Axonal transport of labeled protein and regeneration rate in nerves of streptozocin-diabetic rats

EXPERIMENTAL

Axonal

NEUROLOGY

69, 74-84 (1980)

Transport of Labeled Protein and Regeneration Rate in Nerves of Streptozocin-Diabetic Rats M. A. BISBY’

Division of Medical Physiology, University of Calgary, Calgary, Alberta T.?N IhV, Canada Received December 27, 1979 Axonal transport of3H-labeled protein was studied in the sensory axons of sciatic nerves in normal rats and in rats made diabetic by administration of streptozocin. Within 8 days diabetic rats showed a reduction in tail nerve conduction velocity. Transport studies 2 to 6 weeks after streptozocin administration revealed no significant differences between normal and diabetic rats in fast transport velocity in vitro, distance travelled in vivo by the wavefront of 3H-protein in a given time interval after injection of precursor [3H]leucine, slow axonal transport velocity in vivo, and regeneration rate. Nerves from diabetic animals took significantly longer to commence regeneration after a nerve crush (3.05 f 0.51 days vs. 1.71 2 0.24 days). I conclude that alterations in velocity of axonal transport are not involved in the conduction velocity changes occurring in the nerves of streptozocindiabetic rats.

INTRODUCTION A reduction in action potential conduction velocity occurs in the nerves of acutely diabetic experimental animals (6, 12, 14, 25). The underlying cause is unknown. Morphological studies revealed no demyelination or axon degeneration (15,25), but one author reported axon “dwindling” (15, 16), a decrease in axonal diameter which could account for the reduced conduction velocity. However, this finding has been disputed (27). Because the maintenance of the axon requires continuous axonal transport of macromolecules synthesized in the cell body, a defect in transport might produce the axonal “dwindling.” It was of interest, therefore, that a reduction in the amounts of acetylcholinesterase and choline acetyltransferase accumulating at a nerve crush was observed in diabetic rats (24). Such reductions in accumulation could be due to either a decrease in 1 This work was supported by the Medical Research Council of Canada. 74 00144886/80/070074-11$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

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transport velocity or a decrease in amount of enzyme in transit. Because acetylcholinesterase is transported in the fast phase (approximately 400 mm/day) (19, 22) whereas choline acetyltransferase is transported in the slow phase (approximately 4 mm/day) (23,32), the objectives of the present experiments were to compare the velocities of the fast and slow phases of transport of labeled proteins in normal and experimentally diabetic rats. Regeneration rate was also studied because of the close relationships between regeneration rate and velocity of slow transport (7, 10). METHODS Male and female Sprague-Dawley rats of initial weight 250 to 350 g were obtained from the Medical Vivarium, University of Calgary. Litter-mates were randomly assigned to normal or diabetic groups. The diabetic group was injected with streptozocin (kindly donated by The Upjohn Company, Kalamazoo, Mich.) at a dose of 50 to 75 mg/kg body weight. The drug was dissolved in 0.3 ml sodium citrate buffer, 0.1 mol/liter, pH 4-5, immediately prior to injection into the femoral vein of the lightly anesthetized rat (pentobarbital, 30 mg/kg, i.p.). Mortality rate was 10 to 50% within 2 days of injection, but thereafter animals survived for as long as 60 days. Development of diabetes was monitored by daily measurements of weight, water consumption, and urine glucose (Clinistix). In addition, blood glucose determinations were made on tail vein blood at the time of the transport experiments, using the o-toluidine method (5). Injected rats were included in the diabetic group only if they met the following criteria: a + -I- + reading with the Clinistix test; daily water intake in excess of 100 ml (normal range 20 to 40 ml); whole blood glucose in excess of 300 mg/dl (normal range 90 to 150 mg/dl). No attempt was made to correlate the severity of diabetes with changes in conduction velocity or axonal transport. Injected rats which failed to meet the criteria were discarded. Conduction velocity was measured in the motor nerves of the tail (20). This method permitted serial measurements at approximately weekly intervals, eliminated temperature effects, and provided a long length of nerve. Axonal transport was studied by injecting [3H]L-leucine (New England Nuclear) in saline into the L5 dorsal root ganglia of the anesthetized rat (sodium pentobarbital, 50 mg/kg). After injection the wound was sutured and the animals were kept warm with an infrared lamp. Both sciatic nerves were removed 1.5 to 2.75 h after precursor injection. Previous work showed that fast-transported protein entered the sciatic nerve during this interval. One nerve was placed on a cardboard strip and immediately

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frozen with solid COZ. The other nerve was incubated in oxygenated Locke’s solution (NaCl, 153 mmol/liter; KCl, 5.6 mmolfliter; CaClz, 2.2 mmolfliter; NaHCO,, 2.1 mmol/liter; dextrose, 5.6 mmol/liter) at 37.O”C for a measured period of approximately 1 h. Nerves from diabetic and normal rats were incubated at the same time in the same water bath. After incubation, the nerve was placed on a cardboard strip and frozen. The frozen nerves were cut into 2- or 4-mm segments, solubilized, and assayed for 3H-protein activity as previously described (2). Proties of activity along the nerve were constructed, and the velocity of transport in vitro at 37°C was calculated as (distance between fronts of activity in the two nerves) + (duration of incubation). An indication of transport in vivo was given by the position of the wavefront in the nonincubated nerve which was removed 1.5 to 2.75 h after precursor injection. Wavefront position was plotted against elapsed time (Fig. 3). Slow transport velocity was measured in I&JO, as it was not possible to maintain the nerves in vitro for the long post-injection intervals required. Sciatic nerves were removed 1 to 6 weeks postinjection and profiles of activity were constructed. The position of the peak of 3H-protein was plotted against elapsed time since precursor injection. Regeneration rate was determined by the pinch-reflex test (13) carried out on the lightly anesthetized rat. Regeneration was induced by crushing the sciatic nerve several days earlier with a loop of 2/O thread pulled tight against a glass rod. The position of the crush was marked with a loose ligature. The distance regenerated was plotted against days since crush, and regeneration rate determined as the slope of the regression line of distance regenerated on days since crush. RESULTS Changes in weight and tail nerve conduction velocity for one group of normal and age-matched streptozocin-treated rats are shown in Fig. 1. Normal rats gained weight and showed a parallel increase in conduction velocity, whereas diabetic rats initially lost weight, then maintained a fairly constant weight. The conduction velocity of diabetic rats was significantly reduced by 8 days after injection, and showed a slight further decline thereafter. These data showed that the reduction in conduction velocity was well developed 2 weeks after drug administration, so all experiments on axonal transport were carried out between 2 and 6 weeks after drug administration. Figure 2 gives an example of the profile of activity obtained from an incubated and nonincubated pair of nerves from a single animal and the calculation of fast axonal transport velocity from these data. There was

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I

0

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10

20 30 DAYS

40

FIG. 1. Changes in body weight and tail nerve conduction velocity in normal and diabetic rats. Results are shown for a group of diabetic rats (0) and a group of age-matched control rats (II). Mean f SE are shown, N 2 5. Open symbols and broken lines-weight, closed symbols and solid lines-conduction velocity. y axis-weight or conduction velocity as a percentage of initial value, x axis-elapsed time since day when streptozocin was administered to diabetic group of rats.

a slight decrease in the in vitro velocity in diabetic rats, but this was not significant (Table 1). Because of the variability in the results a difference between normal and diabetic velocities 1 8% would have been required before statistical significance was attained (t test, P 5 0.05). An indication of transport in vivo was found by plotting the position of the wavefront in the nonincubated nerve against the time elapsed since precursor injection (Fig. 3). The regression line of distance on time did not differ significantly between normal and diabetic nerves with respect either to slope or to extrapolated intercept on the distance = 0 axis (Table l), suggesting that in vivo velocity and latent period between precursor injection and appearance of transported protein in the axons were unaltered. However, there is a large scatter in these data, probably due to lack of precise body temperature control in the anesthetized rats during the postinjection period. Because of this large scatter a difference in slopes of 240% would have been required to establish a significant difference (P zs 0.05). The large scatter also means that the true velocity is not the slope of the distance on time regression line, but the slope of the line bisecting the angle between the two regression lines (distance on time) and (time on distance). The calculated velocities were 16.2 mm/h for normal rats, and 15.5 mm/h for diabetic rats. Intercept on the zero distance axis occurred at 0.47 h for both normal and diabetic rats. These values are

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M. A. BISBY

a.:.+, _..,.. 3.x 8’ i 0

I

t

4-12.5-

mm

FIG. 2. Activity profiles from fast axonal transport velocity experiments. y axis-log cpm per 2-mm segment of nerve, x axis-distance. Pair of sciatic nerves from a diabetic rat. l O-Left nerve removed after 1.8 h in viva and frozen, V - - - V-right nerve, incubated for a further 1 h in vitro. The method of determining the position of the wavefront as the intercept between the slope of the wave of activity and the background activity is shown. In this experiment the incubated nerve wavefront was 12.5 mm further along the nerve, so velocity of transport was estimated as 12.5 mm/h.

similar to those obtained in an earlier study (2) where body temperature was controlled and a wider range of time and distance values was obtained. For slowly transported protein the slope of the regression line for distance on time was not significantly different for normal and diabetic groups (Fig. 4 and Table 1). In the nerves removed 7 and 19 days after precursor injection there was evidence for a faster-moving wave of slowly transported activity (component b) (18) which had traveled beyond the distal end of the analyzed segments at later times. A difference of ~35% in component a velocity and 230% in component b would have been required to establish a statistically significant difference. Again, slow-transport velocity is the slope of the line bisecting the two regression lines for the scattered data of Fig. 4. These estimated velocities were: component a, normal = 1.18 mm/h, diabetic = 1.10 mm/h; component b, normal = 3.1 mm/h, diabetic = 2.8 mm/h. Regeneration rate, as measured by the pinch-reflex test, was also unchanged in the nerves of diabetic rats (Fig. 5 and Table 1). A difference of

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TRANSPORT TABLE

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IN DIABETES 1

Transport Velocities and Regeneration Rate Normal

Fast transport velocity, in vitro (mm/h) Fast transported wavefront position x time, in viva: Slope (mm/h) Intercept on distance = 0 axis (h) Slowly transported wavecrest position x time: Slope (mm/h), component a Slope (mm/h), component b Regeneration rate (mm/day) Delay before onset of regeneration (days)

Diabetic

N”

Mean 2

12

14.3

+ 1.4

15

13.4

16 16

13.5 r 2.2 0.34 k 0.21

22 22

12.1 2 2.0 0.14 t- 0.35

14 9 83b 83b

1.08 3.00 4.07 1.71

+ + lr f

SD

0.10 0.23 0.13 0.24

N

8 4 69 69

Mean

1.10 2.83 3.73 3.05

2 SD

t- 1.4

e t2 -c

0.14 0.08 0.20 0.51c

(1 Number of nerves. b Data from previous study (4). r Significantly different (P < 0.05) from normal value.

~21% in regeneration rate would have been required to establish a significant difference. Extrapolation of the regression line to the distance = 0 axis indicated that there was an increased delay in the onset of regeneration in these nerves, compared with nerves from an earlier series of normal animals (4) with the same weight range as the initial weight of the diabetic animals.

lo-

3 HOURS

FIG. 3. Position of wavefront of fast-transported protein (y-axis) plotted against elapsed time since precursor injections (x-axis). 0 - - - O-Normal animals, 0 O-diabetic animals. Regression lines of position on time, calculated by method of least squares, are shown. Slope values are shown in Table 1.

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M. A. BISBY

01

I 10

1

I

I

20

30

40

4

60

DAYS

FIG. 4. Slow axonal transport: profiles ofactivity in nerves from diabetic (A) and normal (B) animals. y axis-cpm/2-mm nerve segment divided by mean cpm per segment for entire nerve, x axis-distance along nerve. One result is shown for each time period after precursor injection, indicated by the number of days shown above each profile. In the 19-day profiles “component b” is located toward the distal end of the nerve. C-velocity of slow axonal transport components a and b. Vertical axis-position of wavecrest, horizontal axis-days after precursor injection. 0 O-diabetic, 0 - - - O-normal. Regression lines calculated by the method of least squares. Slope values are shown in Table 1.

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DAYS

FIG. 5. Regeneration rate of sciatic nerve. y axis-position offastestgrowing sensory axons as determined by the pinch-reflex test,.r axis- days since making injury. The solid regression line represents data obtained in an earlier study (4) from 83 normal rats of similar age and weight range as those of the diabetic rats used in this study. Data points (means t SD) and broken regression line were obtained from 69 diabetic rats. Values for slope (regeneration rate) and intercept (extrapolated delay before onset of regeneration) are shown in Table 1.

DISCUSSION Axonal transport was examined in the sensory axons of the rat sciatic nerve, and the decrease in conduction velocity was detected in motor axons of the tail nerve. Transport determinations are more accurately made in sensory axons, because dorsal root ganglia provide a “point-source” for the origin of transported protein. The pinch-reflex test, which relies on the sensitivity of regenerating sensory axons, is the easiest and most accurate method for determining regeneration rate. What evidence is there that changes in axon conduction velocity affect both sensory and motor axons in experimentally diabetic rats? The time course and magnitude of the reduction in tail nerve conduction velocity were similar to values previously reported for rat sciatic nerve (6, 12), in which there were parallel changes in motor and sensory axons (6). The velocity of axonal transport and the mechanism of transport are identical in motor and sensory axons (2 l), as are the major types of transported protein (1, 3, 29). Regeneration rates are also identical (11). Thus it seems likely that defects in tail nerve motor axon conduction velocity also occur in the sensory axons of the sciatic nerve in which transport was studied, and conversely that any changes in axonal transport velocity in sensory axons would also affect motor axons. An in vitro technique was used to measure fast axonal transport velocity

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principally to eliminate variations due to imprecise body temperature control during the postinjection period. In addition, an in vitro technique eliminated the possible effects of microcirculatory changes in “diabetic” nerves (31) which could have resulted in local anoxia and disturbance of axonal transport. The in vitro velocity is lower than the in vivo velocity previously determined from regression analysis of wavefront position vs. elapsed time since precursor injection (2) (14.3 mm/h in vitro, 16.4 mm/h in vivo). This difference is significant. Possibly the nerves become temporarily anoxic during their removal and transfer so that there is a delay before transport resumes in vitro. No significant changes in fast axonal transport velocity in vitro were found. Similarly, no significant differences in in vivo position of wavefront vs. elapsed time were detected. Another study of axonal transport in sciatic nerves of streptozocin-diabetic rats which appeared recently also concluded that fast transport velocity was unaffected (28). Those measurements were made in viva, with precise body temperature control. An increase in latent period of about 30% was reported for both [3H]leucine-labeled proteins and [ 14C]glucosamine-labeled macromolecules, but the increase was not significant for [3H]leucine. Reduced accumulation of norepinephrine at a sciatic nerve ligature was reported in spontaneously diabetic mice (9), but, as in the earlier study of Schmidt et al. (24), this result did not permit distinction between reduced velocity and reduced quantity of transported material. A reduced incorporation of [3H]leucine into the nerves of diabetic rats was observed (28) but this cannot be interpreted as reduced quantity of transported protein because specific activity was unknown. Altough the absence of any measurable changes in transport velocity indicates that reduced accumulations of transported materials in nerves of diabetic rats must be due to reduced quantities of materials in transit, there is no direct evidence that this occurs. Slow transport velocity had to be determined in vivo because of the long postinjection intervals required. Deficient thermoregulation during the hrst 2 h of a 7- to 42-day period would have little effect. No significant changes in velocity were detected. The unaltered regeneration rate is consistent with an unchanged velocity of component b, as the two processes have a similar velocity (18). Component b, which includes microtuble protein, increases in quantity during regeneration, and may provide the structural substrate for axonal elongation (18). An earlier study, in which regeneration was assessed morphometrically 3 weeks and later after a crush lesion, also concluded that regeneration was unchanged in nerves of diabetic rats (26). The increased delay in onset of regeneration would not have been detected. The delay in onset of regeneration depends largely on

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the type of lesion (crush, cut, resection, etc.) used to induce it (30), and probably represents the time taken by the axon sprouts to penetrate the zone of tissue damage. Nerves from diabetic animals appeared swollen compared with normal nerves, presumably as a result of their enlarged endoneurial space (17). Possibly the swollen “diabetic” nerves were more severely damaged by the crush lesion than compact normal nerves so that the outgrowth of regenerating axons was more delayed. The normal regeneration rate and unchanged velocity of slow axonal transport provided no evidence for a defect in slow axonal transport in nerves of the diabetic rats. Thus the reduced accumulation of slowly transported choline acetyltransferase at a ligature (24) must have resulted from a decreased amount of enzyme undergoing transport. I conclude that changes in axonal transport velocity do not occur in experimental diabetes in rats, and so cannot be responsible for the associated reduction in axonal conduction velocity. There may be alterations in the cell body synthesis of materials required for normal conduction which are transported into the axon, or the defect may lie outside the axon (8). REFERENCES 1. BARKER, J. L., J. H. NEALE, AND H. GAINER. 1976. Rapidly transported proteins in sensory, motor and sympathetic nerves of the isolated frog nervous system. Brain Res. 105: 497-515.

2. BISBY, M. A. 1977. Retrograde axonal transport of endogenous protein: differences between motor and sensory axons. J. Neurochem. 28: 249-251. 3. BISBY, M. A. 1977. Similar polypeptide composition of fast-transported proteins in rat motor and sensory axons. J. Neurobiol. 8: 303-314. 4. BISBY, M. A. 1978. Fast axonal transport of labeled protein in sensory axons during regeneration. Exp. Neural. 61: 281-300. 5. DUBOWSKI, K. M. 1962. An o-toluidine method for body-fluid glucose determination. Clin.

Chem.

8: 215-235.

6. ELIASSON, S. G. 1964. Nerve conduction Invest.

changes in experimental

diabetes. J. Clin.

43: 2353-2358.

7. FRIZELL, M., AND J. SJOSTRAND. 1976. The axonal transport of slowly migrating (3H)-leucine labeled proteins and the regeneration rate in regenerating hypoglossal and vagus nerves of the rabbit. Brain Res. 81: 267-284. 8. FUKUMA, M., J. L. CARPENTIER, L. ORCI, D. A. GREENE, AND A. I. WINEGRAD. 1978. An alteration in internodal myelin membrane structure in large sciatic nerve tibres in rats with acute streptozotocin diabetes and impaired nerve conduction velocity. Diabetologia

15: 65-72.

9. GIACHETTI, A. 1979. Axoplasmic transport of noradrenaline in the sciatic nerves of spontaneously diabetic mice. Diabetologia 16: 191-194. 10. GRAFSTEIN, B. 1971. Role of slow axonal transport in nerve regeneration. Acta Neuropathol. (Berlin) Suppl. 5: 144- 152. 11. GRAFSTEIN, B., AND I. G. MCQUARRIE. 1978. Role of the nerve cell body in axonal

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