Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin

In vivo confocal Raman spectroscopy is a noninvasive optical method to obtain detailed information about the molecular composition of the skin with high spatial resolution. In vivo confocal scanning laser microscopy is an imaging modality that provides optical sections of the skin without physically dissecting the tissue. A combination of both techniques in a single instrument is described. This combination allows the skin morphology to be visualized and (subsurface) structures in the skin to be targeted for Raman measurements. Novel results are presented that show detailed in vivo concentration profiles of water and of natural moisturizing factor for the stratum corneum that are directly related to the skin architecture by in vivo cross-sectional images of the skin. Targeting of skin structures is demonstrated by recording in vivo Raman spectra of sweat ducts and sebaceous glands in situ. In vivo measurements on dermal capillaries yielded high-quality Raman spectra of blood in a completely noninvasive manner. From the results of this exploratory study we conclude that the technique presented has great potential for fundamental skin research, pharmacology (percutaneous transport), clinical dermatology, and cosmetic research, as well as for noninvasive analysis of blood analytes, including glucose.     

In Vivo Blood Glucose Quantification Using Raman Spectroscopy

We here propose a novel Raman spectroscopy method that permits the noninvasive measurement of blood glucose concentration. To reduce the effects of the strong background signals produced by surrounding tissue and to obtain the fingerprint Raman lines formed by blood analytes, a laser was focused on the blood in vessels in the skin. The Raman spectra were collected transcutaneously. Characteristic peaks of glucose (1125 cm^-1) and hemoglobin (1549 cm^-1) were observed. Hemoglobin concentration served as an internal standard, and the ratio of the peaks that appeared at 1125 cm^-1 and 1549 cm^-1 peaks was used to calculate the concentration of blood glucose. We studied three mouse subjects whose blood glucose levels became elevated over a period of 2 hours using a glucose test assay. During the test, 25 Raman spectra were collected transcutaneously and glucose reference values were provided by a blood glucose meter. Results clearly showed the relationship between Raman intensity and concentration. The release curves were approximately linear with a correlation coefficient of 0.91. This noninvasive methodology may be useful for the study of blood glucose in vivo.     

Electroosmosis in transdermal iontophoresis: implications for noninvasive and calibration-free glucose monitoring

Reverse iontophoresis uses a small low electric current to noninvasively extract blood analytes, e.g., glucose, across the skin. The simultaneous quantification of the analyte extracted and of an additional endogenous substance of fixed and known concentration in the body permits the blood level of interest to be found without the need for an invasive calibration procedure. The transport phenomena underlying this approach, applied to glucose monitoring, has been investigated in vitro, using Na+ and neutral model solutes as endogenous "internal standards" (specifically, urea, glycerol, mannitol, and sucrose). The cathodal extracted fluxes of glucose under conditions of modified skin permselectivity were related to those of the different, potential internal standards. Flux ratios depended upon the iontophoretic conditions and the size of the neutral internal standards, whereas high variability was observed with Na+. Constant flux ratios were obtained with mannitol, glycerol, urea, and sucrose for which the mechanism of electrotransport was identical to that of glucose. The advantage of using a neutral internal standard, however, must be weighed against the need to identify and validate the marker under physiological conditions and the additional analytical chemistry necessary for the practical quantification of this substance.     

Noninvasive in vivo glucose detection in human finger interstitial fluid using wavelength‐modulated differential photothermal radiometry

We present a noninvasive and noncontacting biosensor using Wavelength Modulated Differential Photothermal Radiometry (WM‐DPTR) to monitor blood glucose concentration (BGC) through interstitial fluid (ISF) probing in human middle fingers. WM‐DPTR works in the interference‐free mid‐infrared range with differential wavelengths at the peak and baseline of the fundamental glucose molecule absorption band, giving rise to high glucose sensitivity and specificity. In vivo WM‐DPTR measurements and simultaneous finger pricking BGC reference measurements were performed on diabetic and nondiabetic volunteers during oral glucose tolerance testing. The measurement results demonstrated high resolution and large dynamic range (~80 deg) change in phase signal in the normal‐to‐hyperglycemia BGC range (5 mmol/L to higher than 33.2 mmol/L), which were supported by negative control measurements. The immunity to temperature variation of WM‐DPTR yields precise and accurate noninvasive glucose measurements in the ISF.      

Influence of osmolytes on in vivo glucose monitoring using optical coherence tomography

Diabetes mellitus and its complications are the third leading cause of death in the world, exceeded only by cardiovascular disease and cancer. Tighter monitoring and control of blood glucose could minimize complications associated with diabetes. Recently, optical coherence tomography (OCT) for noninvasive glucose monitoring was proposed and tested in vivo. The aim of this work was to investigate the influence of changes in blood glucose concentration ([glu]) and sodium concentration ([Na+]) on the OCT signal. We also investigated the influence of other important analytes on the sensitivity of glucose monitoring with OCT. The experiments were carried out in anesthetized female pigs. The OCT images were acquired continuously from skin, while [glu] and [Na+] were experimentally varied within their physiological ranges. Correlations of the OCT signal slope with [glu] and [Na+] were studied at different tissue depths. The tissue area probed with OCT was marked and cut for histological examination. The correlation of blood [glu] and [Na+] with the OCT signal slope was observed in separate tissue layers. On average, equimolar changes in [glu] produced 2.26 +/- 1.15 greater alterations of the OCT signal slope than changes in [Na+]. Variation of concentrations of other analytes did not influence the OCT signal slope. The influence of [Na+] on relative changes in the OCT signal slope was generally less than [glu]-induced changes. OCT is a promising method for noninvasive glucose monitoring because of its ability to track the influence of changing [glu] on individual tissue layers.     

Assessment of transcapillary glucose exchange in human skeletal muscle and adipose tissue

We studied the kinetics of glucose exchange between plasma and interstitial fluid (ISF) in human skeletal muscle and adipose tissue under fasting conditions. Five normal human subjects received an intravenous [6,6-2H2]glucose infusion in a prime-continuous fashion. During the tracer infusion, the open-flow microperfusion technique was employed to frequently sample ISF from quadriceps muscle and subcutaneous adipose tissue. The tracer glucose kinetics observed in muscle and adipose tissue ISF were found to be well described by a capillary-tissue exchange model. As a measure of transcapillary glucose exchange efficiency, the 95% equilibrium time was calculated from the identified model parameters. This time constant was similar for skeletal muscle and adipose tissue (28.6 +/- 3.2 vs. 26.8 +/- 3.6 min; P = 0.60). Furthermore, we found that the (total) interstitial glucose concentration was significantly lower (P < 0.01) in muscle (3.32 +/- 0.46 mmol/l) and adipose tissue (3.51 +/- 0.17 mmol/l) compared with arterialized plasma levels (5.56 +/- 0.13 mmol/l). Thus the observed gradients and dynamic relationships between plasma and ISF glucose in muscle and adipose tissue provide evidence that transcapillary exchange of glucose is limited in these two tissues under fasting conditions.     

Low-frequency sonophoresis: a noninvasive method of drug delivery and diagnostics

Transdermal drug delivery offers an attractive alternative to injections and oral medications. However, applications of transdermal drug delivery are limited to only a few drugs as a result of low skin permeability. Application of low-frequency ultrasound enhances skin permeability, a phenomenon referred to as low-frequency sonophoresis. In this method, a short application of ultrasound is used to permeabilize skin for a prolonged period of time. During this period, ultrasonically permeabilized skin may be utilized for drug delivery. In addition, a sample of interstitial fluid or its components may be extracted through permeabilized skin for diagnostic applications. In this paper, we report our in vivo studies that demonstrate the principles of both of these concepts. Detailed studies on drug delivery are performed using inulin and mannitol as model drugs. Studies on diagnostics are performed using glucose as a model analyte. Applications of this technology to drug delivery and diagnostics are discussed.     

Brain distribution of 6-mercaptopurine is regulated by the efflux transport system in the blood-brain barrier

6-mercaptopurine (6-MP) has been used clinically for 40 years to maintain remission in patients with acute lymphoblastic leukemia (ALL). However, central nervous system (CNS) relapses frequently occur in patients with ALL who continuously receive anticancer drugs, including 6-MP, during remission maintenance therapy. The cause of such CNS relapse is not well understood. One possible reason may involve the restricted distribution of 6-MP in the brain. This study, therefore, investigates the blood-brain barrier (BBB) transport which largely regulates 6-MP distribution in the brain using a quantitative microdialysis technique and centers on the efflux transport of 6-MP across the BBB. The brain tissue, cerebrospinal fluid (CSF), or hippocampal interstitial fluid (ISF) concentration of 6-MP was very low compared with the unbound plasma concentration, suggesting that 6-MP distribution in the brain is highly restricted. Kinetic analyses of this BBB transport showed that the efflux clearance from brain ISF to plasma across the BBB (CLout) is approximately 20-times greater than the influx clearance from plasma to brain (CLin). The CLout was significantly reduced by 1mM N-ethylmaleimide (NEM), a sulfhydryl-modifying agent, suggesting the participation of transport protein in the efflux of 6-MP across the BBB. In addition, efflux transport was inhibited by an intracerebral infusion of probenecid (1.5 mM), p-aminohippuric acid (PAH, 3.0 mM), benzoate (3.6 mM), or salicylate (3.7 mM) administered through a microdialysis probe, but neither choline (0.8 mM) nor tetraethylammonium (TEA, 0.7 mM) had any effect. These data suggest that the restricted 6-MP brain distribution may be ascribed to efficient efflux from the brain, possibly via both the organic anion transport system, shared with probenecid and PAH, and the monocarboxylic acid transport system, shared with benzoate and salicylate.     

A geometrical model of dermal capillary clearance

A new microscopic model is developed to describe the dermal capillary clearance process of skin permeants. The physiological structure is represented in terms of a doubly periodic array of absorbing capillaries. Convection-dominated transport in the blood flow within the capillaries is coupled with interstitial diffusion, the latter process being quantified via a slender-body-theory approach. Convection across the capillary wall and in the interstitial phase is treated as a perturbation which may be added to the diffusive transport. The model accounts for the finite permeability of the capillary wall as well as for the geometry of the capillary array, based on realistic values of physiological parameters. Calculated dermal concentration profiles for permeants having the size and lipophilicity of salicylic acid and glucose illustrate the power and general applicability of the model. Furthermore, validation of the model with published in vivo experimental results pertaining to human skin permeation of hydrocortisone is presented. The model offers the possibility for in-depth theoretical understanding and prediction of subsurface drug distribution in the human skin following topical application, as well as rates of capillary clearance into the systemic circulation. A simpler approach that treats the capillary bed as a homogeneously absorbing zone is also employed. The latter may be used in conjunction with the capillary exchange model to estimate measurable dermal transport and clearance parameters in a straightforward manner.     

Transport systems of serine at the brain barriers and in brain parenchymal cells

D-Serine is a co-agonist for NMDA-type glutamate receptors. Although D-serine levels in CSF and interstitial fluid (ISF) affect CNS function, the regulatory system remains to be fully understood. Therefore, the purpose of this study was to investigate d-serine transport across the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) and in brain parenchymal cells. D-Serine microinjected into the cerebrum was not eliminated, suggesting a negligible contribution of D-serine efflux transport at the BBB. In contrast, D-serine was taken up from the circulating blood across the BBB via a carrier-mediated process. D-Serine elimination clearance from CSF was fourfold greater than that of d-mannitol, which is considered to reflect CSF bulk flow. The characteristics of D-serine uptake by isolated choroid plexus were consistent with those of Na(+)-independent alanine-serine-cysteine transporter 1 (asc-1). Uptake of D-serine by brain slices appeared to occur predominantly via asc-1 and Na(+)-dependent alanine-serine-cysteine transporter 2. These findings suggest that the regulatory system of D-serine levels in ISF and CSF involves (i) asc-1 at the BCSFB, acting as a major pathway of D-serine elimination from the CSF, (ii) blood-to-brain and blood-to-CSF influx transport of D-serine across the BBB and BCSFB, and (iii) concentrative uptake of D-serine by brain parenchymal cells.     

面向血液葡萄糖测量的生物组织阻抗模型

本文推导了细胞膜电容与血糖浓度之间的关系,并将这种关系应用到Pauly和Schwan提出的弛豫模型中,再将弛豫模型应用于多重Cole-Cole介电模型,最后通过介电模型得出阻抗模型,建立起人体血糖浓度与组织介电谱,阻抗谱之间的联系。通过分析发现,细胞膜电容随着血糖浓度增加而增大,且当血糖浓度趋于无穷大时,细胞膜电容等于血糖浓度为0时细胞膜电容的1.53倍。对介电谱的仿真得出,当外加信号频率小于1 MHz时,介电常数实部随着葡萄糖浓度改变而发生明显变化。阻抗谱的研究结果表明,随着葡萄糖浓度的升高,阻抗的实部减小,阻抗虚部模增大,且分别在频率为1.48 MHz和0.55 MHz时变化得最快。本文建立的模型为血糖无创检测提供了一种新的方法和手段。     

Bone tissue engineering: the role of interstitial fluid flow

It is well established that vascularization is required for effective bone healing. This implies that blood flow and interstitial fluid (ISF) flow are required for healing and maintenance of bone. The fact that changes in bone blood flow and ISF flow are associated with changes in bone remodeling and formation support this theory. ISF flow in bone results from transcortical pressure gradients produced by vascular and hydrostatic pressure, and mechanical loading. Conditions observed to alter flow rates include increases in venous pressure in hypertension, fluid shifts occurring in bedrest and microgravity, increases in vascularization during the injury-healing response, and mechanical compression and bending of bone during exercise. These conditions also induce changes in bone remodeling. Previously, we hypothesized that interstitial fluid flow in bone, and in particular fluid shear stress, serves to mediate signal transduction in mechanical loading- and injury-induced remodeling. In addition, we proposed that a lack or decrease of ISF flow results in the bone loss observed in disuse and microgravity. The purpose of this article is to review ISF flow in bone and its role in osteogenesis.     

Quantitative interrelationship between Gibbs-Donnan equilibrium, osmolality of body fluid compartments, and plasma water sodium concentration.

The presence of negatively charged, impermeant proteins in the plasma space alters the distribution of diffusible ions in the plasma and interstitial fluid (ISF) compartments to preserve electroneutrality. We have derived a new mathematical model to define the quantitative interrelationship between the Gibbs-Donnan equilibrium, the osmolality of body fluid compartments, and the plasma water Na+ concentration ([Na+]pw) and validated the model using empirical data from the literature. The new model can account for the alterations in all ionic concentrations (Na+ and non-Na+ ions) between the plasma and ISF due to Gibbs-Donnan equilibrium. In addition to the effect of Gibbs-Donnan equilibrium on Na+ distribution between plasma and ISF, our model predicts that the altered distribution of osmotically active non-Na+ ions will also have a modulating effect on the [Na+]pw by affecting the distribution of H2O between the plasma and ISF. The new physiological insights provided by this model can for the first time provide a basis for understanding quantitatively how changes in the plasma protein concentration modulate the [Na+]pw. Moreover, this model defines all known physiological factors that may modulate the [Na+]pw and is especially helpful in conceptually understanding the pathophysiological basis of the dysnatremias.     

Hydrogel-Forming Microneedle Arrays Allow Detection of Drugs and Glucose In Vivo: Potential for Use in Diagnosis and Therapeutic Drug Monitoring

We describe, for the first time the use of hydrogel-forming microneedle (MN) arrays for minimally-invasive extraction and quantification of drug substances and glucose from skin in vitro and in vivo. MN prepared from aqueous blends of hydrolysed poly(methyl-vinylether-co-maleic anhydride) (11.1% w/w) and poly(ethyleneglycol) 10,000 daltons (5.6% w/w) and crosslinked by esterification swelled upon skin insertion by uptake of fluid. Post-removal, theophylline and caffeine were extracted from MN and determined using HPLC, with glucose quantified using a proprietary kit. In vitro studies using excised neonatal porcine skin bathed on the underside by physiologically-relevant analyte concentrations showed rapid (5 min) analyte uptake. For example, mean concentrations of 0.16 μg/mL and 0.85 μg/mL, respectively, were detected for the lowest (5 μg/mL) and highest (35 μg/mL) Franz cell concentrations of theophylline after 5 min insertion. A mean concentration of 0.10 μg/mL was obtained by extraction of MN inserted for 5 min into skin bathed with 5 μg/mL caffeine, while the mean concentration obtained by extraction of MN inserted into skin bathed with 15 μg/mL caffeine was 0.33 μg/mL. The mean detected glucose concentration after 5 min insertion into skin bathed with 4 mmol/L was 19.46 nmol/L. The highest theophylline concentration detected following extraction from a hydrogel-forming MN inserted for 1 h into the skin of a rat dosed orally with 10 mg/kg was of 0.363 μg/mL, whilst a maximum concentration of 0.063 μg/mL was detected following extraction from a MN inserted for 1 h into the skin of a rat dosed with 5 mg/kg theophylline. In human volunteers, the highest mean concentration of caffeine detected using MN was 91.31 μg/mL over the period from 1 to 2 h post-consumption of 100 mg Proplus^® tablets. The highest mean blood glucose level was 7.89 nmol/L detected 1 h following ingestion of 75 g of glucose, while the highest mean glucose concentration extracted from MN was 4.29 nmol/L, detected after 3 hours skin insertion in human volunteers. Whilst not directly correlated, concentrations extracted from MN were clearly indicative of trends in blood in both rats and human volunteers. This work strongly illustrates the potential of hydrogel-forming MN in minimally-invasive patient monitoring and diagnosis. Further studies are now ongoing to reduce clinical insertion times and develop mathematical algorithms enabling determination of blood levels directly from MN measurements.     

血糖浓度检测技术的最新进展

本文阐述了血糖浓度检测技术的最新进展.从直接接触式的血糖浓度临床检测方法,以及市面上的各种微创式可随身携带的血糖浓度检测仪的测量技术,到近期正在研究的无创伤血糖浓度检测技术的描述,着重论述了目前国内外正在研究尚没应用于临床的无创伤血糖浓度检测的各种技术原理和方案,并对各种无创伤血糖浓度测量技术的工作原理和存在的临床应用问题进行分析,同时展望了血糖检测技术未来的发展之路.     

A mixture theory model of fluid and solute transport in the microvasculature of normal and malignant tissues. I. Theory

In order to better understand the mechanisms governing transport of drugs, nanoparticle-based treatments, and therapeutic biomolecules, and the role of the various physiological parameters, a number of mathematical models have previously been proposed. The limitations of the existing transport models indicate the need for a comprehensive model that includes transport in the vessel lumen, the vessel wall, and the interstitial space and considers the effects of the solute concentration on fluid flow. In this study, a general model to describe the transient distribution of fluid and multiple solutes at the microvascular level was developed using mixture theory. The model captures the experimentally observed dependence of the hydraulic permeability coefficient of the capillary wall on the concentration of solutes present in the capillary wall and the surrounding tissue. Additionally, the model demonstrates that transport phenomena across the capillary wall and in the interstitium are related to the solute concentration as well as the hydrostatic pressure. The model is used in a companion paper to examine fluid and solute transport for the simplified case of an axisymmetric geometry with no solid deformation or interconversion of mass.     

Fluid mechanics in the perivascular space

Perivascular space (PVS) within the brain is an important pathway for interstitial fluid (ISF) and solute transport. Fluid flowing in the PVS can affect these transport processes and has significant impacts on physiology. In this paper, we carry out a theoretical analysis to investigate the fluid mechanics in the PVS. With certain assumptions and approximations, we are able to find an analytical solution to the problem. We discuss the physical meanings of the solution and particularly examine the consequences of the induced fluid flow in the context of convection-enhanced delivery (CED). We conclude that peristaltic motions of the blood vessel walls can facilitate fluid and solute transport in the PVS.     

Multifrequency Analysis of Single Inductive Coil Measurements Across a Gel Phantom Simulation of Internal Bleeding in the Brain

The present study is part of an ongoing effort to develop a simple diagnostic technology for detecting internal bleeding in the brain, which can be used in lieu or in support of medical imaging and thereby reduce the cost of diagnostics in general, and in particular, would make diagnostics accessible to economically disadvantaged populations. The study deals with a single coil inductive device to be used for detecting cerebral hemorrhage. It presents a first-order experimental study that examines the predictions of our recently published theoretical study. The experimental model employs a homogeneous cylindrical phantom in which internal head bleeding was simulated by way of a fluid inclusion. We measured the changes in amplitude and phase across the coil with a network vector analyzer as a function of frequency (100–1,000 MHz), volume of blood simulating fluid, and the site of the fluid injection. We have developed a new mathematical model to statistically analyze the complex data produced in this experiment. We determined that the resolution for the fluid volume increase following fluid injection is strongly dependent on frequency as well as the location of liquid accumulation. The experimental data obtained in this study supports the predictions of our previous theoretical study, and the statistical analysis shows that the simple single coil device is sensitive enough to detect changes due to fluid volume alteration of two milliliters. Bioelectromagnetics. 2020;41:21–33 © 2019 Bioelectromagnetics Society     

Steady-State Cerebral Glucose Concentrations and Transport in the Human Brain

Abstract: Understanding the mechanism of brain glucose transport across the blood-brain barrier is of importance to understanding brain energy metabolism. The specific kinetics of glucose transport have been generally described using standard Michaelis-Menten kinetics. These models predict that the steady-state glucose concentration approaches an upper limit in the human brain when the plasma glucose level is well above the Michaelis-Menten constant for half-maximal transport, K _t. In experiments where steady-state plasma glucose content was varied from 4 to 30 m M , the brain glucose level was a linear function of plasma glucose concentration. At plasma concentrations nearing 30 m M , the brain glucose level approached 9 m M , which was significantly higher than predicted from the previously reported K _t of ∼4 m M ( p < 0.05). The high brain glucose concentration measured in the human brain suggests that ablumenal brain glucose may compete with lumenal glucose for transport. We developed a model based on a reversible Michaelis-Menten kinetic formulation of unidirectional transport rates. Fitting this model to brain glucose level as a function of plasma glucose level gave a substantially lower K _t of 0.6 ± 2.0 m M , which was consistent with the previously reported millimolar K _m of GLUT-1 in erythrocyte model systems. Previously reported and reanalyzed quantification provided consistent kinetic parameters. We conclude that cerebral glucose transport is most consistently described when using reversible Michaelis-Menten kinetics.     

Isoosmotic transport of fluid across the hamster small intestine in the presence of phlorizin-induced inhibition of sugar transport.

Experiments were performed to investigate whether the fluid transported across the small intestine is isoosmotic with the mucosal solution when the active transport of glucose is partially inhibited. Everted hamster mid small intestine was incubated in one of the following four mucosal solutions: (1) Isotonic control, Krebs-Ringer bicarbonate solution containing 10 mM glucose (KRBSG), (2) Isotonic with phlorizin, KRBSG + 5X10-5 M phlorizin, (3) Hypertonic control, KRBSG + 50 mM mannitol, (4) Hypertonic with phlorizin, KRBSG + 50 MM mannitol + 5x10-5 M phlorizin. The serosal surface of the intestine was not bathed. Results indicate that the transported fluid was always isoosmotic with any of the mucosal solutions used. When the mucosal solution was made hypertonic with mannitol, the concentration of glucose and electrolytes in the absorbate increased, and as a result, the absorbate became hypertonic and isoosmotic with the mucosal solution. The presence of phlorizin either in the isotonic or in the hypertonic mucosal solution decreased the glucose concentration of the absorbate, but the transported fluid became isoosmotic with the mucosal solution due to a higher concentration of Na, K, and their associated anions. Phlorizin caused a decrease in the transmural potential difference. In spite of this, the presence of this glucoside in the mucosal solution increased the transport of sodium in relation to glucose transport. It is suggested that, at the concentrations used, phlorizin inhibits sodium movement through the electrogenic pathway, but increases the transport of this ion through the non-electrogenic route. This increase in neutral sodium transport seems to compensate for the low concentration of glucose in the absorbate, so that the absorbate becomes isoosmotic with the mucosal solution whether the latter is isotonic or hypertonic. It is suggested further that isoosmotic transport of fluid is an inherent property of the small intestine and that there may be an osmoregulatory mechanism in the gut which controls this process.