 min in a solution of a hydrophilic calixarene to lower the LOD of the sensor even further.

In-depth characterization of the neuroinflammatory reaction induced by peripheral surgery in an animal model.

Delirium is a common complication seen after surgery and anesthesia, in particular in older patients. Although the etiology of postoperative delirium is only incompletely understood, various lines of evidence suggest that proinflammatory signaling from the peripheral site of inflammation to central nervous system results in a decrease of cerebral acetylcholine (ACh) levels thereby inducing neuroinflammation. To corroborate this theory, we applied an animal model for characterization of the neuroinflammatory response after partial hepatectomy (HPx). In this model, the surgery-induced decrease in cerebral ACh levels can be prevented by intraoperative application of physostigmine. Thus, ACh-associated changes in the expression and secretion of inflammation-related compounds can be assessed by comparing the results obtained after surgery, in physostigmine-treated and untreated controls. This way we were able to show that the decrease of cerebral ACh triggers increased secretion of IL-1ß, IL-6, TNFα, MIP-2 (CCL3), RANTES, MCP1, IFNgamma, and IP-10. A gene array covering the expression of 370 inflammation-related genes indicated 13 candidates that are induced upon cerebral ACh decrease after HPx. Quantification of the changes in the expression of these candidates by the comparative CT method revealed a significant increase (> 1.5-fold) in the expression of IL-1ß, IL-6, TNFα, MIP2, RANTES, MCP1, TLR2, TLR4, HMGB1, TNFSF6, TNFSF12, IL1R1 and ILR6. Thus, our results suggest that peripheral surgery induces a reduction of cerebral ACh levels which trigger a complex neuroinflammatory response. From a clinical point of view, manipulating cerebral ACh levels by procholinergic drugs such as physostigmine could become an option to therapeutically target this kind of neuroinflammation.

Alteration in amyloid ß42, phosphorylated tau protein, interleukin 6, and acetylcholine during diabetes-accelerated memory dysfunction in diabetic rats: correlation of amyloid ß42 with changes in glucose metabolism.

BACKGROUND: Diabetes accelerates memory dysfunction in a continuous, slowly pathological process. Studies suggest that the time course of certain biomarkers can characterize the pathological course of the disease to provide information for early intervention. Thus, there is an urgent need for validated biomarkers to characterize the cognitive impairment induced by DM. We aimed to detect changes in cerebrospinal fluid biomarkers such as amyloid ß42, phosphorylated tau protein, interleukin 6, and acetylcholine in diabetic rats over time, and to analyse the relationship between diabetes and cognitive impairment. METHODS: Rats were injected once intraperitoneally with 1% of streptozotocin to establish a diabetic model. Index changes were investigated longitudinally and all were measured at the end of the experiment at day 75. Aß42, P-tau, IL-6, and ACh levels in CSF, insulin levels in plasma, and Aß42 levels in plasma and brain tissue were measured by ELISA. RESULTS: Compared with control, the diabetic model showed ACh in CSF to be decreased by day 15, continuing lower out to day 75. Aß42 changes in brain and blood showed the same trends but exhibited minima at different time points: day 30 in CSF and day 15 in plasma. After the minimum, Aß42 in cerebrospinal fluid rose and levelled off lower than in the control group, whereas Aß42 in plasma rose and went above the controls at day 30, slowly trending upwards for the remainder of the experiment. P-tau protein in CSF in diabetic rats showed an increasing trend, becoming significantly different from the controls at day 60 and day 75. Aß42 in CSF was strongly negatively correlated with blood glucose at day 15 and was negatively correlated with insulin in serum, particularly at day 45. CONCLUSION: Our longitudinal research model suggest that changes in the measured biomarkers appear before learning and memory impairments do. Aß42 and ACh in the diabetes model group clearly changed from day 0 to day 45, and then P-tau and IL-6 varied significantly from day 45 to day 75. The reduced ACh levels observed possibly correlated with the factors common to changes in Aß42, P-tau, and IL-6.

Serial CSF sampling over a period of 30 h via an indwelling spinal catheter in healthy volunteers: headache, back pain, tolerability and measured acetylcholine profile.

BACKGROUND/AIM: Timed interval cerebrospinal fluid (CSF) sampling by indwelling catheterization can be a valuable corroborative tool for the pharmacokinetic and pharmacodynamic assessment of drugs. CSF sampling in studies on drug candidates for Alzheimer's disease have been conducted in evaluations of the biomarkers acetylcholine (ACh), tau proteins, amyloid precursor protein and beta-amyloid fragments. The primary aim of this study was to study the feasibility and the burden on the healthy volunteers of serial CSF sampling within the contract research organization environment in order to establish a standardized research tool for future drug development studies. MATERIALS AND METHODS: This study is a validation study in healthy subjects: eight healthy male subjects aged 55-75 years were enrolled. After eligibility had been confirmed, the subjects were admitted to the clinical pharmacology unit 2 days before starting the CSF sampling procedure. Hydration by drip infusion of 2 L saline was performed for 24 h before starting the CSF sampling procedure, and for antithrombotic purposes, Fraxiparine (nadroparine calcium) was given 12 and 36 h after intradural catheterization. CSF catheterization was performed by board-certified anesthesiologists with experience in inserting indwelling intrathecal catheters. Subjects only required to remain in a horizontal position for the first 24 h after removal of the catheter. CSF and blood samples were collected by interval sampling over a 30-h period. RESULTS: The study was completed by seven of the eight subjects. Six subjects who completed the study reported adverse effects (AEs) which were all mild and from which they recovered during their stay in the clinic. A total of 25 AEs were reported of which 13 were considered to be procedure-related. The procedure was well tolerated by all participating subjects, and the VAS scale scores for headache and back pain were low. CSF samples were analyzed for ACh. All values were above the lowest limit of quantification. On average, the ACh concentration started at a low level but rose between 1 and 2 h after insertion of the catheter and then remained high during the whole sampling period up to 30 h. CONCLUSION: Serial sampling of CSF in seven healthy volunteers up to 30 h occurred without serious complications and was well tolerated. The CSF collected was of good quality and facilitated the assessment of an Alzheimer's disease-sensitive biomarker. We conclude that this validation study can form the basis for future patient studies aimed at elucidating disease mechanisms and the pharmacodynamics of drugs in the developmental stage.

Relation between pro-inflammatory cytokines and acetylcholine levels in relapsing-remitting multiple sclerosis patients.

Multiple sclerosis (MS) is a chronic inflammatory, demyelinating and neurodegenerative disorder. Since acetylcholine (ACh) is known to participate in the inflammatory response, we investigated the possible relationship between pro-inflammatory cytokines and acetylcholine levels in relapsing-remitting multiple sclerosis (RR-MS) patients. Levels of ACh and pro-inflammatory cytokines IL1-β and IL-17 were measured both in cerebrospinal fluid (CSF) and sera of 22 RR-MS patients in the relapsing phase and in 17 control subjects affected by other non-neurological diseases (OND). We observed higher levels of pro-inflammatory cytokines such as IL-1β and IL-17 in both CSF and serum of RR-MS patients compared to control subjects. Moreover, ACh levels were lower in CSF and serum of RR-MS patients compared to levels of control subjects. Although the relationship between high inflammatory cytokine levels and low ACh levels need to be further investigated in the future, our data suggest that IL-1β, and cytokines induced by it, such as IL-17 and ACh, may be involved in the pathogenesis of MS.

Development and validation of a sample stabilization strategy and a UPLC-MS/MS method for the simultaneous quantitation of acetylcholine (ACh), histamine (HA), and its metabolites in rat cerebrospinal fluid (CSF).

A UPLC-MS/MS assay was developed and validated for simultaneous quantification of acetylcholine (ACh), histamine (HA), tele-methylhistamine (t-mHA), and tele-methylimidazolacetic acid (t-MIAA) in rat cerebrospinal fluid (CSF). The biological stability of ACh in rat CSF was investigated. Following fit-for-purpose validation, the method was applied to monitor the drug-induced changes in ACh, HA, t-mHA, and t-MIAA in rat CSF following administration of donepezil or prucalopride. The quantitative method utilizes hydrophilic interaction chromatography (HILIC) Core-Shell HPLC column technology and a UPLC system to achieve separation with detection by positive ESI LC-MS/MS. This UPLC-MS/MS method does not require extraction or derivatization, utilizes a stable isotopically labeled internal standard (IS) for each analyte, and allows for rapid throughput with a 4 min run time. Without an acetylcholinesterase (AChE) inhibitor present, ACh was found to have 1.9±0.4 min in vitro half life in rat CSF. Stability studies and processing modification, including the use of AChE inhibitor eserine, extended this half life to more than 60 min. The UPLC-MS/MS method, including stabilization procedure, was validated over a linear concentration range of 0.025-5 ng/mL for ACh and 0.05-10 ng/mL for HA, t-mHA, and t-MIAA. The intra-run precision and accuracy for all analytes were 1.9-12.3% CV and -10.2 to 9.4% RE, respectively, while inter-run precision and accuracy were 4.0-16.0% CV and -5.3 to 13.4% RE, respectively. By using this developed and validated method, donepezil caused increases in ACh levels at 0.5, 1, 2, and 4h post dose as compared to the corresponding vehicle group, while prucalopride produced approximately 1.6- and 3.1-fold increases in the concentrations of ACh and t-mHA at 1h post dose, respectively, compared to the vehicle control. Overall, this methodology enables investigations into the use of CSF ACh and HA as biomarkers in the study of these neurotransmitter systems and related drug discovery efforts.

[Effect of huperzine A on cerebral cholinesterase and acetylcholine in elderly patients during recovery from general anesthesia].

OBJECTIVE: To observe the effect of huperzine A on cerebral cholinergic system in elderly patients during recovery from general anesthesia. METHODS: Thirty elderly patients undergoing elective surgery under general anesthesia were randomized in a double-blind manner into group I (n=15) to receive huperzine A (0.3 mg/2 ml) and group II (n=15) with normal saline (2 ml) given intravenously. Huperzine A or normal saline was administered 30 min before completion of the operation, and acetylcholine (Ach) concentration in the cerebral spinal fluid (CSF) of the patients was determined using high-performance liquid chromatography with electrochemical detector (HPLC-ECD) and the activity of cholinesterase inhibitors (ChE) evaluated with automatic biochemistry analyzer before general anesthesia induction (T1) and 5 h after operation completion (T2). RESULTS: In both the groups, Ach concentration in the CSF were lower at T2 than that at T1 (P<0.01), and at T2, CSF Ach concentration was significantly higher in group I than in group II (P<0.01); in group I, the activity of CSF ChE at T2 was lower than that at T1 (P<0.01), and also lower than at T2 in group II (P<0.01). CONCLUSION: Huperzine A can inhibit cholinesterase to increase Ach, which has a positive effect on cerebral cholinergic system in elderly patients during recovery from general anesthesia.

Intraperitoneal administration of 340 kDa human plasma butyrylcholinesterase increases the level of the enzyme in the cerebrospinal fluid of rats.

Human butyrylcholinesterase (BuChE) is being developed as a new therapeutic for protection against the toxicity of organophosphorus agents and cocaine. The purified BuChE consists predominantly of 340 kDa tetramers and contains less than 5% monomers and dimers. Our goal was to determine whether BuChE crosses the blood-cerebrospinal fluid (CSF) barrier. Rats were injected intraperitoneally with 1mg of purified human BuChE. Plasma BuChE activity increased nearly 400-fold, while BuChE activity in the CSF increased three-fold. Sucrose density centrifugation showed that the human BuChE molecule in the rat CSF was a tetramer. Immunoprecipitation confirmed the identity of the CSF BuChE as human BuChE. The lower amount of human BuChE in the CSF (0.04%) than of smaller proteins (0.1-1%), with respect to their levels in plasma, supports the idea that passage through the blood-CSF barrier depends on molecular size. BuChE in the CSF could serve to protect the brain from the neurotoxicity of organophosphorus pesticides and cocaine.

Differential acetylcholine and choline concentrations in the cerebrospinal fluid of patients with Alzheimer's disease and vascular dementia.

BACKGROUND: An important aspect of Alzheimer's disease (AD) is loss or impairment of cholinergic neurons. It is controversial whether there is a similar cholinergic impairment and cerebral deficit of acetylcholine (ACh) in the case of vascular dementia (VD). The purpose of this study was to explore the levels of ACh and choline (Ch) in the cerebrospinal fluid (CSF) of patients with AD and VD, and their possible relationship with cognitive impairment. METHODS: Twenty-two AD patients, twenty-two VD patients, and twenty normal controls were recruited and scored with a Mini-Mental State Examination (MMSE). CSF concentrations of ACh and Ch were measured using high-performance liquid chromatography with an electrochemical detector (HPLC-ECD) and the results were then compared to cognitive status. RESULTS: ACh concentrations in CSF of AD patients [(10.7 +/- 5.1) nmol/L] and VD patients [(16.8 +/- 7.4) nmol/L] were both significantly lower than in controls [(34.5 +/- 9.0) nmol/L, t = 10.67, P < 0.001; t = 6.91, P < 0.001]. Both results correlated positively with MMSE scores (rs = 0.88 and rs = 0.85, respectively, P < 0.01). The CSF concentration of Ch was significantly higher in VD patients [(887.4 +/- 187.4) nmol/L] compared to AD patients [(627.6 +/- 145.1) nmol/L, t = 6.4, P < 0.001] and controls [(716.0 +/- 159.4) nmol/L, t = 4.2, P = 0.002]. CSF Ch concentration showed no difference between AD patients and normal controls, nor did it correlate with MMSE score in any of the three groups. CONCLUSIONS: The positive correlation between ACh deficit and cognitive impairment suggests that ACh is an important neurotransmitter for memory. The similar decrease in ACh concentration in AD and VD patients may imply a similar pathogenesis for the process of cognitive impairment involved in these two disorders. The elevated CSF levels of Ch in VD patients compared to AD patients may be useful diagnostically. Cholinesterase inhibitors may be helpful not only for AD patients, but also for VD patients.

Responder characteristics to a single oral dose of cholinesterase inhibitor: a double-blind placebo-controlled study with tacrine in Alzheimer patients.

A proportion of Alzheimer's disease (AD) patients treated for several months with cholinesterase (ChE) inhibitors have shown some favorable response on cognition, but the characteristics of the responders are still unclear. This study attempts to identify the characteristics of individuals with a positive behavioral response after a double-blind randomized administration of a single oral dose of tacrine (40 mg) and placebo to AD patients. Furthermore, the relationship between single-dose and long-term responders are examined. Twenty-four mildly to very mildly demented AD patients participated in the study. They all fulfilled the diagnosis of probable AD according to NINCDS-ADRDA criteria. Active treatment (tacrine 40 mg) and placebo was administered in random order on 2 consecutive days, and the effects were evaluated within 2 h using neuropsychological tests (assessing visuospatial ability, episodic memory and attention), registration of EEG activity and measurement of red blood cells (RBC) acetylcholinesterase (AChE), ChE activity and concentrations of tacrine and its metabolites in plasma. Results demonstrated significant improvement, tacrine compared to placebo, in measures of attention, but not in episodic memory or visuospatial ability. A single-dose response was therefore defined in terms of improvement in attention. The tacrine plasma concentration (pcTHA) showed a positively skewed distribution (mean +/- SD: 10.5 +/- 11.8, range: 1.0-51.8 ng/ml). There were no significant differences between single-dose responders compared to nonresponders in pcTHA, metabolites of tacrine, inhibition of AChE in RBC, tau levels in CSF, AChE activity in CSF or plasma and demographic variables. However, single-dose responders showed a higher right frontal alpha/theta ratio on EEG and had lower glucose metabolism in the parietal-temporal association cortex at baseline. In addition, the frequency of apolipoprotein E (APOE) epsilon 4 alleles was higher in responders. Interestingly, the single-dose response was related to the long-term response, although not significantly, which probably was due to lack of power. To conclude, the present study identified single-dose responders in terms of improved attentional performance associated with a relatively higher alpha/theta activity in the right frontal regions of the brain measured on EEG and predominance of APOE epsilon 4 allele.

Acetylcholine in human CSF: methodological considerations and levels in dementia of Alzheimer type.

Four different methods to measure acetylcholine (ACh) and choline (Ch) concentration, i.e. thermospray/mass spectroscopy (TS/MS), high pressure liquid chromatography/mass spectroscopy (HPLC/MS), high pressure liquid chromatography/electrochemical detection (HPLC/ECD) and gas chromatography/mass spectroscopy (GC/MS), both latter methods coupled to a solid phase extraction system were compared for their applicability to human lumbar cerebrospinal fluid (CSF). Furthermore, samples from 15 control persons and 11 patients with dementia of Alzheimer-type (DAT) were compared to search for an ACh deficit in the CSF in DAT. GC/MS was the most sensitive, but most laborious method, and HPLC/ECD was acceptably sensitive, reliable and more specific. TS/MS was not specific enough for CSF extracts and HPLC/MS was more specific, but far less sensitive. Thus, only GC/MS and HPLC/ECD were qualified to detect ACh in human CSF extracts. Comparison of GC/MS and HPLC/ECD revealed highly correlated levels of ACh (r = 0.999). Using HPLC/ECD, ACh concentrations were greatly reduced in the DAT group (3.75 +/- 1.40 pmol/ml CSF) as compared to the controls (6.14 +/- 1.39 pmol/ml CSF), but the difference between controls and DAT patients was not statistically significant due to the number of samples below detection limit (8 out of 11 samples in DAT, 7 out of 15 in controls). Ch concentrations were not statistically significant between the two groups. The data show that methodological limitations preclude a widespread clinical application of determining ACh in the human CSF. Despite of reductions of ACh in the CSF in DAT, the determination of Ach in the CSF is not suitable for diagnostic purposes.

Cerebrospinal fluid pharmacokinetics and pharmacodynamics of intrathecal neostigmine methylsulfate in humans.

BACKGROUND: This study defines the cerebrospinal fluid (CSF) pharmacokinetics of neostigmine after intrathecal injection in humans and its effect on CSF acetylcholine, and it correlates physiologic effects with neostigmine dose and CSF acetylcholine concentrations. METHODS: The CSF was sampled via an indwelling spinal catheter in 12 volunteers receiving intrathecal neostigmine (50-750 microg) and analyzed for neostigmine and acetylcholine. Pharmacokinetic and pharmacodynamic analyses were performed with NONMEM. Effect-site models linked the time course of the neostigmine concentration with the time course of analgesia. RESULTS: Acetylcholine concentrations increased from <20 pmol/ml at baseline to >100 pmol/ml within 15 min of neostigmine injection. The pharmacokinetics of intrathecal neostigmine were best described by a triexponential function with an absorption phase. Individual predicted concentrations varied 100-fold. Post hoc Bayesian estimates described the observed neostigmine concentrations with a median error of 22% and did not show systematic model misspecification. Individual estimates of effect site concentration producing a 50% maximal effect for foot visual analog scale analgesia correlated with the magnitude of individual CSF neostigmine concentrations. CONCLUSIONS: Intrathecal neostigmine concentrations can be well described by a triexponential disposition function, but the intersubject variability is large. The correlation between intersubject variability in concentration and intersubject variability in 50% maximal effect for foot analgesia suggests that both are offset by a common scalar, possibly the distance from the site of injection to the sampling and effect sites. These data provide the basis for the hypothesis of "observation at a distance" to describe the pharmacodynamics of intrathecally administered drugs.

Cerebrospinal fluid free choline in movement disorders of paediatric onset.

We measured free choline in cerebrospinal fluid (CSF) of 78 patients with movement disorders of paediatric onset and various controls as a putative index of central phospholipid metabolism. Most of the disorders studied were myoclonic disorders, such as progressive myoclonus epilepsy, the opsoclonus-myoclonus syndrome, and essential myoclonus, but other movement disorders, interictal seizure disorders, and different neurological and nonneurological disorders were also included. There were no significant differences in CSF choline concentrations in myoclonic disorders or other movement disorders compared with controls. The CSF choline levels were lowest in children with seizure disorders including progressive myoclonus epilepsy. In progressive myoclonus epilepsy, the CSF choline values resembled other epileptic disorders rather than other myoclonic disorders. When all the data were analysed collectively, no significant relation of CSF choline was found to patient age, gender, aliquot of CSF measured, or the length of time the sample was stored at -70 degrees C. Separate analyses of data from children and adults showed a trend toward a biphasic relation between patient age and CSF choline which could be pursued in developmental studies of normal subjects. Reduced CSF choline may indicate increased choline incorporation into brain phospholipids, disturbances of choline metabolism, decreased choline release, or non-neural factors.

Enhancement of analgesia from systemic opioid in humans by spinal cholinesterase inhibition.

Intravenous opioids cause analgesia and increase release of ACh in spinal cord dorsal horn in animals, and these effects are enhanced by intrathecal neostigmine injection. The purpose of the current study was to test whether intrathecal neostigmine enhanced analgesia and increased cerebrospinal fluid concentrations of ACh over those induced by i.v. alfentanil in volunteers, and also to test whether neostigmine enhanced alfentanil-induced side effects. After human studies committee approval, 40 healthy volunteers received an intrathecal injection of saline or neostigmine (50, 100 or 200 microg) followed in 60 min by a computer-controlled, stepped i.v. infusion of alfentanil to escalating targeted plasma concentrations. Pain report to hand and foot immersion in ice water, sedation, nausea, weakness, vital signs, end-tidal CO2 and oxyhemoglobin saturation were measured 60 min after spinal injection and at the end of each 20-min alfentanil infusion. Cerebrospinal fluid was sampled once after drug administration. Intrathecal neostigmine alone caused analgesia in the foot but not in the hand, and was accompanied by leg weakness, whereas IV alfentanil alone caused equivalent analgesia in both the hand and the foot and was accompanied by nausea, sedation, increased end-tidal CO2 and decreased oxyhemoglobin saturation. Neostigmine enhanced analgesia but not respiratory effects induced by i.v. alfentanil; it also enhanced nausea and sedation. Intravenous alfentanil increased cerebrospinal fluid ACh concentration, and neostigmine enhanced this change. These data in humans are consistent with a spinal cholinergic mechanism of i.v. opioid analgesia. Because neostigmine enhances both analgesia and side effects induced by i.v. alfentanil, the clinical utility of their use in combination will depend on the relative strength of these interactions.

Analgesic doses of intrathecal but not intravenous clonidine increase acetylcholine in cerebrospinal fluid in humans.

Epidural clonidine increases acetylcholine (ACh) concentrations in cerebrospinal fluid (CSF) in humans, and experiments in animals support a cholinergic link in spinal alpha2-adrenoceptor-mediated antinociception. The purpose of the present study was to evaluate whether intravenous (I.V.) clonidine is also able to increase CSF ACh in humans. Accordingly, we studied 20 patients scheduled for resection of an acoustic neuroma under general anesthesia. Anesthesia was induced with propofol and maintained with propofol and N2O. After induction, an intrathecal catheter was inserted at the L3-4 interspace. Patients were then assigned, in a random, blind manner to receive either a bolus of 1 microg/kg intrathecal (I.T.) clonidine and an I.V. infusion of saline (n = 10) or an I.V. infusion of 4 microg/kg clonidine given in 20 min and an I.T. injection of saline (n = 10). CSF samples for ACh and clonidine concentration determination were drawn immediately before I.T. injection (time -20), at the end of the I.V. injection (time 0), then every 10 min thereafter. CSF ACh concentrations were determined by high-pressure liquid chromatography and CSF clonidine by radioimmunoassay. There was no significant difference between the groups with respect to age, gender, weight, and ASA physical status. I.T. but not I.V. administration of clonidine increased the CSF ACh concentration. We conclude that I.V. administration of four times the dose of clonidine delivered spinally failed to induce a significant increase of ACh in the CSF. These observations indicate that the analgesic effects observed after I.V. clonidine administration are not mediated by a cholinergic mechanism at the spinal level.

Interaction between intrathecal neostigmine and epidural clonidine in human volunteers.

BACKGROUND: alpha 2-Adrenergic agonists are thought to produce analgesia, in part, by activating spinal acetylcholine release. The purpose of the current study was to examine the interaction between intrathecal neostigmine and epidural clonidine for analgesia and side effects in humans. METHODS: A total of 58 volunteers received an intrathecal injection of 5% dextrose in normal saline (D5NS) or neostigmine (50, 100, or 200 micrograms in D5NS), followed in 1 h by epidural saline or clonidine (computer-controlled infusion targeted to 50, 100, 200, or 400 ng/ml in cerebrospinal fluid) using an isobolographic design. Visual analog scale pain to a noxious cold stimulus, nausea, weakness, sedation, and other safety variables was measured before and at specified intervals after drug administration. RESULTS: The first 21 volunteers randomized to receive intrathecal hyperbaric neostigmine rather than D5NS received the drug while in the sitting position, and had none-to-minimal analgesia 1 h later. The remaining volunteers received the drug while in the lateral position, and demonstrated dose-dependent analgesia in the foot 1 h later. Epidural clonidine also caused dose-dependent analgesia. The combination of neostigmine and clonidine resulted in an additive enhancement for analgesia, but no enhancement of each drug's side effects, and a reduction in clonidine-induced hypotension. Neostigmine injected into subjects in the lateral position diminished clonidine-induced reductions in blood pressure and plasma norepinephrine. CONCLUSION: These results support enhancement of alpha 2-adrenergic analgesia by intrathecal neostigmine, but do not demonstrate synergy, as observed in animals. Lack of enhancement of side effects suggests this combination may be clinically useful.

Bolus metoclopramide does not enhance morphine analgesia after cesarean section.

Intravenous metoclopramide potentiates the analgesic effects of opioids in postoperative patients. We speculate that increased spinal concentrations of acetylcholine from metoclopramide-induced acetylcholinesterase inhibition is the mechanism responsible for enhanced morphine analgesia from metoclopramide. Sixty patients undergoing elective cesarean section with subarachnoid anesthesia were randomized to receive either 20 mg metoclopramide or saline intravenously 30-60 min prior to subarachnoid injection. Prior to subarachnoid injection of local anesthetic, 2 mL of cerebrospinal fluid (CSF) was aspirated for cholinesterase activity measurement. Visual analog scale (VAS) scores for pain were obtained prior to drug administration, at first patient request for analgesia, and at discharge from the postanesthesia care unit. Total morphine use was recorded in the recovery room and for 24 h postoperatively. There were no significant differences in VAS scores, morphine use, or CSF cholinesterase activity between groups. CSF cholinesterase activity was similar to values in nonpregnant patients demonstrated previously. This study failed to confirm the morphine-enhancing action of 20 mg intravenous metoclopramide in postoperative patients. Furthermore, this dose of metoclopramide does not inhibit CSF acetylcholinesterase.

Cerebrospinal fluid norepinephrine and acetylcholine concentrations during acute pain.

Painful stimulation increases spinal cord norepinephrine (NE) in animals, and spinally released NE induces acetylcholine (ACh) release to cause analgesia. The purpose of this study was to determine the relationship between NE and ACh in cerebrospinal fluid (CSF) in sheep and humans during painful stimulation. CSF was sampled in anesthetized sheep before and during electrical nerve stimulation at an intensity sufficient to increase mean arterial pressure 15%-20%. To determine whether spinally released NE caused ACh release by stimulation of alpha(2)-adrenoceptors, seven sheep received intrathecal (IT) idazoxan whereas seven sheep received IT saline before stimulation. To examine the effect of pain on CSF NE and ACh in humans, CSF was sampled in 33 women after at least 4 h of painful labor and in 22 pregnant women without pain. Painful stimulation in sheep increased CSF NE and ACh. IT idazoxan blocked the increase in both NE and ACh. Although mean concentrations of CSF NE and ACh did not differ between parturients with and without pain, there was a significant correlation between NE and ACh concentrations only in those with pain. These data provide evidence in animals for activation of spinal cord noradrenergic-cholinergic systems in response to pain. There is only weak evidence for such activation, however, in women with painful labor.

Elevation of cerebrospinal fluid choline levels by nicotinamide involves the enzymatic formation of N1-methylnicotinamide in brain tissue.

Nicotinamide administration can elevate plasma and brain choline levels and produce a marginal increase in striatal acetylcholine levels in the rat. We now report that subcutaneous nicotinamide produces a substantial and long-lasting rise in cisternal cerebrospinal fluid (CSF) levels of choline in free-moving rats, possibly through the enzymatic formation of N1-methylnicotinamide (NMN) in brain. CSF choline levels peaked 2 hours after nicotinamide administration and were accompanied by increases in striatal, cortical, hippocampal and plasma choline levels. The enzymatic formation of [3H]NMN in rat brain was evaluated by incubating aliquots of rat brain cytosol with unlabelled nicotinamide and the methyl donor [3H]S-adenosylmethionine. High performance liquid chromatography and radiochemical detection demonstrated that [3H]NMN was specifically formed by a brain cytosolic enzyme. The production of [3H]NMN was dependent on exogenous nicotinamide and could be prevented by denaturing the cytosol. The metabolism of nicotinamide to NMN in rat brain may explain the rise in CSF choline levels since NMN, a quaternary amine, can inhibit choline transport at the choroid villus and reduce choline clearance.