Publications by authors named "Matilde Cordero-Erausquin"

13 Publications

  • Page 1 of 1

Enhanced analgesic cholinergic tone in the spinal cord in a mouse model of neuropathic pain.

Neurobiol Dis 2021 Apr 18;155:105363. Epub 2021 Apr 18.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, 67000 Strasbourg, France; University of Strasbourg Institute for Advanced Study (USIAS), 67000 Strasbourg, France. Electronic address:

Endogenous acetylcholine (ACh) is an important modulator of nociceptive sensory processing in the spinal cord. An increased level of spinal ACh induces analgesia both in humans and rodents while interfering with cholinergic signaling is allodynic, demonstrating that a basal tone of spinal ACh modulates nociceptive responses in naïve animals. The plasticity undergone by this cholinergic system in chronic pain situation is unknown, and the mere presence of this tone in neuropathic animals is controversial. We have addressed these issues in mice through behavioral experiments, histology, electrophysiology and molecular biology, in the cuff model of peripheral neuropathy. Our behavior experiments demonstrate the persistence, and even increased impact of the analgesic cholinergic tone acting through nicotinic receptors in cuff animals. The neuropathy does not affect the number or membrane properties of dorsal horn cholinergic neurons, nor specifically the frequency of their synaptic inputs. The alterations thus appear to be in the neurons receiving the cholinergic signaling, which is confirmed by the fact that subthreshold doses of acetylcholinesterase (AChE) inhibitors in sham animals become anti-allodynic in cuff mice and by the altered expression of the β2 nicotinic receptor subunit. Our results demonstrate that endogenous cholinergic signaling can be manipulated to relieve mechanical allodynia in animal models of peripheral neuropathy. Until now, AChE inhibitors have mainly been used in the clinics in situations of acute pain (parturition, post-operative). The fact that lower doses (thus with fewer side effects) could be efficient in chronic pain conditions opens new avenues for the treatment of neuropathic pain. SIGNIFICANCE STATEMENT: Chronic pain continues to be the most common cause of disability that impairs the quality of life, accruing enormous and escalating socio-economic costs. A better understanding of the plasticity of spinal neuronal networks, crucially involved in nociceptive processing, could help designing new therapeutic avenues. We here demonstrate that chronic pain modifies the spinal nociceptive network in such a way that it becomes more sensitive to cholinergic modulations. The spinal cholinergic system is responsible for an analgesic tone that can be exacerbated by acetylcholinesterase inhibitors, a property used in the clinic to relief acute pain (child birth, post-op). Our results suggest that lower doses of acetylcholinesterases, with even fewer side effects, could be efficient to relieve chronic pain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nbd.2021.105363DOI Listing
April 2021

Absence of Subcerebral Projection Neurons Is Beneficial in a Mouse Model of Amyotrophic Lateral Sclerosis.

Ann Neurol 2020 10 11;88(4):688-702. Epub 2020 Jul 11.

Inserm UMR_S 1118, Mécanismes centraux et périphériques de la neurodégénérescence, Faculté de Médecine, Université de Strasbourg, Strasbourg, France.

Objective: Recent studies carried out on amyotrophic lateral sclerosis patients suggest that the disease might initiate in the motor cortex and spread to its targets along the corticofugal tracts. In this study, we aimed to test the corticofugal hypothesis of amyotrophic lateral sclerosis experimentally.

Methods: Sod1 and Fezf2 knockout mouse lines were crossed to generate a model that expresses a mutant of the murine Sod1 gene ubiquitously, a condition sufficient to induce progressive motor symptoms and premature death, but genetically lacks corticospinal neurons and other subcerebral projection neurons, one of the main populations of corticofugal neurons. Disease onset and survival were recorded, and weight and motor behavior were followed longitudinally. Hyper-reflexia and spasticity were monitored using electromyographic recordings. Neurodegeneration and gliosis were assessed by histological techniques.

Results: Absence of subcerebral projection neurons delayed disease onset, reduced weight loss and motor impairment, and increased survival without modifying disease duration. Absence of corticospinal neurons also limited presymptomatic hyper-reflexia, a typical component of the upper motoneuron syndrome.

Interpretation: Major corticofugal tracts are crucial to the onset and progression of amyotrophic lateral sclerosis. In the context of the disease, subcerebral projection neurons might carry detrimental signals to their downstream targets. In its entirety, this study provides the first experimental arguments in favor of the corticofugal hypothesis of amyotrophic lateral sclerosis. ANN NEUROL 2020;88:688-702.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ana.25833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540428PMC
October 2020

The rat corticospinal system is functionally and anatomically segregated.

Brain Struct Funct 2017 Dec 20;222(9):3945-3958. Epub 2017 May 20.

Departamento de Neurobiología del Desarrollo y Neurofisiología, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus UNAM-Juriquilla, Querétaro, Mexico.

The descending corticospinal (CS) projection has been considered a key element for motor control, which results from direct and indirect modulation of spinal cord pre-motor interneurons in the intermediate gray matter of the spinal cord, which, in turn, influences motoneurons in the ventral horn. The CS tract (CST) is also involved in a selective and complex modulation of sensory information in the dorsal horn. However, little is known about the spinal network engaged by the CST and the organization of CS projections that may encode different cortical outputs to the spinal cord. This study addresses the issue of whether the CS system exerts parallel control on different spinal networks, which together participate in sensorimotor integration. Here, we show that in the adult rat, two different and partially intermingled CS neurons in the sensorimotor cortex activate, with different time latencies, distinct spinal cord neurons located in the dorsal horn and intermediate zone of the same segment. The fact that different populations of CS neurons project in a segregated manner suggests that CST is composed of subsystems controlling different spinal cord circuits that modulate motor outputs and sensory inputs in a coordinated manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00429-017-1447-6DOI Listing
December 2017

Neuronal networks and nociceptive processing in the dorsal horn of the spinal cord.

Neuroscience 2016 Dec 3;338:230-247. Epub 2016 Sep 3.

Institut des Neurosciences Cellulaires et Intégratives, UPR3212, Centre National de la Recherche Scientifique, 67084 Strasbourg, France; Université de Strasbourg, 67084 Strasbourg, France. Electronic address:

The dorsal horn (DH) of the spinal cord receives a variety of sensory information arising from the inner and outer environment, as well as modulatory inputs from supraspinal centers. This information is integrated by the DH before being forwarded to brain areas where it may lead to pain perception. Spinal integration of this information relies on the interplay between different DH neurons forming complex and plastic neuronal networks. Elements of these networks are therefore potential targets for new analgesics and pain-relieving strategies. The present review aims at providing an overview of the current knowledge on these networks, with a special emphasis on those involving interlaminar communication in both physiological and pathological conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuroscience.2016.08.048DOI Listing
December 2016

Sensorimotor Integration by Corticospinal System.

Front Neuroanat 2016 9;10:24. Epub 2016 Mar 9.

Departamento de Neurobiología del Desarrollo y Neurofisiología, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus UNAM-Juriquilla Querétaro, México.

The corticospinal (CS) tract is a complex system which targets several areas of the spinal cord. In particular, the CS descending projection plays a major role in motor command, which results from direct and indirect control of spinal cord pre-motor interneurons as well as motoneurons. But in addition, this system is also involved in a selective and complex modulation of sensory feedback. Despite recent evidence confirms that CS projections drive distinct segmental neural circuits that are part of the sensory and pre-motor pathways, little is known about the spinal networks engaged by the corticospinal tract (CST), the organization of CS projections, the intracortical microcircuitry, and the synaptic interactions in the sensorimotor cortex (SMC) that may encode different cortical outputs to the spinal cord. Here is stressed the importance of integrated approaches for the study of sensorimotor function of CS system, in order to understand the functional compartmentalization and hierarchical organization of layer 5 output neurons, who are key elements for motor control and hence, of behavior.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnana.2016.00024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4783411PMC
March 2016

Loss of inhibitory tone on spinal cord dorsal horn spontaneously and nonspontaneously active neurons in a mouse model of neuropathic pain.

Pain 2016 07;157(7):1432-1442

Institut des Neurosciences Cellulaires et Intégratives, CNRS UPR3212, Strasbourg, France.

Plasticity of inhibitory transmission in the spinal dorsal horn (SDH) is believed to be a key mechanism responsible for pain hypersensitivity in neuropathic pain syndromes. We evaluated this plasticity by recording responses to mechanical stimuli in silent neurons (nonspontaneously active [NSA]) and neurons showing ongoing activity (spontaneously active [SA]) in the SDH of control and nerve-injured mice (cuff model). The SA and NSA neurons represented 59% and 41% of recorded neurons, respectively, and were predominantly wide dynamic range (WDR) in naive mice. Nerve-injured mice displayed a marked decrease in the mechanical threshold of the injured paw. After nerve injury, the proportion of SA neurons was increased to 78%, which suggests that some NSA neurons became SA. In addition, the response to touch (but not pinch) was dramatically increased in SA neurons, and high-threshold (nociceptive specific) neurons were no longer observed. Pharmacological blockade of spinal inhibition with a mixture of GABAA and glycine receptor antagonists significantly increased responses to innocuous mechanical stimuli in SA and NSA neurons from sham animals, but had no effect in sciatic nerve-injured animals, revealing a dramatic loss of spinal inhibitory tone in this situation. Moreover, in nerve-injured mice, local spinal administration of acetazolamide, a carbonic anhydrase inhibitor, restored responses to touch similar to those observed in naive or sham mice. These results suggest that a shift in the reversal potential for anions is an important component of the abnormal mechanical responses and of the loss of inhibitory tone recorded in a model of nerve injury-induced neuropathic pain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/j.pain.0000000000000538DOI Listing
July 2016

A novel population of cholinergic neurons in the macaque spinal dorsal horn of potential clinical relevance for pain therapy.

J Neurosci 2013 Feb;33(9):3727-37

Institut des Neurosciences Cellulaires et Intégratives, Unité Propre de Recherche 3212, Centre National de la Recherche Scientifique, Department Nociception et Douleur, Strasbourg Cedex 67084, France.

Endogenous acetylcholine (ACh) is a well-known modulator of nociceptive transmission in the spinal cord of rodents. It arises mainly from a sparse population of cholinergic interneurons located in the dorsal horn of the spinal cord. This population was thought to be absent from the spinal cord of monkey, what might suggest that spinal ACh would not be a relevant clinical target for pain therapy. In humans, however, pain responses can be modulated by spinal ACh, as evidenced by the increasingly used analgesic procedure (for postoperative and labor patients) consisting of the epidural injection of the acetylcholinesterase inhibitor neostigmine. The source and target of this ACh remain yet to be elucidated. In this study, we used an immunolabeling for choline acetyltransferase to demonstrate, for the first time, the presence of a plexus of cholinergic fibers in laminae II-III of the dorsal horn of the macaque monkey. Moreover, we show the presence of numerous cholinergic cell bodies within the same laminae and compared their density and morphological properties with those previously described in rodents. An electron microscopy analysis demonstrates that cholinergic boutons are presynaptic to dorsal horn neurons as well as to the terminals of sensory primary afferents, suggesting that they are likely to modulate incoming somatosensory information. Our data suggest that this newly identified dorsal horn cholinergic system in monkeys is the source of the ACh involved in the analgesic effects of epidural neostigmine and could be more specifically targeted for novel therapeutic strategies for pain management in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.3954-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6619320PMC
February 2013

Nociceptive thresholds are controlled through spinal β2-subunit-containing nicotinic acetylcholine receptors.

Pain 2011 Sep 26;152(9):2131-2137. Epub 2011 Jul 26.

Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique, Strasbourg, France Faculté des Sciences de la Vie, Université de Strasbourg, Strasbourg, France Division of Molecular Psychiatry, Abraham Ribicoff Research Facilities, Connecticut Mental Health Center, Yale University School of Medicine, New Haven, CT, USA.

Although cholinergic drugs are known to modulate nociception, the role of endogenous acetylcholine in nociceptive processing remains unclear. In the current study, we evaluated the role of cholinergic transmission through spinal β(2)-subunit-containing nicotinic acetylcholine receptors in the control of nociceptive thresholds. We show that mechanical and thermal nociceptive thresholds are significantly lowered in β(2)(∗)-knockout (KO) mice. Using nicotinic antagonists in these mice, we demonstrate that β(2)(∗)-nAChRs are responsible for tonic inhibitory control of mechanical thresholds at the spinal level. We further hypothesized that tonic β(2)(∗)-nAChR control of mechanical nociceptive thresholds might implicate GABAergic transmission since spinal nAChR stimulation can enhance inhibitory transmission. Indeed, the GABA(A) receptor antagonist bicuculline decreased the mechanical threshold in wild-type but not β(2)(∗)-KO mice, and the agonist muscimol restored basal mechanical threshold in β(2)(∗)-KO mice. Thus, β(2)(∗)-nAChRs appeared to be necessary for GABAergic control of nociceptive information. As a consequence of this defective inhibitory control, β(2)(∗)-KO mice were also hyperresponsive to capsaicin-induced C-fiber stimulation. Our results indicate that β(2)(∗)-nAChRs are implicated in the recruitment of inhibitory control of nociception, as shown by delayed recovery from capsaicin-induced allodynia, potentiated nociceptive response to inflammation and neuropathy, and by the loss of high-frequency transcutaneous electrical nerve stimulation (TENS)-induced analgesia in β(2)(∗)-KO mice. As high-frequency TENS induces analgesia through Aβ-fiber recruitment, these data suggest that β(2)(∗)-nAChRs may be critical for the gate control of nociceptive information by non-nociceptive sensory inputs. In conclusion, acetylcholine signaling through β(2)(∗)-nAChRs seems to be essential for setting nociceptive thresholds by controlling GABAergic inhibition in the spinal cord.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pain.2011.05.022DOI Listing
September 2011

Morphological and functional characterization of cholinergic interneurons in the dorsal horn of the mouse spinal cord.

J Comp Neurol 2011 Nov;519(16):3139-58

Institut des Neurosciences Cellulaires et Intégratives, UPR3212 CNRS, Dept. Nociception et Douleur, 67084 Strasbourg, France.

Endogenous acetylcholine is an important modulator of sensory processing, especially at the spinal level, where nociceptive (pain-related) stimuli enter the central nervous system and are integrated before being relayed to the brain. To decipher the organization of the local cholinergic circuitry in the spinal dorsal horn, we used transgenic mice expressing enchanced green fluorescent protein specifically in cholinergic neurons (ChAT::EGFP) and characterized the morphology, neurochemistry, and firing properties of the sparse population of cholinergic interneurons in this area. Three-dimensional reconstruction of lamina III ChAT::EGFP neurons based either on their intrinsic fluorescence or on intracellular labeling in live tissue demonstrated that these neurons have long and thin processes that grow preferentially in the dorsal direction. Their dendrites and axon are highly elongated in the rostrocaudal direction, beyond the limits of a single spinal segment. These unique morphological features suggest that dorsal horn cholinergic interneurons are the main contributors to the plexus of cholinergic processes located in lamina IIi, just dorsal to their cell bodies. In addition, immunostainings demonstrated that dorsal horn cholinergic interneurons in the mouse are γ-aminobutyric acidergic and express nitric oxide synthase, as in rats. Finally, electrophysiological recordings from these neurons in spinal cord slices demonstrate that two-thirds of them have a repetitive spiking pattern with frequent rebound spikes following hyperpolarization. Altogether our results indicate that, although they are rare, the morphological and functional features of cholinergic neurons enable them to collect segmental information in superficial layers of the dorsal horn and to modulate it over several segments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cne.22668DOI Listing
November 2011

Dorsal horn neurons presynaptic to lamina I spinoparabrachial neurons revealed by transynaptic labeling.

J Comp Neurol 2009 Dec;517(5):601-15

Centre de Recherche Université Laval Robert-Giffard, Québec G1J 2G3, QC, Canada.

Sensory input to supraspinally projecting lamina I (LI) neurons arises both directly from primary afferents and via neurons intrinsic to the spinal dorsal horn. The types of neurons presynaptic to those projection neurons remain poorly known. To address this question we used retrogradely transported adenoviral vectors encoding green fluorescent protein (GFP) and a GFP-TTC (fragment C of the tetanus toxin) fusion protein, labeling respectively spinoparabrachial projection neurons and neurons presynaptic to them. The expression of GFP by infected neurons labeled the entire dendritic tree, enabling a more complete and quantitative morphological description of spinoparabrachial neurons than previous methods. These neurons were located in spinal LI, with dendritic arbors oriented extensively in the rostrocaudal axis (1,089.8 +/- 91.5 microm) and displaying low spine density. In contrast, their dendrites did not extend significantly ventrally (29.2 +/- 3.5 microm). The use of transynaptic tracer GFP-TTC revealed a population of local circuit LI neurons presynaptic to LI projection neurons. These local circuit LI neurons had distinct morphological properties, in particular significantly longer ventrally oriented dendrites (80.1 +/- 10.1 microm). The transynaptic tracer also revealed a population of stalked cells, some being highly spiny, directly in contact with spinal projection neurons. However, stalked cells were not the only lamina II cells in direct contact with projection neurons. Intracellular injections with Lucifer yellow in parasagittal slices of fixed tissue confirmed the above observations. Overall, these experiments demonstrated that neurons projecting to the parabrachial nucleus had their dendritic branching almost exclusively in LI and had sparse dendritic spines, in contrast with local circuit neurons that often extended ventrally and could be very spiny.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cne.22179DOI Listing
December 2009

Differential maturation of GABA action and anion reversal potential in spinal lamina I neurons: impact of chloride extrusion capacity.

J Neurosci 2005 Oct;25(42):9613-23

Division de Neurobiologie Cellulaire, Centre de Recherche Université Laval Robert-Giffard, Québec, G1J 2G3, Canada.

A deficit in inhibition in the spinal dorsal horn has been proposed to be an underlying cause of the exaggerated cutaneous sensory reflexes observed in newborn rats. However, the developmental shift in transmembrane anion gradient, potentially affecting the outcome of GABAA transmission, was shown to be completed within 1 week after birth in the spinal cord, an apparent disparity with the observation that reflex hypersensitivity persists throughout the first 2-3 postnatal weeks. To further investigate this issue, we used several approaches to assess the action of GABA throughout development in spinal lamina I (LI) neurons. GABA induced an entry of extracellular calcium in LI neurons from postnatal day 0 (P0) to P21 rats, which involved T- and N-type voltage-gated calcium channels. Gramicidin perforated-patch recordings revealed that the shift in anion gradient was completed by P7 in LI neurons. However, high chloride pipette recordings demonstrated that these neurons had not reached their adult chloride extrusion capacity by P10-P11. Simultaneous patch-clamp recordings and calcium imaging revealed that biphasic responses to GABA, consisting of a primary hyperpolarization followed by a rebound depolarization, produced a rise in [Ca2+]i. Thus, even if Eanion predicts GABAA-induced hyperpolarization from rest, a low chloride extrusion capacity can cause a rebound depolarization and an ensuing rise in [Ca2+]i. We demonstrate that GABA action in LI neurons matures throughout the first 3 postnatal weeks, therefore matching the time course of maturation of withdrawal reflexes. Immature spinal GABA signaling may thus contribute to the nociceptive hypersensitivity in infant rats.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.1488-05.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6725724PMC
October 2005

Nicotine differentially activates inhibitory and excitatory neurons in the dorsal spinal cord.

Pain 2004 Jun;109(3):308-318

Récepteurs et Cognition, CNRS URA2182, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France.

Nicotinic agonists have well-documented antinociceptive properties when administered subcutaneously or intrathecally in mice. However, secondary mild to toxic effects are observed at analgesic doses, as a consequence of the activation of the large family of differentially expressed nicotinic receptors (nAChRs). In order to elucidate the action of nicotinic agonists on spinal local circuits, we have investigated the expression and function of nAChRs in functionally identified neurons of neonate mice spinal cord. Molecular markers, amplified at the single-cell level by RT-PCR, distinguished two neuronal populations in the dorsal horn of the spinal cord: GABAergic/glycinergic inhibitory interneurons, and calbindin (CA) or NK1 receptor (NK1-R) expressing, excitatory interneurons and projection neurons. The nicotinic response to acetylcholine of single cells was examined, as well as the pattern of expression of nAChR subunit transcripts in the same neuron. Beside the most expressed subunits alpha4, beta2 and alpha7, the alpha2 subunit transcript was found in 19% of neurons, suggesting that agonists targeting alpha2* nAChRs may have specific actions at a spinal level without major supra-spinal effects. Both inhibitory and excitatory neurons responded to nicotinic stimulation, however, the nAChRs involved were markedly different. Whereas GABA/glycine interneurons preferentially expressed alpha4alpha6beta2* nAChRs, alpha3beta2alpha7* nAChRs were preferentially expressed by CA or NK1-R expressing neurons. Recorded neurons were also classified by firing pattern, for comparison to results from single-cell RT-PCR studies. Altogether, our results identify distinct sites of action of nicotinic agonists in circuits of the dorsal horn, and lead us closer to an understanding of mechanisms of nicotinic spinal analgesia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pain.2004.01.034DOI Listing
June 2004

Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice.

J Neurosci 2003 Aug;23(21):7820-9

Laboratoire de Neurobiologie Moléculaire, Centre National de la Recherche Scientifique Unité de Recherche Associée 2182 Récepteurs et Cognition, Institut Pasteur, 75724 Paris Cedex 15, France.

Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic (DA) neurons have long been considered as potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine and cocaine addiction or Parkinson's disease. However, DA neurons express mRNAs coding for most, if not all, neuronal nAChR subunits, and the subunit composition of functional nAChRs has been difficult to establish. Immunoprecipitation experiments performed on mouse striatal extracts allowed us to identify three main types of heteromeric nAChRs (alpha4beta2*, alpha6beta2*, and alpha4alpha6beta2*) in DA terminal fields. The functional relevance of these subtypes was then examined by studying nicotine-induced DA release in striatal synaptosomes and recording ACh-elicited currents in DA neurons fromalpha4, alpha6, alpha4alpha6, and beta2 knock-out mice. Our results establish that alpha6beta2* nAChRs are functional and sensitive to alpha-conotoxin MII inhibition. These receptors are mainly located on DA terminals and consistently do not contribute to DA release induced by systemic nicotine administration, as evidenced by in vivo microdialysis. In contrast, (nonalpha6)alpha4beta2* nAChRs represent the majority of functional heteromeric nAChRs on DA neuronal soma. Thus, whereas a combination of alpha6beta2* and alpha4beta2* nAChRs may mediate the endogenous cholinergic modulation of DA release at the terminal level, somato-dendritic (nonalpha6)alpha4beta2* nAChRs most likely contribute to nicotine reinforcement.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6740613PMC
August 2003