Anorectal manometry assessment of sphincter relaxation after local-regional anesthesia with posterior perineal block.

INTRODUCTION: Since the 1970s, in the USA, we witnessed a progressive increase of one-day surgical procedures. This attitude soon gained ground in Europe as well. In proctology, this kind of clinical approach has always been limited by the acute sensitivity of the anal- perineal area and by difficulties in attaining a complete sphincter relaxation with local anesthesia. Posterior perineal block seems to be associated with both a good pain control and an effective sphincter relaxation. MATERIAL AND METHODS: Between January 2017 and January 2018, we enrolled in our study 33 patients suffering from hemorrhoidal disease. They were all subjected to posterior perineal block. We measured anal resting pressure and squeeze pressure before and after anesthesia. Measurements where taken 5 minutes before and 15 minutes after the administration of local. RESULTS: We registered an average decrease of 39,2% of resting pressure and of 45,4% of squeeze pressure. CONCLUSIONS: We may state that perineal posterior block, while reducing striated muscle contractile activity, also causes a relevant reduction of anal basal tone. During surgical procedures done under regional anesthesia, we experienced a good sphincter relaxation, which was comparable, if not equal, to that induced by general anesthesia. In fact, 10 to 15 minutes after performing the block you could observe the elevation of the inferior margin of the exterior sphincter and the concomitant descent of the inferior margin of the internal sphincter (coaxial dislocation). KEY WORDS: Anorectal manometry, Anesthesia, Local-regional, Perineal block.

Assessment of the Airway Smooth Muscle Relaxant Effect of Drotaverine.

BACKGROUND: Drotaverine, a type 4 cyclic nucleotide phosphodiesterase (PDE4) inhibitor, blocks the degradation of 3',5'-cyclic adenosine monophosphate. However, published receptor binding data showed that drotaverin also binds to the L-type voltage-operated calcium channel (L-VOCC). Based on these molecular mechanisms of action, a direct and indirect (by blocking the constrictor response) relaxant effect on airway smooth muscle can be predicted, which has not yet been assessed. SUMMARY: Accordingly, drotaverine and reference agents were tested both on the histamine-, methacholine-, or KCl-induced contraction response and on precontracted guinea pig tracheal preparations. It was found that drotaverine not only relaxed the precontracted tracheal preparations but also decreased mediator-induced contraction. These effects of drotaverine were concentration dependent, with a significantly higher potency on the KCl-induced response, than on either the histamine or methacholine induced one. A similar result was noted for nifedipine. The PDE inhibitor, theophylline, also relaxed the precontracted preparations but was ineffective on the mediator-induced contraction in a physiologically relevant concentration range. Moreover, theophylline did not show selectivity and was the least potent relaxant among the 3 tested molecules. Key Message: These results show that drotaverine is a more potent airway smooth muscle relaxant molecule than theophylline. This enhanced potency on relaxation and inhibition of the constrictor response, at least partly, may be explained by the combined L-VOCC blocking and PDE inhibitory potential of drotaverine.

MR-FLIP: a new method that combines a functional lumen imaging probe with anatomical information for spatial compliance assessment of the anal sphincter muscles.

AIM: Continence results from a complex interplay between anal canal (AC) muscles and sensorimotor feedback mechanisms. The passive ability of the AC to withstand opening pressure - its compliance - has recently been shown to correlate with continence. A functional lumen imaging probe (FLIP) is used to assess AC compliance, although it provides no anatomical information. Therefore, assessment of the compliance specific anatomical structures has not been possible, and the anatomical position of critical functional zones remains unknown. In addition, the FLIP technique assumes a circular orifice cross-section, which has not been shown for the AC. To address these shortcomings, a technique combining FLIP with a medical imaging modality is needed. METHOD: We implemented a new research method (MR-FLIP) that combines FLIP with MR imaging. Twenty healthy volunteers underwent MR-FLIP and conventional FLIP assessment. MR-FLIP was validated by comparison with FLIP results. Anatomical markers were identified, and the cross-sectional shape of the orifice was investigated. RESULTS: MR-FLIP provides compliance measurements identical to those obtained by conventional FLIP. Anatomical analysis revealed that the least compliant AC zone was located at the proximal end of the external anal sphincter (EAS). The cross-sectional shape of the AC was found to deviate only slightly from circularity in healthy volunteers. CONCLUSION: The proposed method is equivalent to classical FLIP. It establishes for the first time direct mapping between local tissue compliance and anatomical structure, which is key to gaining novel insights into (in)continence. In addition, MR-FLIP provides a tool for better understanding conventional FLIP measurements in the AC by quantifying its limitations and assumptions.

In vitro evaluation of ureteral contractility: a comparative assessment of human, porcine and sheep ureteral response to vardenafil.

OBJECTIVES: Basic science studies of ureteral physiology and pathophysiology are commonly performed on animal ureters due to several limitations associated with human ureteral sampling. In this work we question whether animal ureters are good replicas of human ureteral behavior for pharmacological studies. MATERIALS AND METHODS: Ureteral rings from human, porcine and ovine ureters underwent the same organ bath protocol. After stimulation with KCl, ureters were subjected to different doses of vardenafil. Basic contractility and ureteral response to vardenafil were analyzed. RESULTS: A different pattern of basic contractility was evidenced between species. Vardenafil administration induced a dose-dependent reduction in KCl-induced amplitude increase in human ureters and a dose-dependent reduction in autonomic contractile rhythm of porcine and ovine ureters. Although animal ureters could predict the relaxant response of human samples to vardenafil, its effect would have been overestimated using only animal models. CONCLUSIONS: Human ureteral investigations cannot entirely be replaced by existing animal models since results of the latter will vary significantly according to the tested pharmaceutical agent.

Anal fissure nanocarrier of lercanidipine for enhanced transdermal delivery: formulation optimization, ex vivo and in vivo assessment.

OBJECTIVE: Pathogenesis of anal fissure (AF) is associated with raised resting anal pressures (RAP) involving contraction of smooth muscle. Therefore, the drug delivery strategy should be customized to reduce this raised RAP. In this investigation, in order to achieve this task, a transdermal nanoemulsion (NE) gel of lercanidipine (LER) was developed and optimized to evaluate its permeation ability and in vivo performance. Further, the same formulation was explored for droplet size analysis, zeta potential measurement and stability studies. METHODS: Pseudo-ternary phase diagram was constructed to determine NE region. The NE was optimized (OPT) by employing three-factor, three-level Box-Behnken design expert software; the independent variables decided were composition of oil, Smix and water and dependent variables, that is, responses were cumulative amount of drug permeated across rat abdominal skin in 24 h (Q24), steady-state flux (Js) and viscosity. The in vivo efficacy was assessed by measuring anorectal pressure in male Wistar rats. RESULTS: The OPT NE formulation, composed of Capryol 90 (12.70% w/w), Cremophor EL (18.0% w/w), Transcutol HP (18.0% w/w) and water (60.00%) w/w was found to have permeation flux of 60.27 µg/cm(2)/h, release of 1699.52 µg/24 h and 491.95 cP viscosity. In addition, a small average droplet size (82.71 ± 9.96 nm) and long-term stability at room temperature (1.666 years) was observed. The in vivo investigation demonstrated direct evidence on significant reduction (27.75%) in anorectal pressure over a period of 4 h. CONCLUSION: These preliminary finding suggested that NE-based gel system of LER may provide promising perspective in management of AF.

Inference of mechanical states of intestinal motor activity using hidden Markov models.

BACKGROUND: Contractions and relaxations of the muscle layers within the digestive tract alter the external diameter and the internal pressures. These changes in diameter and pressure move digesting food and waste products. Defining these complex relationships is a fundamental step for neurogastroenterologists to be able define normal and abnormal gut motility. RESULTS: Utilising an in vitro technique that allows for the simultaneous recording of intraluminal pressure (manometry) and gut diameter (video) in an isolated section of rabbit colon, we developed a technique to help define the mechanical states of the muscle at any point in space and time during actual peristaltic movements. This was achieved by directly relating the changes in pressure to the changes in diameter along the length of the gut studied. For each individual measure of pressure or diameter, 3 dynamic state components were identified; increasing or decreasing changes or a stable period. Two additional static state components, fully contracted and fully distended, were defined for the diameter. Then qualitative mechanical states of the muscle activity were defined as combinations of these state components. A hidden Markov model was used to correlate adjacent-in-time samples, and the Viterbi algorithm was used to infer the most likely sequence of mechanical states based on the observed data. From this a spatiotemporal map of the mechanical states was produced, showing the regions of active contractions, active relaxations, or passive states along the length of the gut throughout the entire recording period. CONCLUSIONS: The identification of mechanical muscles states based on gut diameter and intraluminal pressure was possible by modelling muscle activation with a hidden Markov model.

Assessment of airway hyperresponsiveness in murine tracheal rings.

Isolated tracheal rings have often been used to directly measure the contractile output of airway smooth muscle (ASM). Here, we describe the method for excising murine tracheas, mounting tracheal rings in organ baths, and measuring the isometric forces generated by the ASM when stimulated by drug additions or electric field stimulation. The apparatus for the setup and the pathways responsible for stimulation are detailed. Examples of the responses and analyses of two types of ASM stimulation are illustrated: (1) the carbachol concentration-response curve and (2) the frequency-response curve elicited by electric field stimulation.

Assessment of levator ani morphology and function in asymptomatic nulliparous women via static and dynamic magnetic resonance imaging.

OBJECTIVE: To evaluate levator ani morphology and function in healthy nulliparous women using static and dynamic magnetic resonance imaging. METHODS: Eighty asymptomatic, healthy nulliparous Chinese women (mean age, 25.3±3.5years) volunteered for the present study. Static T2-weighted fast spin-echo images were employed to evaluate levator ani morphology; dynamic T2-weighted fast imaging employing steady-state acquisition was used to evaluate its function. A 2 samples t test was employed to compare groups. RESULTS: No morphologic abnormality was detected in the 80 healthy nulliparous women. However, 15% (12/80) of women had various degrees of pelvic organ descent below the pubococcygeal line. In these women, the width of the pubic portion of the levator ani was significantly reduced during straining, whereas the levator plate angle, the levator hiatus area, and the H and M line lengths were enlarged. These changes were associated with weakened levator ani function and pelvic floor laxity. CONCLUSION: Functional abnormality of the levator ani muscle was noted in nulliparous women at static and dynamic magnetic resonance imaging. Further follow-up investigation is needed to confirm whether women with functional abnormality are more likely to develop a prolapse after vaginal birth.

Assessment of biological activity of novel peptide analogues of angiotensin IV.

OBJECTIVES: Angiotensin IV (Ang IV) is a metabolite of angiotensin II which acts on specific AT(4) receptors identified as the enzyme insulin regulated aminopeptidase (IRAP). The transduction process of these receptors is unresolved, but Ang IV inhibits the aminopeptidase activity. Ang IV improves cognition in animal models thus there is a desire to develop metabolically stable analogues for further development. METHODS: Peptide analogues of Ang IV were obtained commercially or synthesised. Each peptide was tested in vitro for its ability to inhibit the aminopeptidase activity (IRAP) of mouse brain homogenates and for its effects on isolated rat uterine smooth muscle. KEY FINDINGS: [Des-Val(1) ]-Ang IV, acetylated-Ang IV-amide, Ang IV-amide and [des-His(4) ]-Ang IV all inhibited IRAP. [Sar(1) , Ile(8) ]-Angiotensin II (10 µm) had an effect greater than that of Ang IV or any of the other analogues studied. In isolated uterine smooth muscle, angiotensins II and IV induced contractions, which could be antagonised by an AT(1) -receptor antagonist. None of the novel peptides induced uterine smooth muscle contractions, but [Sar(1) , des Arg(2) -Gly(8) ]-angiotensin II showed significant antagonism of the contractile effects of angiotensin II and carboxyamide-terminated Ang IV-NH(2) showed antagonism of Ang IV-induced contractions. CONCLUSIONS: This study provides five novel inhibitors of IRAP worthy of assessment in behavioural models of learning and memory. The analogues are devoid of AT(1) receptor agonist properties, and the carboxyamide analogue presents an opportunity to elucidate the mechanism of action of Ang IV as, like Ang IV, it inhibits IRAP, but antagonises the effects of Ang IV on isolated smooth muscle.

Geometry assessment of anal sphincter muscle based on monopolar multichannel surface EMG signals.

Anatomical studies on the external anal sphincter (EAS) indicate that superficial muscle fibres are circular at low depth within the anal canal. A more complex geometry of the fibres is documented for increasing depth within the muscle and along the anal canal. Monopolar intra-anal EMG signals recorded using an array of electrodes placed in circular direction have no common mode components if the muscle fibres are circular, with constant depth within the muscle and parallel to the detection array. Thus, the presence of common mode signals may provide indications about the geometry of muscle fibres of EAS. Intra-anal EMG signals were recorded from EAS of 12 subjects using an anal probe carrying three circumferential arrays of 16 electrodes at three depths within the anal canal. Contribution of common mode components in single MUAPs was lower for MUs located superficially in the muscle (Pearson correlation coefficient: R=-0.75, p≪0.001) and at a lower depth within the anal canal (non-parametric one way Kruskal-Wallis ANOVA, Χ=17.3, p<0.001), in line with EAS anatomy. A large contribution of common mode components was found in the interference signal, suggesting that the signal receives contributions from far, large muscles (e.g. puborectalis, glutei).

Assessment of gastrointestinal motility using three different assays in vitro.

The protocols detailed in this unit are designed to assess the motor activity of different gastric and intestinal muscle preparations in vitro and the effects of drugs that modulate gastrointestinal motility. The preparations described are characterized by different contractile behaviors, consisting of spontaneous (duodenum), neurogenic (ileum), and drug-stimulated (fundus, ileum) motility; these reproduce motility patterns occurring in the gut wall in vivo. These protocols document the variety of factors that can influence the responses of isolated tissues and describe how such tissues can be used for testing substances that affect gut movements. These preparations allow evaluation of direct interactions with the processes that control contractile machinery, as well as indirect effects resulting from the modification of neurotransmitter release from myenteric neurons. These models can be exploited to assay novel compounds undergoing preclinical development or to evaluate the functional toxicity exerted by environmental or alimentary pollutants, like xenobiotics and naturally occurring toxins, as well as the mechanisms underlying these effects.

The assessment of rat ureteral pressure generation in vitro: regional heterogeneity and influence of distending pressure.

Contraction of ureteral smooth muscle drives the urine bolus to the urinary bladder for storage prior to micturition. This study describes a novel approach to the measurement of ureteral pressure generation in vitro and the influence of distending pressure on acetylcholine-stimulated ureteral lumenal pressure generation. Isolated segments of ureters obtained from Wistar rats were pressurised in a blind-ended sac arrangement and contractile responses were recorded as phasic oscillations in ureteral luminal pressure. Distal segments generated greater luminal pressures than proximal segments (p<0.001) in response to acetylcholine. Increasing baseline distending pressures in the range 2-10 mmHg in proximal segments was associated with greater frequency of contraction (p<0.001) and decreased magnitude of contraction (p<0.001) when expressed as % maximum response. Nifedipine (10(-5) M) or removal of extracellular Ca(2+) abolished the contractions. Isometric contractile responses of ureteral ring preparations were not significantly influenced by pretensions equivalent to distending pressures in the range 2-10 mmHg. This is the first study to fully establish the influence of baseline ureteral distending pressure upon ureteral luminal pressure generation in vitro and demonstrates regional heterogeneity of ureteral contractile responses. It is suggested that this experimental approach may be a useful methodology for the investigation of ureteral function during urinary outflow obstruction.

Assessment and characterization of purinergic contractions and relaxations in the rat urinary bladder.

The aim of the present study was to assess the purinoceptor functional responses of the urinary bladder by using isolated rat urinary bladder strip preparations. ATP elicited a transient bladder contraction followed by a sustained relaxation and ADP, UDP and UTP generated predominantly potent relaxations (relaxatory potencies: ADP = ATP > UDP = UTP). The ATP contractions were desensitized with the P2X(1/3) purinoceptor agonist/desensitizer alpha,beta-meATP and reduced by the P2 purinoceptor antagonist PPADS but unaffected by the P2 purinoceptor antagonist suramin. Electrical field stimulation (1-60 Hz) evoked frequency-dependent bladder contractions that were decreased by incubation with alpha,beta-meATP but not further decreased by PPADS. Suramin antagonized relaxations generated by UDP but not those by ADP, ATP or UTP. PPADS antagonized and tended to antagonize UTP and UDP relaxations, respectively, but did neither affect ADP nor ATP relaxations. ADP relaxations were insensitive to the P2Y(1) purinoceptor antagonist MRS 2179 and the ATP-sensitive potassium channel antagonist glibenclamide. The ATP relaxations were inhibited by the P1 purinoceptor antagonist 8-p-sulfophenyltheophylline but unaffected by the A2A adenosine receptor antagonist 8-(3-chlorostyryl)caffeine and glibenclamide. Adenosine evoked relaxations that were antagonized by the A2B adenosine receptor antagonist PSB 1115. Thus, in the rat urinary bladder purinergic contractions are elicited predominantly by stimulation of the P2X(1) purinoceptors, while UDP/UTP-sensitive P2Y purinoceptor(s) and P1 purinoceptors of the A2B adenosine receptor subtype are involved in bladder relaxation.

Quantitative magnetic resonance imaging assessment of matrix development in cell-seeded natural urinary bladder smooth muscle tissue-engineered constructs.

The approach of cell-seeded natural scaffolds holds great promise for tissue engineering complicated soft-tissue organs such as the urinary bladder and heart. However, relatively little is known about cell-natural scaffold interactions or their influence on magnetic resonance imaging (MRI) characterization, which is valuable for noninvasive monitoring. Ideally, MRI should provide information on tissue biochemistry in addition to structure and function. In this study, quantitative MRI was performed on control and smooth muscle cell-seeded natural bladder matrices at different time points up to 7 days postseeding. Measurements of MR relaxation times (T1 and T2) and diffusion coefficient (D) showed an overall change that was incompatible with cell presence. Multicomponent T2 provided greater specificity, revealing time-course changes in the short T2 fraction that were consistent with biochemically determined matrix degradation from collagenase released from seeded cells. These matrix alterations are noted for the first time, and their relatively early occurrence may be unique to soft-tissue matrices compared with synthetic materials. More importantly, they are not evident on histology but are revealed on quantitative MRI. We conclude that quantitative MRI may provide specific information on cell-matrix interaction and is a promising noninvasive approach to understand and monitor cell-seeded natural scaffold-based regeneration.

An inner medullary concentrating process actuated by renal pelvic/calyceal muscle contractions: assessment and hypothesis.

We propose a mechanism of an inner medullary concentrating process in which water extraction is accomplished by a colloid osmotic mechanism and hydrostatic pressure. There are 3 essential features of the proposal: 1. the fluid compartmental structure of the inner medullary interstitium: owing to molecular exclusion, negatively charged macromolecules, i.e. hyaluronan and extravasated plasma albumin form separate compartments (the HA and the EPA compartments); the resulting Gibbs-Donnan effect governs the movements of both ions and water. 2. NaCl, in high concentration in the inner medulla conditioned by the outer medullary countercurrent processes, significantly reduces the equilibrium colloid osmotic pressure between these compartments. 3. Urea, also accumulated here by special transport mechanisms, increases the mobility of water molecules and the flexibility of the HA fibrils by loosening hydrogen bonds. These features suggest that rhythmic, small pressure increases of the pelvic/calyceal muscles squeeze dilute fluid out of the HA compartment and, at the same time, accelerate the outflow of fluid and albumin into the ascending vasa recta from the EPA compartment. Further, they suggest a mechanism for the phenomenon that living organisms utilize hydrostatic pressure generated by muscle contractions in water economy namely, concentrating and diluting body fluids.

Assessment of muscarinic receptor subtypes in human and rat lower urinary tract by tissue segment binding assay.

Muscarinic receptors in the human and rat lower urinary tract (urinary bladder detrusor muscle and mucosa, and prostate) were identified by intact tissue segment binding assays with two radioligands, and the effects of prolonged receptor activation in vitro on muscarinic receptors were examined. Hydrophilic [(3)H]-NMS and hydrophobic [(3)H]-QNB bound to the detrusor muscle segments with the same density, suggesting that the muscarinic receptors were localized at the plasma membrane. While the density of muscarinic receptor was higher in detrusor muscle than in the bladder mucosa and prostate, there was no species-specific difference either in density or in subtype distribution (M(1), M(2), and M(3) subtypes in detrusor; M(2) and M(3) subtypes in bladder mucosa; and M(1) and M(2) subtypes in prostate). Incubation of detrusor strips with carbachol decreased [(3)H]-NMS binding sites within 20 min, followed by a reduction of [(3)H]-QNB binding sites after a 60-min lag phase. The loss of the binding sites over 3 h after carbachol treatment was the same (approximately 40%) for both radioligands. The present intact tissue segment binding assay reveals tissue-specific and plasma membrane distribution of distinct muscarinic receptor subtypes and their dynamic changes (internalization and down-regulation) in lower urinary tract of humans and rats.

In vivo assessment of safety and mechanisms underlying in vitro relaxation induced by Mikania laevigata Schultz Bip. ex Baker in the rat trachea.

Mikania laevigata, popularly known in Brazil as "guaco", is largely used in folk medicine against respiratory diseases. However, neither the assessment of the toxicity of "guaco" syrup (GS, used by humans) nor its efficacy or mechanisms of action has been properly investigated. Using in vitro procedures, we showed that the hydroalcoholic extract (HE) from Mikania laevigata induces a concentration-dependent relaxation of rat trachea which does not depend on epithelium-derived substances but involves changes in the cellular mobilization of calcium, perhaps due to a direct effect on membrane potassium channels. In addition, we assessed both oral and intraperitoneal acute toxicity, as well as the oral subchronic and chronic toxicity of GS containing controlled amounts of coumarin, the main biological marker of Mikania laevigata preparations used in humans. The calculated LD(50) of GS after intraperitoneal administration was 0.904 g/kg in mice (both sexes) and 0.967 and 0.548 g/kg in male and female rats, respectively. However, the LD(50) values of GS by the oral route were calculated to be up to 10 g/kg, in both male and female mice and rats. Repeated dose 28- or 90-day oral treatment with GS (75, 150 and 300 mg/kg) did not produce any disturbances in the hematological or biochemical parameters of either male or female rats, nor did it provide evidence of toxicity in the hepatic, renal or pancreatic systems. Besides the mechanistic findings, our results provide evidence of the safety of Mikania laevigata in rodents, even after subchronic and chronic administration, at least in relation to the evaluated parameters.

Simultaneous assessment of the intraluminal pressure and transit time of the colon using a telemetry technique.

Colonic motility disorders are common conditions. However, our understanding of normal and pathological motor functions of the colon remains limited, mainly due to the technical difficulties in accessing this organ for study. To investigate colonic motility under normal physiological conditions, we have developed a novel monitoring system based on a telemetry technique. The system is capable of prolonged and noninvasive measurement of intraluminal pressure changes and transit time of intra-colonic contents. To test the in vivo performance of the monitoring system, 15 healthy volunteers and 15 patients with functional constipation (FC) participated in this study. A single-use telemetry capsule embedded with sensors was ingested by the subjects. The capsule is capable of transmitting colonic pressure and temperature wirelessly. The time of the telemetry capsule entering the segmental colon was detected by ultrasonic detection of the batteries in capsule. Pressure recordings confirmed in general a circadian behavior of colonic motility, as well as its response to waking and meals. In the FC patients, the contractile response to morning awakening and meal ingestion was significantly lower compared to the controls. The transit time measured using this method agreed with the time calculated from radiopaque markers (r = 0.89, p < 0.05). The clinical study proved both the reliability and the noninvasiveness of the system. This capsule-style manometric system may represent a useful tool for the study of physiology and pathology of colonic motor disorders.

Skin biopsy for assessment of autonomic denervation in Parkinson's disease.

Autonomic dysfunction in Parkinson's disease (PD) is considered a late complication of the disease or an adverse effect of anti-parkinsonian medications. Morphological changes are demonstrated only by postmortem examination. The study objective was to evaluate peripheral autonomic neural involvement in PD using punch skin biopsy. The study sample included 22 patients (mean age 50 +/- 7.7 years, mean disease duration 5.3 +/- 3.8 years) and 19 controls. Four-millimeter skin biopsies were immunohistochemically stained with anti-PGP 9.5 antibody. Autonomic innervation of the blood vessels, sweat glands, and erector pili muscles was assessed and rated from 0 (normal) to 2 (severe). Cutaneous autonomic innervation was decreased in patients compared to controls. Semi quantitative analysis demonstrated reduced autonomic innervation of the blood vessels (1.0 +/- 0.8 vs. 0.42 +/- 0.8 in controls; p < 0.02), of sweat glands (0.95 +/- 0.67 vs. 0.47 +/- 0.61; p < 0.02) and of the erector pili muscles (1.06 +/- 0.55 vs 0.21 +/- 0.42; p < 0.001). This method demonstrates that the peripheral autonomic system is affected in PD at early stage of the disease and that autonomic involvement in PD may be more prevalent than previously thought.