Hereditary hypertrophic neuropathy in the trembler mouse

Journal of the Neurological Sciences, 1976, 30:327-341 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

HEREDITARY MOUSE

HYPERTROPHIC

NEUROPATHY

IN

THE

327

TREMBLER

Part 1. Histopathological Studies: Light Microscopy

P. A. LOW Department of Medicine, University of Sydney, Sydney, N.S. I4I., 2006 (Australia)

(Received 28 April, 1976)

SUMMARY The Trembler mouse suffers f r o m a dominantly inherited chronic hypertrophic neuropathy. Quantitative light-microscopic studies have been performed on Trembler peripheral nerves from birth to adulthood, and compared with the findings in agematched control mice. Teased fibres of Trembler nerves contained virtually no myelin, nodes of Ranvier were difficult to identify, and supernumerary Schwann cells were prominent. Results of quantitative studies performed on the dorsal root ganglia and spinal cord of Trembler mice did not differ from those in controls. The density of myelinated fibres in the case of Tremblers was reduced at all ages when compared with controls, and there was a predominant loss of large diameter fibres.

INTRODUCTION

The pathology of hereditary hypertrophic neuropathies in man, such as Dejerine-Sottas neuropathy and hypertrophic Charcot-Marie-Tooth disease have been well described (Dyck 1966, 1975; Thomas, Lascelles and Stewart 1975). Dyck, Lambert, Sanders and O'Brien (1971) considered that Dejerine-Sottas neuropathy was due to a primary Schwann cell disorder. Subsequently however, Brimijoin, Capek and Dyck (1973) demonstrated marked slowing of axoplasmic flow and concluded that there was a primary axonal abnormality. Little information is available about the evolution of the pathological changes. This work was supported by grants from the National Health and Medical Research Council of Australia, and from the Postgraduate Medical Foundation, University of Sydney.

328 The Trembler mouse has a dominantly inherited hypertrophic neuropathy similar to that of Dejerine-Sottas neuropathy in man (Ayers and Anderson 1973). Quantitative studies of the dorsal root ganglion, anterior horn cells, and of the peripheral nerves from birth to adulthood may provide insights into the evolution of the neuropathy in the Trembler and similar neuropathies in man. One major difficulty in interpretation of the pathological changes in the newborn animal is that the Trembler mouse is not clinically affected until the age of 7-10 days. In recent qualitative studies on immature mice, Ayers and Anderson (1975) were unable to identify clinically immature Trembler mice. The difficulties have largely been overcome by breeding a strain of mice with physical characte;istics which enable them to be recognized at birth. In the present paper quantitative studies on the peripheral nerves, dorsal root ganglia and spinal cord of control and Trembler mice are described. MATERIALS AND METHODS Animals Control mice: Ten adult and 14 immature mice were studied. The ages of immature mice ranged from 0 to 23 days and of adult mice from 2 to 15 months. The weight of adult mice ranged from 26 to 33.7 g (mean 28.7; SD, 4.8). Trembler mice: Nine adult and 15 immature mice were studied. The ages of immature mice ranged from 0 to 23 days and of adult mice from 1 to 27 months. The weight of adult animals ranged from 16.0 to 28.8 g (mean 23.4; SD, 4.2). The animals were bred at the University of Sydney and the environmental temperature of the breeding chamber was maintained at 22 °C and the humidity held at 65 ~ . As the clinical manifestations of the tremor, convulsions and gait disturbance are not recognizable before 7-10 days, it is not possible to identify the affected mice in the first week after birth. The difficulty has been largely overcome by making use of the Trembler-Rex linkage. The Trembler (Tr) gene has been shown to be linked with the Rex (Re) gene; they are 23 recombination units apart on linkage group VII (Falconer and Sobey 1953). As the Rex gene is dominant, and the Rex characteristics of curly whiskers are recognizable at birth, the Trembler-Rex mouse is also identifiable at birth. The Trembler-Rex linkage was obtained in the following manner: Tr+

(1)--

++

+ Re

+ Re

++

++

+ - - - ~ - -

(a)

Tr+

+ - -

+ Re (b)

Tr+

+ - -

++ (c)

+

+ + ++ (d)

(b) are Trembler-Rex offspring in the repulsion phase, i.e. the Trembler and Rex genes are on different chromosomes. If these Trembler-Rex females are mated with a control male: + Re +-+Tr+ (2)-+ - - - + - Tr+ ++ ++

+ Re + - ++

329 the offspring, in the majority of instances, will be either Tremblers or Rexes but not Trembler-Rexes. However, about 8 ~ of offspring will be Trembler-Rexes due to crossing over (Falconer and Sobey 1953). These offspring, being of known genotype (Tr R e / + + ) are then used to produce offspring for pathological studies: Tr Re (3) ~ ++

x

-

+ + - ++

-

>

Tr Re - ++

+ + -t- - ++

i.e., offspring will either be Trembler-Rexes or neither in the majority of cases, with only approximately 15 ~ of Rex phenotype at birth not being Tremblers as well.

Breeding experiments (Table

1) Experiment 1 was performed using 6 Trembler females which were mated with 6 Rex males. A total of 219 offspring from 28 litters were produced from which 195 survived sufficiently long for phenotyping. Nine of the female Trembler-Rexes were then mated with control males (Exp. 2). There were 121 offspring from 20 litters and 103 of these mice survived long enough to be phenotyped. 10/103 (9.7 ~o) of the offspring were Trembler-Rexes produced by crossing over. One of the Trembler-Rex offspring (linked in the coupling phase, i.e. on the same chromosome; see Exp. 2) was successfully mated with a control mouse (Exp. 3). Twenty-five offspring ensued from 5 litters. As predicted, most of the offspring were either Trembler-Rexes or controls. There were 3 (12 ~ ) mice which were phenotypically Rexes at birth but were subsequently found to be not Tremblers.

Histopathology Fifteen T r e m b l e r - R e x mice of ages 0-23 days were used for histological studies. Ten of these were 7 days or younger and were identified by the curliness of their whiskers. The 5 older mice were clinically recognizable as Trembler mice. It was observed that Trembler nerves could be identified by fine structural changes (Low TABLE 1 RESULTS OF BREEDING EXPERIMENTS Exp.

Parents

Progeny

1

Tr + ++

x

2

Tr+ ++

x

3

Re + ÷+

- -

Total

Recombination (%)

Tr Re

Tr +

Re +

++

45

48

41

61

195

31

42

20

103

29.1

1

3

9

25

16.0

++ 10 ++

Tr Re ++ × 12 ++ ++ Combining 2 -+ 3

26.6

330 1976). One of the 10 Trembler-Rex mice did not have these changes and was excluded from the study.

(1) Peripheral nerves The animal was killed with an overdose of ether or pentobarbitone following which the sciatic and posterior tibial nerves were exposed. The nerves were flooded with ice-cold 3 ~ glutaraldehyde in 0.1 cacodylate buffer for 10 min, following which they were divided into 2 portions each about 1 cm in length and these segments were splinted on cards. Immediately after excision, one segment of each nerve was fixed for 3 hr in the same solution at 4 °C, followed by 2 ~o cacodylate buffered osmium tetroxide for 90 min. The tissue was dehydrated in graded concentrations of ethanol, passed through acetone and embedded in Spurr's resin. Two micron thick sections of Spurr's embedded material were stained with 1 ~ toluidine blue for light microscopy (Prineas, McLeod and Wolfenden 1971). The other segment of each nerve was fixed in 10 ~ formol saline and used later for fibre teasing. For the preparation of teased fibres a part of the nerve was macerated for 24 hr in 2 parts of glycerol to 1 part water, after which it was teased apart into small fascicles and placed in a solution of saturated Sudan Black B in 85 ~ propylene glycol (Chifelle and Putt 1951) for 15-30 min. The dye stains membranes sufficiently well to enable individual fibres to be identified clearly by the pale blue colour of the myelin sheath. Normal myelin appeared blue, and abnormal myelin appeared black. Even very thin myelin sheaths and Schwann cell nuclei were clearly visible under the dissecting microscope. The fibres were teased apart and mounted in glycerine gel following which they were photographed. In about half the animals the roots and lumbosacral plexus were also fixed and processed by the same method. In a few animals a third segment of nerve was placed directly into iced Dalton's chrome-osmium solution (Dalton 1955) at pH 7.34 for 90 min at 4°C, following which it was dehydrated and embedded as for glutaraldehyde-fixed tissue.

(2) Dorsal root ganglia and spinal cord The animal was killed in the same manner as previously described. A long vertical midline incision was made and the abdominal contents removed. The thoracic spinal column was then transected and, with the aid of a dissecting microscope, the vertebral bodies were dissected from the spinal cord from the level of the lower thoracic region to the sacrum. The spinal cord was then flooded with ice cold 3 ~o glutaraldehyde in 0.1 M cacodylate buffer for 10 min, following which the L6 segment of cord was identified, dissected free and immersed for a further 4 hr in glutaraldehydecacodylate solution. The tissue was next dehydrated in graded alcohols, and doubleembedded in paraffin wax. The dorsal root ganglion of the same segment was identified, dissected, fixed and processed in exactly the same way. Seven-/~m sections of these blocks were serially cut transversely and stained with cresyl violet acetate, 0.5 ~ aqueous.

(3) Muscle Small pieces of muscle, approximately 5 mm × 10 mm, were obtained from the

331 gastrocnemius and hamstring muscles of control and Trembler mice. The tissues were snap frozen in isopentane (--70°C), cooled in liquid nitrogen, cut at 10/~m on an American Optical Corporation Cryocut cryostat and stained with haematoxylin and eosin, succinic dehydrogenase (SDH) (Nachlas, Tsou, De Souza, Cheng and Seligman 1957), myosin ATPase (Padykula and Herman 1955), modified Trichrome (Engel and Cunningham 1965), oil red O and PAS. Quantitative studies were not performed.

Quantitative studies (1) Fascicular area Fascicles of control and Trembler sciatic nerve were photographed on 35 mm film with a Leitz orthomat microscope camera, and printed on photographic paper at a final magnification of exactly x 250. The accuracy of the enlargement was checked by photographing a Leitz calibration slide with divisions ruled at 10-#m intervals and subjecting the negative to the same developing, enlarging and printing processes. The intraperineurial area of each fascicle was measured with a planimeter (Dyck, Gutrecht, Bastron, Karnes and Dale 1968).

(2) Myelinated fibre density and distribution Selected fascicles of control and Trembler sciatic nerves were photographed, enlarged and printed to a final magnification of exactly x 1000. The external diameter of all myelinated fibres not undergoing active degeneration was measured and counted with a Zeiss T G Z 3 Particle Size Analyser set in the linear mode. Trembler fibres were all very thinly myelinated and often incomplete circumferentially. With Trembler nerves a fibre was accepted for measurement and counting if greater than 75 ~ of the circumference contained myelin. The area of the fascicles was measured with a planimeter and the fibre density was calculated as the number of fibres/mm z of intraperineurial area (Swallow 1966). Histograms of fibre size were constructed for each nerve according to the method of Swallow (1966) and Fullerton and O'Sullivan (1968) and a mean histogram of fibre diameter distribution was calculated for all control and Trembler nerves. Fibres of external diameter 2/zm or less were grouped together, and larger fibres were subdivided into 1-#m groups.

(3) Dorsal root ganglia All the ganglion cells of one complete dorsal root ganglion were serially sectioned at 7-/~m intervals and measured and counted, using an eye-piece micrometer at a magnification of x 400. There were 174 sections. The area under study was divided into squares by means of an eye-piece graticule, and the squares were examined sequentially from left to right. Because of the small size of each section, the error of counting the same cell twice was not encountered. Ganglion cells were counted only if they contained a nucleolus. Not infrequently, a nucleolus was seen in more than one section. In these instances, when the same nucleolus was seen in more than one section, the dorsal root ganglion cell in which the nucleolus was larger was counted and measured, and the same cell in the other section(s) was rejected. Using this criterion, the error of counting the same cell more than once and of recording an inappropriate diameter because the cell had not been sectioned through the centre, was avoided (Ohta, Offord and Dyck 1974). The adequacy of the sampling procedure was then

332 TABLE 2 DORSAL ROOT GANGLION CELLS AT L6 Comparison of actual counts with those obtained by sampling every 10th section. Actual count

2365 Percent error

Every 10th section beginning with section 3

4

5

6

7

8

9

10

1

2

2180 --7.8

2260 2310 2500 2590 2480 2350 2 3 5 0 2220 2320 --4.4 --2.3 ~ 5.7 ~9.5 t 4.9 --0.6 --0.6 --6.1 --1.9

assessed when the dorsal root ganglion cells in only every 10th section were measured and counted (Table 2). The percentage error of sampling every 10th section was calculated using a method similar to that of Tomlinson, Irving and Rebeiz (1973). These workers counted all the anterior horn cells in the lumbosacral spinal cord and compared the actual count with those obtained when cells were counted in only selected sections. In the present study, the results of the sampling procedure differed depending on at which section the count was commenced. For instance, the scores obtained of sections 1, 11, 21, etc. differed from those obtained on sections 2, 12, 22, etc. ; however, the percentage error did not exceed 10 ~ in any case. Following this, every tenth section of the other dorsal root ganglia was photographed and printed at a final magnification of exactly × 250. The first and last sections were those sections respectively which contained one ganglion cell with a nucleolus. Measurements were made using a Zeiss T G Z 3 Particle Size Analyser. Every ganglion cell containing a nucleolus was compared with the corresponding ganglion cell on the section before and the one after the section in question, and the cell accepted or rejected on the criteria of Ohta et al. (1974). No attempt was made to quantitate separately clear and dark cells (Emery and Singhal 1973). Cells were grouped into 4-ffm groups from 0 to 44 #m and the density and the percent distribution of cell diameters were tabulated. The densities and diameters of the 4 control and 4 Trembler dorsal root ganglia were totalled into a control and Trembler group. Mean cell diameter density and distribution histograms were constructed using the grouped data.

(4) Spinal cord Quantitative studies were performed on 4 control and 4 Trembler spinal cords. The spinal motoneurones were quantitated by measuring every tenth section of 100 serially cut 7-fire sections taken at the L6 level. The following method of assessment was modified from the method of Papapetropoulos and Bradley (1972). The tissue was examined under x 400 magnification. Neurones anterior to a horizontal line passing through the midpoint of the central spinal canal were assessed and the left and right horns counted independently. The largest diameter of each neurone was measured by means of an eyepiece micrometer and the only cells accepted were those whose maximum diameter was equal to or exceeded 25 # m and which contained dark prominent Nissl substance. No attempt was made to classify neurones into Groups I to

333 I I I (Papapetropoulos and Bradley 1972). G r o u p I neurones were those with extremely dense Nissl substance. These cells were frequently rather shrunken in outline. G r o u p II neurones had dark prominent Nissl substance and a maximum diameter of at least 25/~m. G r o u p III neurones were all the remaining neurones. Most of the neurones counted corresponded to G r o u p II as G r o u p I cells were infrequently seen. A major difficulty was the lack of a clear differentiation of motoneurones from other neurones of similar size. In cases of uncertainty, the neurone was accepted if its maximum diameter was greater than 25 #m. RES ULTS The fibres in the sciatic nerve of adult Trembler mice have been shown to be A

Fig. 1. Continuous segments of a teased fibre preparation of a control (A) and Trembler (B) sciatic nerve from adult age-matched sciatic nerve stained with Sudan Black B. Arrow-heads indicate nodes of Ranvier and arrows indicate Schwann cell nuclei.

334 very thinly myelinated when compared with controls (Low and McLeod 1975). Similar abnormalities were also found in the lumbosacral plexus, anterior and posterior roots, median nerve, posterior tibial nerve and nerves of the tail. There was a relatively abrupt transition from severely hypomyelinated posterior roots to well myelinated dorsal columns. However, in the anterior and posterior nerve root entry zone there is a small transitional zone of severely hypomyelinated axons invested with oligodendrocyte processes (Harrison 1976). Single myelinated fibres were teased from control and Trembler nerves. There was an excess of fibrous tissue. Fig. IA shows a single myelinated fibre teased from the sciatic nerve of a 100-day-old adult control mouse. Nodes ot Ranvier are indicated by arrowheads. By contrast, Fig. 1B which is a single fibre teased from the sciatic nerve of an age-matched Trembler mouse, may be seen to be virtually bereft of myelin; nodes of Ranvier cannot be identified and there is a marked increase in the number of Schwann cell nuclei. Fibres were teased from the posterior tibial, lumbosacral plexus, upper limb nerves, and anterior and posterior roots. Similar abnormalities were noted in all the nerves that were studied. Every fibre in every nerve that was examined was markedly abnormal and completely normal internodes were never seen. The presence of denervation atrophy in the gastrocnemius muscle of a 4-monthold Trembler mouse was well seen on haematoxylin and eosin, and modified trichromestained sections. There were clusters of fibres stained well for mitochondria; a similar appearance was noted in control fibres. Well developed fibre type grouping was seen in sections stained for succinic dehydrogenase which provided further evidence for a neurogenic lesion (Fig. 2A) (Dubowitz and Brooke 1973).

Fig. 2. Transverse section of gastrocnemius muscle of 4-month-old Trembler mouse stained with succinic dehydrogenase (A), modified trichrome (B) and haematoxylin and eosin (C). Scale, 50/~m.

400 600 2834 1766 708 625 1221 1055 172 336 692 730

100 166 108 278 22 77

4 3½ 4 6 3 6 12

14 12 5 4 2 4

C13 C16 C33 C42 C44 C43 C51

TI5 TI7 T20 T21 T22 T26

2794 3380 6784 3947 3069 1782 1826

1059 3276 3416 3489 3138 1245 1087

29 628 842 484 1423 150 38

Age (mths)

Animal

471 900 558 222 373 1230

2676 2744 2758 3021 3169 1979 1826 271 734 167 28 299 269

1941 2606 2026 2153 2961 2096 1443

45

105

33

15 34 17

2411 1248 1337 1637 1199 1102 776

57

1735 1628 1284 926 1407 1272 955

11 /~m

12 /~m

15 38

8

15

1941 1 7 3 5 1 7 0 6 1147 1 3 0 7 1102 701 409 1226 716 195 37 1358 616 232 32 1169 669 254 1265 973 742 374 856 856 697 458

2/~m 3/~m 4/~m 5/~m 6/~m 7/~m 8/~m 9/~m 10 /~m

5 102 288

204 341

412 95

14 pm

853 175 5 21

13 /~m

14 106

7

15 /~m

27

16 ~m

Area (mm a)

0.134 0.137 0.190 0.190 0.130 0.147 0.264 Mean 0.070 0.060 0.120 0.076 0.134 0.126 Mean

No. of fibres

2739 2644 3919 3405 2400 1955 3057 135 386 267 213 183 383

SCIATIC NERVE MYELINATED FIBRE DENSITY OF A D U L T CONTROL (C) A N D T R E M B L E R (T) MICE

TABLE 3

1.9 6.4 2.2 2.8 1.4 3.0 3.0

20.4 19.3 20.6 19.9 18.5 13.3 11.6 17.4

m m 2)

Density (10a/

336 C •

T u

0.2-

.

.s

•

•o0

0,1 "

e~ee

::::

.555

Fig. 3. The fascicular area (mm ~) of control (C) and Trembler (T) sciatic nerves. Bars represent mean values.

Quantitative studies (1) Fascicular area (Fig. 3) The fascicular areas o f control sciatic nerves ranged from 0.002 to 0.253 m m z (mean 0.085; SD, 0.092) while those of Tremblers ranged from 0.003 to 0.135 (mean 0.046; SD, 0.043). The difference is highly significant (P < 0.001).

(2) Myelinated fibre density in sciatic nerves of adult mice Myelinated fibre densities and distributions in control and Trembler mice are summarised in Table 3 and Fig. 4. The densities of Trembler myelinated fibres all fall below the control range and it may be seen that there are very few myelinated fibres greater than 6 #m. E E 0

>,-

Z LLI 6

10

14

tu t~

Z

DIAMETER

(vm)

Fig. 4. Mean myelinated fibre diameter distribution in control and Trembler sciatic nerves. Bars represent 2 SD.

337

Fig. 5. Transverse sections of sciatic nerve from control (top column) and Trembler (bottom column) mice at different ages. Numbers indicate age in days.

(3) Myelinated fibre density in sciatic nerves of immature mice Transverse sections f r o m the sciatic nerves of immature control and Trembler mice are shown in Fig. 5 and the results are summarised in Fig. 6. The densities of myelinated fibres of Trembler nerves are reduced when compared with controls at all ages. The diameter distribution at days 2 and 4 are similar but by day 7 there is a difference in that the peak for Trembler nerves is at 3/~m while that for control nerves is at 3-4/zm. By day 14 the difference is more pronounced. Very few fibres exceeded 4 # m in diameter in Tremblers while in the case of control nerves many fibres were as large as 7/~m.

(4) Dorsal root ganglia The appearance of dorsal root ganglion cells at the L6 level were the same for control (Fig. 7A) and Trembler mice (Fig. 7B). The density and diameter distributions of 4 adult control and 4 adult Tremblers were compared. The cell counts and distributions are summarised in Fig. 8, using the method of sampling every 10th section. In none of the ganglia of Trembler mice were cell num-

338 ~-~

day 2

day 4

day 7

day 14

E 14

14

J4 "

I0

I0

I0

0

- R _J

w

k,u

:LL LL LL L 2

< z

2

8

2

8

2

2

8

2

8

~

2

2

8

2

8

2

8

2

8

>-

•

D,,,METER

Fig. 6. Myelinated fibre density and distribution of control (upper column) and Trembler (lower column) sciatic nerves at different ages.

Fig. 7. Dorsal root ganglion cells of age-matched control (A) and Trembler (B) mice at the L6 level.

339 12.

8-

-o

.c: -I- , 8 o~

, 12

, 16

, 20

, 24

,-I28

I 32

12

:E Z

uJ (J

8

12

16

20

DIAMETER

24

28

32

36

(#m)

Fig. 8. Distribution of diameters of dorsal root ganglion cells in adult control and Trembler (stippled) dorsal root ganglion at the L6 level. Bars represent 2 SD.

bers below the control range. The cell diameter distribution was similar in the 2 groups. (5) Spinal cord (Fig. 9) The ventral horns of control and Trembler mice at the L6 level did not appear significantly different. The motoneurone counts for control mice ranged from 430 to 750 (mean 623; SD, 136), while those for Trembler mice ranged from 510 to 910 (mean 638; SD, 186). The difference is not significant.

C

"- i

U

z

T •

800 I

o

-I-

II o¢

o

600 -

•

,=,. D,Z <

400'

Fig. 9. Control (C) and Trembler (T) motoneurone counts of adult mice at the L6 level. Bars represent mean values.

340 DISCUSSION The presence of severe hypomyelination in the peripheral nerves of the Trembler mouse was first described by Ayers and Anderson (1973). These workers reported that the spinal cord, dorsal ioot ganglia and brain appeared normal and that denervation atrophy was present in muscle. Segmental demyelination was described as a prominent feature on longitudinal section. Quantitative studies were not performed. In the present study the findings in adult mice of a markedly reduced density of myelinated fibres with a predominant loss of large diameter fibres, a normal density and diameter distribution of dorsal root ganglion cells and a normal density of spinal cord motoneurones confirm the qualitative findings of Ayers and Anderson (1973). In the quantitation of spinal motoneurones, the criteria of size over 25 pm and prominent Nissl staining are the same as those used by Papapetropoulos and Bradley (1972). It is likely that not all the neurones counted were limb motoneurones since some cells satisfying the criteria of size and Nissl staining may have other functions (Romanes 1964). Teased fibre preparations from the peripheral nerves of mice of all ages showed appearances quite different from those found in human demyelinating neuropathies like hypertrophic Charcot-Marie-Tooth disease (Gutrecht and Dyck 1966). In the majority of fibres myelin was virtually absent and discontinuous along the length of the fibre; nodes of Ranvier were not usually discernible and supernumerary Schwann cells were prominent. The changes were similar to those seen in Dejerine-Sottas neuropathy where severe hypomyelination overshadows the features of myelin breakdown (Dyck and Gomez 1968). The mean fascicular area of Trembler sciatic nerve was reduced when compared with controls. Collagen in mice appears to occupy a smaller proportion of the fascicle than it does in man, and although it is increased in Trembler nerves, it does not compensate for the severe loss of myelin and the smaller size of the remaining axons (Low 1976). The myelinated fibre density is reduced from birth in the nerves of Trembler mice when compared with those of controls, and the difference becomes more pronounced with age. These findings suggest an abnormality in myelin formation, and the defect in myelination has been further defined by electron microscope studies (Low 1976). ACKNOWLEDGEMENTS The author was in receipt of the Roche Research Fellowship of the Royal Australasian College of Physicians. The helpful advice and criticism of Professor J. G. McLeod in the preparation of the paper was much appreciated.

REFERENCES Ayers, M. M. and R. McD. Anderson (1973) A model of hypertrophic interstitial neuropathy (Dejerine-Sottas) in man, Acta neuropath. (Berl.), 25: 54-70.

341 Ayers, M. M. and R. McD. Anderson (1975) Development of onion bulb neuropathy in the Trembler mouse - - Comparison with normal nerve maturation, Acta neuropath. (BerL), 32: 43-59. Brimijoin, S., P. Capek and P. J. Dyck (1973) Axonal transport of dopamine-fl-hydroxylase by h u m a n sural nerves in vitro, Science, 180: 1295-1297. Chifelle, T. L. and F. A. Putt (1951) Propylene and ethylene glycol as solvents for Sudan IV and Sudan Black B, Stain TechnoL, 26: 51056. Dalton, A. J. (1955) A chrome-osmium fixative for electron microscopy, Anat. Rec., 121 : 281. Dubowitz, V. and M. H. Brooke (1973) Definition of pathological changes seen in muscle biopsies. In: Muscle B i o p s y - A Modern Approach, W. B. Saunders, London, pp. 74--102. Dyck, P. J. (1966) Histologic measurements and fine structure of biopsied sural nerve: normal, and in peroneal muscular atrophy, hypertrophic neuropathy, and congenital sensory neuropathy, Mayo Clin. Proc., 41 : 742-774. Dyck, P. J. (1975) Pathological alterations of the peripheral nervous system in man. In: P. J. Dyck, P. K. Thomas and E. H. Lambert (Eds.), Peripheral Neuropathy, VoL 1, Saunders, Philadelphia: pp. 296-336. Dyck, P. J. and M. R. Gomez (1968) Segmental demyelination in Dejerine-Sottas disease - - Light, phase contrast, and electron microscopic studies, Proc. Mayo Clin., 43: 280-296. Dyck, P. J., E. H. Lambert, K. Sanders and P. C. O'Brien (1971) Severe hypomyelination and marked abnormality of conduction in Dejerine-Sottas hypertrophic neuropathy - - Myelin thickness and compound action potential of sural nerve in vitro, Proc. Mayo Clin., 46: 432436. Dyck, P. J., J. A. Gutrecht, J. A. Bastron, W. E. Karnes and A. J. D. Dale (1968) Histologic and teased-fiber measurements of sural nerve in disorders of lower motor and primary sensory neurons, Proc. Mayo Clin., 43 : 81-123. Emery, J. L. and R. Singhal (1973) Changes associated with growth in the cells of the dorsal root ganglion in children, Develop. reed. Child NeuroL, 15 : 460~,66. Engel, W. K. and G. G. Cunningham (1963) Rapid examination of muscle tissue - - An improved trichrome method for fresh-frozen biopsy sections, Neurology (Minneap.), 13: 909-923. Falconer D. S. and W. R. Sobey (1953) The location of "Trembler" in linkage group VII of the house mouse, J. Hered., 49 : 159-160. Fullerton, P. M. and D. J. O'Sullivan (1968) Thalidomide neuropathy - - A clinical, electrophysiological and histological follow-up study, J. NeuroL Neurosurg. Psychiat., 31 : 543-551. Gutrecht, J. A. and P. J. Dyck (1966) Segmental demyelinization in peroneal muscular atrophy - Nerve fibres teased from sural nerve biopsy specimens, Proc. Mayo Clin., 41 : 775-777. Harrison, B. M. (1976) The spinal cord - Ventral root junction in the Trembler mouse, Submitted for publication. Low, P. A. (1976) Hereditary hypertrophic neuropatby in the Trembler mouse, Part 2 (Histopathological studies: electron microscopy), J. neuroL Sci., 30: 343-368. Low, P. A. and J. G. McLeod (1975) Hereditary demyelinating neuropathy in the Trembler mouse, J. neuroL Sci., 26: 565-574. Nachlas, M. M., K.-C. Tsou, E. De Souza, C. S. Cheng and A. M. Seligman (1957) Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole, J. Histochern. Cytochem., 5: 420--436. Ohta, H., K. Offord and P. J. Dyck (1974) Morphometric evaluation of first sacral ganglia of man, J. neurol. Sci., 22: 73-82. Padykula, H. A. and E. Herman (1955) Factors affecting the activity of adenosine triphosphatase and other phosphatases as measured by histochemical techniques, J. Histochem. Cytochem., 3: 161-169. Papapetropoulos, T. A. and W. G. Bradley (1972) Spinal motor neurones in murine muscular dystrophy and spinal muscular atrophy, J. NeuroL Neurosurg. Psychiat., 35: 60-65. Prineas, J. W., J. G. McLeod and W. H. Wolfenden (1971) Endoneurial deposits of amyloid-like fibrils in a recurrent demyelinating neuropathy - - An electron microscopic study, J. Neuropath. exp. NeuroL, 30: 583-592. Romanes, G. J. (1964) The motor pools of the spinal cord. In: J. C. Eccles and J. P. Schad~ (Eds.), Organization of the Spinal Cord (Progress in Brain Research, Vol. 11), Elsevier, Amsterdam, pp. 93-119. Swallow, M. (1966) Fibre size and content of the anterior tibial nerve of the foot, J. Neurol. Neurosurg. Psychiat., 29: 205-213. Thomas, P. K., R. G. Lascelles and G. Stewart (1975) Hypertrophic neuropathy. In: P. J. Vinken and G. W. Bruyn (Eds.), Handbook of Clinical Neurology, VoL 25 (Injuries of the Spine and Spinal Cord, Part 1), North-Holland Publ. Co., Amsterdam, pp. 145-170. Tomlinson, B. E., D. Irving and J. J. Rebeiz (1973) Total numbers of limb motor neurones in the human lumbosacral cord and an analysis of the accuracy of various sampling procedures, J. neuroL Sci., 20: 313-327.