New method for quantification and statistical analysis of nociceptive reflex receptive fields in humans

A method for quantifying nociceptive withdrawal reflex receptive fields in human volunteers and patients is described. The reflex receptive field (RRF) for a specific muscle denotes the cutaneous area from which a muscle contraction can be evoked by a nociceptive stimulus. The method is based on random stimulations presented in a blinded sequence to 10 stimulation sites. The sensitivity map is derived by interpolating the reflex responses evoked from the 10 sites. A set of features describing the size and location of the RRF is presented based on statistical analysis of the sensitivity map within every subject. The features include RRF area, volume, peak location and center of gravity. The method was applied to 30 healthy volunteers. Electrical stimuli were applied to the sole of the foot evoking reflexes in the ankle flexor tibialis anterior. The RRF area covered a fraction of 0.57+/-0.06 (S.E.M.) of the foot and was located on the medial, distal part of the sole of the foot. An intramuscular injection into flexor digitorum brevis of capsaicin was performed in one spinal cord injured subject to attempt modulation of the reflex receptive field. The RRF area, RRF volume and location of the peak reflex response appear to be the most sensitive measures for detecting modulation of spinal nociceptive processing. This new method has important potential applications for exploring aspects of central plasticity in volunteers and patients. It may be utilized as a new diagnostic tool for central hypersensitivity and quantification of therapeutic interventions.

Omega-3 fatty acids and the neurovascular responses to mental stress in humans

Hypertension is the most prevalent form of cardiovascular disease (CVD) in the world, and is known to increase the risk for developing other diseases. Recently, the American Heart Association introduced a new classification of blood pressure, prehypertension (PHT). The criteria for PHT include a systolic of 120-139 mmHg and/or a diastolic blood pressure of 80-89 mmHg. It has been observed that individuals with PHT have a higher risk of developing hypertension later in life. Therefore, it is important to understand the mechanisms contributing to PHT in order to possibly prevent hypertension. Omega-3 fatty acids found in fish oils have been suggested as a means of lowering blood pressure. However, little is known on the effects of fish oil in PHT humans. Therefore we conducted two studies. In Study 1 we investigated PHT and normotensive (NT) individuals during a mental stress task. Mental stress is known to contribute to the development of hypertension. In Study 2 PHT and NT subjects were placed in an eight week double-blind placebo controlled study in which subjects consumed 9g/day of either fish oil or placebo (olive oil) in addition to their regular diets. Subjects were tested during a resting baseline (seated and supine), 5 minutes of a mental stress task, and 5 minutes of recovery both pre and post supplementation. We measured arterial pressure (AP), heart rate (HR), muscle sympathetic nerve activity (MSNA), and forearm and calf vascular responses. In Study 1 PHT demonstrated augmented AP and blunted vasodilation during mental stress, but MSNA did not change. In Study 2, fish oil did not directly influence blood pressure, MSNA or vascular responses to mental stress. However, it became clear that fish oil had an effect on some but not all subjects (both PHT and NT). Specifically, subjects who experienced a reduced blood pressure response to fish oil also demonstrated a decrease in MSNA and HR during mental stress. Collectively, the investigations in this dissertation had several novel findings. First, PHT individuals demonstrate an augmented pressor and blunted vascular response to mental stress, a response that may be contributing to the development of hypertension. Second, fish oil does not consistently lower resting blood pressure, but the interindividual responses may be related to MSNA. Third, fish oil attenuated the heart rate and MSNA responses and to mental stress in both PHT and NT. In conclusion, we found that there are both similarities and differences in the way PHT and NT individuals respond to mental stress and fish oil.

Acute alcohol ingestion and sympathetic neural responses during orthostatic stress in humans

Acute alcohol consumption has been reported to decrease mean arterial pressure (MAP) during orthostatic challenge, a response that may contribute to alcohol-mediated hypotension and eventually syncope. Muscle sympathetic nerve activity (MSNA) increases during orthostatic stress to help maintain MAP, yet the influence of alcohol on MSNA during orthostatic stress has not been determined. We hypothesized that alcohol ingestion would blunt arterial blood pressure and MSNA responses to progressive lower body negative pressure (LBNP). MAP, MSNA, and heart rate (HR) were recorded during progressive LBNP (-5, -10, -15, -20, -30, and -40 mmHg; 3 min/stage) in 30 subjects(age 24 ± 1 yrs). After an initial progressive LBNP protocol (pre-treatment), subjects were randomly assigned to consume alcohol (0.8g ethanol/kg body mass; n=15) or placebo (n=15) and then repeated the progressive LBNP protocol (post-treatment). Alcohol increased (drug × treatment, P ≤ 0.05) resting HR (59 ± 2 to 65 ± 2 beats/min) and MSNA (13 ± 3 to 19 ± 4 bursts/min) when compared to placebo. While alcohol increased MAP (83 ± 2 to 87 ± 2 mmHg), these increases were also observed with placebo (82 ± 2 to 88 ± 1 mmHg; treatment, P < 0.05; drug × treatment, P > 0.05). During progressive LBNP, a prominent decrease in MAP was observed after alcohol (drug × time × treatment, P < 0.05), but not placebo. There was also a significant attenuated response in forearm vascular resistance (FVR) during progressive LBNP (drug × time × treatment, P < 0.05). MSNA and HR increased during all LBNP protocols, but there were no differences between treatments or groups (drugs). In summary, acute alcohol ingestion induces an attenuation in blood pressure response during an orthostatic challenge, possibly due to the effect that alcohol has on impairing peripheral blood vessel constriction.

INFLUENCE OF 24-HOUR SLEEP DEPRIVATION ON CARDIOVASCULAR AND NEURAL CONTROL AT REST AND DURING ACUTE STRESS IN HUMANS

Recent epidemiological studies report a consistent association between short sleep and incidence of hypertension, as well as short sleep and cardiovascular disease-related mortality. While the association between short sleep and hypertension appears to be stronger in women than men, the mechanisms underlying the relations between sleep deprivation, stress, risks of cardiovascular diseases, and sex remain unclear. We conducted two studies to investigate the underlying neural mechanisms of these relations. In study 1, we examined sympathetic neural and blood pressure responses to experimentally-induced sleep deprivation in men and women. We further investigated the influence of sleep deprivation on cardiovascular reactivity to acute stress. In study 2, we examined the neural and cardiovascular function throughout the ovarian cycle in sleep deprived women. Twenty-eight young healthy subjects (14men and 14 women) were tested twice in study 1, once after normal sleep (NS) and once after 24-h total sleep deprivation (TSD). We measured the blood pressure, heart rate (HR), muscle sympathetic nerve activity (MSNA) and forearm blood flow (FBF) during 10min baseline, 5min of mental stress (MS) and 2 min cold pressor test (CPT). We demonstrated that TSD increased resting arterial blood pressure to a similar extent in both men and women, but MSNA decreased only in men following TSD. This MSNA response was associated with altered baroreflex function in women and divergent testosterone responses to TSD between men and women. Regarding TSD and cardiovascular reactivity, TSD elicited augmented HR reactivity and delayed recovery during both MS and CPT in men and women, and responses between sexes were not statistically different. Fourteen young healthy women participated in study 2. Subjects were tested twice, once during their early follicular (EF) phase after TSD, once during their mid-luteal (ML) phase after TSD. Blood pressure, HR, MSNA, and FBF were recorded during 10min baseline, 5 min MS, and 2 min CPT. We observed an augmented resting supine blood pressure during EF compared to ML in sleep deprived women. In contrast, resting MSNA, as well as cardiovascular responses to stressors, were similar between EF and ML after TSD. In conclusion, we observed sex differences in MSNA responses to TSD that demonstrate reductions of MSNA in men, but not women. TSD elicited augmented HR reactivity and delayed HR recovery to acute stressors similarly in men and women. We also reported an augmented supine blood pressure during EF compared to ML in sleep deprived women. These novel findings provide new and valuable mechanistic insight regarding the complex and poorly understood relations among sleep deprivation, sex, stress, and risk of cardiovascular disease.

Uncrossed cortico-muscular projections in humans are abundant to facial muscles of the upper and lower face, but may differ between sexes

It is a popular concept in clinical neurology that muscles of the lower face receive predominantly crossed cortico-bulbar motor input, whereas muscles of the upper face receive additional ipsilateral, uncrossed input. To test this notion, we used focal transcranial magnetic brain stimulation to quantify crossed and uncrossed cortico-muscular projections to 6 different facial muscles (right and left Mm. frontalis, nasalis, and orbicularis oris) in 36 healthy right-handed volunteers (15 men, 21 women, mean age 25 years). Uncrossed input was present in 78% to 92% of the 6 examined muscles. The mean uncrossed: crossed response amplitude ratios were 0.74/0.65 in right/left frontalis, 0.73/0.59 in nasalis, and 0.54/0.71 in orbicularis oris; ANOVA p>0.05). Judged by the sizes of motor evoked potentials, the cortical representation of the 3 muscles was similar. The amount of uncrossed projections was different between men and women, since men had stronger left-to-left projections and women stronger right-to-right projections. We conclude that the amount of uncrossed pyramidal projections is not different for muscles of the upper from those of the lower face. The clinical observation that frontal muscles are often spared in central facial palsies must, therefore, be explained differently. Moreover, gender specific lateralization phenomena may not only be present for higher level behavioural functions, but may also affect simple systems on a lower level of motor hierarchy.

Insulin signaling and action in cultured skeletal muscle cells from lean healthy humans with high and low insulin sensitivity

The aim of these studies was to investigate whether insulin resistance is primary to skeletal muscle. Myoblasts were isolated from muscle biopsies of 8 lean insulin-resistant and 8 carefully matched insulin-sensitive subjects (metabolic clearance rates as determined by euglycemic-hyperinsulinemic clamp: 5.8 +/- 0.5 vs. 12.3 +/- 1.7 ml x kg(-1) x min(-1), respectively; P < or = 0.05) and differentiated to myotubes. In these cells, insulin stimulation of glucose uptake, glycogen synthesis, insulin receptor (IR) kinase activity, and insulin receptor substrate 1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity were measured. Furthermore, insulin activation of protein kinase B (PKB) was compared with immunoblotting of serine residues at position 473. Basal glucose uptake (1.05 +/- 0.07 vs. 0.95 +/- 0.07 relative units, respectively; P = 0.49) and basal glycogen synthesis (1.02 +/- 0.11 vs. 0.98 +/- 0.11 relative units, respectively; P = 0.89) were not different in myotubes from insulin-resistant and insulin-sensitive subjects. Maximal insulin responsiveness of glucose uptake (1.35 +/- 0.03-fold vs. 1.41 +/- 0.05-fold over basal for insulin-resistant and insulin-sensitive subjects, respectively; P = 0.43) and glycogen synthesis (2.00 +/- 0.13-fold vs. 2.10 +/- 0.16-fold over basal for insulin-resistant and insulin-sensitive subjects, respectively; P = 0.66) were also not different. Insulin stimulation (1 nmol/l) of IR kinase and PI 3-kinase were maximal within 5 min (approximately 8- and 5-fold over basal, respectively), and insulin activation of PKB was maximal within 15 min (approximately 3.5-fold over basal). These time kinetics were not significantly different between groups. In summary, our data show that insulin action and signaling in cultured skeletal muscle cells from normoglycemic lean insulin-resistant subjects is not different from that in cells from insulin-sensitive subjects. This suggests an important role of environmental factors in the development of insulin resistance in skeletal muscle.

In vitro evaluation of differences in phase 1 metabolism of ketamine and other analgesics among humans, horses, and dogs

OBJECTIVE: To investigate cytochrome P450 (CYP) enzymes involved in metabolism of racemic and S-ketamine in various species and to evaluate metabolic interactions of other analgesics with ketamine. SAMPLE POPULATION: Human, equine, and canine liver microsomes. PROCEDURES: An analgesic was concurrently incubated with luminogenic substrates specific for CYP 3A4 or CYP 2C9 and liver microsomes. The luminescence signal was detected and compared with the signal for negative control samples. Ketamine and norketamine enantiomers were determined by use of capillary electrophoresis. RESULTS: A concentration-dependent decrease in luminescence signal was detected for ibuprofen and diclofenac in the assay for CYP 2C9 in human and equine liver microsomes but not in the assay for CYP 3A4 and methadone or xylazine in any of the species. Coincubation of methadone or xylazine with ketamine resulted in a decrease in norketamine formation in equine and canine liver microsomes but not in human liver microsomes. In all species, norketamine formation was not affected by ibuprofen, but diclofenac reduced norketamine formation in human liver microsomes. A higher rate of metabolism was detected for S-ketamine in equine liver microsomes, compared with the rate for the S-enantiomer in the racemic mixture when incubated with any of the analgesics investigated. CONCLUSIONS AND CLINICAL RELEVANCE: Enzymes of the CYP 3A4 family and orthologs of CYP 2C9 were involved in ketamine metabolism in horses, dogs, and humans. Methadone and xylazine inhibited in vitro metabolism of ketamine. Therefore, higher concentrations and diminished clearance of ketamine may cause adverse effects when administered concurrently with other analgesics.

Helium breathing provides modest antiinflammatory, but no endothelial protection against ischemia-reperfusion injury in humans in vivo

BACKGROUND: The noble gas helium is devoid of anesthetic effects, and it elicits cardiac preconditioning. We hypothesized that inhalation of helium provides protection against postocclusive endothelial dysfunction after ischemia-reperfusion of the forearm in humans. METHODS: Eight healthy male subjects were enrolled in this study with a crossover design. Each volunteer was randomly exposed to 15 min of forearm ischemia in the presence or absence of helium inhalation. Helium was inhaled at an end-tidal concentration of 50 vol% from 15 min before ischemia until 5 min after the onset of reperfusion ("helium conditioning"). Hyperemic reaction, a marker of nitric oxide bioavailability and endothelial function, was determined at 15 and 30 min of reperfusion on the forearm using venous occlusion plethysmography. Expression of the proinflammatory markers CD11b, ICAM-1, PSGL-1, and L-selectin (CD62L) on leukocytes and P-selectin (CD62P), PSGL-1, and CD42b on platelets were measured by flow cytometry during reperfusion. RESULTS: Ischemia-reperfusion consistently reduced the postocclusive endothelium-dependent hyperemic reaction at 15 and 30 min of reperfusion. Periischemic inhalation of helium at 50 vol% did not improve postocclusive hyperemic reaction. Helium decreased expression of the proinflammatory marker CD11b and ICAM-1 on leukocytes and attenuated the expression of the procoagulant markers CD42b and PSGL-1 on platelets. CONCLUSIONS: Although inhalation of helium diminished the postischemic inflammatory reaction, our data indicate that human endothelium, which is a component of all vital organs, is not amenable to protection by helium at 50 vol% in vivo. This is in contrast to sevoflurane, which protects human endothelium at low subanesthetic concentrations.

Direct combination of nanoparticle fabrication and exposure to lung cell cultures in a closed setup as a method to simulate accidental nanoparticle exposure of humans

The tremendous application potential of nanosized materials stays in sharp contrast to a growing number of critical reports of their potential toxicity. Applications of in vitro methods to assess nanoparticles are severely limited through difficulties in exposing cells of the respiratory tract directly to airborne engineered nanoparticles. We present a completely new approach to expose lung cells to particles generated in situ by flame spray synthesis. Cerium oxide nanoparticles from a single run were produced and simultaneously exposed to the surface of cultured lung cells inside a glovebox. Separately collected samples were used to measure hydrodynamic particle size distribution, shape, and agglomerate morphology. Cell viability was not impaired by the conditions of the glovebox exposure. The tightness of the lung cell monolayer, the mean total lamellar body volume, and the generation of oxidative DNA damage revealed a dose-dependent cellular response to the airborne engineered nanoparticles. The direct combination of production and exposure allows studying particle toxicity in a simple and reproducible way under environmental conditions.

Comparison of the receptor FGFRL1 from sea urchins and humans illustrates evolution of a zinc binding motif in the intracellular domain

BACKGROUND: FGFRL1, the gene for the fifth member of the fibroblast growth factor receptor (FGFR) family, is found in all vertebrates from fish to man and in the cephalochordate amphioxus. Since it does not occur in more distantly related invertebrates such as insects and nematodes, we have speculated that FGFRL1 might have evolved just before branching of the vertebrate lineage from the other invertebrates (Beyeler and Trueb, 2006). RESULTS: We identified the gene for FGFRL1 also in the sea urchin Strongylocentrotus purpuratus and cloned its mRNA. The deduced amino acid sequence shares 62% sequence similarity with the human protein and shows conservation of all disulfides and N-linked carbohydrate attachment sites. Similar to the human protein, the S. purpuratus protein contains a histidine-rich motif at the C-terminus, but this motif is much shorter than the human counterpart. To analyze the function of the novel motif, recombinant fusion proteins were prepared in a bacterial expression system. The human fusion protein bound to nickel and zinc affinity columns, whereas the sea urchin protein barely interacted with such columns. Direct determination of metal ions by atomic absorption revealed 2.6 mole zinc/mole protein for human FGFRL1 and 1.7 mole zinc/mole protein for sea urchin FGFRL1. CONCLUSION: The FGFRL1 gene has evolved much earlier than previously assumed. A comparison of the intracellular domain between sea urchin and human FGFRL1 provides interesting insights into the shaping of a novel zinc binding domain.

Visibility of vascular phenylalanine in dynamic uptake studies in humans using magnetic resonance spectroscopy

In general, vascular contributions to the in vivo magnetic resonance (MR) brain spectrum are too small to be relevant. In cerebral uptake studies, however, vascular contributions may constitute a major confounder. MR visibility of vascular Phe was investigated by recording localized spectra from fully oxygenated and well-mixed whole blood. Blood Phe levels determined by MR spectroscopy (MRS) and ion-exchange chromatography showed excellent correlation. In addition, effects of blood flow were shown to have a small effect on signal amplitude with the MRS methodology used. Hence, blood Phe is almost completely MR visible at 1.5 T, even though it is severely broadened at higher fields. Without appropriate correction, cerebral Phe influx in studies of brain Phe uptake in phenylketonuria patients or healthy subjects would appear to be faster and lead to higher levels. Similar effects are envisaged for studies of ethanol or glucose uptake across the blood-brain barrier.

Determination of menthol in plasma and urine of rats and humans by headspace solid phase microextraction and gas chromatography--mass spectrometry

A method for the determination of menthol and menthol glucuronide (M-G) after enzymatic hydrolysis in plasma and urine of rats and humans was developed using headspace solid phase microextraction and gas chromatography-mass spectrometry in the selected ion monitoring mode (HS-SPME/GC-MS). The assay linearity for plasma ranged from 5 to 1000 ng/ml. The limit of quantification (LOQ) in plasma was 5 ng/ml. The intra- and inter-day precision for menthol and M-G were < or = 18.1% R.S.D. at the LOQ and < or = 4.0% at higher concentrations. Menthol and M-G were determined in rat and human plasma and urine after administration of menthol.

Development and pharmacokinetic characterization of pulmonal and intravenous delta-9-tetrahydrocannabinol (THC) in humans

The aim of the present study was to develop a physiologically compatible inhalation solution of delta-9-tetrahydrocannabinol (THC), and to compare the pharmacokinetic and analgesic properties of pulmonal THC versus pulmonal placebo and intravenous (iv) THC, respectively. Eight healthy volunteers were included in this randomized, double-blind, crossover study. The aqueous THC formulations were prepared by using a solubilization technique. iv THC (0.053 mg/kg body weight), pulmonal THC (0.053 mg/kg), or a placebo inhalation solution was administered as single dose. At defined time points, blood samples were collected, and somatic and psychotropic side effects as well as vital functions monitored. An ice water immersion test was performed to measure analgesia. Using a pressure-driven nebulizer, the pulmonal administration of the THC liquid aerosol resulted in high THC peak plasma levels within minutes. The bioavailability of the pulmonal THC was 28.7 +/- 8.2% (mean +/- SEM). The side effects observed after pulmonal THC were coughing and slight irritation of the upper respiratory tract, very mild psychotropic symptoms, and headache. The side effects after iv THC were much more prominent. Neither pulmonal nor iv THC significantly reduced experimentally induced pain.