Ignite Creation Date:
2024-05-05 @ 11:58 AM
Last Modification Date:
2024-10-26 @ 9:17 AM
Study NCT ID:
NCT00190073
Status:
UNKNOWN
Last Update Posted:
2005-09-19
First Post:
2005-09-11
Brief Title:
Cutaneous Denervation in Alcoholic Neuropathy
Sponsor:
Far Eastern Memorial Hospital
Organization:
Far Eastern Memorial Hospital
Study Overview
Official Title:
Cutaneous Denervation in Alcoholic Neuropathy
Status:
UNKNOWN
Status Verified Date:
2005-09
Last Known Status:
RECRUITING
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
Peripheral neuropathy is a frequent neurological complication of chronic alcoholism Most studies evaluated large-fiber involvement by nerve conduction studies NCS Since previous studies document the predominant injury of small myelinated and unmyelinated fibers in patients with alcoholic neuropathy it will be imperative to know their prevalence and clinical significance Moreover the pathogenesis of alcoholic neuropathy especially the roles of ethanol and its metabolites and thiamine remains elusive This proposal will be designed to understand the extent and clinical significance of cutaneous nerve degeneration in the skin of alcoholic patients and to investigate its pathogenesis We will investigate cutaneous innervation by 3 mm punch skin biopsies with immunohistochemistry for protein gene product 95 and quantifying epidermal nerve density END in alcoholic patients Patients will undergo clinical evaluation quantitative sensory testing QST nerve conduction studies NCS and tests of sympathetic skin response SSR and beat-to-beat RR interval variability RRIV The prevalence of peripheral neuropathy in chronic alcoholic patients with emphasis on small-fiber involvement will be first evaluated The sensitivity of punch skin biopsy QST SSR and RRIV tests and NCS will be compared and the correlations between END and psychophysic and electrodiagnostic parameters will be discussed Subsequently we will elucidate the clinical significance of END reduction in alcoholic patients Patients with evidences of involvement of central nervous system will be excluded and END will be correlated with clinical manifestations and neurological deficits Finally the role of ethanol and thiamine in alcoholic neuropathy will be further studied To clarify the role of thiamine in alcoholic neuropathy we will examine whether it has influences on small-fiber degeneration This may provide important information in understanding the pathogenesis and designing optimal treatment for alcoholic neuropathy
Detailed Description:
Alcoholic patients and control subjects Alcoholic patients will be recruited from neurologic clinics and ward at the Far Eastern Memorial Hospital Taipei Taiwan A detailed clinical history that includes daily alcohol consumption daily dietary intake lifestyle and occupation will be obtained from patients as well as their families The inclusion criteria include daily uptake of at least 100 g ethanol for more than 3 years prior to the onset of neuropathic symptoms Behse and Buchthal 1977 All patients will undergo clinical and neurologic assessment cranial MRI or CT and neuropsychological evaluation to rule out CNS disorders which interfere the evaluation of neuropathic symptoms and signs and psychophysic test Laboratory investigations will include complete blood count fasting plasma glucose hemoglobin A1C liver and renal function tests ethanol level tumor markers antinuclear antibodies complement factors serum protein electrophoresis thyroid function human immunodeficiency virus hepatitis virus and vitamin B12 level Thiamine status will be assessed at the time of the first referral to the hospital by measuring total thiamine concentration in whole blood by high performance liquid chromatography as described elsewhere Koike et al 2001 and 2003 None of the patients will administrate thiamine at the time of determination Age- and gender-matched control subjects will be retrieved from our database who will be evaluated by detailed questionnaires and neurological examinations to exclude any neurological disorder or clinical neuropathy McCarthy et al 1995
Skin biopsy Skin biopsy will be performed following established procedures after informed consent has been obtained McCarthy et al 1995 Pan et al 2003 Shun et al 2004 Under local anesthesia with 2 lidocaine punches 3mm in diameter will be taken from the following locations 1 the extensor side of the distal forearm 5 cm above the middle point of a line connecting the radial styloid process and the ulnar styloid process and 2 the lateral side of the distal leg 10 cm above the lateral malleolus No suturing will be required and the wounds will be covered with a piece of gauze Wound healing takes 7-10 days the same as would a typical abrasion wound The protocol is under review by the Ethics Committee of Far Eastern Memorial Hospital
Immunohistochemistry For immunohistochemistry on freezing microtome sections the skin samples will be fixed with 4 paraformaldehyde in 01M phosphate-buffered saline PBS pH 74 for 48 h McCarthy et al 1995 Hsieh et al 2000 Sections of 50 mm perpendicular to the dermis will be cut on a sliding microtome model 440E Microm Walldorf Germany They will be quenched with 1 H2O2 blocked with 5 normal goat serum and incubated with rabbit antiserum to PGP 95 UltraClone UK diluted 1 1000 in 1 normal serumTris at 4C for 16-24 h After rinsing in Tris sections will be incubated with biotinylated goat anti-rabbit IgG at room temperature for 1 h followed by incubation with avidin-biotin complex Vector Burlingame CA for another hour The reaction product will be demonstrated with chromogen SG Vector Burlingame CA and counterstained with eosin Sigma St Louis MO
Quantitation of epidermal innervation Epidermal innervation will be quantified following established protocols and slides will be coded to ensure that measurements are blinded Hsieh et al 2000 Pan et al 2003 Shun et al 2004 PGP 95-immunoreactive nerve fibers in the epidermis of each section will be counted at a magnification of 40 with an Olympus BX40 microscope Tokyo Japan through the depth of the entire section Each individual nerve with branching points inside the epidermis will be counted as one For epidermal nerves with branching points in the dermis each individual nerve will be counted separately The length of the epidermis along the upper margin of the stratum corneum in each section will be measured using Image-Pro PLUS Media Cybernetics Silver Spring MD END will therefore be derived and expressed as fibersmm For each tissue there will be 48-50 sections and all sections will be sequentially labeled Every fifth section will be immunostained and quantified The mean of values from these sections will be considered the END of the tissue specimen In the distal leg normative values from our laboratory mean SD 5th percentile of END are 1116 370 588 fibresmm for subjects aged 60 years and 764 308 250 fibresmm for subjects aged 60 years The cut-off point of END is 588 and 250 fibresmm in the two age groups respectively
Quantitative sensory testing We will perform quantitative sensory testing QST with a Thermal Sensory Analyzer and Vibratory Sensory Analyzer Medoc Advanced Medical System Minneapolis MN to measure thresholds of warm cold and vibratory sensations following the established protocol Pan et al 2001 Pan et al 2003 Yarnitsky and Ochoa 1991 Briefly the machine will deliver a stimulus of constant intensity which had been determined by the test algorithm The intensity of the next stimulus will be either increased or decreased by a fixed ratio according to the response of the subject ie whether or not the subject perceives the stimulus Such procedures will be repeated until a pre-determined difference of intensity is reached The mean intensity of the last two stimuli will be the threshold for the level method Thermal thresholds recorded on the toe will be expressed as warm threshold temperature and cold threshold temperature respectively These temperatures will be compared with normative values Pan et al 2003 Vibratory thresholds recorded on the malleolus will be measured with similar algorithms Normative values are documented previously and are similar to those of previous reports Pan et al 2001 Pan et al 2003 Threshold values greater than the 95th percentile value for each age group will be considered abnormal
Nerve conduction studies NCS will be carried out in all patients with a Keypoint electromyographer following standardized methods Pan et al 2003 Amplitudes of compound muscle action potential CMAP and sensory action potential SAP will be analyzed according to the normative data in our laboratory Studied nerves include bilateral median ulnar peroneal tibial and sural nerves Abnormal result on NCS is defined as having abnormalities of 1 or more nerves with reduced amplitude prolonged distal latency or slowed nerve conduction velocity Neurophysiologic criteria of sensorimotor polyneuropathy are defined as having symmetrical abnormalities in motor and sensory nerves Dyck et al 1985
Tests of the autonomic nervous system Sudomotor function will be examined using the sympathetic skin response SSR Ravits 1997 Results of SSR in the palm and sole will be interpreted as present or absent but will not be evaluated quantitatively because of variations in the latencies and amplitudes of SSR Parasympathetic function will be evaluated using the beat-to-beat heart rate variation RR interval variability RRIV at rest and during deep breathing Ravits 1997 Each test will be performed three times and the mean value will be compared with that for the age-matched controls in our laboratory Medication that interferes with sympathetic or parasympathetic functions will not be administered before or during these tests
Statistical analysis Numerical variables following Gaussian distribution will be compared using t-test and expressed as the mean SD for those variables not following Gaussian distribution data will be expressed as the median range and will be analyzed with non-parametric Mann-Whitney U test Results are considered significant if p 005
Patients with evidences of CNS disorders will be excluded first Furthermore those with attributable etiologies for neuropathy such as diabetes uremia autoimmune disorders or neoplasm will be excluded too The rest alcoholic patients will be separated as 2 groups alcoholic patients with normal thiamine level ATN and alcoholic patients with thiamine deficiency ATD In the ATN group patients will be further subgrouped as alcoholic patients without clinical evidence of neuropathy alcoholic patients with sensory symptoms especially burning lightning pains and painful paresthesia and alcoholic patients with sensory and motor symptoms
The first point to address in our study is to evaluate the prevalence of peripheral neuropathy in chronic alcoholic patients with emphasis on small-fiber involvement Since early detection of neural degeneration is important the sensitivity of punch skin biopsy QST NCS and SSR and RRIV tests will be compared The correlations between END and psychophysic and electrodiagnostic parameters will be discussed
Second we will elucidate the clinical significance of END reduction in alcoholic patients END will be correlated with clinical manifestations and neurological deficits Small-fiber sensory symptoms and signs will be correlated with END and autonomic symptoms and signs will be correlated with the presence of sweat gland denervation The degrees of END reduction in the 3 groups of ATN patients and control subjects will be compared Since other systemic disease especially diabetes also causes small-fiber degeneration we will examine whether alcoholic patients with diabetes have further cutaneous denervation
Finally the role of ethanol and thiamine in alcoholic neuropathy will be explored The reduction of END will be correlated with thiamine status lifetime ethanol dose ethanol level and other laboratory parameters If END correlates with ethanol consumption it will support the conclusion that alcoholic neuropathy is caused by direct toxic effect of ethanol or its metabolites Since previous report documented the large-fiber-predominant axonal loss in sural nerve specimens in patients with pure-form of thiamine-deficiency neuropathy Koike et al 2003 we wonder if thiamine level influences small-fiber degeneration ie reduction of END The degrees of END reduction between alcoholic patients with ATD and those with ATN will be compared These will further clarify the role of thiamine in alcoholic neuropathy
Skin biopsy Skin biopsy will be performed following established procedures after informed consent has been obtained McCarthy et al 1995 Pan et al 2003 Shun et al 2004 Under local anesthesia with 2 lidocaine punches 3mm in diameter will be taken from the following locations 1 the extensor side of the distal forearm 5 cm above the middle point of a line connecting the radial styloid process and the ulnar styloid process and 2 the lateral side of the distal leg 10 cm above the lateral malleolus No suturing will be required and the wounds will be covered with a piece of gauze Wound healing takes 7-10 days the same as would a typical abrasion wound The protocol is under review by the Ethics Committee of Far Eastern Memorial Hospital
Immunohistochemistry For immunohistochemistry on freezing microtome sections the skin samples will be fixed with 4 paraformaldehyde in 01M phosphate-buffered saline PBS pH 74 for 48 h McCarthy et al 1995 Hsieh et al 2000 Sections of 50 mm perpendicular to the dermis will be cut on a sliding microtome model 440E Microm Walldorf Germany They will be quenched with 1 H2O2 blocked with 5 normal goat serum and incubated with rabbit antiserum to PGP 95 UltraClone UK diluted 1 1000 in 1 normal serumTris at 4C for 16-24 h After rinsing in Tris sections will be incubated with biotinylated goat anti-rabbit IgG at room temperature for 1 h followed by incubation with avidin-biotin complex Vector Burlingame CA for another hour The reaction product will be demonstrated with chromogen SG Vector Burlingame CA and counterstained with eosin Sigma St Louis MO
Quantitation of epidermal innervation Epidermal innervation will be quantified following established protocols and slides will be coded to ensure that measurements are blinded Hsieh et al 2000 Pan et al 2003 Shun et al 2004 PGP 95-immunoreactive nerve fibers in the epidermis of each section will be counted at a magnification of 40 with an Olympus BX40 microscope Tokyo Japan through the depth of the entire section Each individual nerve with branching points inside the epidermis will be counted as one For epidermal nerves with branching points in the dermis each individual nerve will be counted separately The length of the epidermis along the upper margin of the stratum corneum in each section will be measured using Image-Pro PLUS Media Cybernetics Silver Spring MD END will therefore be derived and expressed as fibersmm For each tissue there will be 48-50 sections and all sections will be sequentially labeled Every fifth section will be immunostained and quantified The mean of values from these sections will be considered the END of the tissue specimen In the distal leg normative values from our laboratory mean SD 5th percentile of END are 1116 370 588 fibresmm for subjects aged 60 years and 764 308 250 fibresmm for subjects aged 60 years The cut-off point of END is 588 and 250 fibresmm in the two age groups respectively
Quantitative sensory testing We will perform quantitative sensory testing QST with a Thermal Sensory Analyzer and Vibratory Sensory Analyzer Medoc Advanced Medical System Minneapolis MN to measure thresholds of warm cold and vibratory sensations following the established protocol Pan et al 2001 Pan et al 2003 Yarnitsky and Ochoa 1991 Briefly the machine will deliver a stimulus of constant intensity which had been determined by the test algorithm The intensity of the next stimulus will be either increased or decreased by a fixed ratio according to the response of the subject ie whether or not the subject perceives the stimulus Such procedures will be repeated until a pre-determined difference of intensity is reached The mean intensity of the last two stimuli will be the threshold for the level method Thermal thresholds recorded on the toe will be expressed as warm threshold temperature and cold threshold temperature respectively These temperatures will be compared with normative values Pan et al 2003 Vibratory thresholds recorded on the malleolus will be measured with similar algorithms Normative values are documented previously and are similar to those of previous reports Pan et al 2001 Pan et al 2003 Threshold values greater than the 95th percentile value for each age group will be considered abnormal
Nerve conduction studies NCS will be carried out in all patients with a Keypoint electromyographer following standardized methods Pan et al 2003 Amplitudes of compound muscle action potential CMAP and sensory action potential SAP will be analyzed according to the normative data in our laboratory Studied nerves include bilateral median ulnar peroneal tibial and sural nerves Abnormal result on NCS is defined as having abnormalities of 1 or more nerves with reduced amplitude prolonged distal latency or slowed nerve conduction velocity Neurophysiologic criteria of sensorimotor polyneuropathy are defined as having symmetrical abnormalities in motor and sensory nerves Dyck et al 1985
Tests of the autonomic nervous system Sudomotor function will be examined using the sympathetic skin response SSR Ravits 1997 Results of SSR in the palm and sole will be interpreted as present or absent but will not be evaluated quantitatively because of variations in the latencies and amplitudes of SSR Parasympathetic function will be evaluated using the beat-to-beat heart rate variation RR interval variability RRIV at rest and during deep breathing Ravits 1997 Each test will be performed three times and the mean value will be compared with that for the age-matched controls in our laboratory Medication that interferes with sympathetic or parasympathetic functions will not be administered before or during these tests
Statistical analysis Numerical variables following Gaussian distribution will be compared using t-test and expressed as the mean SD for those variables not following Gaussian distribution data will be expressed as the median range and will be analyzed with non-parametric Mann-Whitney U test Results are considered significant if p 005
Patients with evidences of CNS disorders will be excluded first Furthermore those with attributable etiologies for neuropathy such as diabetes uremia autoimmune disorders or neoplasm will be excluded too The rest alcoholic patients will be separated as 2 groups alcoholic patients with normal thiamine level ATN and alcoholic patients with thiamine deficiency ATD In the ATN group patients will be further subgrouped as alcoholic patients without clinical evidence of neuropathy alcoholic patients with sensory symptoms especially burning lightning pains and painful paresthesia and alcoholic patients with sensory and motor symptoms
The first point to address in our study is to evaluate the prevalence of peripheral neuropathy in chronic alcoholic patients with emphasis on small-fiber involvement Since early detection of neural degeneration is important the sensitivity of punch skin biopsy QST NCS and SSR and RRIV tests will be compared The correlations between END and psychophysic and electrodiagnostic parameters will be discussed
Second we will elucidate the clinical significance of END reduction in alcoholic patients END will be correlated with clinical manifestations and neurological deficits Small-fiber sensory symptoms and signs will be correlated with END and autonomic symptoms and signs will be correlated with the presence of sweat gland denervation The degrees of END reduction in the 3 groups of ATN patients and control subjects will be compared Since other systemic disease especially diabetes also causes small-fiber degeneration we will examine whether alcoholic patients with diabetes have further cutaneous denervation
Finally the role of ethanol and thiamine in alcoholic neuropathy will be explored The reduction of END will be correlated with thiamine status lifetime ethanol dose ethanol level and other laboratory parameters If END correlates with ethanol consumption it will support the conclusion that alcoholic neuropathy is caused by direct toxic effect of ethanol or its metabolites Since previous report documented the large-fiber-predominant axonal loss in sural nerve specimens in patients with pure-form of thiamine-deficiency neuropathy Koike et al 2003 we wonder if thiamine level influences small-fiber degeneration ie reduction of END The degrees of END reduction between alcoholic patients with ATD and those with ATN will be compared These will further clarify the role of thiamine in alcoholic neuropathy
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None
Secondary IDs
| Secondary ID | Type | Domain | Link |
|---|---|---|---|
| FEMH-94003 | None | None | None |