A chronic high alcohol intake was induced in rats through the use of two procedures: the schedule-induced polydipsia technique and the liquid diet technique. Rats consumed 11 -1 2 g of ethanol per kilogram body weight per day for 16 to 18 weeks. Morphologic evidence of a mild distal axonal neuropathy in the ventral caudal nerve was produced. The red blood cell transketolase levels were normal, indicating that the rats were not deficient in thiamine and suggesting that the axonal degeneration was due to the direct toxic effect of alcohol. Axonal transport studies demonstrated a significant increase in the amount of acetylcholinesterasetransported in an orthograde direction in the sciatic nerves of alcohol-exposed rats, and indicated no change in the transport of choline acetyltransferaseor in the specific binding of colchicine by neurotubulin. MUSCLE & NERVE
2~133-144
1979
ANIMAL MODELS OF ALCOHOLIC NEUROPATHY: MORPHOLOGIC, ELECTROPHYSIOLOGIC, AND BIOCHEMICAL FINDINGS E. P. BOSCH, MD, R. W. PELHAM, PhD, C. G. RASOOL, PhD, A. CHATTERJEE, MA, R. W. LASH, L. BROWN, BS, T. L. MUNSAT, MD, and W. G. BRADLEY, DM, FRCP
F o r 150 years, it has been recognized that chronic alcoholism can cause peripheral nerve damage in man,34but there are still conflicting opinions about the nature and pathogenesis of the pathologic process involving the nerve. Most recent studies of nerve biopsies in alcoholic neuropathy have shown a significant reduction in the density of both small and large myelinated fibers, with evidence of axonal regeneration and some acute axonal degene r a t i ~ n . ~Ho ~ wever, ' ~ ~ ~Denny-Brown14 ~.~~ stated that segmental demyelination was commonly seen in early alcoholic neuropathy. In an electron-microscopic study of 11 cases of alcoholic neuropathy, Bischoff3 found axonal degeneration in every case, From the University of Iowa Hospitals and Cllnlcs, Iowa City, IA (Dr. Bosch) and the Tufts-New England Medical Center, Boston, MA (Drs. Pelham, Rasool, Munsat, and Bradley; Ms. Chatterjee and Ms. Brown; and Mr. Lash). Acknowledgments: The authors wish to thank Mr. Dennis Silber for his technical assistance. This study was supported by a grant from the Muscular Dystrophy Association, Inc., and by NIH grant AA03213-01. It was presented in part at the International Symposium on Peripheral Nerves, Milan, Italy, June 1978. Address reprint requests to Dr. Bradley at the Tufts-New England Medical Center, 171 Harrison Ave., Boston, MA 02111. Received for publication September 22, 1978; accepted for publication November 6, 1978. 0148-639X/0202/0133$OO.OO/O 1979 Houghton Mifflin Professional Publishers
Experimental Alcoholic Neuropathy
although four had predominant segmental demyelination. In man, chronic alcoholic neuropathy has commonly been attributed to n u t r i t i ~ n a l ~ ~ , ~ ~more -or, specifically, thiamine-defi~iency.~'*~'Blood levels of thiamine and other group B vitamins are significantly reduced in alcoholic patients with peripheral neuropathy," and the absorption of thiamine is impaired in chronic alcoholic^.^^ I n addition, experimental thiamine deficiency in man,jg rats, and other s p e ~ i e s ~ produces , ~ ~ , ~ ~a, periph~~ eral neuropathy. On the other hand, Delaney et all3 detected a thiamine-deficient state in only 37% of chronic alcoholics and in only half of their cases with alcoholic neuropathy. Behse and Buchthal' found no evidence of vitamin deficiencies o r other nutritional deficiencies in almost two-thirds of 37 chronic alcoholic patients with peripheral neuropathy. They found the pathologic change in alcoholic neuropathy to consist primarily of axonal degeneration, which differed from the predominant segmental demyelination found in nutritional neuropathy following gastrectomy. Behse and Buchthal concluded that alcohol was directly toxic to the peripheral nervous system. Because of the socioeconomic importance of chronic alcoholism, and because of uncertainties concerning the pathology and pathogenesis of the
MUSCLE & NERVE
MadApr 1979
133
human disease, experimental studies in animals are needed to clarify the picture. Several different experimental models have been developed to induce a high alcohol intake in animals. Two of these were used in the present study: the scheduleinduced polydipsia and the liquid diet model (in which animals received only liquid nourishment and no solid food, and in which alcohol was part of the liquid diet).26 This report describes electrophysiologic, morphologic, and biochemical studies of the peripheral nervous system of rats subjected to 16 weeks of chronic high alcohol intake in the scheduleinduced polydipsia paradigm. Some of these studies were also performed with rats consuming an alcohol-containing liquid diet for 18 weeks. MATERIALS AND METHODS Experimental Models. Schedule-induced polydipsia. Twenty-nine Holtzman rats (16 experimental and 13 control animals) were subjected to the scheduleinduced polydipsia The animals were individually housed in cages with metal-grid floors and food-pellet dispensers. The pellet dispenser (model PD 104 or PD 109, Davis Scientific Co., Los Angeles, CA) released a single 45-mg pellet once every two minutes for the first hour of every hur-hour period around the clock. The food pellets (P. J. Noyes Co., Lancaster, NH) were composed of meat, vegetable meals, and vitamin supplements, resulting in a daily food intake of 8.1 g (36.5 kcal). At the beginning of the experiment, the mean free-feeding weights of experimental and control animals were comparable (135 g and 132 g, respectively). Before they were placed in the experimental cages, the rats were fasted to 80% of their initial free-feeding weight. Experimental rats drank 5% (v/v) absolute alcohol in water containing 0.35% ( w h ) saccharin. Control rats drank saccharin solution alone, plus daily standard laboratory food supplements (Charles River RHM 3000, Wilmington, MA) in amounts necessary to keep their body weight equal to that of the experimental animals. The room was kept at a constant temperature (20"C), with 12-hour-on, 12-hour-off illumination. Fluid intake and body weights were measured twice weekly. The experiment was terminated after 16 weeks. Liquid diet. Five male Sprague-Dawley rats drank a liquid diet similar to that of Freund,26consisting of 6% ( v h ) absolute alcohol, 57% chocolate-flavored Sustacal, and 37% tap water. Five control rats consumed a diet in which ethanol was isocalorically replaced by sucrose, providing 35% of to-
134
Experimental Alcoholic Neuropathy
tal daily calories. Control rats initially weighed 258 9 g, while ethanol rats weighed 269 2 2 g. No weight reduction preceded the institution of these diets. The experiment was terminated after 18 weeks.
&
Electrophysiology. At the end of the study period, maximal motor nerve conduction velocities of the caudal nerves were measured by means of a technique similar to that of Miyoshi and got^.^^ Rats anesthetized with pentobarbital (25 mg/kg IP) and a nitrous oxide-oxygen-halothane mixture were kept in a heated, foil-shielded box (fig. 1). Rectal temperature and skin temperature over the distal end of the rat tail were measured with thermistor probes at the time each conduction velocity was recorded. The tail-skin temperature during the electrophysiologic studies was 36.7 k 0.5"C. Action potentials from a distal segmental tail muscle were recorded through surface ring electrodes (Teca 6032, Teca Corp., White Plains, NY) placed around the distal tail. T h e caudal nerve was stimulated supramaximally with ring electrodes placed at two proximal sites (the rostra1 base of the tail and the midtail). Stimulating and recording electrodes were connected to a Teca TE4 electromyograph. Motor nerve conduction velocities were calculated from paper recordings of evoked muscle action potentials. Axunul trunsport and colchicine binding. After each animal had been placed under anesthesia as delineated above, two ligatures were placed around the right sciatic nerve-the upper at the level of the sciatic notch and the lower at the level of the popliteal fossa. Six hours later, both sciatic nerves were removed and cleaned, and enzyme activities of' acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) were determined in 3-mm segments proximal to and between the ligatures, as well as in corresponding segments from the contralateral sciatic nerve. AChE activity was determined by a modification of the radiometric method of Potter.46Nerve segments were homogenized in 1.0 ml of 50 mM sodium phosphate buffer (pH 7.4) containing 0.5%) Triton X-100 (Sigma Corp., St. Louis, MO), 20 nM diisopropyl fluorophosphate, and 0.05% bovine serum albumin. The assay mixture (100 pl) contained 50 pl of homogenate, 50 mM sodium phosphate buffer, 2.2 mM acetyl- 1-l4C-cholinechloride (specific activity: 0.22 mCi/mmole), and 20 mM magnesium chloride. After 20 min of incubation at 37"C, the r e a d o n was stopped by use of 100 p1 of 0.2 N HCl. 14C-acetate was extracted into Laboratory Studies.
MUSCLE & NERVE
Mar/Apr 1979
t1
\-
rI
I I I
-.
I
I I
k It 2
I I
I I I I
Foil Shielded Box Figure I , Diagram of experimental technique for measuring the maximal motor nerve conduct/on velocity of the caudal nerves of the rat. S, = proximal site of stimuiating electrodes. S , = di.sta/ site of stimuiating = anode. - = cathode. Th, = tail electrodes. R = recording electrodes. G = ground electrode. thermistor. ThR = rectal thermistor. d , = distance between proximal and distal stimulation sites. d 2 = distance between distal stimulation site and recording electrode. t , and t, = intervals between time of stimulation and onset of evoked action potential with proximal and dlstai stimulation, respectively. cv = conduction velocity
+
2 ml of toluene-isoamyl alcohol (5:l v h ) ; and, after centrifugation at 1500 x g for 20 min, 1 ml of the organic phase was counted with 10 ml of toluene-based Permablend I1 (Packard Corp., Downers Grove, IL) in a Packard 2650 liquid scintillation counter. Enzyme activity was expressed as rianomoles of acetylcholine hydrolyzed per 3-mm segment per hour. C h A T activity was detcrniiried by the method of F ~ n n u m . ' ~ The indices of axonal transport were determined according to the following equations (sec Rasool and BradleyY): Apparent transport rate ('I'Ra,,)= (Activity of en(Activity of enzyme in segment - zyme i n contraproximal to lateral control proximal ligation) segment)
24 x L x --nini/24 hih (Activity of enzyme i n contralateral control segment)
where L Length of segrricrit i n millimeters and h = 'Time in hours after ligature. 1
Experimental Alcoholic Neuropathy
Percentagc o f enzyme mobile in the orthograde direction (%M) = (Activity of enzyme in segment proximal to distal ligation)
-
(Activity of enzyme ii contralateral coritrol segment) ~
~
X
(Total en/yme activity of ligated segment)
00%
Absolute trarisport rate ( I'Rab)=
Anioiirit of e n q m e activity t i ansported (AMt,) = (i\cti\itv of enzyme in control segment) pmo1124 hr (I.engt11 of control segment) TRall
Colc hicine binding was determined by the method o f Morgan and Seeds.43The nerve homogenate was centrifuged at 10,000 x g for 10 min at 4"C, and the supernatant was taken for assay. For determinations of total colchicine binding, aliquots were incubated with 2.5 x 10p5M 3H-
MUSCLE & NERVE
MariApr 1979
135
colchicine (specific activity: 0.05 Ciimmol) at 37°C for 90 min. For determination of nonspecific colchicine binding, the incubation was performed in the presence of 1 x lOP3M unlabeled colchicine. Specific colchicinc binding, expressed as picomoles per milligram protein, was determined by deducting nonspecific from total colchicine binding. Morphology. Following removal of both sciatic nerves, the extensor digit orum longus (EDL) and soleus muscles contralateral to the ligatures were removed for histologic and histochemical studies of’ 10-pm-thick cryostat sections. Specimens stained for hematoxylin 8c eosin, NADH diaphorase, and myosin ATPase at p H 9.4 were prepared according to standard histochemical techniques.lS Following removal of these specimens, the animals were hepaririized through a cardiac puncture, and 3 ml of blood was taken for erythrocyte transketolase determinations (Bio-Science Laboratories, Van Nuys, CA). T h e animals w e r e then perfused through the left ventricle with 2% paraformaldehyde in 0.1 M sodium phosphate buffer at pH 7.4, followed by 3.6% glutaraldehyde in the same buffer. Following perfusion. the posterior tibia1 nerve in the calf contralateral to the ligated nerve was removed, as was the ventral caudal nerve in the proximal and distal third of the tail. Further fixation in 3.6% glutaraldehyde for 2 hr was followed by postfixation in 2%) osmium tetroxide in the same buffer for 1 hr. T h e nerves sampled were dehydrated in graded concentrations of ethanol and were embedded in Spurr’s resin. Transverse (1-pm-thick) sections were stained with toluidine blue for light microscopy; ultrathin sections were stained with uranyl acetate and lead citrate and were viewed with a Phillips 200 electron microscope. The densities of myelinated fibers per square millimeter of fascicular area, as well as myelinatedfiber-diameter histograms of caudal nerves at proximal and distal levels, were determined from photographs of whole nerves at a magnification of
Table 1. Rat caudal nerve conduction studies Treatment group Control (N=7) Mean t SD Range Experimental (N = 8) Mean & SD Range
Motor nerve conduction velocity (misec)
Terminal latency (rnsec)
32.3 & 3.8 29 - 39
2.3 ? 0.4 1.9 - 2.8
31.6 -t 6.4” 23 - 46
2.0 2 0.7“ 0.8 - 2.9
‘Not significantly dffferentfrom control values
136
Experimental Alcoholic Neuropathy
x 1000 using a Zeiss ‘l’ZG3 particle-size analyzer in the exponential mode. Two-ce n ii nict er-lon g portions of the caudal nerves from proximal and distal levels were prepared for microdissection of single nerve fibers of at least five internodes’ length, as described e l ~ e w h e r eSingle .~ teased nerve fi hers were typed according to the descriptive categories of‘ Dyck;“ 50 such fibers were used for each level. Internodal-length spectra were determined on 50 single teased fiber^.'^ RESULTS
Ethanol Consumption. During the 16-week study period, experimental animals ( N = 16) and control animals ( N = 13) in the schedule-induced polydipsia model gained an average of 183 g, and their weights at sacrifice were comparable (3 18 g and 3 14 g, respectiTely). Fluid intake remained constant throughout the study. ‘I’he mean daily fluid intake was higher in controls than in alcohol-consuming rats (experimental group: 306 ml/kg/day; controls: 330 mllkgiday). The experimental group drank 12.1 ? 1.4 g of ethanol per kilogram body weight per day, which provided 42% of their total caloric intake. Immediately following a feeding period, the rats in the experimental group appeared drowsy but regained alertness before the next feeding cycle. l h e experiment using the liquid diet model of chronic alcohol intake was terniinatcd after 18 weeks, at which time the control rats weighed 548 & 51 g and the experimental rats weighed 488 ? 20 g. The control rats consumed an average of 270 2 30 ml of diet per kg body weight per day, while the experimental rats consumed 242 ? 19 nil/kg/ day, which provided 11 g of ethanol per kilogram body weight per day. No clinical signs of a peripheral neuropathy were seen in the alcohol-consuming rats in either experiment.
Electrophysiologic Findings. At the conclusion of the study period, maximal motor nerve conduction velocities and terminal latencies of the caudal nerve were determined in eight alcohol-consuming and seven control rats from the schedule-induced polydipsia study (table 1). A reduction in conduction velocity exceeding 30%-i.e., two standard deviations below the mean control value-was seen in only one rat from the experimental group. The mean conduction velocities and terminal latencies of the experimental groups did not differ significantly from control values after 16 weeks.
MUSCLE & NERVE
MarjApr 1979
Figure 2 Transverse section of the d/stal ventral caudal nerve of a rat in the experimental group (scheduleinduced poiydipsia experiment) Two myemated fibers are undergoing axonal degeneration [arrows) Spurr's resin, toluidine blue Bar = 5 prn
degeneration was particularly marked (fig. 2). In the experimental group, the mean density of myelinated fibers in the distal ventral caudal nerve was reduced by 16% of the mean control value (p
2~133-144
1979
ANIMAL MODELS OF ALCOHOLIC NEUROPATHY: MORPHOLOGIC, ELECTROPHYSIOLOGIC, AND BIOCHEMICAL FINDINGS E. P. BOSCH, MD, R. W. PELHAM, PhD, C. G. RASOOL, PhD, A. CHATTERJEE, MA, R. W. LASH, L. BROWN, BS, T. L. MUNSAT, MD, and W. G. BRADLEY, DM, FRCP
F o r 150 years, it has been recognized that chronic alcoholism can cause peripheral nerve damage in man,34but there are still conflicting opinions about the nature and pathogenesis of the pathologic process involving the nerve. Most recent studies of nerve biopsies in alcoholic neuropathy have shown a significant reduction in the density of both small and large myelinated fibers, with evidence of axonal regeneration and some acute axonal degene r a t i ~ n . ~Ho ~ wever, ' ~ ~ ~Denny-Brown14 ~.~~ stated that segmental demyelination was commonly seen in early alcoholic neuropathy. In an electron-microscopic study of 11 cases of alcoholic neuropathy, Bischoff3 found axonal degeneration in every case, From the University of Iowa Hospitals and Cllnlcs, Iowa City, IA (Dr. Bosch) and the Tufts-New England Medical Center, Boston, MA (Drs. Pelham, Rasool, Munsat, and Bradley; Ms. Chatterjee and Ms. Brown; and Mr. Lash). Acknowledgments: The authors wish to thank Mr. Dennis Silber for his technical assistance. This study was supported by a grant from the Muscular Dystrophy Association, Inc., and by NIH grant AA03213-01. It was presented in part at the International Symposium on Peripheral Nerves, Milan, Italy, June 1978. Address reprint requests to Dr. Bradley at the Tufts-New England Medical Center, 171 Harrison Ave., Boston, MA 02111. Received for publication September 22, 1978; accepted for publication November 6, 1978. 0148-639X/0202/0133$OO.OO/O 1979 Houghton Mifflin Professional Publishers
Experimental Alcoholic Neuropathy
although four had predominant segmental demyelination. In man, chronic alcoholic neuropathy has commonly been attributed to n u t r i t i ~ n a l ~ ~ , ~ ~more -or, specifically, thiamine-defi~iency.~'*~'Blood levels of thiamine and other group B vitamins are significantly reduced in alcoholic patients with peripheral neuropathy," and the absorption of thiamine is impaired in chronic alcoholic^.^^ I n addition, experimental thiamine deficiency in man,jg rats, and other s p e ~ i e s ~ produces , ~ ~ , ~ ~a, periph~~ eral neuropathy. On the other hand, Delaney et all3 detected a thiamine-deficient state in only 37% of chronic alcoholics and in only half of their cases with alcoholic neuropathy. Behse and Buchthal' found no evidence of vitamin deficiencies o r other nutritional deficiencies in almost two-thirds of 37 chronic alcoholic patients with peripheral neuropathy. They found the pathologic change in alcoholic neuropathy to consist primarily of axonal degeneration, which differed from the predominant segmental demyelination found in nutritional neuropathy following gastrectomy. Behse and Buchthal concluded that alcohol was directly toxic to the peripheral nervous system. Because of the socioeconomic importance of chronic alcoholism, and because of uncertainties concerning the pathology and pathogenesis of the
MUSCLE & NERVE
MadApr 1979
133
human disease, experimental studies in animals are needed to clarify the picture. Several different experimental models have been developed to induce a high alcohol intake in animals. Two of these were used in the present study: the scheduleinduced polydipsia and the liquid diet model (in which animals received only liquid nourishment and no solid food, and in which alcohol was part of the liquid diet).26 This report describes electrophysiologic, morphologic, and biochemical studies of the peripheral nervous system of rats subjected to 16 weeks of chronic high alcohol intake in the scheduleinduced polydipsia paradigm. Some of these studies were also performed with rats consuming an alcohol-containing liquid diet for 18 weeks. MATERIALS AND METHODS Experimental Models. Schedule-induced polydipsia. Twenty-nine Holtzman rats (16 experimental and 13 control animals) were subjected to the scheduleinduced polydipsia The animals were individually housed in cages with metal-grid floors and food-pellet dispensers. The pellet dispenser (model PD 104 or PD 109, Davis Scientific Co., Los Angeles, CA) released a single 45-mg pellet once every two minutes for the first hour of every hur-hour period around the clock. The food pellets (P. J. Noyes Co., Lancaster, NH) were composed of meat, vegetable meals, and vitamin supplements, resulting in a daily food intake of 8.1 g (36.5 kcal). At the beginning of the experiment, the mean free-feeding weights of experimental and control animals were comparable (135 g and 132 g, respectively). Before they were placed in the experimental cages, the rats were fasted to 80% of their initial free-feeding weight. Experimental rats drank 5% (v/v) absolute alcohol in water containing 0.35% ( w h ) saccharin. Control rats drank saccharin solution alone, plus daily standard laboratory food supplements (Charles River RHM 3000, Wilmington, MA) in amounts necessary to keep their body weight equal to that of the experimental animals. The room was kept at a constant temperature (20"C), with 12-hour-on, 12-hour-off illumination. Fluid intake and body weights were measured twice weekly. The experiment was terminated after 16 weeks. Liquid diet. Five male Sprague-Dawley rats drank a liquid diet similar to that of Freund,26consisting of 6% ( v h ) absolute alcohol, 57% chocolate-flavored Sustacal, and 37% tap water. Five control rats consumed a diet in which ethanol was isocalorically replaced by sucrose, providing 35% of to-
134
Experimental Alcoholic Neuropathy
tal daily calories. Control rats initially weighed 258 9 g, while ethanol rats weighed 269 2 2 g. No weight reduction preceded the institution of these diets. The experiment was terminated after 18 weeks.
&
Electrophysiology. At the end of the study period, maximal motor nerve conduction velocities of the caudal nerves were measured by means of a technique similar to that of Miyoshi and got^.^^ Rats anesthetized with pentobarbital (25 mg/kg IP) and a nitrous oxide-oxygen-halothane mixture were kept in a heated, foil-shielded box (fig. 1). Rectal temperature and skin temperature over the distal end of the rat tail were measured with thermistor probes at the time each conduction velocity was recorded. The tail-skin temperature during the electrophysiologic studies was 36.7 k 0.5"C. Action potentials from a distal segmental tail muscle were recorded through surface ring electrodes (Teca 6032, Teca Corp., White Plains, NY) placed around the distal tail. T h e caudal nerve was stimulated supramaximally with ring electrodes placed at two proximal sites (the rostra1 base of the tail and the midtail). Stimulating and recording electrodes were connected to a Teca TE4 electromyograph. Motor nerve conduction velocities were calculated from paper recordings of evoked muscle action potentials. Axunul trunsport and colchicine binding. After each animal had been placed under anesthesia as delineated above, two ligatures were placed around the right sciatic nerve-the upper at the level of the sciatic notch and the lower at the level of the popliteal fossa. Six hours later, both sciatic nerves were removed and cleaned, and enzyme activities of' acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) were determined in 3-mm segments proximal to and between the ligatures, as well as in corresponding segments from the contralateral sciatic nerve. AChE activity was determined by a modification of the radiometric method of Potter.46Nerve segments were homogenized in 1.0 ml of 50 mM sodium phosphate buffer (pH 7.4) containing 0.5%) Triton X-100 (Sigma Corp., St. Louis, MO), 20 nM diisopropyl fluorophosphate, and 0.05% bovine serum albumin. The assay mixture (100 pl) contained 50 pl of homogenate, 50 mM sodium phosphate buffer, 2.2 mM acetyl- 1-l4C-cholinechloride (specific activity: 0.22 mCi/mmole), and 20 mM magnesium chloride. After 20 min of incubation at 37"C, the r e a d o n was stopped by use of 100 p1 of 0.2 N HCl. 14C-acetate was extracted into Laboratory Studies.
MUSCLE & NERVE
Mar/Apr 1979
t1
\-
rI
I I I
-.
I
I I
k It 2
I I
I I I I
Foil Shielded Box Figure I , Diagram of experimental technique for measuring the maximal motor nerve conduct/on velocity of the caudal nerves of the rat. S, = proximal site of stimuiating electrodes. S , = di.sta/ site of stimuiating = anode. - = cathode. Th, = tail electrodes. R = recording electrodes. G = ground electrode. thermistor. ThR = rectal thermistor. d , = distance between proximal and distal stimulation sites. d 2 = distance between distal stimulation site and recording electrode. t , and t, = intervals between time of stimulation and onset of evoked action potential with proximal and dlstai stimulation, respectively. cv = conduction velocity
+
2 ml of toluene-isoamyl alcohol (5:l v h ) ; and, after centrifugation at 1500 x g for 20 min, 1 ml of the organic phase was counted with 10 ml of toluene-based Permablend I1 (Packard Corp., Downers Grove, IL) in a Packard 2650 liquid scintillation counter. Enzyme activity was expressed as rianomoles of acetylcholine hydrolyzed per 3-mm segment per hour. C h A T activity was detcrniiried by the method of F ~ n n u m . ' ~ The indices of axonal transport were determined according to the following equations (sec Rasool and BradleyY): Apparent transport rate ('I'Ra,,)= (Activity of en(Activity of enzyme in segment - zyme i n contraproximal to lateral control proximal ligation) segment)
24 x L x --nini/24 hih (Activity of enzyme i n contralateral control segment)
where L Length of segrricrit i n millimeters and h = 'Time in hours after ligature. 1
Experimental Alcoholic Neuropathy
Percentagc o f enzyme mobile in the orthograde direction (%M) = (Activity of enzyme in segment proximal to distal ligation)
-
(Activity of enzyme ii contralateral coritrol segment) ~
~
X
(Total en/yme activity of ligated segment)
00%
Absolute trarisport rate ( I'Rab)=
Anioiirit of e n q m e activity t i ansported (AMt,) = (i\cti\itv of enzyme in control segment) pmo1124 hr (I.engt11 of control segment) TRall
Colc hicine binding was determined by the method o f Morgan and Seeds.43The nerve homogenate was centrifuged at 10,000 x g for 10 min at 4"C, and the supernatant was taken for assay. For determinations of total colchicine binding, aliquots were incubated with 2.5 x 10p5M 3H-
MUSCLE & NERVE
MariApr 1979
135
colchicine (specific activity: 0.05 Ciimmol) at 37°C for 90 min. For determination of nonspecific colchicine binding, the incubation was performed in the presence of 1 x lOP3M unlabeled colchicine. Specific colchicinc binding, expressed as picomoles per milligram protein, was determined by deducting nonspecific from total colchicine binding. Morphology. Following removal of both sciatic nerves, the extensor digit orum longus (EDL) and soleus muscles contralateral to the ligatures were removed for histologic and histochemical studies of’ 10-pm-thick cryostat sections. Specimens stained for hematoxylin 8c eosin, NADH diaphorase, and myosin ATPase at p H 9.4 were prepared according to standard histochemical techniques.lS Following removal of these specimens, the animals were hepaririized through a cardiac puncture, and 3 ml of blood was taken for erythrocyte transketolase determinations (Bio-Science Laboratories, Van Nuys, CA). T h e animals w e r e then perfused through the left ventricle with 2% paraformaldehyde in 0.1 M sodium phosphate buffer at pH 7.4, followed by 3.6% glutaraldehyde in the same buffer. Following perfusion. the posterior tibia1 nerve in the calf contralateral to the ligated nerve was removed, as was the ventral caudal nerve in the proximal and distal third of the tail. Further fixation in 3.6% glutaraldehyde for 2 hr was followed by postfixation in 2%) osmium tetroxide in the same buffer for 1 hr. T h e nerves sampled were dehydrated in graded concentrations of ethanol and were embedded in Spurr’s resin. Transverse (1-pm-thick) sections were stained with toluidine blue for light microscopy; ultrathin sections were stained with uranyl acetate and lead citrate and were viewed with a Phillips 200 electron microscope. The densities of myelinated fibers per square millimeter of fascicular area, as well as myelinatedfiber-diameter histograms of caudal nerves at proximal and distal levels, were determined from photographs of whole nerves at a magnification of
Table 1. Rat caudal nerve conduction studies Treatment group Control (N=7) Mean t SD Range Experimental (N = 8) Mean & SD Range
Motor nerve conduction velocity (misec)
Terminal latency (rnsec)
32.3 & 3.8 29 - 39
2.3 ? 0.4 1.9 - 2.8
31.6 -t 6.4” 23 - 46
2.0 2 0.7“ 0.8 - 2.9
‘Not significantly dffferentfrom control values
136
Experimental Alcoholic Neuropathy
x 1000 using a Zeiss ‘l’ZG3 particle-size analyzer in the exponential mode. Two-ce n ii nict er-lon g portions of the caudal nerves from proximal and distal levels were prepared for microdissection of single nerve fibers of at least five internodes’ length, as described e l ~ e w h e r eSingle .~ teased nerve fi hers were typed according to the descriptive categories of‘ Dyck;“ 50 such fibers were used for each level. Internodal-length spectra were determined on 50 single teased fiber^.'^ RESULTS
Ethanol Consumption. During the 16-week study period, experimental animals ( N = 16) and control animals ( N = 13) in the schedule-induced polydipsia model gained an average of 183 g, and their weights at sacrifice were comparable (3 18 g and 3 14 g, respectiTely). Fluid intake remained constant throughout the study. ‘I’he mean daily fluid intake was higher in controls than in alcohol-consuming rats (experimental group: 306 ml/kg/day; controls: 330 mllkgiday). The experimental group drank 12.1 ? 1.4 g of ethanol per kilogram body weight per day, which provided 42% of their total caloric intake. Immediately following a feeding period, the rats in the experimental group appeared drowsy but regained alertness before the next feeding cycle. l h e experiment using the liquid diet model of chronic alcohol intake was terniinatcd after 18 weeks, at which time the control rats weighed 548 & 51 g and the experimental rats weighed 488 ? 20 g. The control rats consumed an average of 270 2 30 ml of diet per kg body weight per day, while the experimental rats consumed 242 ? 19 nil/kg/ day, which provided 11 g of ethanol per kilogram body weight per day. No clinical signs of a peripheral neuropathy were seen in the alcohol-consuming rats in either experiment.
Electrophysiologic Findings. At the conclusion of the study period, maximal motor nerve conduction velocities and terminal latencies of the caudal nerve were determined in eight alcohol-consuming and seven control rats from the schedule-induced polydipsia study (table 1). A reduction in conduction velocity exceeding 30%-i.e., two standard deviations below the mean control value-was seen in only one rat from the experimental group. The mean conduction velocities and terminal latencies of the experimental groups did not differ significantly from control values after 16 weeks.
MUSCLE & NERVE
MarjApr 1979
Figure 2 Transverse section of the d/stal ventral caudal nerve of a rat in the experimental group (scheduleinduced poiydipsia experiment) Two myemated fibers are undergoing axonal degeneration [arrows) Spurr's resin, toluidine blue Bar = 5 prn
degeneration was particularly marked (fig. 2). In the experimental group, the mean density of myelinated fibers in the distal ventral caudal nerve was reduced by 16% of the mean control value (p
