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Ultrastructural abnormalities of myelinated fibres in the tibial nerve of streptozotocin-diabetic rats
Journal of the Neurological Sciences, 1990, 98:327-345 Elsevier
327
JNS 03382
Ultrastructural abnormalities of myelinated fibres in the tibial nerve of streptozotocin-diabetic rats P. D o c k e r y i and A . K . S h a r m a 2 'Department of Biomedical Science, University of Sheffield, Sheffield SIO 2TN (U.K.) and 2Department of Anatomy. University" of the United Arab Emirates. (United Arab Emirates) (Received 7 November, 1989) (Revised, received 1 May, 1990) (Accepted 1 May, 1990)
SUMMARY
In this paper we have examined the ultrastructural changes in myelinated fibre structure after the administration of streptozotocin to Sprague-Dawley rats which had passed the rapid growth period. Myelinated fibre size in the tibial nerve was found to be less in diabetic animals 4 and 6 months after the induction of diabetes, when compared to age-matched controls, but not less than onset. The relative contributions of axon and myelin to this reduction in fibre dimensions were examined. When myelin area was plotted against axon area (derived from perimeter) it showed that the pathological insult of diabetes had a greater effect on the rate of myelin production. The incidence of axonal glycogenosomes was also assessed. These results are discussed in detail.
Key words: Streptozotocin; Diabetes; Rat tibial nerve; Myelinated fibres; Morphometry
INTRODUCTION
Myelinated fibre size has been found to be decreased in the peripheral nerves of streptozotocin-induced diabetic rats (Sharma etal. 1977, 1985; Jakobsen and Correspondence to: Dr. P. Dockery, Department of Anatomy, University of Hong Kong, Faculty of Medicine, Li Shu Fan Building, 5 Sassoon Road, Hong Kong. 0022-510X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
328 Lundbaek 1976; Jakobsen 1976, 1979; and several others). Sharma et al. i i 977. 198l) demonstrated that myelinated fibre size increased at a slower rate in diabetic animals as compared with controls, suggesting a maturational deficit secondary to the metabolic effects of diabetes. However, the extent to which axon and Schwann cells :Ire affected has been disputed. The effects reported have ranged from a primary Schwann cell lesion (Bestetti et al. 198 la,b; Zemp et al., 1981), axonopathy (Jakobsen and Lundbaek, 1976; J akobsen 1976, 1979) to axonal shrinkage possibly due to interstitial h yperosmolarity (Sugimura et al. 1980). These discrepancies may have been due to methodological differences between investigators such as the use of animals of different strains, ages and the duration and severity of diabetes. The light microscopical techniques employed by some workers (Jakobsen and Lundbaek 1976; Jakobsen 1976; Bestetti et al. 1981a, b; Zemp et al. 1981 ; Matingley and Fischer 1985; Cameron et al. 1986) may be inadequate for assessing the relative contributions of axon and myelin to fibre dimensions. Electron microscopy is considered to be more accurate in assessing axon size and myelin sheath thickness (Landon and Hall 1976; Thomas and Ochoa 1984). Therefore, in this study we have used electron microscopy to establish relative contributions of axon and myelin sheath in the reduction ofmyelinated fibre size in the tibial nerve of streptozotocin-induced rats which had passed the rapid growth period.
METHODS
Induction of diabetes The male albino Sprague-Dawley rats used in this study were aged 20-21 weeks and weighed between 525 and 755 g at the onset of the experiment. A group of 7 animals served as onset controls, to provide baseline morphological parameters for this age and strain. Six further groups were studied and observations were made on diabetic and age-matched controls at 2, 4 and 6 months after the induction of diabetes. Diabetes was induced by a single intraperitoneal injection of a streptozotocin solution at a dosage of 45 mg/kg body weight. Body weight and blood glucose levels used were recorded at regular intervals and at the time of death. Experimental and control animals were maintained on sawdust in plastic metabolic cages with water and Oxoid breeding diet (Oxoid Ltdl, Hampshire) available ad libitum.
Histological and morphometric procedures Biopsies were performed under Sagital (May and Baker Ltd.) anaesthesia (at 60 mg/kg body weight). The left tibial nerves were removed at a standard site between knee and ankle after removal of connective tissues. The nerves were then processed for electron microscopy according to previously described standardised methods (Sharma et al. 1985). Ultrathin sections of the whole transected nerve were cut on a Reichert OMU4 ultramicrotome and examined on a Philips EM201 electron microscope. A systematic random sampling procedure was employed (see Mayhew and
329
Fig. 1. Lower power micrograph of tibial nerve used in the present study. Note the 200-mesh grid bars provide a convenient lattice for systematic random sampling.
330 Sharma 1984; Fig. 1) and ultrastructural morphometric observations were made on 200-300 myelinated nerve fibres per animal on micrographs at a final magnification of × 5000. A magnification standard was taken on each film. In order to exclude a sampling bias (Gundersen 1977) a celluloid overlay half the diameter of the largest fibre was superinaposed prior to any measurements being made, and only those fibres whose centres lay within these bounds were included in the study, The area and length of perimeter of myelinated nerve fibres and their axon were obtained by programmed digitisation using a sonic digitiser linked to a microcomputer, An index of circularity was also calculated from these measurements (Sugimura et al. 1980). Fibres sectioned through paranodal and pen.nuclear regions and at Schmidt-Lantermann incisures were excluded. The initial sampling strategy provided information on the size and shape of myelinated nerve fibres and their axons. However in order to assess the relative contributions of axon and myelin to fibre dimensions a further series of micrographs
Fig. 2. Myelinated nerve fibres sectioned through a perinuclear region to illustrate the degree of structural preservation obtained in the present study. Bar = 1 #m.
331
Fig. 3. Detail of myelinated nerve fibre showing myelin lamellae. Lamellaewere counted and the width of the sheath measured at its most compact region. Bar = 0.5/~m. were taken at a higher initial magnification ( x 10000). The same sampling regime described earlier was used. Counts of the number of myelin lamellae and measurements of axolemmal lengths were made on tracings of the negative using a Zeiss projecting microscope (see Figs. 2 and 3). A data array (for 100 randomly sampled fibres per animal) was set up for the following variables in each animal using the University Honeywell 66/80 mainframe computer: (1) Axon diameter (of circles of equivalent area). (2) Number of myelin lamellae. (3) Axonal index of circularity. (4) Width of myelin at its most compact region. Features 1 and 3 allowed the retrieval of corresponding perimeter and area measurements. A datafile was created for each animal containing the measured variables. A further series of variables was created.
332 (5) Periodicity: this was calculated by taking the slope of the line w = b" nl + c,
where w = width of myelin at its most compact region, nl = number of myelin lamellae comprising the myelin sheath, and b and c = regression coefficients, slope and intercept, respectively. (6) Axon diameter (perimeter): axon diameter derived from perimeter measurements. It was obtained by dividing axon diameter (of circle of equivalent area) by index circularity (I.C.): d ( p ) = d/I.C.
(7) Fibre D (perimeter): fibre diameter derived from perimeter measurements of axon ( d ( p ) ) and number of myelin lamellae (nl) also accounting for periodicity (b in variable 5). Fibre D (perimeter) = d(p) + (2 x nl x b). (8) Axon area (perimeter) = n(d(p) / 2) z (9) Fibre area (perimeter) = n(fibre D (perimeter)/2) z (10) Myelin area: this is in effect a measurement of the actual cross-sectional area of the myelin sheath, as the measurements are derived from the perimeter of the axon and number of myelin lamellae with periodicity being accounted for. Myelin area = fibre area (perimeter) - axon area (perimeter). The incidence of axonal glycogenosomes was also assessed in each nerve studied. This was carried out on the microscope at a magnification of about x 10000 and the counts were expressed as percentage of fibres containing the organelle. Statistical analysis. All of the data were initially calculated on a "per animal" basis, so the n value was the number of rats. A one-way analysis of variance was employed routinely to test for differences in or between the various parameters. A two-way analysis of variance was used to detect any effects of age, diabetes or their interaction on a given parameter (Sokal and Rohlf 1978). Regression coefficients were compared by Student's t-test. Histograms of size frequency distributions of axon and fibre populations were constructed. The various distributions were analysed by a Z2 test.
333 RESULTS
Blood glucose and body weight
Diabetes developed within 24 h, polydipsia and polyuria were the most obvious features of the diabetic animals. The mean blood glucose levels of controls and diabetic groups are shown in Table 1, the diabetic animals had significantly higher blood glucose levels than controls at all survival periods. The body weight growth curves for control and diabetic animals are shown in Fig. 4. The control animals increased in weight throughout the period of study, however
TABLE 1 S T R E P T O Z O T O C I N I N D U C E D D I A B E T I C RATS
This table shows mean ( + SE) blood glucose levels (mmol/1) for onset, control and diabetic groups 2, 4 and 6 months after induction of diabetes.
2 months
4 months 6 months
Diabetic
Control
30.0 + 2.6 33.1 + 1.2 35.5 + 1.3
8.4 + 0.4* 9.9 +_ 0.5* 9.8 + 0.4*
* P < 0.05.
2 MONTHS
8OO
CONTROL 600 DIABETIC 400~
~
-
4 MONTHS 800
CONTROL
-r"
DIABETIC >. t",, 0 m
4
0
% CONTROL
8001'
6
Tr
0
0
40(~ 8
I •
~ .
.
6 MONTHS
DIABETIC .
.
12
.
.
.
. ~,I
24
WEEKS Fig. 4. Body weights (mean _+ SE) for control and diabetic animals 2, 4 and 6 months after the induction of diabetes.
334 the weight of the diabetic animals decreased by about 3090. At 4 and o months the diabetic animals appeared severely emaciated, at 6 months some of thc animals had developed cataracts. No obvious signs of neuropathy were observed at any survival period.
Myelinated fibre area The cross-sectional areas of myelinated fibres in the tibial nerve are shown in Fig. 5. Cross-sectional myelinated fibre area increased significantly in controls over the period of study. Myelinated fibre area in the tibial nerve of diabetic animals was found to be significantly less than age-matched controls at 4 and 6 months but not less than onset. When the data for 2, 4 and 6 months control and diabetic groups were subjected to a two-way analysis of variance there was found to be a significant effect of age and diabetes at the 1 ~ level and a significant interaction at the 5 ~o level (Table 2), implying that the longer the duration of diabetes the greater the effect on the nerve fibre. Fibre size-frequency distributions for control and diabetic groups are shown in Fig. 6. All of the distributions were subjected to a X2 analysis. The fibre diameter spectra of control animals was significantly different from onset only at 4 and 6 months survival periods. No difference in the distributions of fibre diameter was detected when any of the diabetic groups were compared with onset. However, a significant difference was detected in the diabetic animals only at 6 months when compared with age-matched controls. FIBRE AREA [~
pm 2
P<0.01
CONTROL
1771 DIABETIC
50p~0.01
40
n.s.
I
13.8.
/A ,
i A
/// ~ n.s. ONSET
2 MONTHS
¢ / 1 / / p~ 0.01 4 MONTHS
p
Fig. 5. Myelinated fibre area (mean _+SE) for onset, control and diabetics at 2, 4 and 6 months after the induction of diabetes. Values above respective columns show level of significance between column and onset. Values below columns show the comparison between diabetic and age matched control This format is repeated in all similar figures.
335 TABLE 2 T W O WAY ANALYSIS OF V A R I A N C E The table shows the F values and levels of significance after performing a two-way analysis of variance on fibre area, axon and myelin area. Age d.f. Fibre area Axon area Myelin area
P
2,36 15.85 3.54 0.54
Diabetes
P
1,36 35.61 6.81 55.23
<0.01 < 0.05 n.s.
<0.01 < 0.05 <0.01
Interaction
P
1,36 3.82 0.309 1.32
0.05 0.05 n.s, n.s.
d.f. = degrees of freedom numerator, denominator.
CONTROL
ONSET
o
"J~'~-
0
~ .
DIABETIC
2 MONTHS 09
u J - u
_.~,=..-_
in tt.
4 MONTHS m
;D
6 MONTHS
o
]5~ 0
5
I0
15
,.~" 0
'3h~, 5
lO
15
FIBRE DIAMETER (A) i~m Fig. 6. Fibre-size frequency distribution (diameter based on circle of equivalent area) for onset, control and diabetic groups at 2, 4 and 6 months after induction of diabetes.
336 AXON,
pm
AREA
2
p <0.05 I--'I 15
+
rI,S,
14
n.s,
+
13
t n.s,
T
12
CL$,
r/V
CONTROL DIABETIC
13,s,
IlIA IlIA IlIA
//A Ilia
11
IlIA n.S.
ONSET
2 MONTHS
n.S.
4 MONTHS
p< ).o5 6 MONTHS
Fig. 7. Myelinated axonal area (mean + SE) for onset, control and diabetics at 2, 4 and 6 months after induction of diabetes.
Myelinated axonal area The cross-sectional axonal area of myelinated fibres in the tibial nerve is shown in Fig. 7. Using a one-way analysis of variance there was found to be no overall change in axonal area with age in the control animals, however at 6 months the axonal area of the control animals was significantly larger than onset (at 5 % level). No significant difference was found when each individual diabetic group was compared with onset. No significant differences in cross-sectional axonal area were detected between diabetic and age-matched controls at 2 and 4 months but axonal area was significantly less in diabetic animals at 6 months when compared with age matched controls. The data for 2, 4 and 6 month control and diabetic groups were subjected to a two-way analysis of variance. There was a significant effect of age and diabetes at the 5')~, level but no interaction (Table 2). Age and diabetes have slight but significant effects on axonal area. The axon diameter spectra (Fig. 8) of control animals was significantly different from onset only at 4 and 6 months. When each diabetic group was compared with age-matched controls, no significance was observed at any survival period. Index of circularity The mean index of circularity is shown in Fig. 9. Mean axonal index of circularity was not found to be different between diabetic and age-matched controls and 4 and 6 months. It was significantly greater in diabetics
337 CONTROL
DIABETIC
ONSET
2 MONTHS tO I.U n" IJ_
~o
U-
o
4 MONTHS
n" ILl t~
o
Z
6 MONTHS o
4
B
o
4
o
AXON DIAMETER (A) pm
Fig. 8. Axon size-frequency distribution (diameter based on circle of equivalent area) for onset, control and diabetic groups at 2, 4 and 6 months after induction of diabetes. A X O N A L INDEX
OF
CIRCULARITY
F'-ICONTROL W'~ D I A B E T I C
0.8-
p
~0.05 I P
0.7-
p
'///
p
i/tO, I
///i I i
0.5
'///
///,
I
p
2 MONTHS
n.s.
4 MONTHS
fI.S.
6 MONTHS
Fig. 9. Axonal index of circularity (mean + SE) for onset, control and diabetics at 2, 4 and 6 months after the induction of diabetes.
338 at 2 months as compared with age-matched controls. Both diabetic and control axons were found to be less circular than onset at all survival periods. This shape index was examined in more detail. The plots of axonal index of circularity against axon diameter (perimeter) are shown in Fig. 10. There appears to be no strong size related difference in shape between large and small axons. There is a wide scatter of points throughout the fibre spectra. Fibre spectra based upon axonal index of circularity are presented in Fig. 11• A ~:" analysis was performed on these data. The distributions for controls were found to be significantly different from onset at 4 and 6 months survival periods. Diabetic groups were also found to be significantly different from onset at 4 and 6 months. When diabetics were compared with age-matched controls no significant differences in the distributions were detected at any survival period• Changes in axonal shape do not seem to be an important factor in streptozotocin-induced diabetes in the rat.
Relationship between myelin sheath thickness and axon size In order to assess the relative contributions of axon and myelin to fibre dimensions, the number of myelin lamellae was related in various ways to axonal dimensions CONTROL
1
'.
DIABETIC
. . . . ':.;l;;;;::.'-,,, • :.:t:;:'|l::.'=;::::::;;:;l;;:
, ,.,.. ,.; •, . . . . . .
" : ,"
, %""
ONSET
1
.. ~ • ,.
,-r,::;: .gl"r ,g~,'lt'F;~lrl ..;
ik[~iE;::'~'~2~:'2.:':'
,.- ,. •,
2 MONTHS
,., ........
• 1:'.""~l~llltll~gl,~li;';%' ,,
>I-
,,; •
. :..'
.
g:,o 1 ii 0
:
x u.I
-
2
'~7;':i~L=--".~:::!r'I::
• ,':,•,
,.....
4 MONTHS -
._1 ,< z
0 x
0
1
.... •,;ii,. I,...' .. •
t , l l l p H I l , l l e l l ilk i . . . . . . . .
, .. ' : •
:|
;~'~I:I:P":P.".;,;'~:. '
": ":,.',':1,1;; ;,~.~i~|.l':';, •,
6 MONTHS
• "."|
,..,;.
,,. ,",
0 0
t0
AXON
DIAMETER
0
10
(P)llm
Fig. 10. Plots (pooled data) of axonal index of circularity against axon diameter (by circle of equivalent perimeter) for all groups studied.
339 CONTROL
DIABETIC
20
ONSET
2 MONTHS
03 LU
r'r
4 MONTHS
2O
U. U. O nu.I
ID Z
20
6 MONTHS
0 ~ti'1~ 0.025
0.975
O. 025
0.975
A X O N A L INDEX OF C I R C U L A R I T Y
Fig. 11. Pooled fibre spectra based on axonal index of circularity for onset, control and diabetic groups at 2, 4 and 6 months after induction of diabetes.
(see Dockery 1986). The most appropriate method was found to be myelin area plotted against axon area based on perimeter measurements. Myelin area vs. a x o n area
The plots of pooled data from each group are shown in Fig. 12. The slopes of the linear regression lines (myelin area = C O + C~ axon area) are shown in Table 3. The slopes of the control groups were significantly less steep than onset at 4 months (P < 0.05). Diabetic groups were found to have significantly less steep slopes than onset at 4 and 6 months. The slopes of diabetic groups at 4 (P < 0.05) and 6 months (P < 0.001) were significantly less steep than age-matched controls. Myelin area
The mean myelin cross-sectional area for all the control and diabetic groups is shown in Fig. 13. A one-way analysis of variance performed on the data from all of the control groups showed no significant effect of age. However, myelin cross-sectional area was
340 DIABETIC
CONTROL
100
ONSET
,oo] 2 MONTHS
....:,:;::i£~ii:i:ii.'i'.:!""
:"
o - ':::-:;~!
ILl tr ,,( Z
4 MONTHS .~
100 1 : .!:!ii:i;;i~ii;¢~.~i~i:'~i'~% ' O"
6 MONTHS
o
.:iiiii::;i:;;""';:':"";....
0
100
0
100
A X O N AREA ( P ) Ilrn 2
Fig. 12. Plots of myelin area against area (perimeter) for onset, control and diabetic groups at 2, 4 and 6 months after induction of diabetes.
TABLE 3 SLOPES OF L I N E A R R E G R E S S I O N FOR M Y E L I N A R EA VERSUS A X O N A R EA Myelin area = Co + CI axon area (p)
Slope C1
Onset 2 months 4 months 6 months
Control
Diabetic
0.450 0.501 0.385 0.448
0.430 +_ 0.026 0.302 _+ 0.021 **~ 0.295 + 0.037 **+ t
_+ 0.018 + 0.025 + 0.017" _+ 0.017
* P < 0.05 compared with onset; **P < 0.001 compared with onset; + P < 0.05 compared with age-matched controls; + ÷ P < 0.001 compared with age-matched controls.
341 MYELIN AREA
iJrn 2
p
P
[-""1
CONTROL
P'~O.01 [77]
DIABETIC
+
--t24
I 13.5.
20
n.s°
rI.S.
ONSET
p
P
~
2 MONTHS
4 MONTHS
6 MONTHS
Fig. 13. Myelin area for onset, control and diabetic groups at 2, 4 and 6 months after induction of diabetes.
found to be significantly greater in control groups at 2, 4 and 6 months survival periods when compared with the onset group. Although there was an apparent decrease in mean myelin area, no significant difference was detected between onset and diabetic groups at any survival period. The mean myelin cross-sectional area of diabetic animals was found to be significantly less than age-matched controls at all survival periods. A two-way analysis of variance revealed that diabetes had a significant effect (P < 0.01) but there was no effect of age or interaction on cross-sectional myelin are (Table 2).
Axonal glycogenosomes The number of fibres containing axonal glycogenosomes was found to be increased at 4 and 6 months in controls and at 2, 4 and 6 months in diabetic groups when each group was compared with onset (Table 4). Although the diabetics consistently appeared to have a greater number of fibres containing these organelles, there was no significant difference between diabetic and age-matched controls. At 6 months, there was a border line difference between diabetic and age-matched controls (P < 0.06).
342 TABLE 4 PERCENTAGE INCIDENCE (MEAN _+SE) OF MYELINATEDFIBRES CONTAININGAXONAL GLYCOGENOSOMES IN TIBIAL NERVE 2 months
4 months
~ months
Onset controls End controls P-level
0.166 _+0.079 1.263 ± 0.513 n.s.
0.166 _+0.079 1.840 ± 0.590 < 0.05
I}166 ± 0.079 3170 ± 0.990 <~(}.05
Onset controls Diabetic P-level
0,166 _+0.079 2.420 _+0,620 < 0.05
0.166 ± 0.079 3,970 _+1.040 < 0.05
0,166 _+0,079 7,320 ± 1,520 < 0.01
Controls Diabetic P-level
1.263 _+0.513 2.420 _+0.620 n.s.
1.840 _+0.590 3.970 + 1.040 n.s.
3.170 ± 0.990 ,'.320 ± 1.520 < ((}.06)
DISCUSSION Myelinated fibre area was found to be reduced in diabetic animals when compared with age-matched controls but not different from onset controls. This deficit in fibre size is in line with most previous studies on peripheral nerve trunks (Jakobsen 1976; Sharma et al. 1977, 1985; Jakobsen and Lundbaek 1976; Bestetti et al. 1981a,b; Zemp et al. 1981). Myelinated fibre size increased throughout the period of study in the control animals, confirming the earlier work of Sharma et al. (1981). Sharma et al. (1977) found that fibre size diameter in diabetic animals failed to show the age-related increase exhibited by the controls. A similar situation has been encountered in the present study. Sugimura et al. (1980) also found that external fibre diameter in rats after 20 weeks of streptozotocin-induced diabetes was intermediate between onset and end controls. This raises the possibility that the pathological insult of diabetes may affect the normal maturation of the myelinated nerve fibre. In light microscopical studies Jakobsen and Lundbaek (1976) and Jakobsen (1976) reported that axonal diameter was reduced to a greater extent than external fibre diameter, however, this was not demonstrated in a convincing manner as the author did not include an onset group which would provide baseline morphological parameters for the age and strain of rat used. The concept of axonal "dwindling" proposed by these authors may therefore be misnamed, as all that they have demonstrated is that axon size is less in the diabetic when compared with age-matched controls at a given survival period. In a subsequent study, J akobsen (1979)did include a group of animals that acted as an onset control group. These animals were 24 weeks old, diabetic and age-matched controls survived for a further 4 weeks. The light microscopical data presented do not show any evidence for the concept of axonal "dwindling". The axonal cross-sectional area in their study was found not to be significantly less in the diabetic animals when compared with the 28-week-old onset group.
343 Nevertheless, axonal area has been shown to be smaller in diabetics when compared with age-matched controls examined by electron microscopy (Jakobsen, 1979). Sugimura et al. (1980) examined the sural nerve in streptozotocin-induced Sprague-Dawley rats by light and electron microscopy. The light microscopical data showed that axonal size measured from the "inner edge" of myelin sheath from diabetic animals was found not to be significantly smaller "axis cylinders" than the end control group. Examination of the material by electron microscopy showed that axonal area of animals diabetic for 20 weeks was less than age-matched controls, but not less than onset. They also reported that axonal perimeter was not different between any of the groups. However, axons from diabetic animals were found to be significantly less circular than onset and end controls. They suggested that this was due to a dehydration shrinkage which may be a consequence of interstitial hyperosmolarity. The onset control animals in the Sugimura et al. (1980) study weighed only 275-300 g compared with the 20-21-week-old animals used in the present study which were 525-755 g at the start of the experiment. The differences in the age of animals used may account for the discrepancies found in the index of circularity results between these two studies. In the present study no overall effect of age on axonal area was observed when either controls or diabetics were subjected to a one-way analysis of variance test. A slight but significant increase in axonal area was observed in control animals at 6 months when compared with onset. No significant difference was observed in axonal area when diabetic animals at any survival period were compared with onset. Diabetics were only significantly less than age-matched controls at 6 months. Changes in axonal shape do not seem to be an important factor in streptozotocin-induced diabetes in the rat. The relative contributions of axon and myelin to fibre dimensions in the streptozotocin-induced diabetic rat have been investigated by a number of workers. Bestetti et al. ( 1981 a,b) and Zemp et al. (1981) reported that there was a reduction in the myelin axon ratio and provided evidence that the myelin sheath is in fact affected to a greater extent than axonal area. The recent work of Mattingley and Fischer (1985) and Cameron et al. (1986) revealed that there is a decrease in axonal area with a concomitant increase in sheath thickness. The fact that all of these studies have employed light microscopy (which has much poorer resolving power than the electron microscope) to examine axon and myelin must cast doubts on any interpretations of their results. Myelin and axonal contributions to fibre size in the experimental diabetic rats examined by electron microscopy have been reported by Jakobsen (1979) and Sugimura et al. (1980). Sugimura et al. (1980) reported that there was an increase in the median number of myelin lamellae in the diabetic animals, although the difference was not statistically significant. Plotting myelin area against axon area (of circle of equivalent perimeter) suggested a greater vulnerability of the Schwann cell to the pathologic insult of diabetes. As myelin production is still a fairly active process at this age in control rats, and axonal growth seems to be rather slow, this may make myelin production a sensitive indicator of any slowing of metabolic rate induced by diabetes. It is of considerable interest to note that control animals 4 weeks after the onset of the experiment by Jakobsen (1979), showed an increase in axonal area (measured by light
344 microscopy). It seems that the active growth of the axon in these animals had not in fact passed, thus making it especially liable to the pathologic insults. In the present study, the number of fibres containing axonal glycogenosomes appeared slightly greater in the diabetic animals but the differences did not reach the level of statistical significance. Moore et al. (1981) reported that fibres containing axonal glycogenosomes were found to be significantly more numerous in streptozotocin diabetic rats than controls 2 weeks and 2 months after the induction of diabetes. Intra-axonal glycogen accumulation has also been observed in the nerves of atloxaninduced diabetic rats (Powell et al. 1979; Moore et al. 1981). The difference between the action of the two diabetogenic agents, especially the toxic effect of alloxan may be important in the accumulation of glycogen. However, Moore et al. (I 981) reported th at at 4, 8 and 12 months, control animals had significantly greater numbers of fibres containing these organelles than age-matched streptozotocin diabetic animals. Therefore, the authors suggested, that it seems doubtful than hyperglycemia is directly responsible for axonal glycogen accumulation o f glycogen. Furthermore, an age related phenomenon has previously been proposed to explain the increase in axonal glycogenosomes (Powell et al. 1979; T h o m a s et al. 1980; Grover-Johnson and Spencer 1981 ; Moore et al. 1981). It has also been suggested that glycogen accumulation might be related to distal trauma or ischaemia from focal entrapment of the plantar nerve (Thomas et al. 1980; Grover-Johnson and Spencer 1981). The animals used by Moore et al. (1981) were maintained on wire mesh flooring and an early involvement of the diabetic nerves may be due to an increased susceptibility to the ageing phenomenon or pressure related injuries. Whatever the etiology of this disorder in this model we have shown that the reduction in myelinated fibre size is primarily due to a decreased rate o f myelin production.
REFERENCES Bestetti, G., G.L. Rossi and P.C. Zemp (1981a) Changes in peripheral nerves of rats 4months after induction of streptozotocin diabetes. Acta Neuropathol. (Bed.), 54: 129-134. Bestetti, G., P.C. Zemp, D. Probst and G.L. Rossi (1981b) Neuropathy and myopathy in the diaphragm of rats after 12 months of streptozotocin induced diabetes mellitus. Acta Neuropathol., (Bed.), 55: 11-20.
Cameron, N.E., M.B. Leonard, I. S. Ross and P.H. Whiting (1986) The effects of Sorbinil on peripheral nerve conduction velocity, polyol concentrations and morphology in the streptozotocin-diabetic rat. Diabetologia, 29: 168-174. Dockery, P. (1986) Ultrastructural abnormalities in the peripheral nerves of streptozotocin induced diabetic rats and genetically diabetic (db/db) mice. PhD Thesis, University of Aberdeen, Aberdeen. Gundersen, H. J. G. (1977) Notes on the estimation of numerical density of arbitrary profiles- the edge effect. J. Microsc., 111: 219-223. Jakobsen, J. (1976) Axonal dwindling in early experimental diabetes, I. A study of cross-sectioned nerves. Diabetologia, 12: 539-546. Jakobsen, J. (1979) Early and preventable changes of peripheral nerve structure and function in insulindeficient diabetic rats. J. Neurol. Neurosurg. Psychiat., 42: 509-518. J akobsen, J. and K. Lundbaek (1976) Neuropathy in experimental diabetes. An animal model. Br. Med. J., 2: 278-279.
345 Landon, D.N. and S. Hall (1976) The myelinated nerve fibre. In D.N. Landon (Ed.), The Peripheral Nerve, Chapman and Hall, London, pp. 11-105. Mattingley, G. E. and V. W. Fischer (1985) Peripheral nerve axon dwindling with concomitant myelin sheath hypertrophy in experimentally induced diabetes. Acta Neuropathol. (Berl.) 68: 149-154. Mayhew, T. M., A. K.Sharma (1984) Sampling schemes for estimating nerve fibre size, I. Methods for nerve trunks of mixed fasicularity. J. Anat., 139: 45-48. Moore, S.A., R.G. Peterson, D.L. Felten and B.L. O'Connor (1981) Glycogen accumulation in the tibial nerves of experimentally diabetic and aging control rats. J. Neurol. Sci., 52: 289-303. Powell, H.C., H.W. Ward, R. S. Garrett, M.J. Orloff and P.W. Lampert (1979) Glycogen accumulation in the nerves and kidney of chronically diabetic rats. J. Neuropathol. Exp. Neurol., 38: 114-127. Sharma, A. K., P. K. Thomas and A. F. DeMolina (1977) Peripheral nerve fibre size in experimental diabetes. Diabetes, 26: 689-692. Sharma, A. K., S. Bajada, P. K. Thomas (1981 ) Influence of streptozotocin-induced diabetes on myelinated nerve fibre maturation and on body growth in the rat. Acta Neuropathol. (Berl.), 53: 257-265. Sharma, A.K., I.G. Duguid, D. S. Blanchard and P. K. Thomas (1985) The effect of insulin treatment on myelinated nerve fibre maturation and integrity and on body growth in streptozotocin diabetic rats. J. Neurol. Sci., 67: 285-297. Sokal, R.R. and F.J. Rohlf (1978)Introduction to Biostatistics, W.H. Freeman and Co., San Francisco, CA. Sugimura, K., A.J. Windbank, V. Natjarajan, E.H, Lambert, H.H.O. Schmid and P.J. Dyck (1980) Interstitial hyperosmolarity may cause axis cylinder shrinkage in streptozotocin diabetic nerve. J. Neuropathol. Exp. Neurol., 39: 710-721. Thomas, P. K. and J. Ochoa (1984), The microscopic anatomy of peripheral nerve fibres. In P.J. Dyck, P. K. Thomas, E.H. Lambert and R.P. Bunge (Eds.), Peripheral Neuropathy, Vol. 1, 2rid edn. Saunders, Philadelphia, PA. Zemp, C., G. Bestetti and G.L. Rossi (1981) Morphological and morphometric study of peripheral nerves from rats with streptozotocin-induced diabetes mellitus. Acta Neuropathol. (Berl.), 53: 99-106.
327
JNS 03382
Ultrastructural abnormalities of myelinated fibres in the tibial nerve of streptozotocin-diabetic rats P. D o c k e r y i and A . K . S h a r m a 2 'Department of Biomedical Science, University of Sheffield, Sheffield SIO 2TN (U.K.) and 2Department of Anatomy. University" of the United Arab Emirates. (United Arab Emirates) (Received 7 November, 1989) (Revised, received 1 May, 1990) (Accepted 1 May, 1990)
SUMMARY
In this paper we have examined the ultrastructural changes in myelinated fibre structure after the administration of streptozotocin to Sprague-Dawley rats which had passed the rapid growth period. Myelinated fibre size in the tibial nerve was found to be less in diabetic animals 4 and 6 months after the induction of diabetes, when compared to age-matched controls, but not less than onset. The relative contributions of axon and myelin to this reduction in fibre dimensions were examined. When myelin area was plotted against axon area (derived from perimeter) it showed that the pathological insult of diabetes had a greater effect on the rate of myelin production. The incidence of axonal glycogenosomes was also assessed. These results are discussed in detail.
Key words: Streptozotocin; Diabetes; Rat tibial nerve; Myelinated fibres; Morphometry
INTRODUCTION
Myelinated fibre size has been found to be decreased in the peripheral nerves of streptozotocin-induced diabetic rats (Sharma etal. 1977, 1985; Jakobsen and Correspondence to: Dr. P. Dockery, Department of Anatomy, University of Hong Kong, Faculty of Medicine, Li Shu Fan Building, 5 Sassoon Road, Hong Kong. 0022-510X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
328 Lundbaek 1976; Jakobsen 1976, 1979; and several others). Sharma et al. i i 977. 198l) demonstrated that myelinated fibre size increased at a slower rate in diabetic animals as compared with controls, suggesting a maturational deficit secondary to the metabolic effects of diabetes. However, the extent to which axon and Schwann cells :Ire affected has been disputed. The effects reported have ranged from a primary Schwann cell lesion (Bestetti et al. 198 la,b; Zemp et al., 1981), axonopathy (Jakobsen and Lundbaek, 1976; J akobsen 1976, 1979) to axonal shrinkage possibly due to interstitial h yperosmolarity (Sugimura et al. 1980). These discrepancies may have been due to methodological differences between investigators such as the use of animals of different strains, ages and the duration and severity of diabetes. The light microscopical techniques employed by some workers (Jakobsen and Lundbaek 1976; Jakobsen 1976; Bestetti et al. 1981a, b; Zemp et al. 1981 ; Matingley and Fischer 1985; Cameron et al. 1986) may be inadequate for assessing the relative contributions of axon and myelin to fibre dimensions. Electron microscopy is considered to be more accurate in assessing axon size and myelin sheath thickness (Landon and Hall 1976; Thomas and Ochoa 1984). Therefore, in this study we have used electron microscopy to establish relative contributions of axon and myelin sheath in the reduction ofmyelinated fibre size in the tibial nerve of streptozotocin-induced rats which had passed the rapid growth period.
METHODS
Induction of diabetes The male albino Sprague-Dawley rats used in this study were aged 20-21 weeks and weighed between 525 and 755 g at the onset of the experiment. A group of 7 animals served as onset controls, to provide baseline morphological parameters for this age and strain. Six further groups were studied and observations were made on diabetic and age-matched controls at 2, 4 and 6 months after the induction of diabetes. Diabetes was induced by a single intraperitoneal injection of a streptozotocin solution at a dosage of 45 mg/kg body weight. Body weight and blood glucose levels used were recorded at regular intervals and at the time of death. Experimental and control animals were maintained on sawdust in plastic metabolic cages with water and Oxoid breeding diet (Oxoid Ltdl, Hampshire) available ad libitum.
Histological and morphometric procedures Biopsies were performed under Sagital (May and Baker Ltd.) anaesthesia (at 60 mg/kg body weight). The left tibial nerves were removed at a standard site between knee and ankle after removal of connective tissues. The nerves were then processed for electron microscopy according to previously described standardised methods (Sharma et al. 1985). Ultrathin sections of the whole transected nerve were cut on a Reichert OMU4 ultramicrotome and examined on a Philips EM201 electron microscope. A systematic random sampling procedure was employed (see Mayhew and
329
Fig. 1. Lower power micrograph of tibial nerve used in the present study. Note the 200-mesh grid bars provide a convenient lattice for systematic random sampling.
330 Sharma 1984; Fig. 1) and ultrastructural morphometric observations were made on 200-300 myelinated nerve fibres per animal on micrographs at a final magnification of × 5000. A magnification standard was taken on each film. In order to exclude a sampling bias (Gundersen 1977) a celluloid overlay half the diameter of the largest fibre was superinaposed prior to any measurements being made, and only those fibres whose centres lay within these bounds were included in the study, The area and length of perimeter of myelinated nerve fibres and their axon were obtained by programmed digitisation using a sonic digitiser linked to a microcomputer, An index of circularity was also calculated from these measurements (Sugimura et al. 1980). Fibres sectioned through paranodal and pen.nuclear regions and at Schmidt-Lantermann incisures were excluded. The initial sampling strategy provided information on the size and shape of myelinated nerve fibres and their axons. However in order to assess the relative contributions of axon and myelin to fibre dimensions a further series of micrographs
Fig. 2. Myelinated nerve fibres sectioned through a perinuclear region to illustrate the degree of structural preservation obtained in the present study. Bar = 1 #m.
331
Fig. 3. Detail of myelinated nerve fibre showing myelin lamellae. Lamellaewere counted and the width of the sheath measured at its most compact region. Bar = 0.5/~m. were taken at a higher initial magnification ( x 10000). The same sampling regime described earlier was used. Counts of the number of myelin lamellae and measurements of axolemmal lengths were made on tracings of the negative using a Zeiss projecting microscope (see Figs. 2 and 3). A data array (for 100 randomly sampled fibres per animal) was set up for the following variables in each animal using the University Honeywell 66/80 mainframe computer: (1) Axon diameter (of circles of equivalent area). (2) Number of myelin lamellae. (3) Axonal index of circularity. (4) Width of myelin at its most compact region. Features 1 and 3 allowed the retrieval of corresponding perimeter and area measurements. A datafile was created for each animal containing the measured variables. A further series of variables was created.
332 (5) Periodicity: this was calculated by taking the slope of the line w = b" nl + c,
where w = width of myelin at its most compact region, nl = number of myelin lamellae comprising the myelin sheath, and b and c = regression coefficients, slope and intercept, respectively. (6) Axon diameter (perimeter): axon diameter derived from perimeter measurements. It was obtained by dividing axon diameter (of circle of equivalent area) by index circularity (I.C.): d ( p ) = d/I.C.
(7) Fibre D (perimeter): fibre diameter derived from perimeter measurements of axon ( d ( p ) ) and number of myelin lamellae (nl) also accounting for periodicity (b in variable 5). Fibre D (perimeter) = d(p) + (2 x nl x b). (8) Axon area (perimeter) = n(d(p) / 2) z (9) Fibre area (perimeter) = n(fibre D (perimeter)/2) z (10) Myelin area: this is in effect a measurement of the actual cross-sectional area of the myelin sheath, as the measurements are derived from the perimeter of the axon and number of myelin lamellae with periodicity being accounted for. Myelin area = fibre area (perimeter) - axon area (perimeter). The incidence of axonal glycogenosomes was also assessed in each nerve studied. This was carried out on the microscope at a magnification of about x 10000 and the counts were expressed as percentage of fibres containing the organelle. Statistical analysis. All of the data were initially calculated on a "per animal" basis, so the n value was the number of rats. A one-way analysis of variance was employed routinely to test for differences in or between the various parameters. A two-way analysis of variance was used to detect any effects of age, diabetes or their interaction on a given parameter (Sokal and Rohlf 1978). Regression coefficients were compared by Student's t-test. Histograms of size frequency distributions of axon and fibre populations were constructed. The various distributions were analysed by a Z2 test.
333 RESULTS
Blood glucose and body weight
Diabetes developed within 24 h, polydipsia and polyuria were the most obvious features of the diabetic animals. The mean blood glucose levels of controls and diabetic groups are shown in Table 1, the diabetic animals had significantly higher blood glucose levels than controls at all survival periods. The body weight growth curves for control and diabetic animals are shown in Fig. 4. The control animals increased in weight throughout the period of study, however
TABLE 1 S T R E P T O Z O T O C I N I N D U C E D D I A B E T I C RATS
This table shows mean ( + SE) blood glucose levels (mmol/1) for onset, control and diabetic groups 2, 4 and 6 months after induction of diabetes.
2 months
4 months 6 months
Diabetic
Control
30.0 + 2.6 33.1 + 1.2 35.5 + 1.3
8.4 + 0.4* 9.9 +_ 0.5* 9.8 + 0.4*
* P < 0.05.
2 MONTHS
8OO
CONTROL 600 DIABETIC 400~
~
-
4 MONTHS 800
CONTROL
-r"
DIABETIC >. t",, 0 m
4
0
% CONTROL
8001'
6
Tr
0
0
40(~ 8
I •
~ .
.
6 MONTHS
DIABETIC .
.
12
.
.
.
. ~,I
24
WEEKS Fig. 4. Body weights (mean _+ SE) for control and diabetic animals 2, 4 and 6 months after the induction of diabetes.
334 the weight of the diabetic animals decreased by about 3090. At 4 and o months the diabetic animals appeared severely emaciated, at 6 months some of thc animals had developed cataracts. No obvious signs of neuropathy were observed at any survival period.
Myelinated fibre area The cross-sectional areas of myelinated fibres in the tibial nerve are shown in Fig. 5. Cross-sectional myelinated fibre area increased significantly in controls over the period of study. Myelinated fibre area in the tibial nerve of diabetic animals was found to be significantly less than age-matched controls at 4 and 6 months but not less than onset. When the data for 2, 4 and 6 months control and diabetic groups were subjected to a two-way analysis of variance there was found to be a significant effect of age and diabetes at the 1 ~ level and a significant interaction at the 5 ~o level (Table 2), implying that the longer the duration of diabetes the greater the effect on the nerve fibre. Fibre size-frequency distributions for control and diabetic groups are shown in Fig. 6. All of the distributions were subjected to a X2 analysis. The fibre diameter spectra of control animals was significantly different from onset only at 4 and 6 months survival periods. No difference in the distributions of fibre diameter was detected when any of the diabetic groups were compared with onset. However, a significant difference was detected in the diabetic animals only at 6 months when compared with age-matched controls. FIBRE AREA [~
pm 2
P<0.01
CONTROL
1771 DIABETIC
50p~0.01
40
n.s.
I
13.8.
/A ,
i A
/// ~ n.s. ONSET
2 MONTHS
¢ / 1 / / p~ 0.01 4 MONTHS
p
Fig. 5. Myelinated fibre area (mean _+SE) for onset, control and diabetics at 2, 4 and 6 months after the induction of diabetes. Values above respective columns show level of significance between column and onset. Values below columns show the comparison between diabetic and age matched control This format is repeated in all similar figures.
335 TABLE 2 T W O WAY ANALYSIS OF V A R I A N C E The table shows the F values and levels of significance after performing a two-way analysis of variance on fibre area, axon and myelin area. Age d.f. Fibre area Axon area Myelin area
P
2,36 15.85 3.54 0.54
Diabetes
P
1,36 35.61 6.81 55.23
<0.01 < 0.05 n.s.
<0.01 < 0.05 <0.01
Interaction
P
1,36 3.82 0.309 1.32
0.05 0.05 n.s, n.s.
d.f. = degrees of freedom numerator, denominator.
CONTROL
ONSET
o
"J~'~-
0
~ .
DIABETIC
2 MONTHS 09
u J - u
_.~,=..-_
in tt.
4 MONTHS m
;D
6 MONTHS
o
]5~ 0
5
I0
15
,.~" 0
'3h~, 5
lO
15
FIBRE DIAMETER (A) i~m Fig. 6. Fibre-size frequency distribution (diameter based on circle of equivalent area) for onset, control and diabetic groups at 2, 4 and 6 months after induction of diabetes.
336 AXON,
pm
AREA
2
p <0.05 I--'I 15
+
rI,S,
14
n.s,
+
13
t n.s,
T
12
CL$,
r/V
CONTROL DIABETIC
13,s,
IlIA IlIA IlIA
//A Ilia
11
IlIA n.S.
ONSET
2 MONTHS
n.S.
4 MONTHS
p< ).o5 6 MONTHS
Fig. 7. Myelinated axonal area (mean + SE) for onset, control and diabetics at 2, 4 and 6 months after induction of diabetes.
Myelinated axonal area The cross-sectional axonal area of myelinated fibres in the tibial nerve is shown in Fig. 7. Using a one-way analysis of variance there was found to be no overall change in axonal area with age in the control animals, however at 6 months the axonal area of the control animals was significantly larger than onset (at 5 % level). No significant difference was found when each individual diabetic group was compared with onset. No significant differences in cross-sectional axonal area were detected between diabetic and age-matched controls at 2 and 4 months but axonal area was significantly less in diabetic animals at 6 months when compared with age matched controls. The data for 2, 4 and 6 month control and diabetic groups were subjected to a two-way analysis of variance. There was a significant effect of age and diabetes at the 5')~, level but no interaction (Table 2). Age and diabetes have slight but significant effects on axonal area. The axon diameter spectra (Fig. 8) of control animals was significantly different from onset only at 4 and 6 months. When each diabetic group was compared with age-matched controls, no significance was observed at any survival period. Index of circularity The mean index of circularity is shown in Fig. 9. Mean axonal index of circularity was not found to be different between diabetic and age-matched controls and 4 and 6 months. It was significantly greater in diabetics
337 CONTROL
DIABETIC
ONSET
2 MONTHS tO I.U n" IJ_
~o
U-
o
4 MONTHS
n" ILl t~
o
Z
6 MONTHS o
4
B
o
4
o
AXON DIAMETER (A) pm
Fig. 8. Axon size-frequency distribution (diameter based on circle of equivalent area) for onset, control and diabetic groups at 2, 4 and 6 months after induction of diabetes. A X O N A L INDEX
OF
CIRCULARITY
F'-ICONTROL W'~ D I A B E T I C
0.8-
p
~0.05 I P
0.7-
p
'///
p
i/tO, I
///i I i
0.5
'///
///,
I
p
2 MONTHS
n.s.
4 MONTHS
fI.S.
6 MONTHS
Fig. 9. Axonal index of circularity (mean + SE) for onset, control and diabetics at 2, 4 and 6 months after the induction of diabetes.
338 at 2 months as compared with age-matched controls. Both diabetic and control axons were found to be less circular than onset at all survival periods. This shape index was examined in more detail. The plots of axonal index of circularity against axon diameter (perimeter) are shown in Fig. 10. There appears to be no strong size related difference in shape between large and small axons. There is a wide scatter of points throughout the fibre spectra. Fibre spectra based upon axonal index of circularity are presented in Fig. 11• A ~:" analysis was performed on these data. The distributions for controls were found to be significantly different from onset at 4 and 6 months survival periods. Diabetic groups were also found to be significantly different from onset at 4 and 6 months. When diabetics were compared with age-matched controls no significant differences in the distributions were detected at any survival period• Changes in axonal shape do not seem to be an important factor in streptozotocin-induced diabetes in the rat.
Relationship between myelin sheath thickness and axon size In order to assess the relative contributions of axon and myelin to fibre dimensions, the number of myelin lamellae was related in various ways to axonal dimensions CONTROL
1
'.
DIABETIC
. . . . ':.;l;;;;::.'-,,, • :.:t:;:'|l::.'=;::::::;;:;l;;:
, ,.,.. ,.; •, . . . . . .
" : ,"
, %""
ONSET
1
.. ~ • ,.
,-r,::;: .gl"r ,g~,'lt'F;~lrl ..;
ik[~iE;::'~'~2~:'2.:':'
,.- ,. •,
2 MONTHS
,., ........
• 1:'.""~l~llltll~gl,~li;';%' ,,
>I-
,,; •
. :..'
.
g:,o 1 ii 0
:
x u.I
-
2
'~7;':i~L=--".~:::!r'I::
• ,':,•,
,.....
4 MONTHS -
._1 ,< z
0 x
0
1
.... •,;ii,. I,...' .. •
t , l l l p H I l , l l e l l ilk i . . . . . . . .
, .. ' : •
:|
;~'~I:I:P":P.".;,;'~:. '
": ":,.',':1,1;; ;,~.~i~|.l':';, •,
6 MONTHS
• "."|
,..,;.
,,. ,",
0 0
t0
AXON
DIAMETER
0
10
(P)llm
Fig. 10. Plots (pooled data) of axonal index of circularity against axon diameter (by circle of equivalent perimeter) for all groups studied.
339 CONTROL
DIABETIC
20
ONSET
2 MONTHS
03 LU
r'r
4 MONTHS
2O
U. U. O nu.I
ID Z
20
6 MONTHS
0 ~ti'1~ 0.025
0.975
O. 025
0.975
A X O N A L INDEX OF C I R C U L A R I T Y
Fig. 11. Pooled fibre spectra based on axonal index of circularity for onset, control and diabetic groups at 2, 4 and 6 months after induction of diabetes.
(see Dockery 1986). The most appropriate method was found to be myelin area plotted against axon area based on perimeter measurements. Myelin area vs. a x o n area
The plots of pooled data from each group are shown in Fig. 12. The slopes of the linear regression lines (myelin area = C O + C~ axon area) are shown in Table 3. The slopes of the control groups were significantly less steep than onset at 4 months (P < 0.05). Diabetic groups were found to have significantly less steep slopes than onset at 4 and 6 months. The slopes of diabetic groups at 4 (P < 0.05) and 6 months (P < 0.001) were significantly less steep than age-matched controls. Myelin area
The mean myelin cross-sectional area for all the control and diabetic groups is shown in Fig. 13. A one-way analysis of variance performed on the data from all of the control groups showed no significant effect of age. However, myelin cross-sectional area was
340 DIABETIC
CONTROL
100
ONSET
,oo] 2 MONTHS
....:,:;::i£~ii:i:ii.'i'.:!""
:"
o - ':::-:;~!
ILl tr ,,( Z
4 MONTHS .~
100 1 : .!:!ii:i;;i~ii;¢~.~i~i:'~i'~% ' O"
6 MONTHS
o
.:iiiii::;i:;;""';:':"";....
0
100
0
100
A X O N AREA ( P ) Ilrn 2
Fig. 12. Plots of myelin area against area (perimeter) for onset, control and diabetic groups at 2, 4 and 6 months after induction of diabetes.
TABLE 3 SLOPES OF L I N E A R R E G R E S S I O N FOR M Y E L I N A R EA VERSUS A X O N A R EA Myelin area = Co + CI axon area (p)
Slope C1
Onset 2 months 4 months 6 months
Control
Diabetic
0.450 0.501 0.385 0.448
0.430 +_ 0.026 0.302 _+ 0.021 **~ 0.295 + 0.037 **+ t
_+ 0.018 + 0.025 + 0.017" _+ 0.017
* P < 0.05 compared with onset; **P < 0.001 compared with onset; + P < 0.05 compared with age-matched controls; + ÷ P < 0.001 compared with age-matched controls.
341 MYELIN AREA
iJrn 2
p
P
[-""1
CONTROL
P'~O.01 [77]
DIABETIC
+
--t24
I 13.5.
20
n.s°
rI.S.
ONSET
p
P
~
2 MONTHS
4 MONTHS
6 MONTHS
Fig. 13. Myelin area for onset, control and diabetic groups at 2, 4 and 6 months after induction of diabetes.
found to be significantly greater in control groups at 2, 4 and 6 months survival periods when compared with the onset group. Although there was an apparent decrease in mean myelin area, no significant difference was detected between onset and diabetic groups at any survival period. The mean myelin cross-sectional area of diabetic animals was found to be significantly less than age-matched controls at all survival periods. A two-way analysis of variance revealed that diabetes had a significant effect (P < 0.01) but there was no effect of age or interaction on cross-sectional myelin are (Table 2).
Axonal glycogenosomes The number of fibres containing axonal glycogenosomes was found to be increased at 4 and 6 months in controls and at 2, 4 and 6 months in diabetic groups when each group was compared with onset (Table 4). Although the diabetics consistently appeared to have a greater number of fibres containing these organelles, there was no significant difference between diabetic and age-matched controls. At 6 months, there was a border line difference between diabetic and age-matched controls (P < 0.06).
342 TABLE 4 PERCENTAGE INCIDENCE (MEAN _+SE) OF MYELINATEDFIBRES CONTAININGAXONAL GLYCOGENOSOMES IN TIBIAL NERVE 2 months
4 months
~ months
Onset controls End controls P-level
0.166 _+0.079 1.263 ± 0.513 n.s.
0.166 _+0.079 1.840 ± 0.590 < 0.05
I}166 ± 0.079 3170 ± 0.990 <~(}.05
Onset controls Diabetic P-level
0,166 _+0.079 2.420 _+0,620 < 0.05
0.166 ± 0.079 3,970 _+1.040 < 0.05
0,166 _+0,079 7,320 ± 1,520 < 0.01
Controls Diabetic P-level
1.263 _+0.513 2.420 _+0.620 n.s.
1.840 _+0.590 3.970 + 1.040 n.s.
3.170 ± 0.990 ,'.320 ± 1.520 < ((}.06)
DISCUSSION Myelinated fibre area was found to be reduced in diabetic animals when compared with age-matched controls but not different from onset controls. This deficit in fibre size is in line with most previous studies on peripheral nerve trunks (Jakobsen 1976; Sharma et al. 1977, 1985; Jakobsen and Lundbaek 1976; Bestetti et al. 1981a,b; Zemp et al. 1981). Myelinated fibre size increased throughout the period of study in the control animals, confirming the earlier work of Sharma et al. (1981). Sharma et al. (1977) found that fibre size diameter in diabetic animals failed to show the age-related increase exhibited by the controls. A similar situation has been encountered in the present study. Sugimura et al. (1980) also found that external fibre diameter in rats after 20 weeks of streptozotocin-induced diabetes was intermediate between onset and end controls. This raises the possibility that the pathological insult of diabetes may affect the normal maturation of the myelinated nerve fibre. In light microscopical studies Jakobsen and Lundbaek (1976) and Jakobsen (1976) reported that axonal diameter was reduced to a greater extent than external fibre diameter, however, this was not demonstrated in a convincing manner as the author did not include an onset group which would provide baseline morphological parameters for the age and strain of rat used. The concept of axonal "dwindling" proposed by these authors may therefore be misnamed, as all that they have demonstrated is that axon size is less in the diabetic when compared with age-matched controls at a given survival period. In a subsequent study, J akobsen (1979)did include a group of animals that acted as an onset control group. These animals were 24 weeks old, diabetic and age-matched controls survived for a further 4 weeks. The light microscopical data presented do not show any evidence for the concept of axonal "dwindling". The axonal cross-sectional area in their study was found not to be significantly less in the diabetic animals when compared with the 28-week-old onset group.
343 Nevertheless, axonal area has been shown to be smaller in diabetics when compared with age-matched controls examined by electron microscopy (Jakobsen, 1979). Sugimura et al. (1980) examined the sural nerve in streptozotocin-induced Sprague-Dawley rats by light and electron microscopy. The light microscopical data showed that axonal size measured from the "inner edge" of myelin sheath from diabetic animals was found not to be significantly smaller "axis cylinders" than the end control group. Examination of the material by electron microscopy showed that axonal area of animals diabetic for 20 weeks was less than age-matched controls, but not less than onset. They also reported that axonal perimeter was not different between any of the groups. However, axons from diabetic animals were found to be significantly less circular than onset and end controls. They suggested that this was due to a dehydration shrinkage which may be a consequence of interstitial hyperosmolarity. The onset control animals in the Sugimura et al. (1980) study weighed only 275-300 g compared with the 20-21-week-old animals used in the present study which were 525-755 g at the start of the experiment. The differences in the age of animals used may account for the discrepancies found in the index of circularity results between these two studies. In the present study no overall effect of age on axonal area was observed when either controls or diabetics were subjected to a one-way analysis of variance test. A slight but significant increase in axonal area was observed in control animals at 6 months when compared with onset. No significant difference was observed in axonal area when diabetic animals at any survival period were compared with onset. Diabetics were only significantly less than age-matched controls at 6 months. Changes in axonal shape do not seem to be an important factor in streptozotocin-induced diabetes in the rat. The relative contributions of axon and myelin to fibre dimensions in the streptozotocin-induced diabetic rat have been investigated by a number of workers. Bestetti et al. ( 1981 a,b) and Zemp et al. (1981) reported that there was a reduction in the myelin axon ratio and provided evidence that the myelin sheath is in fact affected to a greater extent than axonal area. The recent work of Mattingley and Fischer (1985) and Cameron et al. (1986) revealed that there is a decrease in axonal area with a concomitant increase in sheath thickness. The fact that all of these studies have employed light microscopy (which has much poorer resolving power than the electron microscope) to examine axon and myelin must cast doubts on any interpretations of their results. Myelin and axonal contributions to fibre size in the experimental diabetic rats examined by electron microscopy have been reported by Jakobsen (1979) and Sugimura et al. (1980). Sugimura et al. (1980) reported that there was an increase in the median number of myelin lamellae in the diabetic animals, although the difference was not statistically significant. Plotting myelin area against axon area (of circle of equivalent perimeter) suggested a greater vulnerability of the Schwann cell to the pathologic insult of diabetes. As myelin production is still a fairly active process at this age in control rats, and axonal growth seems to be rather slow, this may make myelin production a sensitive indicator of any slowing of metabolic rate induced by diabetes. It is of considerable interest to note that control animals 4 weeks after the onset of the experiment by Jakobsen (1979), showed an increase in axonal area (measured by light
344 microscopy). It seems that the active growth of the axon in these animals had not in fact passed, thus making it especially liable to the pathologic insults. In the present study, the number of fibres containing axonal glycogenosomes appeared slightly greater in the diabetic animals but the differences did not reach the level of statistical significance. Moore et al. (1981) reported that fibres containing axonal glycogenosomes were found to be significantly more numerous in streptozotocin diabetic rats than controls 2 weeks and 2 months after the induction of diabetes. Intra-axonal glycogen accumulation has also been observed in the nerves of atloxaninduced diabetic rats (Powell et al. 1979; Moore et al. 1981). The difference between the action of the two diabetogenic agents, especially the toxic effect of alloxan may be important in the accumulation of glycogen. However, Moore et al. (I 981) reported th at at 4, 8 and 12 months, control animals had significantly greater numbers of fibres containing these organelles than age-matched streptozotocin diabetic animals. Therefore, the authors suggested, that it seems doubtful than hyperglycemia is directly responsible for axonal glycogen accumulation o f glycogen. Furthermore, an age related phenomenon has previously been proposed to explain the increase in axonal glycogenosomes (Powell et al. 1979; T h o m a s et al. 1980; Grover-Johnson and Spencer 1981 ; Moore et al. 1981). It has also been suggested that glycogen accumulation might be related to distal trauma or ischaemia from focal entrapment of the plantar nerve (Thomas et al. 1980; Grover-Johnson and Spencer 1981). The animals used by Moore et al. (1981) were maintained on wire mesh flooring and an early involvement of the diabetic nerves may be due to an increased susceptibility to the ageing phenomenon or pressure related injuries. Whatever the etiology of this disorder in this model we have shown that the reduction in myelinated fibre size is primarily due to a decreased rate o f myelin production.
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