In-line Raman imaging of mixing by herringbone grooves in microfluidic channels.

The control over fluid flow achievable in microfluidic devices creates opportunities for applications in many fields. In simple microchannels, flow is purely laminar when one solvent is used, and hence, achieving reliable mixing is an important design consideration. Integration of structures, such as grooves, into the channels to act as static mixers is a commonly used approach. The mixing induced by these structures can be validated by determining concentration profiles in microfluidic channels following convergence of solvent streams from separate inlets. Spatially resolved characterisation is therefore necessary and requires in-line analysis methods. Here we report a line-focused illumination approach to provide operando, spatially resolved Raman spectra across the width of channels in the analysis of single- and multi-phase liquid systems and chemical reactions. A scientific complementary metal oxide semiconductor (sCMOS) sensor is used to overcome smearing encountered during spectral readout of images with CCD sensors. Isotopically labelled probes, in otherwise identical flow streams, show that z-confocality limits the spatial resolution and certainty as to the extent of mixing that can be achieved. These limitations are overcome using fast chemical reactions between reagents entering a microchannel in separate solvent streams. We show here that the progression of a chemical reaction, for which only the product is observable, is a powerful approach to determine the extent of mixing in a microchannel. Specifically resonance enhancement of Raman scattering from a product formed allows for determination of the true efficiency of mixing over the length and width of microchannels. Raman spectral images obtained by line-focused illumination show onset of mixing by observing the product of reagents entering from the separate inlets. Mixing is initially off-centre and immediately before the apex of the first groove of the static mixer, and then evolves along the entire width of the channel after a full cycle of grooves.

Selective Analysis of Redox Processes at the Electrode Interface with Time-Resolved Raman Spectroscopy.

Electrochemistry and electrochemical reactions are increasingly important in the transition to a sustainable chemical industry. The electron transfer that drives such reactions takes place within nanometers of the electrode surface, and follow-up chemical reactions take place within the diffusion layer. Hence, understanding electrochemical reactions requires time-, potential-, and spatially resolved analysis. The confocal nature of Raman spectroscopy provides high spatial resolution, in addition to detailed information on molecular structure. The intrinsic weakness of nonresonant Raman scattering, however, is not sensitive enough for relatively minor changes to the solution resulting from reactions at the electrode interface. Indeed, the limit of detection is typically well above the concentrations used in electrochemical studies. Here, we show that surface-enhanced Raman scattering (SERS) and resonance Raman (rR) spectroscopy allow for spatially and time-resolved analysis of solution composition at (<1-2 nm) and near (within 5 µm) the electrode surface, respectively, in a selective manner for species present at low (<1 mM) concentrations. We show changes in concentration of species at the electrode surface, without the need for labels, specific adsorption, or resonance enhancement, using a SERS-active gold electrode prepared readily by electrochemical surface roughening. A combination of smooth and roughened gold electrodes is used to distinguish between surface and resonance enhancement using the well-known redox couples ferrocene and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). We discuss the impact of specific adsorption on the spectral analysis with the ruthenium(II) polypyridyl complex, [Ru(bpy)3]2+. The dual function of the electrode (surface enhancement and electron transfer) in the analysis of solution processes is demonstrated with the reversible oxidation of TMA (4,N,N-trimethylaniline), where transient soluble species are identified in real time, with rapid spectral acquisition, making use of localized enhancement. We anticipate that this approach will find use in elucidating electro(catalytic) reactions at electrode interfaces.

Single wavelength colour tuning of spiropyran and dithienylethene based photochromic coatings.

Controlling the transmission of thin films with external stimuli is an important goal in functional optical materials and devices. Tuning is especially challenging where both broad band (neutral density filtering) and spectrally varied (colour) transmission are required. The external control provided by photochemically driven switching, between transmission levels and colours, is functionally simple from a device perspective. The limits due to the spectral ranges of individual photochromic compounds can be overcome by combining several photochromes within one material or device. Here we show that a combination of photochromic molecular switches immobilised in a PMMA polymer matrix enables tuning of colour and transparency. We show that only a single excitation wavelength is required through the use of the primary inner filter effect and the layered construction of the films in which the photochromes nitrospiropyran (NSP), and nitrothiospiropyran (TSP) or 1,2-bis-terthienyl-hexafluorocyclopentene (DTE) are separated spatially. The approach taken circumvents the need to match photochemical quantum yields and thermal reactivity of the component photochromes. The photochemical switching of the films was characterised by UV/vis absorption spectroscopy and shows that switching rates and photostationary states are limited by inner filter effects rather than the intrinsic properties of photochromes, such as photochemical quantum yields and thermal stability. The photochemical behaviour and stability of the photochromes in solution and in the PMMA films were compared and the concentration range over which self-inhibition of photochemical switching occurs was established. The rate of photochemical switching and the difference in transmission between the spiropyran and merocyanine forms in solution enable prediction of the performance in the films and enable rational design of colour tuning ranges and responsivity in thin film filters.

Minimal blocking concentrations of bupivacaine and procaine in an exclusively nociceptive system in humans.

BACKGROUND AND OBJECTIVES: Prior to this investigation, there was no approach to compare both the potency of local anesthetics and their time course of action in a reproducible nociceptive system in humans. We tested whether the vascularly isolated vein segment is appropriate for such an approach. METHODS: In six healthy men, a hand vein segment was vascularly isolated and intraluminally stimulated with electropulses of constant current intensity. The subjects rated pain between threshold and maximally tolerable pain on a visual analogue scale. For determining minimal blocking concentrations (a measure of potency), the vein segment was continuously perfused with Tyrode's solution with increasing concentrations of bupivacaine or procaine for at least 10 minutes each until pain was completely blocked. Subsequently, the respective local anesthetic was rinsed off with Tyrode's solution to determine the time course of recovery. RESULTS: Both bupivacaine and procaine blocked pain in a concentration-related fashion, the minimal blocking concentrations being 1.6 (0.6-1.9; median and range) mmol/L for bupivacaine and 15.0 (7.5-22.5) mmol/L for procaine. Whereas the onset of block (time of 50% block) did not differ significantly between bupivacaine and procaine [43 s (range, 3-80) vs 53 s (range, 30-115)], local anesthesia lasted significantly longer after application of bupivacaine [278 s (range, 215-325)] than after procaine [183 s (range, 125-225)]. CONCLUSIONS: The vascularly isolated vein segment is well suited to compare in vivo the properties of local anesthetics with a minimally invasive approach at a reproducible nociceptive system in humans.

[Chronopharmacology. Time of day variations in the action of bupivacaine are not manifested in nerves]. / Chronopharmakologie. Tageszeitabhängige Schwankungen in der Wirksamkeit von Bupivacain manifestieren sich nicht am Nerven.

UNLABELLED: To test the hypothesis that the efficacy of local anaesthetics to block nerve conduction is related the time of day (TOD) of drug application, we retrospectively analysed data from previous experiments on tachyphylaxis. During the course of these experiments, as a measure of drug efficacy we determined the minimal blocking concentration (c(m)) of bupivacaine in rabbit aortic nerves at several TODs. The special in situ preparation used permitted study of local anaesthetic pharmacodynamics without the influence of pharmacokinetics, making it possible to investigate the chronopharmacodynamics of these drugs by relating c(m)s to the respective TODs of their measurement. METHODS: In 43 New Zealand rabbits anaesthetised with urethane, the aortic nerve was dissected and partly placed in a double-lumen perfusion chamber (Fig. 1A), which was continuously perfused with tyrode solution or bupivacaine. Spike activity was continuously recorded by bipolar platinum-iridium electrodes caudad to the chamber for control and cephalad for registration of blocking effects. As a measure of drug efficacy, by increasing the bupivacaine concentration stepwise we determined the smallest concentration that blocks spike activity i.e., c(m). After each determination bupivacaine was rinsed off to confirm intact nerve function (Fig. 18). RESULTS: Forty-nine determinations of bupivacaine c(m) were performed between 12:25 p.m. and 2:35 a.m. Data were pooled into groups of 2 h (Fig. 2). There was no significant difference between groups (ANOVA). In particular, c(m) at 3:00 p.m. was not lower than at 11:00 p.m., times at which local anaesthetics have been found to be most and least effective, respectively. CONCLUSIONS: The c(m) of bupivacaine, and thus its efficacy to block nerve conduction, does not depend on TOD of drug application. Therefore, it is suggested that chronopharmacodynamics does not play an important role in the well-known circadian rhythm of the action of bupivacaine and probably of local anaesthetics in general.

[Conduction block in man is stimulation frequency dependent]. / Leitungsblockaden beim Menschen sind stimulationsfrequenzabhängig.

The action of local anaesthetics on isolated nerves is enhanced by high stimulation frequencies. The aim of our study was to investigate whether high-frequency stimulation enhances regional anaesthesia in man. METHODS. Seven healthy volunteers underwent three ulnar nerve blocks while non-noxious electrical stimulation with high (10, 50 Hz) or low (0.2 Hz) frequencies was applied via a widespread cutaneous electrode in the area supplied by the ulnar nerve. Perception was monitored continuously by means of a visual analogue scale. Skin temperature (infrared telethermometry) was evaluated every minute as an indicator of vasomotor block. After complete loss of perception or after 45 min, the spread of anaesthesia was determined by sharp-dull discrimination. The nerve block was preceded by a control stimulation of 45 min under otherwise identical conditions. RESULTS. During stimulation with 10 and 50 Hz, perception was lost significantly earlier than with 0.2 Hz. The spread of sensory block at the end of the experiments was also enhanced by stimulation with high frequencies, whereas the onset of vasomotor block (rise in skin temperature) remained unaltered. CONCLUSION. Non-oxious electrical stimulation with high frequencies significantly accelerates the onset of anaesthesia and extends the spread of sensory block.

The nociceptive systems of skin, paravascular tissue and hand veins of humans and their sensitivity to bradykinin.

To study the pain-evoking properties of bradykinn on the nociceptive systems of skin, paravascular tissue and hand veins of humans, bradykinin was injected intracutaneously, retrogradially into occluded finger veins for reaching the paravascular tissue or into vascularly isolated hand vein segments of seven subjects. Regardless of the injection site, bradykinin always evoked pain of concentration-related intensity within nearly similar concentration ranges of 0.1-10.0 microM, yielding congruent pain intensity-concentration relations. Thus, in humans, the nociceptive systems of skin, deep tissue and hand veins are equally sensitive to the endogenous algetic bradykinin.

Dermal anaesthesia: comparison of EMLA cream with iontophoretic local anaesthesia.

We have compared the efficacy of two non-invasive methods of transdermal anaesthesia: application of EMLA cream and iontophoresis of 5% lignocaine with adrenaline 1:50,000 in six healthy subjects. We tested depth of tissue penetration (pinprick) and effect on pain evoked by i.v. injection. After iontophoresis, pain on i.v. injection was abolished in five of six volunteers, whereas EMLA had no effect. We conclude that local anaesthetics penetrate deeply enough to numb both veins and skin with iontophoresis only.

Effects of arginine-vasopressin on regional blood volume distribution in supine humans.

In healthy humans, the increase in arterial blood pressure seen in patients with autonomic dysfunction in response to exogenous vasopressin (AVP) is abolished. We tested the hypothesis that redistribution of blood from the intra- to the extrathoracic vascular compartment might contribute to this buffer response. Regional distribution of 99mTc labeled autologous red cells was assessed in healthy supine volunteers (n = 7) during arginine-vasopressin administration (1 ng.kg-1 bolus i.v. followed by a 14-min infusion of 3 ng.kg-1 x min-1), along with arterial and central venous pressures, and heart rate. Exogenous vasopressin increased plasma vasopressin concentration from 4.0 +/- 1.4 SEM to 91 pg.ml-1 +/- 12. Thoracic counts increased slightly but significantly by 2.2% +/- 0.9, while global abdominal counts remained unchanged. Most surprisingly, counts in the liver markedly increased (+8.1% +/- 1.8, p = 0.02), but significantly decreased in the spleen (-3.1% +/- 1.4). Intestinal (-2.5% +/- 2.4) and limb counts did not change significantly. Consistent with the increase in thoracic counts central venous pressure increased from 3.6 mmHg +/- 1 to 4.7 +/- 1 (p = 0.02), while arterial pressure and heart rate did not change. All changes reversed towards baseline when vasopressin administration ceased. Thus, in humans with an intact autonomic system, vasopressin, at concentrations observed during hypotension, increases liver and, albeit to a small extent, also thoracic blood volume, but decreases splenic blood content. These results: 1) are incompatible with the hypothesis that AVP induces a shift of blood from intra- to extrathoracic capacitance vessels, and 2) show that AVP increases rather than decreases central blood volume.

In vivo determination of concentration-effect curves of local anaesthetics in man.

In clinical use, the concentrations of local anaesthetics at the site of action are unknown. With the method described here, concentrations of local anaesthetics can be predetermined and kept constant at the site of action. In six volunteers, a blister was raised on the ventral surface of the forearm. After removal of the epithelium, the blister base was rinsed continuously with carbogenated Tyrode's solution with and without increasing concentrations of bupivacaine (Carbostesin) for 15 min each. The effects of bupivacaine were determined by changes in the perception (tactile sensation) of drops falling on the blister base from increasing heights. The minimal height at which the drops were just perceived characterized the threshold of perception. With increasing bupivacaine concentrations, threshold increased until the drops were no longer perceived, at a median concentration of 2.48 mmol litre-1 (range 1.24-3.10 mmol litre-1). After the blister base was rinsed with Tyrode's solution, threshold of perception reached baseline values, which was in accordance with an intact blister base.

Capsaicin did not evoke pain from human hand vein segments but did so after injections into the paravascular tissue.

1. To see if pain from veins is mediated by C fibre endings, the C fibre stimulant capsaicin was applied intravenously, and, for comparison, paravenously and intracutaneously. 2. Capsaicin, dissolved in the fat emulsion Intralipid, was applied intravenously by continuous perfusion of vascularly isolated hand vein segments as well as by injections into occluded finger veins. Using the latter approach chemicals reach the paravascular space. 3. Pain intensities were recorded continuously with an electronically controlled visual analogue scale for deriving capsaicin concentration-pain intensity relations and the time course of pain (latencies, pain durations). 4. Capsaicin always evoked pain upon injection into skin and paravenous tissue (0.3-6.5 microM) and into occluded finger veins (3.3-33 microM), whereas it had no effect whatsoever when perfused through hand vein segments even at a concentration of 650 microM. 5. Pain intensity increased with concentration and usually reached the tolerance maximum at the fivefold threshold concentration, so that the concentration-pain intensity relations were congruent for the various routes of drug application. 6. The latencies and pain durations were independent of the capsaicin concentration, but were substantially longer with injections into occluded finger veins (latency 10-30 s, pain duration 60-120 s) than with intradermal or paravenous injections (2-9 s, 10-28 s). 7. These observations show for the first time a functional similarity between the nociceptive C fibre system of the skin and the paravascular tissues, and by inference, they dismiss the possibility that C fibre endings mediate pain in cutaneous veins.

Pain and inflammation evoked in human skin by bradykinin receptor antagonists.

We showed the intrinsic effects of the bradykinin (BK) receptor antagonists, NPC 567 (D-Arg-[Hyp3,D-Phe7]BK) and NPC 349 (D-Arg-[Hyp3,Thi5,8,D-Phe-7]BK), in the skin of humans. NPC 567 and NPC 349 caused dose-dependent pain, wheal, and flare on intracutaneous injection. After bradykinin, only the pain, but not the wheal and flare reactions were dose-dependent. The intravenous application of antihistamines (dimetidinmaleate and ranitidine) had little effect on pain intensity and reduced both wheal and flare response to the antagonists but not to bradykinin. We conclude that NPC 567 and NPC 349 have pain-evoking and histamine-releasing properties in the skin of humans which may limit their therapeutic and experimental use.

The role of nociceptors of cutaneous veins in the mediation of cold pain in man.

1. To test the hypothesis that nociceptors of cutaneous veins mediate cold pain, we studied in man the time course of pain intensity and skin sensibility in relation to both intracutaneous and vein wall temperature during cooling of the dorsum of the hand by ice water before and after perivenous and intravenous nerve block. 2. Upon exposure to cold, intracutaneous temperatures fell exponentially (half-life/45-75s) within 10 min to a median of 4 degrees C (range 2-9 degrees C) and returned to baseline with a similar time course during rewarming (half-life/40-85 s). 3. Skin sensitivity to pin prick disappeared and returned at almost the same intracutaneous temperatures (16-26 degrees C). Pain, however, occurred and eve increased when the skin was already numb. 4. Pain occurred during cooling and disappeared during rewarming at vein wall temperatures between 23 and 28 degrees C and its intensity increased to a maximum of 72-100% of visual analogue scale as vein wall temperature decreased to a minimum of 9 degrees C (range 7-10.5 degrees C). 5. the pain intensity-vein wall temperature relations derived from skin cooling with threshold temperature changes between -5.5 and -9 degrees C and slopes between 2.2 and 3.3 were congruent to those derived from intravenous cooling in a previous study to ours. 6. Perivenous and intravenous nerve block, which did not alter the sensitivity of skin and periosteum, relieved cold pain markedly (perivenous block) or completely (intravenous block). 7. These observations are consistent with the hypothesis that nociceptors of cutaneous veins mediate cold pain in humans.

Adenosine does not evoke pain from venous and paravascular nociceptors in the human.

OBJECTIVE: The pain evoking and pain modulating properties of adenosine were studied at venous and paravascular nociceptors in humans. METHODS: In six volunteers, adenosine (3 to 15.10(-3) M) was perfused continuously through vascularly isolated segments of dorsal hand veins or injected into occluded finger veins. The effects of adenosine on pain evoked by intravenous electrostimulation of hand veins were also studied. The subjects rated the pain intensities on a visual analogue scale. RESULTS: Adenosine neither evoked pain nor altered the intensities of electrically evoked pain. CONCLUSIONS: Adenosine does not mediate or modulate pain via venous or paravascular nociceptors. Therefore these nociceptors are unlikely to be involved in adenosine related ischaemia pain.

Nalbuphine does not act analgetically in electrical painful tooth pulp stimulation in man.

Nalbuphine is a mixed opioid agonist/antagonist, the analgesic properties of which are still open to debate. In a randomized and placebo-controlled protocol, we compared its effects (0.2 mg/kg) on man's perception of multimodal stimuli (i.e., nociceptive, acoustic and visual) to those of aspirin (acetylsalicylic acid, 10 mg/kg) and meperidine (0.5 mg/kg). Amplitudes and latencies of the evoked potentials (EP), tolerance maxima to painful tooth pulp stimuli, and subjective intensity ratings were measured as indicators of drug induced perception differences. After nalbuphine, the EP amplitudes markedly decreased while the stimuli of each modality were rated by the subjects to be of higher intensity. Also, the tolerance maxima of painful tooth pulp stimulation were reduced by nalbuphine, and naloxone did not have additional effects. In contrast, after aspirin and meperidine, the subjective pain ratings corresponded to the reduction of nociceptive EP amplitudes. Tolerance maxima to painful stimulation were also increased by both drugs. While aspirin did not influence acoustic and visual perception, meperidine caused a slight increase in EP amplitudes as well as in the intensity ratings of these stimuli relative to placebo. Thus, at the dose studied, nalbuphine did not act analgetically. The amplitude reduction of nociceptive EP suggested that nalbuphine had analgetic properties. These were, however, not confirmed by subjective pain ratings nor by changes in tolerance maxima.

Pain on injection of propofol: effects of concentration and diluent.

The emulsion formulation of propofol (Diprivan) evokes pain on i.v. injection, although its pH and osmolality are close to those of blood. The pain induced by serial dilutions of propofol in Intralipid and 5% glucose was examined in isolated vein segments and after intracutaneous injection. Propofol evoked pain in a concentration-related manner in six of eight subjects after i.v. perfusion and in all eight subjects after intracutaneous injections. Pain was maximal with propofol 56 x 10(-3) mol litre-1 when visual analogue pain scale was 60% of maximum (range 20-92%) for venous perfusion and 89% (range 66-100%) for intracutaneous injection. Dilution with 10% Intralipid reduced pain more than that with 5% glucose. We conclude that the intensity of pain after i.v. injection of propofol was related to its free aqueous concentration.

[Paraplegia following removal of an epidural catheter]. / Paraplegie nach Entfernung eines Epiduralkatheters.

Acute paraplegia caused by an epidural hematoma developed in a patient following the removal of an epidural catheter. This catheter had been used for 3 days for postoperative pain relief with no apparent complications. Heparin (10,000 units/day) had been infused for thrombosis prophylaxis and was associated with a normal activated partial thromboplastin time (aPTT) for the first two postoperative days. However, test results from blood drawn prior to catheter removal revealed, in retrospect, an unexpected prolongation of the aPTT (75 s) and PT (56%, Quick's method). An epidural hematoma extending from T12 to L4 was evacuated during emergency laminectomy and neurologic deficits resolved completely over the next days. Thus, the removal of an epidural catheter has the potential for inducing formation of an epidural hematoma. Accordingly, it may be safest to leave epidural catheters in place if test results demonstrate a bleeding diathesis or if a potential for bleeding is suspected on clinical grounds.

Pain but no temperature sensations are evoked by thermal stimulation of cutaneous veins in man.

To test the hypothesis that the sensory innervation of veins subserves nociception exclusively, we studied pain and temperature sensations on intravenously applied thermal stimuli before and after blockade of either cutaneous or venous afferents in man. Intravenous cooling or warming of a cutaneous vein segment of the hand was associated with corresponding changes in skin temperature and evoked pain only after numbing the skin but temperatures sensations only after intravenous local anesthesia. Thus, the sensory elements of veins, probably polymodal nociceptors, convey pain only on thermal stimulation while coactivated cutaneous thermoreceptors are responsible for concomitant temperature sensations.

Pain on i.v. injection of some anaesthetic agents is evoked by the unphysiological osmolality or pH of their formulations.

We have studied the intensity and time-course of pain during and after injection into an isolated vein segment in seven normal subjects of saline or glucose of different osmolalities (0-6 osmol kg-1) or pH (2-13). Pain scores were recorded continuously by a modified visual analogue scale apparatus. With osmolar stimulation, pain occurred at 1.0 osmol kg-1 during perfusion and 3.0 osmol kg-1 with rapid injection and increased with osmolar concentration of both saline and glucose solutions. Acidic and alkaline solutions evoked pain at a pH value less than 4 or greater than 11. We conclude that pain on i.v. injection of some sedative and hypnotic drugs is likely to be caused by formulations of extremely unphysiological osmolalities or pH values.