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Pathology and time relationship of peripheral nerve changes in experimental diabetes
Journal of the Neurological Sciences, 1977, 32:53-67 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
53
P A T H O L O G Y A N D T I M E R E L A T I O N S H I P OF P E R I P H E R A L N E R V E C H A N G E S I N E X P E R I M E N T A L DIABETES*
J. S. CHOPRA, B. B. SAWHNEY and R. N. CHAKRAVORTY Department of Neurology, Postgraduate Institute of Medical Education and Research, Chandigarh (India 160011)
(Received 1 September, 1976)
SUMMARY Structural changes have been studied in peripheral nerves in streptozotocininduced diabetic monkeys. Teased single nerve fibre preparations were most informative. Abnormalities were present in 32 of 58 nerves examined from 15 diabetic monkeys with varying degree of hyperglycaemia. The earliest change was an increase in the gap at nodes of Ranvier, seen 12 weeks after the diabetic state had been established. Well marked segmental demyelination was seen in distal nerves at 14-16 weeks. Later similar changes were seen in proximal, larger nerves. Evidence of remyelination was present at a later date. Wallerian degeneration was seen in only 4 nerves. Changes in the myelin sheath were more prominant than axonal abnormalities at all times. There was no abnormality in the vasa nervorum and only a mild increase in endoneurial and perineurial fibrous tissue. A direct correlation was present between the extent and degree of pathology and both severity as well as duration of hyperglycaemia.
INTRODUCTION Structural changes of segmental demyelination and some degree of axonal loss have been clearly demonstrated in peripheral nerves in patients with diabetes mellitus (Thomas and Lascelles 1966; Chopra, Hurwitz and Montgomery 1969). The relationship of the pathological lesions to the severity and duration of diabetes however, remains controversial. Greenbaum (1964), Gregersen (1967) and Chopra et al. (1969) observed a direct relationship between the structural changes, reduced nerve conduction velocity and degree and duration of the hyperglycaemic state.
* Presented in part at the 3rd International Congress of Muscle Diseases, Newcastle upon Tyne, September 1974.
54 Since it is almost impossible to know, with any degree of certainty, the onset of the diabetic state in a particular individual, no idea can be had as to the time taken for the pathological changes to appear in the peripheral nerves. This can only be studied by closely following an experimental model where one is certain about the onset of the diabetic state. It has been observed that pathological changes in peripheral nerves similar to those seen in human diabetics are seen in alloxan-induced diabetic animals (Preston 1967; Hildebrand, Joffroy, Graft and CoOrs (1968). Question was raised if such changes were not a direct toxic effect of alloxan (Lovelace 1968). Although Sharma and Thomas (1974) showed reduced nerve conduction in streptozotocininduced diabetic rats, no morphological changes were seen to explain this electrophysiological abnormality. Jakobsen and I_undb~ek (1976), however, thought that the fibre size was diminished and that the reduction in size of axons was twice that of the myelin sheath in streptozotocin-induced diabetic rats. The present study was designed to find out the earliest time period in which pathological changes are established in peripheral nerves in experimental diabetes using a different species of animals. The relationship between the intensity of the diabetic state and the severity of histological changes could also be studied. MATERIAL AND METHODS Twenty-five Rhesus monkeys, both male and female, were used in the experiment. These healthy monkeys were kept in the animal house for 2-3 months before being subjected to the experiments. Each animal received a standard mixed diet of 900 calories daily. This consisted of 25 ~ proteins, 65 % carbohydrates, and 10 ~ fats, with appropriate amounts of salts and vitamins. All animals were kept in separate metabolic cages under similar environmental conditions. The estimated age of the group was 3-4 years and their weight varied between 2 and 5.5 kg. Fasting blood sugar levels in all the animals were in the normal range (Tables 1 and 2). The blood for examination was collected from the anterior cubital or femoral veins and serum glucose content estimated by Nelson-Somogyi's method. None of the animals showed evidence of any reducing agent in the urine tested by the Benedict's qualitative method. Six monkeys served as controls and 19 received a single intravenous injection of
TABLE1 CONTROL GROUP Monkey No. Sex
Weight(kg) Fasting blood sugar (mg/100 ml)
Duration alive (months)
5 6 9 10 17 20
2.0 6.0 2.0 4.5 2.8 3.0
4 20 20 20 14 1
M M M M M F
76 70 80 85 80 90
55 TABLE 2 EXPERIMENTAL GROUP Fasting blood sugar values refer to initial figures before streptozotocin was given. Monkey No. Sex
Weight Fasting blood sugar (kg) (mg/100ml)
Range of blood sugar (rag/100 ml)
Durationalive (months)
1 2 3 4 7 8 I1 12 13 14 15 16 18 19 2l 22 23 24 25
3.1 3.4 2.5 4.0 2.5 5.5 5.0 5.0 4.2 5.0 4.5 4.2 3.0 3.5 2.5 6.5 2.0 4.2 5.0
250-390 200-425 245-295 210 430 237-386 300-312 230-420 200-375 165-180 150-170 230-430 230-400 100-110 85-115 225-320 75-130 100-110 116-320 200-320
10 4½ 2 16 2 17 days 10 13 12 2 9 8 14 days 11 18 days (escaped) 1l 10 5½ 3
F F M M M M M F M M M M M M M M M F F
71 70 100 88 88 78 70 75 80 106 80 85 80 83 80 70 85 113 80
streptozotocin in the dose of 50 mg/kg body weight according to the method adopted from Pitkin and Reynolds (1970). The drug was injected after the animals had been kept lasting overnight. Two monkeys at a time were made diabetic and stabilized for 10-15 days, before a new pair was incorporated into the experiment. The first blood sugar was tested 48 hr after the streptozotocin injection and subsequent serial estimations carried out fortnightly. Daily urinalysis for glycosuria and acetone was done. The animals in whom the blood sugar level remained above 200 mg/100 ml were labelled as severely diabetic. There were 13 such animals (Table 2). The animals in whom the blood sugar levels were between 150 and 200 mg/100 ml were classified as mildly diabetic (Nos. 13, 14). Four monkeys (Nos. 18, 19, 22, 23) did not become diabetic. One monkey (No. 21) from the first group escaped after 18 days and further studies could not be carried out. Severely diabetic animals were given 2-6 units of plain insulin when there was orange reduction in urine with Benedict's test or when acetone was present. This procedure was adopted to prevent the complications of ketosis and coma. However, no attempt was made to control the diabetic state with insulin therapy. All diabetic and control animals were weighed fortnightly. A reduction of 0.25-1 kg in body weight was observed in all diabetic animals over the period that these were kept alive. None of the control monkeys lost any weight during this period.
Nerve biopsy Serial nerve biopsies were taken under general anaesthesia from each monkey
56 TABLE 3 NUMBER OF NERVES EXAMINED AT VARIOUS TIME PERIODS Time interval (months)
Diabetic
Control
1-2 2- 4 4-6 6-8 8-10 10-12 12-14 14-16
4 23 12 5 5 5 2 2
8 5 8 1 3 1 2 4
Total
58
27
(diabetic or control) at an interval of 1-4 months. At each instance a sural nerve (at the level of the lateral matleolus) or a radial nerve (at the level of the wrist) was biopsied. Larger nerves such as the femoral, median and/or ulnar could be examined following the death or the sacrifice of the animal. Table 3 shows the total number of nerves studied and the duration of the diabetic state at the time of the study o f a particular nerve. Each nerve was appropriately stained to study myelin (Fleming's solution fixed specimen stained with Kulchitsky's stain), axons (silver impregnation technique) and with haematoxylin and eosin (H & E), periodic acid-Schiff (PAS) and haematoxylinvan Gieson stains as described earlier (Chopra and Hurwitz 1967; Chopra et al. 1969). Over 20 nerve fibres of varying diameter were teased from the formol-saline fixed specimens and measurements of internodal lengths and fibre diameters ptotted from these graphically (Thomas and Lascelles 1966; Chopra et al. 1969). Internodal lengths were also plotted against the diameter of the widest internode on each fibre. This gave a convenient assessment of the pattern of the variations in internodal distances. RESULTS Fifty-eight nerves from the diabetic animals were compared with 27 nerves from control monkeys at different time periods. Myelinated nerve fibres Figure 1 shows the density of the myelinated fibres in a control monkey kept for 6 months under the same conditions as the experimental group. The number of nerves examined in the experimental group at a different time interval after the onset of the diabetic state is given in Table 4. Of a total of 58 nerves examined, 19 showed a varying degree of loss of the myelinated fibres (Figs. 2-4). This was equally distributed in fibres of all sizes. The extent of the changes at different time periods is discussed below. Axonal loss As compared with the loss of myelinated fibres, the axons were relatively well
57
Fig. 1. Sural nerve (transverse section, TS) from a control monkey (No. 9). Kulschitsky's haematoxylin (KH), × 200.
preserved. Only a mild loss of axons was seen in monkeys Nos. 4, 12 and 13 in median and femoral nerves after 14-16 months (Table 4, Fig. 5). Loss of myelinated nerve fibres or axons was not observed in nerves from the control group at any stage. Connective tissue Only mild perineurial fibrosis was seen in 3 animals in the diabetic group. Monkey
N o . 12 s h o w e d t h e s e c h a n g e s i n t h e s u r a l n e r v e a f t e r 3 m o n t h s a n d i n t h e
r a d i a l n e r v e a f t e r 8 m o n t h s o f t h e d i a b e t i c state. M o n k e y s N o s . 1 a n d 13 s h o w e d c h a n ges i n t h e r a d i a l n e r v e s a f t e r 4 a n d 11 m o n t h s , r e s p e c t i v e l y . O n e b i o p s y f r o m t h e c o n trol group also showed similar changes in the radial nerve after 8 months. TABLE 4 H I S T O P A T H O L O G I C A L C H A N G E S IN DIABETIC MONKEYS Time Myelinated interval fibre loss (months)
Axonal loss
Segmental demyelination
Wallerian degeneration
Perineurial fibrosis
2- 4 4- 6 6- 8 8-10 10-12 12-14 14-16
4s 12~ 13s 16s lr l l s 15s 16s 24r 4r 12r l l r 12r 15~ 13r lu 13f 4r,m
-----12r,m 13t 4t,m
4s l l s 12s 13s 15s 16s 2r 2r,r 4r l l s 15s 16s 24s,~ l l r 12r 13r 16rd 4r l l r 12r 15rd lu 12t, m 13t,m 4t,m
---15t -12t 4r,m
12s 12r lr 12r -13r ---
Total
19
5
32
4
5
Figure indicates monkey No., s = sural nerve, r = radial nerve, f = femoral nerve, m ~ median nerve, u = ulnar nerve.
58
Fig. 2. Sural nerve (TS), diabetic monkey (No. 13) at 4 months, showing a moderate loss of myelinated nerve fibres. KH, :. 100.
Single nerve fibre preparations Morphological changes in the single nerve fibre preparations were observed in 32 of the 58 (55.2 °,~i) nerves studied f r o m the diabetic animals. The earliest change was an increase in the gap at the nodes of Ranvier due to retraction of myelin from the nodes. This was observed as early as 12 weeks after the diabetic state had been established and was seen in 7 nerves. Table 4 shows the changes of segmental demyelination in different nerves related to the duration of the diabetic state (Figs. 6 and 7). Changes of segmental demyelination and remyelination were not only confined to the
Fig. 3. Radial nerve (TS), diabetic monkey (No. 11) at 8 months, showing moderately severe loss of myelinated nerve fibres. KH, × 100.
59
Fig. 4. Ulnar nerve (TS), diabetic monkey (No. 1) at 10 months, showing severe loss of myelinated nerve fibres. KH, ~ 100.
distal nerves but also seen in the proximal nerves, viz. femoral, ulnar and the median (Figs. 8-10). Changes of Wallerian degeneration were seen in only 3 animals of the diabetic group. This abnormality was seen in the femoral nerve of monkey No. 15 after 9 months, in the median nerve of monkey No. 12 after 13 months and in both the femoral and median nerves of monkey No. 4 after 16 months (Fig. lla, b, c). Thus these abnormalities appeared later and were minimal as compared to the pathology of segmental demyelination. Internodal length and fibre diameters were measured and
Fig. 5. Femoral nerve (TS), diabetic monkey (No. 13) at 14 months, showing only a mild loss of axis cylinders. Glees Marsland silver impregnation, .~ 100. (Compare with earlier biopsy above Fig. 2.)
60
Figs. 6 and 7. Teased single nerve fibres from diabetic monkeys Nos. 15 and 16 at 4 and 6 months respectively, showing areas of segmental demyelination. Osmium tetroxide.
Figs. 8 and 9. Teased single nerve fibres from the femoral nerves, diabetic monkeys Nos. 24 and 13 at 5½ and 14 months respectively, showing areas of segmental demyelination. Osmium tetroxide.
61
Fig. 10a, b, c. Teased single fibres from the femoral nerves, diabetic monkeys Nos. 15, 12 and 4 at 9, 14 and 16 months respectively, showing areas of segmental demyelination. Osmium tetroxide. p l o t t e d graphically. These o b s e r v a t i o n s also showed a wide scatter indicating segmental d e m y e l i n a t i o n a n d r e m y e l i n a t i o n (Figs. 12-14). M o r p h o l o g i c a l changes in teased fibre p r e p a r a t i o n s were seen in 9 m o n k e y s o f the diabetic g r o u p a n d were absent f r o m the c o n t r o l group. It was noticed t h a t in single fibre p r e p a r a t i o n s p a t h o l o g i c a l changes were observed even in nerves in which transverse sections h a d shown no definite a b n o r m a l i t y .
Vasa nervorum There was r e m a r k a b l e p r e s e r v a t i o n o f the vasa n e r v o r u m in all the nerves examined. N o d e p o s i t i o n o f P A S - p o s i t i v e m a t e r i a l was seen in a n y o f the sections.
Relationship of severity and duration of the diabetic state to the pathological lesions In 5 animals (Nos. 2, 4, 11, 15, 16) the usual b l o o d sugar levels were 400 mg/100
III
Q
~
b
c Fig. l l a , b, c. Teased single fibres from diabetic monkey 4 at 16 months (femoral nerve in l l a and median nerve in 1lb) and femoral nerve at 9 months (monkey No. 15), showing Wallerian degeneration. Osmium tetroxide.
62 1,4" 1.2"
E
1.0"
,-rI.z
0.8" 0.6
o z Iz
0.4 0.2
2
4
6
8
10
12
D I A M ETE R(,.~ )
Fig. 12. SuraI nerve of diabetic monkey No. 16 at 6 months. In this and subsequent Figs: 13 and 14, lengths of the internodes on individual fibres are plotted against the diameter of the widest internode of the respective fibre and the points joined by vertical lines. This indicates nerve fibres with both long and short internodal segments.
ml or above and in all these animals pathological lesions in the distal nerves could be demonstrated as early as 3 months after hyperglycaemia had been established. There were mild to moderate changes of segmental demyelination. Similar changes were also seen in the proximal nerves in these animals at a later date. In 6 animals (Nos. 1, 7, 8, 12, 24, 25) the usual blood sugar levels were below 400 mg/100 ml. In this group, early changes at 3 months were only seen in 1 animal (No. 12) in the sural nerve. However, lesions were observed in the radial nerve at 6 months and in proximal nerves after 14 I-Z'
1.0' E E
O.8.
"II,--
0.6'
~ 0.4, Z
ul ~ 0.2.
DIAMETER(.~) Fig. 13. Radial nerve of diabetic monkey No. 4, at 7½ months.
63 1"4
1"2
I0
0'8
0"6
04
0'2
2
4
6
8
DIAMETER
I0
12
14
t~LI)
Fig. 14. Femoral nerve of diabetic monkey No. 24, at 5½ months.
months in this animal. In monkey No. 24 of this group, no abnormality was seen at biopsy at 3 months but at autopsy at 5½ months, changes were present in the other sural nerve and in the femoral nerve. In monkey No. l, no lesion was detected in 4 biopsies taken till 8 months of hyperglycaemia, and only mild segmental demyelination was seen in the ulnar nerve at autopsy after 10 months. Blood sugar levels remained between 200 and 300 mg/100 ml in monkey No. 13. Early changes in 1 sural and 1 radial nerve were seen at 4 and 7 months, respectively. Autopsy after 14 months, however, showed severe segmental demyelination and remyelination. These observations indicate a direct relationship between the severity of the diabetic state (as judged by hyperglycaemia) and the pathological lesions in the peripheral nerves. In addition to the intensity of the diabetic state, its duration also affected the extent and severity of the morphological changes. This was seen in monkeys Nos. 2, 4, 11, 12, 13 and 16 in whom pathological alterations increased with the lengthening of the diabetic state. DISCUSSION It has been established that diabetic neuropathy in man is associated with a significant reduction in both motor and sensory nerve conduction velocities (Downie and Newell 1961 ; Lawrence and Locke 1961 ; Mulder, Lambert, Bastron and Sprague 1961 ; Chopra and Hurwitz 1968). Morphologically this has been thought to be due to predominant segmental demyelination and some axonal loss seen in biopsies from the sural nerves (Thomas and Lascelles 1966; Chopra et al. 1969) and from the larger nerves at autopsy (Chopra and Fanin 1971). These pathological changes indicated either a primary or a secondary disorder of Schwann cells, where metabolic alterations have been demonstrated in experimental models (Gabbay and O'Sullivan 1968; Ward, Baker and Davis 1972). Recently, however, the importance of segmental demyelination has been questioned, and Bischoff (1973) and Brown, Martin and Asbury (1976) have re-emphasized axonal involvement.
64 Alloxan-induced diabetic rats have almost exclusively been used as an experimental model for the study of diabetic neuropathy. Nearly all authors have shown a reduction in nerve conduction either in vitro or in vivo in such animals (Eliasson 1964; Preston 1967; Hildebrand et al. 1968; Lovelace 1968; Seneviratne and Peiris 1969; Miyoshi and Goto 1973). Slow nerve conduction was observed as early as 2 weeks following an established diabetic state by Eliasson (1964). Preston (1967) and Hildebrand et al. (1968) however, found significant changes only after 3-4 months, kovelace (1968) thought that the early delay observed by Eliasson (1964) could be due to toxic or hypersensitivity reactions to alloxan itself and not due to the metabolic changes. This, however, can be eliminated from the follow-up studies by Miyoshi and Goto (1973). These authors found progressive decrease in serial conduction velocity from 2-6 weeks in rats which were made diabetic by alloxan but observed no changes in the same period in rats who did not become diabetic. In pathological studies, segmental demyelination has been observed in alloxan-induced diabetic rats (Preston 1967; Lovelace 1968). Variations in nodal myelin architecture have also been described (Hildebrand et al. 1968; Seneviratne and Peiris 1969). These pathological abnormalities are similar to those seen in diabetic neuropathy in man referred to above, it is interesting to note that similar pathological alterations have also been seen in an inbred strain of chinese hamsters who develop spontaneous diabetes (Schlaepfer, Gerritsen and Dulin 1974). Streptozotocin has been considered to have a selective action on beta cells of the pancreas without other toxic effects and has a wider margin of safety than alloxan (Junod, Lambert, Orci, Pickett, Gonot and Renold 1967). Pitkin and Reynolds (1970) and Salazar, Chez and Pardo (1973) have produced diabetes in monkeys using this drug but have not studied peripheral nerves either electrophysiologically or pathologically. Ward (1973) studied pylol pathways in neuropathy of early diabetes in rats induced by streptozotocin. He observed that nerves were already damaged, often severely, when the diagnosis of diabetes was made. Sharma and Thomas (1974) studied the structure and function of peripheral nerves in streptozotocin-induced diabetes in rats. Motor nerve conduction velocity was reduced within a few days in severely diabetic animals and remained so during survival times of up to 1 year. These authors, however, did not find any pathological changes in peripheral nerves of these animals to explain the electrophysiological alterations recorded. There was no evidence of segmental demyelination in teased fibre preparations. Jakobsen and Lundb~ek (1976) have recently shown changes, mainly affecting the axons, in morphometric studies of similar experimental animals. Thus it would appear that there exists some degree of controversy regarding the pathological lesions in experimental diabetes. Whereas definite morphological changes have been observed in aUoxan-induced diabetic rats (Preston 1967; Hildebrand et al. 1968), no such lesions were seen in streptozotocin-induced diabetic animals (Sharma and Thomas 1974). That this is not due to toxic effects of alloxan itself has been discussed above. The present study, however, has shown unequivocal pathological changes in streptozotocin-induced diabetic monkeys. It has been observed that the nature of these abnormalities was essentially the same as seen in human diabetic
65 neuropathy. It was seen that distal nerves showed alterations in myelin architecture as early as 3 months after the diabetic state was established. The earliest abnormality in these diabetic nerves was an increase in nodal gap, and later demyelination of the whole internode was followed by remyelination. Similar nodal widening and paranodal swelling were amongst the early changes observed in alloxan-induced diabetic rats by Seneviratne and Weerasuriya (1974) and seen 2-5 weeks after onset of the diabetes. It can be presumed that the differences in time interval in the appearance of pathological changes in alloxan-induced diabetic rats (Seneviratne and Weerasuriya 1974) and streptozotocin-induced diabetic monkeys (present study) is due to a difference in the evolutionary scale of the animals used and not due to differences in the methods used to induce diabetes. It is possible that the same species difference may explain why no morphological changes were seen in an earlier study of streptozotocin-induced diabetic rats (Sharma and Thomas 1974). For the same reason it is easier to extrapolate the findings of the present study to explain the pathogenesis of human diabetic neuropathy. The present study has also demonstrated that peripheral nerve changes in experimental diabetes in monkeys are widespread. Distal nerves are affected earlier and the proximal nerves show similar pathological abnormalities at a later date. In this respect too, there is a similarity between the present experimental study and an earlier demonstration of similar pathology in human diabetic neuropathy (Chopra and Fanin 1971). The abnormality of Wallerian degeneration or loss of axis cylinders was mild as compared with disruption of myelin. These changes were seen only in nerves which had borne the brunt of metabolic disturbances for a longer period. Though it has recently been suggested that axonal alterations may be the predominant lesion in human diabetic neuropathy (Bischoff 1973; Brown et al. 1976; Jakobsen and Lundbmk 1976), the present study supports the earlier view that axonal changes appeared late and were possibly secondary to persistent demyelination. It has been pointed out that in some circumstances segmental demyelination (especially paranodal) may be secondary to axonal damage (Dyck, Johnson, Lambert and O'Brien 1971). However, as far as diabetes is concerned, the mass of biochemical evidence also favours metabolic disturbances in the Schwann cells. The relationship of the pathological lesions to the severity and duration of diabetes mellitus has remained a disputed point in human diabetes. Greenbaum (1964), Gregerson (1967) and Chopra et al. (1969) observed a direct relationship between the structural changes, reduced nerve conduction and known duration of the hyperglycaemic state. Faerman, Fox, Jadzinsky, Glocer and Cibeira (1973) also concluded that duration and severity of clinical diabetes could contribute to the intensification of neuropathy. Similar conclusions could be drawn from this study. Monkeys with a higher level of hyperglycaemia developed a structural abnormality in the peripheral nerves earlier as compared with mildly or moderately severe diabetic monkeys. It was also seen that the longer the duration of diabetes, the greater was the tendency of nerves to be involved, irrespective of the degree of hyperglycaemia. Thus, in some monkeys, no pathological changes were seen at early biopsy but later biopsies or
66 autopsy showed widespread changes although the degree of hyperglycaemia had rem a i n e d a b o u t the same. This progress of pathological lesions, suggesting a n i m p o r t a n t role of both the severity a n d d u r a t i o n of hyperglycaemia in the development of neuropathic changes has been a u n i q u e feature of this study and is not reported earlier. As is true for h u m a n diabetics, some experimental animals develop severe pathological lesions in peripheral nerves whereas others with a similar degree and d u r a t i o n of hyperglycaemia do n o t develop any n e u r o p a t h i c changes at all. Scientific reasons for this individuality, if any, r e m a i n elusive. ACKNOWLEDGEMENT The authors would like to t h a n k Professor P. K. T h o m a s for a critical review of the m a n u s c r i p t a n d helpful suggestions.
REFERENCES Bischoff, A. (1973) Ultrastructural pathology of the peripheral nervous system in early diabetes. In : R. A. Camerini-Davalos and H. S. Cole (Eds.), Vascular and Neurologic Changes in Early Diabetes, Academic Press, New York, N. Y., pp. 441-449. Brown, M. J., J. R. Martin and A. K. Asbury 0976) Painful diabetic neuropathy, Arch. Neurol. (Chic.), 33: 164-171. Chopra, J. S. and T. Fanin (1971) Pathology of diabetic neuropathy, J. path. Bact., 104: 175-t84. Chopra, J. S. and L. J. Hurwitz (1967) Internodal length of sural nerve fibres in chronic occlusive vascular disease, J. Neurol. Neurosurg. Psychiat., 30: 207-214. Chopra, J. S. and L. J. Hurwitz (1968) Femoral nerve conduction in diabetes and chronic occlusive vascular disease, J. Neurol. Neurosurg. Psychiat., 31: 28-33. Chopra, J. S., L. J. Hurwitz and D. A. D. Montgomery (1969) Pathogenesis of sural nerve changes in diabetes mellitus, Brain, 92: 391-418, Downie, A. N. and D. J. NeweU (1961) Sensory nerve conduction in patients with diabetes mellitus and controls, Neurology (Minneap.), 11 : 876-882. Dyck, P. J., W. J. Johnson, E. H. Lambert and P. C. O'Brien (1971) Segmental demyelination secondary to axonal degeneration in uremic neuropathy, Proc. Mayo Clin., 46: 400-423. Eliasson, S. G. (1964) Nerve conduction changes in experimental diabetes, J. olin. Invest., 43: 23532358. Faerman, 1., D. Fox, M. N. Jadzinsky, L. Glocer and J. B. Cibeira (1973) Neurological findings in chemical diabetes. In: R. A. Camerini-Davalos and H. S. Cole (Eds.), Vascular and Neurologic Changes in Early Diabetes, Academic Press, New York, N.Y., pp. 451-457. Gabbay, K. H. and J. B. O'Sullivan (1968) The sorbital pathway - - Enzyme localization and content in normal and diabetic nerve and cord, Diabetes, 17: 239-243. Greenbaum, D. (1964) Observations on the homogeneous nature and pathogenesis of diabetic neuropathy, Brain, 87: 215-232. Gregerson, G. (1967) Diabetic neuropathy - - Influence of age, sex, metabolic control and duration of diabetes on motor nerve conduction, Neurology (Minneap.), 17: 972-980. Hildebrand, J., A. Joffroy, G. Graft and C. C~rs (1968) Neuromuscular changes with alloxan hypergtycaemia, Arch. Neurol. (Chic.), 18: 633-641. Jakobsen, J. and K. Lundb~ek (1976) Neuropathy in experimental diabetes - - An animal model, Brit. med. J., 2: 278-279. Junod, A., A. E. Lambert, L. Orci, R. Pickett, A. E. Gonot and A. E. Renold (1967) Studies of the diabetogenic action of streptozotocin, Proc. Soc. exp. Biol. Med., 126: 201-205. Lawrence, D. G. and S. Locke (1961) Motor nerve conduction velocity in diabetes, Arch. Neurol. (Chic.), 5: 483-489. Lovelace, R. E. (1968) Experimental neuropathy in rats made diabetic with alloxan, Electroenceph. clin. NeurophysioL, 25 : 393.
67 Miyoshi, T. and I. Goto (1973) Serial in vivo determinations of nerve conduction velocity in rat tails, Electroenceph. clin. Neurophysiol., 35: 125-131. Mulder, D. W., E. H. Lambert, J. A. Bastron and R. G. Sprague (1961) The neuropathies associated with diabetes mellitus, Neurology (Minneap.), 11: 275-284. Pitkin, R. M. and W. A. Reynolds (1970) Diabetogenic effects of streptozotocin in Rhesus monkeys, Diabetes, 19: 85-90. Preston, G. M. (1967) Peripheral neuropathy in alloxan-diabetic rat, J. PhysioL (Lond.), 189: 49-50P. Salazar, H. R., A. Chez and M. Pardo (1973) Absence of ultrastructural changes in the basement membrane of muscle capillaries in streptozotocin-induced carbohydrate intolerance in Rhesus monkeys, Amer. J. Path., 71 : 437-445. Schlaepfer, W. W., G. C. Gerritsen and W. E. Dulin (1974) Segmental demyelination in the distal peripheral nerves of chronically diabetic chinese hamsters, Diabetologia, 10: 541-548. Seneviratne, K. N. and O. A. Peiris (1969) The effects of hypoxia on the excitability of the isolated peripheral nerves of the alloxan-diabetic rats, J. Neurol. Neurosurg. Psychiat., 32: 462-469. Seneviratne, K. N. and A. Weerasuriya (1974) Nodal gap substance in the diabetic nerve, J. NeuroL Neurosurg. Psychiat., 37: 503-513. Sharma, A. K. and P. K. Thomas (1974) Peripheral nerve structure and function in experimental diabetes, J. neurol. Sci., 23: 1-15. Thomas, P. K. and R. G. Lascelles (1966) The pathology of diabetic neuropathy, Quart. J. Med., 35: 489-509. Ward, J. D. (1973) The pylol pathway in the neuropathy of early diabetes. In: R. A. Camerini-Davalos and H. S. Cole (Eds.), Vascular and Neurologic Changes in Early Diabetes, Academic Press, New York, N.Y., pp. 426-429. Ward, J. D., R. W. R. Baker and B. H. Davis (1972) Effect of blood sugar control on the accumulation of sorbitol and fructose in nervous tissue, Diabetes, 21 : 1173-1178.
53
P A T H O L O G Y A N D T I M E R E L A T I O N S H I P OF P E R I P H E R A L N E R V E C H A N G E S I N E X P E R I M E N T A L DIABETES*
J. S. CHOPRA, B. B. SAWHNEY and R. N. CHAKRAVORTY Department of Neurology, Postgraduate Institute of Medical Education and Research, Chandigarh (India 160011)
(Received 1 September, 1976)
SUMMARY Structural changes have been studied in peripheral nerves in streptozotocininduced diabetic monkeys. Teased single nerve fibre preparations were most informative. Abnormalities were present in 32 of 58 nerves examined from 15 diabetic monkeys with varying degree of hyperglycaemia. The earliest change was an increase in the gap at nodes of Ranvier, seen 12 weeks after the diabetic state had been established. Well marked segmental demyelination was seen in distal nerves at 14-16 weeks. Later similar changes were seen in proximal, larger nerves. Evidence of remyelination was present at a later date. Wallerian degeneration was seen in only 4 nerves. Changes in the myelin sheath were more prominant than axonal abnormalities at all times. There was no abnormality in the vasa nervorum and only a mild increase in endoneurial and perineurial fibrous tissue. A direct correlation was present between the extent and degree of pathology and both severity as well as duration of hyperglycaemia.
INTRODUCTION Structural changes of segmental demyelination and some degree of axonal loss have been clearly demonstrated in peripheral nerves in patients with diabetes mellitus (Thomas and Lascelles 1966; Chopra, Hurwitz and Montgomery 1969). The relationship of the pathological lesions to the severity and duration of diabetes however, remains controversial. Greenbaum (1964), Gregersen (1967) and Chopra et al. (1969) observed a direct relationship between the structural changes, reduced nerve conduction velocity and degree and duration of the hyperglycaemic state.
* Presented in part at the 3rd International Congress of Muscle Diseases, Newcastle upon Tyne, September 1974.
54 Since it is almost impossible to know, with any degree of certainty, the onset of the diabetic state in a particular individual, no idea can be had as to the time taken for the pathological changes to appear in the peripheral nerves. This can only be studied by closely following an experimental model where one is certain about the onset of the diabetic state. It has been observed that pathological changes in peripheral nerves similar to those seen in human diabetics are seen in alloxan-induced diabetic animals (Preston 1967; Hildebrand, Joffroy, Graft and CoOrs (1968). Question was raised if such changes were not a direct toxic effect of alloxan (Lovelace 1968). Although Sharma and Thomas (1974) showed reduced nerve conduction in streptozotocininduced diabetic rats, no morphological changes were seen to explain this electrophysiological abnormality. Jakobsen and I_undb~ek (1976), however, thought that the fibre size was diminished and that the reduction in size of axons was twice that of the myelin sheath in streptozotocin-induced diabetic rats. The present study was designed to find out the earliest time period in which pathological changes are established in peripheral nerves in experimental diabetes using a different species of animals. The relationship between the intensity of the diabetic state and the severity of histological changes could also be studied. MATERIAL AND METHODS Twenty-five Rhesus monkeys, both male and female, were used in the experiment. These healthy monkeys were kept in the animal house for 2-3 months before being subjected to the experiments. Each animal received a standard mixed diet of 900 calories daily. This consisted of 25 ~ proteins, 65 % carbohydrates, and 10 ~ fats, with appropriate amounts of salts and vitamins. All animals were kept in separate metabolic cages under similar environmental conditions. The estimated age of the group was 3-4 years and their weight varied between 2 and 5.5 kg. Fasting blood sugar levels in all the animals were in the normal range (Tables 1 and 2). The blood for examination was collected from the anterior cubital or femoral veins and serum glucose content estimated by Nelson-Somogyi's method. None of the animals showed evidence of any reducing agent in the urine tested by the Benedict's qualitative method. Six monkeys served as controls and 19 received a single intravenous injection of
TABLE1 CONTROL GROUP Monkey No. Sex
Weight(kg) Fasting blood sugar (mg/100 ml)
Duration alive (months)
5 6 9 10 17 20
2.0 6.0 2.0 4.5 2.8 3.0
4 20 20 20 14 1
M M M M M F
76 70 80 85 80 90
55 TABLE 2 EXPERIMENTAL GROUP Fasting blood sugar values refer to initial figures before streptozotocin was given. Monkey No. Sex
Weight Fasting blood sugar (kg) (mg/100ml)
Range of blood sugar (rag/100 ml)
Durationalive (months)
1 2 3 4 7 8 I1 12 13 14 15 16 18 19 2l 22 23 24 25
3.1 3.4 2.5 4.0 2.5 5.5 5.0 5.0 4.2 5.0 4.5 4.2 3.0 3.5 2.5 6.5 2.0 4.2 5.0
250-390 200-425 245-295 210 430 237-386 300-312 230-420 200-375 165-180 150-170 230-430 230-400 100-110 85-115 225-320 75-130 100-110 116-320 200-320
10 4½ 2 16 2 17 days 10 13 12 2 9 8 14 days 11 18 days (escaped) 1l 10 5½ 3
F F M M M M M F M M M M M M M M M F F
71 70 100 88 88 78 70 75 80 106 80 85 80 83 80 70 85 113 80
streptozotocin in the dose of 50 mg/kg body weight according to the method adopted from Pitkin and Reynolds (1970). The drug was injected after the animals had been kept lasting overnight. Two monkeys at a time were made diabetic and stabilized for 10-15 days, before a new pair was incorporated into the experiment. The first blood sugar was tested 48 hr after the streptozotocin injection and subsequent serial estimations carried out fortnightly. Daily urinalysis for glycosuria and acetone was done. The animals in whom the blood sugar level remained above 200 mg/100 ml were labelled as severely diabetic. There were 13 such animals (Table 2). The animals in whom the blood sugar levels were between 150 and 200 mg/100 ml were classified as mildly diabetic (Nos. 13, 14). Four monkeys (Nos. 18, 19, 22, 23) did not become diabetic. One monkey (No. 21) from the first group escaped after 18 days and further studies could not be carried out. Severely diabetic animals were given 2-6 units of plain insulin when there was orange reduction in urine with Benedict's test or when acetone was present. This procedure was adopted to prevent the complications of ketosis and coma. However, no attempt was made to control the diabetic state with insulin therapy. All diabetic and control animals were weighed fortnightly. A reduction of 0.25-1 kg in body weight was observed in all diabetic animals over the period that these were kept alive. None of the control monkeys lost any weight during this period.
Nerve biopsy Serial nerve biopsies were taken under general anaesthesia from each monkey
56 TABLE 3 NUMBER OF NERVES EXAMINED AT VARIOUS TIME PERIODS Time interval (months)
Diabetic
Control
1-2 2- 4 4-6 6-8 8-10 10-12 12-14 14-16
4 23 12 5 5 5 2 2
8 5 8 1 3 1 2 4
Total
58
27
(diabetic or control) at an interval of 1-4 months. At each instance a sural nerve (at the level of the lateral matleolus) or a radial nerve (at the level of the wrist) was biopsied. Larger nerves such as the femoral, median and/or ulnar could be examined following the death or the sacrifice of the animal. Table 3 shows the total number of nerves studied and the duration of the diabetic state at the time of the study o f a particular nerve. Each nerve was appropriately stained to study myelin (Fleming's solution fixed specimen stained with Kulchitsky's stain), axons (silver impregnation technique) and with haematoxylin and eosin (H & E), periodic acid-Schiff (PAS) and haematoxylinvan Gieson stains as described earlier (Chopra and Hurwitz 1967; Chopra et al. 1969). Over 20 nerve fibres of varying diameter were teased from the formol-saline fixed specimens and measurements of internodal lengths and fibre diameters ptotted from these graphically (Thomas and Lascelles 1966; Chopra et al. 1969). Internodal lengths were also plotted against the diameter of the widest internode on each fibre. This gave a convenient assessment of the pattern of the variations in internodal distances. RESULTS Fifty-eight nerves from the diabetic animals were compared with 27 nerves from control monkeys at different time periods. Myelinated nerve fibres Figure 1 shows the density of the myelinated fibres in a control monkey kept for 6 months under the same conditions as the experimental group. The number of nerves examined in the experimental group at a different time interval after the onset of the diabetic state is given in Table 4. Of a total of 58 nerves examined, 19 showed a varying degree of loss of the myelinated fibres (Figs. 2-4). This was equally distributed in fibres of all sizes. The extent of the changes at different time periods is discussed below. Axonal loss As compared with the loss of myelinated fibres, the axons were relatively well
57
Fig. 1. Sural nerve (transverse section, TS) from a control monkey (No. 9). Kulschitsky's haematoxylin (KH), × 200.
preserved. Only a mild loss of axons was seen in monkeys Nos. 4, 12 and 13 in median and femoral nerves after 14-16 months (Table 4, Fig. 5). Loss of myelinated nerve fibres or axons was not observed in nerves from the control group at any stage. Connective tissue Only mild perineurial fibrosis was seen in 3 animals in the diabetic group. Monkey
N o . 12 s h o w e d t h e s e c h a n g e s i n t h e s u r a l n e r v e a f t e r 3 m o n t h s a n d i n t h e
r a d i a l n e r v e a f t e r 8 m o n t h s o f t h e d i a b e t i c state. M o n k e y s N o s . 1 a n d 13 s h o w e d c h a n ges i n t h e r a d i a l n e r v e s a f t e r 4 a n d 11 m o n t h s , r e s p e c t i v e l y . O n e b i o p s y f r o m t h e c o n trol group also showed similar changes in the radial nerve after 8 months. TABLE 4 H I S T O P A T H O L O G I C A L C H A N G E S IN DIABETIC MONKEYS Time Myelinated interval fibre loss (months)
Axonal loss
Segmental demyelination
Wallerian degeneration
Perineurial fibrosis
2- 4 4- 6 6- 8 8-10 10-12 12-14 14-16
4s 12~ 13s 16s lr l l s 15s 16s 24r 4r 12r l l r 12r 15~ 13r lu 13f 4r,m
-----12r,m 13t 4t,m
4s l l s 12s 13s 15s 16s 2r 2r,r 4r l l s 15s 16s 24s,~ l l r 12r 13r 16rd 4r l l r 12r 15rd lu 12t, m 13t,m 4t,m
---15t -12t 4r,m
12s 12r lr 12r -13r ---
Total
19
5
32
4
5
Figure indicates monkey No., s = sural nerve, r = radial nerve, f = femoral nerve, m ~ median nerve, u = ulnar nerve.
58
Fig. 2. Sural nerve (TS), diabetic monkey (No. 13) at 4 months, showing a moderate loss of myelinated nerve fibres. KH, :. 100.
Single nerve fibre preparations Morphological changes in the single nerve fibre preparations were observed in 32 of the 58 (55.2 °,~i) nerves studied f r o m the diabetic animals. The earliest change was an increase in the gap at the nodes of Ranvier due to retraction of myelin from the nodes. This was observed as early as 12 weeks after the diabetic state had been established and was seen in 7 nerves. Table 4 shows the changes of segmental demyelination in different nerves related to the duration of the diabetic state (Figs. 6 and 7). Changes of segmental demyelination and remyelination were not only confined to the
Fig. 3. Radial nerve (TS), diabetic monkey (No. 11) at 8 months, showing moderately severe loss of myelinated nerve fibres. KH, × 100.
59
Fig. 4. Ulnar nerve (TS), diabetic monkey (No. 1) at 10 months, showing severe loss of myelinated nerve fibres. KH, ~ 100.
distal nerves but also seen in the proximal nerves, viz. femoral, ulnar and the median (Figs. 8-10). Changes of Wallerian degeneration were seen in only 3 animals of the diabetic group. This abnormality was seen in the femoral nerve of monkey No. 15 after 9 months, in the median nerve of monkey No. 12 after 13 months and in both the femoral and median nerves of monkey No. 4 after 16 months (Fig. lla, b, c). Thus these abnormalities appeared later and were minimal as compared to the pathology of segmental demyelination. Internodal length and fibre diameters were measured and
Fig. 5. Femoral nerve (TS), diabetic monkey (No. 13) at 14 months, showing only a mild loss of axis cylinders. Glees Marsland silver impregnation, .~ 100. (Compare with earlier biopsy above Fig. 2.)
60
Figs. 6 and 7. Teased single nerve fibres from diabetic monkeys Nos. 15 and 16 at 4 and 6 months respectively, showing areas of segmental demyelination. Osmium tetroxide.
Figs. 8 and 9. Teased single nerve fibres from the femoral nerves, diabetic monkeys Nos. 24 and 13 at 5½ and 14 months respectively, showing areas of segmental demyelination. Osmium tetroxide.
61
Fig. 10a, b, c. Teased single fibres from the femoral nerves, diabetic monkeys Nos. 15, 12 and 4 at 9, 14 and 16 months respectively, showing areas of segmental demyelination. Osmium tetroxide. p l o t t e d graphically. These o b s e r v a t i o n s also showed a wide scatter indicating segmental d e m y e l i n a t i o n a n d r e m y e l i n a t i o n (Figs. 12-14). M o r p h o l o g i c a l changes in teased fibre p r e p a r a t i o n s were seen in 9 m o n k e y s o f the diabetic g r o u p a n d were absent f r o m the c o n t r o l group. It was noticed t h a t in single fibre p r e p a r a t i o n s p a t h o l o g i c a l changes were observed even in nerves in which transverse sections h a d shown no definite a b n o r m a l i t y .
Vasa nervorum There was r e m a r k a b l e p r e s e r v a t i o n o f the vasa n e r v o r u m in all the nerves examined. N o d e p o s i t i o n o f P A S - p o s i t i v e m a t e r i a l was seen in a n y o f the sections.
Relationship of severity and duration of the diabetic state to the pathological lesions In 5 animals (Nos. 2, 4, 11, 15, 16) the usual b l o o d sugar levels were 400 mg/100
III
Q
~
b
c Fig. l l a , b, c. Teased single fibres from diabetic monkey 4 at 16 months (femoral nerve in l l a and median nerve in 1lb) and femoral nerve at 9 months (monkey No. 15), showing Wallerian degeneration. Osmium tetroxide.
62 1,4" 1.2"
E
1.0"
,-rI.z
0.8" 0.6
o z Iz
0.4 0.2
2
4
6
8
10
12
D I A M ETE R(,.~ )
Fig. 12. SuraI nerve of diabetic monkey No. 16 at 6 months. In this and subsequent Figs: 13 and 14, lengths of the internodes on individual fibres are plotted against the diameter of the widest internode of the respective fibre and the points joined by vertical lines. This indicates nerve fibres with both long and short internodal segments.
ml or above and in all these animals pathological lesions in the distal nerves could be demonstrated as early as 3 months after hyperglycaemia had been established. There were mild to moderate changes of segmental demyelination. Similar changes were also seen in the proximal nerves in these animals at a later date. In 6 animals (Nos. 1, 7, 8, 12, 24, 25) the usual blood sugar levels were below 400 mg/100 ml. In this group, early changes at 3 months were only seen in 1 animal (No. 12) in the sural nerve. However, lesions were observed in the radial nerve at 6 months and in proximal nerves after 14 I-Z'
1.0' E E
O.8.
"II,--
0.6'
~ 0.4, Z
ul ~ 0.2.
DIAMETER(.~) Fig. 13. Radial nerve of diabetic monkey No. 4, at 7½ months.
63 1"4
1"2
I0
0'8
0"6
04
0'2
2
4
6
8
DIAMETER
I0
12
14
t~LI)
Fig. 14. Femoral nerve of diabetic monkey No. 24, at 5½ months.
months in this animal. In monkey No. 24 of this group, no abnormality was seen at biopsy at 3 months but at autopsy at 5½ months, changes were present in the other sural nerve and in the femoral nerve. In monkey No. l, no lesion was detected in 4 biopsies taken till 8 months of hyperglycaemia, and only mild segmental demyelination was seen in the ulnar nerve at autopsy after 10 months. Blood sugar levels remained between 200 and 300 mg/100 ml in monkey No. 13. Early changes in 1 sural and 1 radial nerve were seen at 4 and 7 months, respectively. Autopsy after 14 months, however, showed severe segmental demyelination and remyelination. These observations indicate a direct relationship between the severity of the diabetic state (as judged by hyperglycaemia) and the pathological lesions in the peripheral nerves. In addition to the intensity of the diabetic state, its duration also affected the extent and severity of the morphological changes. This was seen in monkeys Nos. 2, 4, 11, 12, 13 and 16 in whom pathological alterations increased with the lengthening of the diabetic state. DISCUSSION It has been established that diabetic neuropathy in man is associated with a significant reduction in both motor and sensory nerve conduction velocities (Downie and Newell 1961 ; Lawrence and Locke 1961 ; Mulder, Lambert, Bastron and Sprague 1961 ; Chopra and Hurwitz 1968). Morphologically this has been thought to be due to predominant segmental demyelination and some axonal loss seen in biopsies from the sural nerves (Thomas and Lascelles 1966; Chopra et al. 1969) and from the larger nerves at autopsy (Chopra and Fanin 1971). These pathological changes indicated either a primary or a secondary disorder of Schwann cells, where metabolic alterations have been demonstrated in experimental models (Gabbay and O'Sullivan 1968; Ward, Baker and Davis 1972). Recently, however, the importance of segmental demyelination has been questioned, and Bischoff (1973) and Brown, Martin and Asbury (1976) have re-emphasized axonal involvement.
64 Alloxan-induced diabetic rats have almost exclusively been used as an experimental model for the study of diabetic neuropathy. Nearly all authors have shown a reduction in nerve conduction either in vitro or in vivo in such animals (Eliasson 1964; Preston 1967; Hildebrand et al. 1968; Lovelace 1968; Seneviratne and Peiris 1969; Miyoshi and Goto 1973). Slow nerve conduction was observed as early as 2 weeks following an established diabetic state by Eliasson (1964). Preston (1967) and Hildebrand et al. (1968) however, found significant changes only after 3-4 months, kovelace (1968) thought that the early delay observed by Eliasson (1964) could be due to toxic or hypersensitivity reactions to alloxan itself and not due to the metabolic changes. This, however, can be eliminated from the follow-up studies by Miyoshi and Goto (1973). These authors found progressive decrease in serial conduction velocity from 2-6 weeks in rats which were made diabetic by alloxan but observed no changes in the same period in rats who did not become diabetic. In pathological studies, segmental demyelination has been observed in alloxan-induced diabetic rats (Preston 1967; Lovelace 1968). Variations in nodal myelin architecture have also been described (Hildebrand et al. 1968; Seneviratne and Peiris 1969). These pathological abnormalities are similar to those seen in diabetic neuropathy in man referred to above, it is interesting to note that similar pathological alterations have also been seen in an inbred strain of chinese hamsters who develop spontaneous diabetes (Schlaepfer, Gerritsen and Dulin 1974). Streptozotocin has been considered to have a selective action on beta cells of the pancreas without other toxic effects and has a wider margin of safety than alloxan (Junod, Lambert, Orci, Pickett, Gonot and Renold 1967). Pitkin and Reynolds (1970) and Salazar, Chez and Pardo (1973) have produced diabetes in monkeys using this drug but have not studied peripheral nerves either electrophysiologically or pathologically. Ward (1973) studied pylol pathways in neuropathy of early diabetes in rats induced by streptozotocin. He observed that nerves were already damaged, often severely, when the diagnosis of diabetes was made. Sharma and Thomas (1974) studied the structure and function of peripheral nerves in streptozotocin-induced diabetes in rats. Motor nerve conduction velocity was reduced within a few days in severely diabetic animals and remained so during survival times of up to 1 year. These authors, however, did not find any pathological changes in peripheral nerves of these animals to explain the electrophysiological alterations recorded. There was no evidence of segmental demyelination in teased fibre preparations. Jakobsen and Lundb~ek (1976) have recently shown changes, mainly affecting the axons, in morphometric studies of similar experimental animals. Thus it would appear that there exists some degree of controversy regarding the pathological lesions in experimental diabetes. Whereas definite morphological changes have been observed in aUoxan-induced diabetic rats (Preston 1967; Hildebrand et al. 1968), no such lesions were seen in streptozotocin-induced diabetic animals (Sharma and Thomas 1974). That this is not due to toxic effects of alloxan itself has been discussed above. The present study, however, has shown unequivocal pathological changes in streptozotocin-induced diabetic monkeys. It has been observed that the nature of these abnormalities was essentially the same as seen in human diabetic
65 neuropathy. It was seen that distal nerves showed alterations in myelin architecture as early as 3 months after the diabetic state was established. The earliest abnormality in these diabetic nerves was an increase in nodal gap, and later demyelination of the whole internode was followed by remyelination. Similar nodal widening and paranodal swelling were amongst the early changes observed in alloxan-induced diabetic rats by Seneviratne and Weerasuriya (1974) and seen 2-5 weeks after onset of the diabetes. It can be presumed that the differences in time interval in the appearance of pathological changes in alloxan-induced diabetic rats (Seneviratne and Weerasuriya 1974) and streptozotocin-induced diabetic monkeys (present study) is due to a difference in the evolutionary scale of the animals used and not due to differences in the methods used to induce diabetes. It is possible that the same species difference may explain why no morphological changes were seen in an earlier study of streptozotocin-induced diabetic rats (Sharma and Thomas 1974). For the same reason it is easier to extrapolate the findings of the present study to explain the pathogenesis of human diabetic neuropathy. The present study has also demonstrated that peripheral nerve changes in experimental diabetes in monkeys are widespread. Distal nerves are affected earlier and the proximal nerves show similar pathological abnormalities at a later date. In this respect too, there is a similarity between the present experimental study and an earlier demonstration of similar pathology in human diabetic neuropathy (Chopra and Fanin 1971). The abnormality of Wallerian degeneration or loss of axis cylinders was mild as compared with disruption of myelin. These changes were seen only in nerves which had borne the brunt of metabolic disturbances for a longer period. Though it has recently been suggested that axonal alterations may be the predominant lesion in human diabetic neuropathy (Bischoff 1973; Brown et al. 1976; Jakobsen and Lundbmk 1976), the present study supports the earlier view that axonal changes appeared late and were possibly secondary to persistent demyelination. It has been pointed out that in some circumstances segmental demyelination (especially paranodal) may be secondary to axonal damage (Dyck, Johnson, Lambert and O'Brien 1971). However, as far as diabetes is concerned, the mass of biochemical evidence also favours metabolic disturbances in the Schwann cells. The relationship of the pathological lesions to the severity and duration of diabetes mellitus has remained a disputed point in human diabetes. Greenbaum (1964), Gregerson (1967) and Chopra et al. (1969) observed a direct relationship between the structural changes, reduced nerve conduction and known duration of the hyperglycaemic state. Faerman, Fox, Jadzinsky, Glocer and Cibeira (1973) also concluded that duration and severity of clinical diabetes could contribute to the intensification of neuropathy. Similar conclusions could be drawn from this study. Monkeys with a higher level of hyperglycaemia developed a structural abnormality in the peripheral nerves earlier as compared with mildly or moderately severe diabetic monkeys. It was also seen that the longer the duration of diabetes, the greater was the tendency of nerves to be involved, irrespective of the degree of hyperglycaemia. Thus, in some monkeys, no pathological changes were seen at early biopsy but later biopsies or
66 autopsy showed widespread changes although the degree of hyperglycaemia had rem a i n e d a b o u t the same. This progress of pathological lesions, suggesting a n i m p o r t a n t role of both the severity a n d d u r a t i o n of hyperglycaemia in the development of neuropathic changes has been a u n i q u e feature of this study and is not reported earlier. As is true for h u m a n diabetics, some experimental animals develop severe pathological lesions in peripheral nerves whereas others with a similar degree and d u r a t i o n of hyperglycaemia do n o t develop any n e u r o p a t h i c changes at all. Scientific reasons for this individuality, if any, r e m a i n elusive. ACKNOWLEDGEMENT The authors would like to t h a n k Professor P. K. T h o m a s for a critical review of the m a n u s c r i p t a n d helpful suggestions.
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67 Miyoshi, T. and I. Goto (1973) Serial in vivo determinations of nerve conduction velocity in rat tails, Electroenceph. clin. Neurophysiol., 35: 125-131. Mulder, D. W., E. H. Lambert, J. A. Bastron and R. G. Sprague (1961) The neuropathies associated with diabetes mellitus, Neurology (Minneap.), 11: 275-284. Pitkin, R. M. and W. A. Reynolds (1970) Diabetogenic effects of streptozotocin in Rhesus monkeys, Diabetes, 19: 85-90. Preston, G. M. (1967) Peripheral neuropathy in alloxan-diabetic rat, J. PhysioL (Lond.), 189: 49-50P. Salazar, H. R., A. Chez and M. Pardo (1973) Absence of ultrastructural changes in the basement membrane of muscle capillaries in streptozotocin-induced carbohydrate intolerance in Rhesus monkeys, Amer. J. Path., 71 : 437-445. Schlaepfer, W. W., G. C. Gerritsen and W. E. Dulin (1974) Segmental demyelination in the distal peripheral nerves of chronically diabetic chinese hamsters, Diabetologia, 10: 541-548. Seneviratne, K. N. and O. A. Peiris (1969) The effects of hypoxia on the excitability of the isolated peripheral nerves of the alloxan-diabetic rats, J. Neurol. Neurosurg. Psychiat., 32: 462-469. Seneviratne, K. N. and A. Weerasuriya (1974) Nodal gap substance in the diabetic nerve, J. NeuroL Neurosurg. Psychiat., 37: 503-513. Sharma, A. K. and P. K. Thomas (1974) Peripheral nerve structure and function in experimental diabetes, J. neurol. Sci., 23: 1-15. Thomas, P. K. and R. G. Lascelles (1966) The pathology of diabetic neuropathy, Quart. J. Med., 35: 489-509. Ward, J. D. (1973) The pylol pathway in the neuropathy of early diabetes. In: R. A. Camerini-Davalos and H. S. Cole (Eds.), Vascular and Neurologic Changes in Early Diabetes, Academic Press, New York, N.Y., pp. 426-429. Ward, J. D., R. W. R. Baker and B. H. Davis (1972) Effect of blood sugar control on the accumulation of sorbitol and fructose in nervous tissue, Diabetes, 21 : 1173-1178.