Acta Neuropathol (1991) 81:649 - 656
Acta Neuropathologlca 9 Spri~ger-Verlag 1991
Nerve growth factor receptor immunostaining in the spinal cord and peripheral nerves in amyotrophic lateral sclerosis* H. Kerkhoff 1, F. G. I. Jennekens 1, D. Troost 2, and H. Veldman 1 1 Laboratory for Neuromuscular Diseases, Department of Neurology, Rudolf Magnus Research Institute, University of Utrecht, Utrecht, Heidelberglaan 100, NL-3508 GA Utrecht, The Netherlands 2 Laboratory for Neuropathology, Academic Medical Center, Amsterdam, The Netherlands Received August 14, 1990/Revised, accepted December 24, 1990
Summary. In animal experiments, nerve transection is followed by expression of nerve growth factor receptors (NGFR) on Schwann cells of both motor and sensory nerve fibres distally to the site of the lesion. To determine whether denervated Schwann cells in amyotrophic lateral sclerosis (ALS) similarly express NGFR, a study was made of post-mortem material of peripheral nerves and ventral roots from ALS cases and age-matched controls, using immunolabelling methods. Dorsal roots and spinal cords were also examined for the presence of NGFR. In all the ALS cases and controls, NGFR immunostaining was seen in the outer layer of vessel walls, perineurial sheaths, connective tissue surrounding fascicles in nerve roots and in the substantia gelatinosa of the spinal cord. In ALS, NGFR staining was also present in the Schwann cells of degenerated nerve fibres in mixed peripheral nerves, in ventral roots and, to a lesser extent, in dorsal roots. NGFR immunoreactivity was also seen in elongated cells extending from the perifascicular connective tissue into the nerve fascicles. It is concluded that denervated Schwann cells in ALS express NGFR and that NGFR immunostaining on Schwann cells may be used as an indicator of axonal degeneration. The NGFR labelling in the dorsal roots supports the notion that ALS is not a pure motor syndrome. Key words: Nerve growth factors - Amyotrophic lateral sclerosis - Schwann cells
Amyotrophic lateral sclerosis (ALS) is a degenerating disease of the lower and upper motor neurons [28, 29, 34]. Changes in this disease are not entirely restricted to
* Supported by a grant from the Foundation for Research of ALS and Spinal Muscular Atrophy. Offprint requests to: H. Kerkhoff (address see above)
the motor neurons, as evidenced by a reduction in the number of large neurons in the dorsal root ganglia and of thick axons in the dorsal roots [18]. Loss of alpha motor neurons is compensated for, in part, by collateral sprouting of intramuscular nerve fibres and by reinnervation of denervated muscle fibres resulting in fibre-type grouping and enlarged motor units [3, 32]. Development, maintenance and regeneration of nerve cells are influenced by trophic factors. These effects are brought about by nerve growth factor (NGF) on sensory and sympathetic neurons. NGF is released by target cells, binds itself to the NGF receptor (NGFR) at the presynaptic membrane of NGF-dependent nerve cells and is then internalised and transported to the cell bodies [2, 8, 9, 11, 12]. In tissue cultures of sensory or sympathetic neurons, exposure of NGF to NGF antibodies leads to destruction of the neurons. This does not happen, however, with lower motor neurons (LMNs) which are, therefore, considered to be NGF independent [15, 19, 20, 33]. Seemingly contradictory to the supposed NGF independence is the recent finding of NGFR expression by motor neurons in the developing chick and rat [25, 36]. In addition, h:igh levels of the NGFR mRNA have been found in rat spinal cord motor neurons following crush lesion of the sciatic nerve [7] and NGFR has been observed in regenerating hypoglossal motor neurons [35]. It has been demonstrated in animal experiments that nerve transection is followed by expression of NGFR by Schwann cells of motor and sensory nerve fibres distally to the site of the lesion [24, 30, 31]. Denervated Schwann cells also express NGF (for review [27]). In view of these findings, Johnson et al. suggested that prior to target innervation, both sensory and lower motor neurons might depend on Schwann cell-produced NGF [16]. In this study, we examined denervated Schwann cells in ventral roots and peripheral nerves from ALS patients, to determine whether these cells expressed NGFR similarly to Schwann cells in experimentally transected nerves. Dorsal roots and spinal cords were also examined for the presence of NGFR.
650
Materials and m e t h o d s Post-mortem material of ventral and dorsal roots, together with the corresponding portion of the lumbar spinal cord, were obtained from 4 ALS patients and from 3 age-matched controls. Sciatic and femoral nerves were obtained from 11 ALS patients. The ALS material was provided by the ALS-SMA-Bank, Academic Medical Center, Amsterdam,The Netherlands.The specimens were fixed in a 4 % buffered formaldehyde solution. For the purpose of morphometry, part of the material was postfixed in 1% osmium tetroxide for 2 h at room temperature, dehydrated and embedded in Epon according to routine procedures. Transverse, 1-~tm sections were stained with p-phenylenediamine and micrographs were made at a final magnification x 1000. Nerve fibre diameters and fibre densities were measured using a digitizer (Calcomp 2000) coupled to an IBM-PC. Histo-
303
20-
10--
g
30--
g
20-
5
10
15 diameter (~m)
5
10
15 diameter (JJm)
10--
g
grams showing frequency distributions of myelinated-fibre diameters were made and the percentages of large and small myelinated fibres were calculated in both ventral and dorsal roots in each case. Large myelinated fibres were arbitrarily defined as fibres with diameters larger than 7 ,am; small myelinated fibres were defined as fibres with diameters less than or equal to 7 ~tm. To demonstrate NGFR immunoreactivity part of the material was cryoprotected by immersion in graded (7,5 %, 15 %, 25 %, 35 %) sucrose in 0.05 M phosphate buffer; this material was frozen in isopentane cooled in liquid nitrogen. For light microscopy, 6-,urn cryostat sections of the spinal cords and ventral roots were rinsed with 100 % acetone and phosphate-buffered saline (PBS) containing 0.2 % Triton X-100. The sections were incubated overnight with mouse monoclonal antibodies against NGFR (clone ME20-4) diluted 1:10 in PBS containing 0.2% bovine serum albumin (PBS/BSA), rinsed with PBS, and incubated for 1 h with horseradish-peroxidase (HRP)-conjugated rabbit anti-mouse antibodies diluted 1:100 in PBS/BSA. Sections were stained for peroxidase according to Adams [1] for 15 min. After rinsing the sections were dehydrated and mounted in DPX. After photographing, the sections were rehydrated and counterstained with hematoxylin. For Epon embedding, 20-~tm cryostat sections of the roots and peripheral nerves were similarly treated with the first step (8 h) and second step (overnight) antibodies and stained for peroxidase. After intensive rinsing the sections were postfixed in 1% osmium tetroxide for 60 min at room temperature, dehydrated and embedded in Epon as in routine procedures. One-micrometer sections were cut and mounted in DPX. To examine axons in NGFR-stained Epon sections, the postembedding method described by Hilderink et al. was employed [14]. Sections measuring 1 ~m were cut following NGFR immunostaining and Epon embedding (see above). Epon was removed using a saturated sodium ethoxide solution in 100 % alcohol. The sections were rinsed consecutively in 100 %, 95 % and 80 % alcohol and treated with 1% hydrogen peroxide in 100 % alcohol for 5 min. Aldehyde groups were blocked with a 0.05 % sodium borohydride solution for 1 h. The sections were incubated overnight with mouse monoclonal antibodies to phosphorylated neurofilament (clone 2 F l l , Dako, Copenhagen, Denmark), diluted 1:40 in PBS/BSA, rinsed with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG (1:200, Dako, Copenhagen, Denmark) for 1 h. The sections were then mounted in 90 % glycerol in veronal-buffered saline containing 10 % 1,4-diazabicyclo (2.2.2) octane [17]. For electron microscopic examination, sections were stained with NGFR antibodies and embedded in Epon as described. Ultrathin sections were contrasted with lead citrate for i min and viewed in an electron microscope (Zeiss EM 109, Oberkochen, Germany).
30-Results
3
g
20-
Frequency distributions of myelinated-fibre diameters in v e n t r a l a n d d o r s a l r o o t s
,o_ r 5
10
15 diameter (JJm)
Fig. 1. Myelinated-fibre-frequencydistributions in ventral roots of a control case (upper) and two cases of amyotrophic lateral sclerosis (ALS; middle and lower). In ALS, minor changes in the frequency distribution are seen in one group (middle), marked changes with a shift to fibres with small diameters are seen in the other case
(lower)
A l l t h e r o o t s o b t a i n e d f r o m t h e A L S p a t i e n t s s h o w e d an i n c r e a s e in s m a l l - f i b r e p e r c e n t a g e s a n d a d e c r e a s e in l a r g e - f i b r e p e r c e n t a g e s (Table 1, Fig. 1). Two g r o u p s w e r e d i s c e r n e d ; o n e s h o w e d m a r k e d c h a n g e s in t h e f r e q u e n c y d i s t r i b u t i o n a n d an i n c r e a s e in t h e fibre d e n s i t y (Fig. 1, l o w e r ) , w h i l s t t h e o t h e r s h o w e d m i n o r c h a n g e s in t h e f r e q u e n c y d i s t r i b u t i o n a n d a d e c r e a s e in t h e fibre d e n s i t y (Fig. 1, m i d d l e ) . I n t h e d o r s a l r o o t s , t h e f i b r e d e n s i t y was d e c r e a s e d in all A L S cases, w i t h f r e q u e n c y d i s t r i b u t i o n s s h o w i n g no d i f f e r e n c e s w h e n c o m p a r e d w i t h c o n t r o l s (Fig. 2).
651 Table 1. Morphometric quantification of ventral and dorsal roots in ALS and controls Ventral roots % of fibres - 7 ~ t m
fibre density (1/mm2)
Dorsal roots % of fibres 7 am
fibre density (1/mm2)
Control-1 Control-2 Control-3
13.9 23.5 21.8
86.1 76.5 78.2
5490 6739 4638
74.8 75.8 69.7
25.2 24.2 30.3
13998 14951 13933
ALS-1 ALS-2 ALS-3 ALS-4
92.6 92.7 36.8 32.4
7.4 7.3 63.2 67.6
15588 14103 2189 2602
73.1 69.2 66.3 75.6
26.9 30.8 33.7 24.4
7172 12085 11732 11060
30-
o- 20--
10--
rll 5
10
5
10
15 diameter (jJm)
30-
g
&
20-
10-
ri 15
diameter (IJm) Fig. 2. Myelinated-fibre-frequency distributions in dorsal roots of a control case (upper) and a case of ALS (lower). Myelinated fibres are similarly distributed
N G F R i m m u n o s t a i n i n g in the peripheral nerves I n all cases, N G F R i m m u n o s t a i n i n g was visible in the o u t e r layers o f the vessel walls a n d in the perineurial sheaths (Fig. 3). I n the A L S material, N G F R i m m u n o s taining was also seen inside the fascicles; the i m m u n o s taining h a d a s p o t t e d aspect and an irregular distribution p a t t e r n . I n s o m e areas within the fascicles, a dense p a t t e r n o f N G F R i m m u n o s t a i n i n g was seen, whilst in o t h e r areas little or no staining was detectable (Fig. 3). D o u b l e labelling with antibodies to p h o s p h o r y l a t e d n e u r o f i l a m e n t , r e v e a l e d a n o r m a l o c c u r r e n c e of axons in areas with little or no N G F R i m m u n o s t a i n i n g . I n areas
Fig. 3. Nerve growth factor receptor (NGFR) immunoreactivity in a sciatic nerve in ALS. Immunostaining with a spotted aspect and irregular distribution is seen within the fascicles. There is also immunostaining in the outer layers of vessel walls (arrows) and in the perineurial sheaths. Bar = 50 ~tm
with a dense N G F R i m m u n o s t a i n i n g p a t t e r n , however, p h o s p h o r y l a t e d n e u r o f i l a m e n t i m m u n o l a b e l l i n g was i n f r e q u e n t (Fig. 4). E l e c t r o n m i c r o s c o p y revealed N G F R i m m u n o s t a i n ing l o c a t e d at the m e m b r a n e s and in the cytoplasms of cells containing a basal lamina, indicative of S c h w a n n cells.
652
Fig. 4. A Phosphorylated neurofilament immunostaining in a sciatic nerve fascicle in ALS. B N G F R immunostaining of the same area in the same section. Many NGFR-positive spots are seen in
areas with little or no phosphorylated neurofilament immunostaining and vice versa. Bar = 25 ,am
NGFR immunostaining in the ventral roots In the ventral roots, NGFR immunoreactivity was present in all cases in the outer layer of the vessel walls and in the connective tissue surrounding the fascicles (Fig. 5). In ALS, NGFR immunostaining was also seen within the fascicles and had the same spotted aspect as in the peripheral nerves (Fig. 6a). In addition, in the ALS cases positive staining of elongated cells extending from the perifascicular connective tissue into the nerve root fascicles was also evident (Fig. 6c). These cells had a spindle-like nucleus and electron microscopy showed that they were not covered by basal laminas.
]i"
~S
~7
Fig. 5. N G F R immunostaining in a control ventral root. Immunolabelling is seen in the connective tissue surrounding the fascicles but is absent within the fascicles. Bar = 50 ~tm
653
Fig. 6A-C. NGFR immunostaining in nerve roots of a case of ALS. A Ventral root. Throughout the whole of the fascicles immunolabelling with a spotted aspect is present. Positive labelling of the perifascicular connective tissue can also be seen. B Dorsal
N G F R immunostaining in the dorsal roots N G F R immunostaining was seen in all cases in the vessel walls and in the perifascicular connective tissue, exactly as was seen in the ventral roots. In A L S cases, N G F R labelling was also positive within the fascicles, but the distribution was less dense than in the ventral roots (Fig. 6 b). Again, positively stained elongated cells were seen, but were less numerous here than in the ventral roots.
N G F R immunostaining in the spinal cord In all cases, N G F R immunoreactivity was detected in the substantia gelatinosa of the dorsal horns. In the r e m a i n d e r of the spinal cord N G F R immunostaining was absent b o t h in A L S and in controls. Neither m o t o r neurons nor glial cells revealed any immunoreactivity (Fig. 7). T h e transitions between the spinal cords and the ventral and dorsal roots were also studied. N G F R immunostaining was negative in areas with central
root. Immunolabelling is present, but is seen much less frequently than in the ventral root. C Detail of elongated cells in a ventral root extending from the perifascicular connective tissue into the nerve root fascicle. Bars A, B = 50 gm; C = 10 am
myelin. In contrast, areas with peripheral myelin showed N G F R immunolabelling c o m p a r a b l e to the nerve roots m o r e distally as described above.
Discussion The two groups apparent in the ventral roots in the A L S material using frequency distributions and fibre densities, have previously b e e n described and discussed [10, 22, 28, 29, 34]. In contrast to prior observations [18], the decrease in fibre densities in the dorsal roots of the A L S cases was distributed equally over all fibre diameters. O u r results indicate that d e n e r v a t e d Schwann cells in A L S do express N G F R . A disorder in the functioning of Schwann cells in A L S has previously b e e n suggested [10]. A r g u m e n t s in favour of Schwann cell involvement are the presence of m a n y fibres with a variety of internodal lengths [10], a relatively high frequency of segmental demyelination in the sural aerves [5] and a decrease in the m o t o r nerve conduction velocity, not
654
Fig. 7. A Ventral horn of the spinal cord of a case of ALS, immunostained for NGFR. Immunolabelling is seen in a vessel wall, but is absent from motor neuron cell bodies and glial cells. B Same area of the same section counterstained with hematoxylin,
showing the presence of motor neuron cell bodies and nonneuronal cells. C Positive NGFR immunostaining in the ventral root in the same section. Bar = 100 ~tm
only in the distal parts but also in the proximal parts of the peripheral nerves (discussed by H a n y u et al. [10]). Our results, however, do not support the theory of a primary Schwann cell disorder in ALS, as there is no apparent difference between the expression of N G F R by denervated Schwann cells in ALS and by Schwann cells in experimentally injured nerves [30]. The results of our double-labelling studies with antibodies against phosphorylated neurofilament and N G F R suggest that N G F R immunostaining may be used as an indicator of axonal degeneration, because positive N G F R immunostaining correlates with the absence of intact axons. Positive N G F R immunoreactivity in dorsal roots points to axonal loss. This may explain the decreased fibre density in the dorsal roots and supports the notion that ALS is not a pure m o t o r syndrome [6, 18]. In view of the frequency distributions mentioned earlier, we must conclude that the axonal loss in the dorsal roots is not confined to large axons, but is distributed equally over all fibre densities. N G F R immunostaining in the substantia gelatinosa of the dorsal horns confirms previous observations by Yip and Johnson [38] in rats. These authors demonstrated that N G F R immunoreactivity in the substantia gelatinosa was absent following dorsal root section, suggesting localisation of N G F R in the central processes of sensory nerve fibres.
In contrast to Schwann cells, glial cells in the spinal cord revealed no N G F R immunostaining. Experimentally, N G F R have been detected in several astrocytic cell lines in tissue cultures and in retinal Mtiller cells, a modified astrocyte type [21, 26], but N G F R immunostaining on the glial cells of animal spinal cords has never been detected. N G F R immunostaining is present in developing m o t o r neurons [25, 36]. N G F R m R N A and N G F R have also been detected in m o t o r neurons during regeneration of their damaged axons [7, 35]. One might, therefore, expect to find N G F R in neurons forming collateral axonal sprouts. Collateral sprouting is a feature of ALS [3, 32] and expression of N G F R in m o t o r neurons in the spinal cord of ALS patients is, therefore, conceivable. N G F R immunostaining, however, was absent in the m o t o r neurons in the ventral horns. T h e r e are several possible explanations for this phenomenon. Firstly, the hypothesis that m o t o r neurons forming collateral sprouts show increased N G F R expression may be wrong. Regeneration as is seen following crush or transection of axons does not occur in ALS. This may explain the difference between our results and the findings of Wood et al. [35]. Secondly, the sensitivity of our detection m e t h o d may be too low to be able to demonstrate N G F R in the spinal m o t o r neurons. Thirdly, the absence of N G F R in m o t o r neurons may be the
655 expression of a defect, characteristic for ALS. A d d i t i o n al e x p e r i m e n t s are r e q u i r e d to c o n f i r m or disprove this latter hypothesis. A c c o r d i n g to S c h a t t e m a n et al. [26], two factors c o n t r i b u t e to the p r e s e n c e o f N G F R in vessel walls. Firstly, N G F R i m m u n o s t a i n i n g has b e e n o b s e r v e d on vascular pericytes and secondly, s y m p a t h e t i c nerve fibres i n n e r v a t i n g t h e vessel walls m a y also carry N G F R . Perineurial N G F R i m m u n o l a b e l l i n g has b e e n described by C h e s a et al. [4]. T h e f u n c t i o n o f N G F R on perineurial cells is at present unclear. Obviously, the perifascicular cells in the ventral and dorsal roots are c o m p a r a b l e to those in the perineurial sheaths as far as N G F R expression is c o n c e r n e d . T h e N G F R - p o s i t i v e e l o n g a t e d cells e x t e n d i n g f r o m the perifascicular c o n n e c t i v e tissue into the fascicles of the nerve roots in A L S are p r o b a b l y fibroblasts. Fibroblasts in tissue culture do n o t show N G F R i m m u n o r e a c tivity [37, 39]; n e i t h e r have fibroblasts in the distal s e g m e n t of a t r a n s e c t e d nerve b e e n r e p o r t e d as showing any N G F R immunoreactivity. N G F m R N A , however, has b e e n d e t e c t e d in fibroblast-like cells in the distal s e g m e n t o f e x p e r i m e n t a l l y transected nerves [13] and this m a y indicate a functional role for s o m e specialised fibroblasts in the r e g e n e r a t i o n o f nerve fibres. M a c r o p h a g e s in lesioned peripheral nerves m a y also be e l o n g a t e d and c o m e to r e s e m b l e fibroblasts [23]. T h e cells in o u r material, however, were longer and less stellate t h a n the m a c r o p h a g e s described by P e r r y et al. [23].
11.
12. 13.
14.
15. 16. 17.
18.
19. 20. 21.
References 22. 1. Adams JC (1981) Heavy metal intensification of DAB-based HRP reaction products. J Histochem Cytochem 29: 775-780 2. Banerjee SR Snyder SH, Cuatrecasas R Green LA (1973) Binding of nerve growth factor in superior cervical ganglia. Proc Natl Acad Sci USA 70:2519-2523 3. Bonduelle M (1975) Amyotrophic lateral sclerosis. Handb Clin Neurol 22:281-338 4. Chesa PG, Rettig WJ, Thomson TM, Old L J, Melamed MR (1988) Immunohistochemical analysis of nerve growth factor receptor expression in normal and malignant human tissues. J Histochem Cytochem 36:383-389 5. Dayan AD, Graveson GS, Illis LS, Robinson PK (1969) Schwann cell damage in motoneuron disease. Neurology 19: 242-246 6. Dyck PJ, Stevens JC, Mulder DW, Espinosa RE (1975) Frequency of nerve fiber degeneration of peripheral motor and sensory neurons in amyotrophic lateral sclerosis: morphometry of deep and superficial peroneal nerves. Neurology 25: 781-787 7. Ernfors E Henschen A, Olson L, Persson H (1989) Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons. Neuron 2:1605-1613 8. Frazier WA, Boyd LE Bradshaw RA (1974) Properties of specific binding of lZSI-nerve growth factor to responsive peripheral neurons. J Biol Chem 249:5513-5519 9. Greene LA, Shooter EM (1980) The nerve growth factor: biochemistry, synthesis, and mechanism of action. Annu Rev Neurosci 3:353-402 10. Hanyu N. Oguchi K, Yanagisawa N, Tsukagoshi H (1982) Degeneration and regeneration of ventral root motor fibers in
23.
24.
25. 26. 27. 28.
29.
30.
amyotrophic lateral sclerosis. Morphometric studies of cervical ventral roots. J Neurol Sci 55:99-115 Hefti E Hartikka J, Salvatierra A,Weiner WJ, Mash DC (1986) Localization of nerve growth factor receptors in cholinergic neurons of the human basal forebrain. Neurosci Lett 69: 37-41 Herrup K, Shooter EM (1973) Properties of the B-nerve growth factor receptor of avian dorsal root ganglia. Proc Natl Acad Sci USA 70:3884-3888 Heumann R, Korsching S, Bandtlow C, Thoenen H (1987) Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection. J Cell Biol 104: 1623-1631 Hilderink PH, Veldman H, Jennekens FGI (1989) A simple method for immunostaining of axons in plastic embedded material. Proceedings of the 30th Dutch Federation meeting. Dutch Foundation Federation of Medical Scientific Societies, Maastricht. Abstr no 169 Johnson EM, Rich KM, Yip HK (1986) The role of NGF in sensory neurons in vivo. Trends Neurosci 9:33-37 Johnson EM,TaniuchiM, DiStefanoPS (1988) Expression and possible function of nerve growth factor receptors in Schwann cells. Trends Neurosci 11:299-304 Johnson GD, Davidson RS, McNamee KC, Russell G, Goodwin D, Holborow EJ (1982) Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy. J Immuuol Methods 55:231-242 KawamuraY, Dyck PJ, Masatake S, Okazaki H,Tateishi J, Doi H (1981) Morphometric comparison of the vulnerability of peripheral motor and sensory neurons in amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 40:(;67-675 Levi-Montalcini R (1987) The Nerve Growth Factor 35 years later. Science 237:1154-1162 Levi-Montalcini R, Angeletti P (1968) Nerve growth factor. Physiol Rev 48:534-569 Lindsay RM (1979) Adult rat brain astrocytes support survival of both NGF-dependent and NGF-insensitive neurones. Nature 282:80-82 Ohnishi Y, Makifuchi T, Ikuta F (1980) Morphometric investigation of the myelinated fibers of the anterior spinal roots in amyotrophic lateral sclerosis and Shy-Drager syndrome. Clin Neurol 20:809-815 Perry VH, Brown MC, Gordon S (1987) The macrophage response to central and peripheral nerve injury. A possible role for macrophages in regeneration. J Exp Med 165: 1218-1223 Raivich G, Kreutzberg GW (1987) Expression of growth factor receptors in injured nervous tissue. I. Axotomy leads to ashift in the cellular distribution of specific t3-nerve growth factor binding in the injured and regenerating PNS. J Neurocytol 16:689-700 Raivich G, Zimmermann A, Sutter A (1985) The spatial and temporal pattern of [3NGF receptor expression in the developing chick embryo. EMBO J 4:637-644 Schatteman GC, Gibbs L, Lanahan AA, Claude P, Bothwell M (1988) Expression of NGF receptor in the developing and adult primate central nervous system. J Neurosci 8:860-873 Snider WD, Johnson EM (1989) Neurotrophic molecules. Ann Neurol 26:489-506 Sobue G, Matsuoka Y, Mukai E, Takayanagi T, Sobue I, Hashizume Y (1981) Spinal and cranial motor nerve roots in amyotrophic lateral sclerosis and X-linked recessive bulbospihal muscular atrophy: morphometric and teased fiber study. Acta Neuropathol (Berl) 55:227-235 Sobue G, MatsuokaY, Mukai E,Takayanagi T, Sobue t (1981) Pathology of myelinated fibers in cervical and lumbar ventral spinal roots in amyotrophic lateral sclerosis. J Neurol Sci 50: 413-421 Taniuchi M, Clark HB, Johnson EM (1986) Induction of nerve growth factor receptor in Schwann cells after axotomy. Proc Natl Acad Sci USA 83:4094-4098
656 31. Taniuchi M, Clark HB, Schweitzer JB, Johnson EM (1988) Expression of nerve growth factor receptors by Schwann cells of axotomized peripheral nerves: ultrastmctural location, suppression by axonal contact, and binding properties. J Neurosci 8:664-681 32. Telerman-Toppet N, CoOrs C (1978) Motor innervation and fiber-type pattern in amyotrophic lateral sclerosis and in Charcot-Marie-Tooth disease. Muscle Nerve 1:133-139 33. Thoenen H, Barde YA (1980) Physiology of nerve growth factor. Physiol Rev 60:1284-1335 34. Wohlfart G, Swank PL (1941) Pathology of amyotrophic lateral sclerosis, fiber analysis of the ventral roots and pyramidal tracts of the spinal cord. Arch Neurol Psychiatry 46: 783 -799 35. Wood SJ, Pritchard J, Sofroniew MV (1990) Re-expression of nerve growth factor receptor after axonal injury recapitulates a
36. 37. 38. 39.
developmental event in motor neurons: differential regulation when regeneration is allowed or prevented. Eur J Neurosci 2: 650-657 Yah Q, Johnson EM (1988) An immunohistochemical study of the nerve growth factor receptor in developing rats. J Neurosci 8:3481-3498 Yasuda T, Sobue G, Mokuno K, Kreider B, Pleasure D (1987) Cultured rat Schwann cells express low affinity receptors for nerve growth factor. Brain Res 436:113-119 Yip HK, Johnson EM (1987) Nerve growth factor receptors in rat spinal cord: an autoradiographic and immunohistochemical study. Neuroscience 22:267-279 Zimmermann A, Sutter A (1983) B-Nerve growth factor (~NGF) receptors on glial cells. Cell-cell interaction between neurones and Schwann cells in cultures of chick sensory ganglia. EMBO J 2:879-885
Acta Neuropathologlca 9 Spri~ger-Verlag 1991
Nerve growth factor receptor immunostaining in the spinal cord and peripheral nerves in amyotrophic lateral sclerosis* H. Kerkhoff 1, F. G. I. Jennekens 1, D. Troost 2, and H. Veldman 1 1 Laboratory for Neuromuscular Diseases, Department of Neurology, Rudolf Magnus Research Institute, University of Utrecht, Utrecht, Heidelberglaan 100, NL-3508 GA Utrecht, The Netherlands 2 Laboratory for Neuropathology, Academic Medical Center, Amsterdam, The Netherlands Received August 14, 1990/Revised, accepted December 24, 1990
Summary. In animal experiments, nerve transection is followed by expression of nerve growth factor receptors (NGFR) on Schwann cells of both motor and sensory nerve fibres distally to the site of the lesion. To determine whether denervated Schwann cells in amyotrophic lateral sclerosis (ALS) similarly express NGFR, a study was made of post-mortem material of peripheral nerves and ventral roots from ALS cases and age-matched controls, using immunolabelling methods. Dorsal roots and spinal cords were also examined for the presence of NGFR. In all the ALS cases and controls, NGFR immunostaining was seen in the outer layer of vessel walls, perineurial sheaths, connective tissue surrounding fascicles in nerve roots and in the substantia gelatinosa of the spinal cord. In ALS, NGFR staining was also present in the Schwann cells of degenerated nerve fibres in mixed peripheral nerves, in ventral roots and, to a lesser extent, in dorsal roots. NGFR immunoreactivity was also seen in elongated cells extending from the perifascicular connective tissue into the nerve fascicles. It is concluded that denervated Schwann cells in ALS express NGFR and that NGFR immunostaining on Schwann cells may be used as an indicator of axonal degeneration. The NGFR labelling in the dorsal roots supports the notion that ALS is not a pure motor syndrome. Key words: Nerve growth factors - Amyotrophic lateral sclerosis - Schwann cells
Amyotrophic lateral sclerosis (ALS) is a degenerating disease of the lower and upper motor neurons [28, 29, 34]. Changes in this disease are not entirely restricted to
* Supported by a grant from the Foundation for Research of ALS and Spinal Muscular Atrophy. Offprint requests to: H. Kerkhoff (address see above)
the motor neurons, as evidenced by a reduction in the number of large neurons in the dorsal root ganglia and of thick axons in the dorsal roots [18]. Loss of alpha motor neurons is compensated for, in part, by collateral sprouting of intramuscular nerve fibres and by reinnervation of denervated muscle fibres resulting in fibre-type grouping and enlarged motor units [3, 32]. Development, maintenance and regeneration of nerve cells are influenced by trophic factors. These effects are brought about by nerve growth factor (NGF) on sensory and sympathetic neurons. NGF is released by target cells, binds itself to the NGF receptor (NGFR) at the presynaptic membrane of NGF-dependent nerve cells and is then internalised and transported to the cell bodies [2, 8, 9, 11, 12]. In tissue cultures of sensory or sympathetic neurons, exposure of NGF to NGF antibodies leads to destruction of the neurons. This does not happen, however, with lower motor neurons (LMNs) which are, therefore, considered to be NGF independent [15, 19, 20, 33]. Seemingly contradictory to the supposed NGF independence is the recent finding of NGFR expression by motor neurons in the developing chick and rat [25, 36]. In addition, h:igh levels of the NGFR mRNA have been found in rat spinal cord motor neurons following crush lesion of the sciatic nerve [7] and NGFR has been observed in regenerating hypoglossal motor neurons [35]. It has been demonstrated in animal experiments that nerve transection is followed by expression of NGFR by Schwann cells of motor and sensory nerve fibres distally to the site of the lesion [24, 30, 31]. Denervated Schwann cells also express NGF (for review [27]). In view of these findings, Johnson et al. suggested that prior to target innervation, both sensory and lower motor neurons might depend on Schwann cell-produced NGF [16]. In this study, we examined denervated Schwann cells in ventral roots and peripheral nerves from ALS patients, to determine whether these cells expressed NGFR similarly to Schwann cells in experimentally transected nerves. Dorsal roots and spinal cords were also examined for the presence of NGFR.
650
Materials and m e t h o d s Post-mortem material of ventral and dorsal roots, together with the corresponding portion of the lumbar spinal cord, were obtained from 4 ALS patients and from 3 age-matched controls. Sciatic and femoral nerves were obtained from 11 ALS patients. The ALS material was provided by the ALS-SMA-Bank, Academic Medical Center, Amsterdam,The Netherlands.The specimens were fixed in a 4 % buffered formaldehyde solution. For the purpose of morphometry, part of the material was postfixed in 1% osmium tetroxide for 2 h at room temperature, dehydrated and embedded in Epon according to routine procedures. Transverse, 1-~tm sections were stained with p-phenylenediamine and micrographs were made at a final magnification x 1000. Nerve fibre diameters and fibre densities were measured using a digitizer (Calcomp 2000) coupled to an IBM-PC. Histo-
303
20-
10--
g
30--
g
20-
5
10
15 diameter (~m)
5
10
15 diameter (JJm)
10--
g
grams showing frequency distributions of myelinated-fibre diameters were made and the percentages of large and small myelinated fibres were calculated in both ventral and dorsal roots in each case. Large myelinated fibres were arbitrarily defined as fibres with diameters larger than 7 ,am; small myelinated fibres were defined as fibres with diameters less than or equal to 7 ~tm. To demonstrate NGFR immunoreactivity part of the material was cryoprotected by immersion in graded (7,5 %, 15 %, 25 %, 35 %) sucrose in 0.05 M phosphate buffer; this material was frozen in isopentane cooled in liquid nitrogen. For light microscopy, 6-,urn cryostat sections of the spinal cords and ventral roots were rinsed with 100 % acetone and phosphate-buffered saline (PBS) containing 0.2 % Triton X-100. The sections were incubated overnight with mouse monoclonal antibodies against NGFR (clone ME20-4) diluted 1:10 in PBS containing 0.2% bovine serum albumin (PBS/BSA), rinsed with PBS, and incubated for 1 h with horseradish-peroxidase (HRP)-conjugated rabbit anti-mouse antibodies diluted 1:100 in PBS/BSA. Sections were stained for peroxidase according to Adams [1] for 15 min. After rinsing the sections were dehydrated and mounted in DPX. After photographing, the sections were rehydrated and counterstained with hematoxylin. For Epon embedding, 20-~tm cryostat sections of the roots and peripheral nerves were similarly treated with the first step (8 h) and second step (overnight) antibodies and stained for peroxidase. After intensive rinsing the sections were postfixed in 1% osmium tetroxide for 60 min at room temperature, dehydrated and embedded in Epon as in routine procedures. One-micrometer sections were cut and mounted in DPX. To examine axons in NGFR-stained Epon sections, the postembedding method described by Hilderink et al. was employed [14]. Sections measuring 1 ~m were cut following NGFR immunostaining and Epon embedding (see above). Epon was removed using a saturated sodium ethoxide solution in 100 % alcohol. The sections were rinsed consecutively in 100 %, 95 % and 80 % alcohol and treated with 1% hydrogen peroxide in 100 % alcohol for 5 min. Aldehyde groups were blocked with a 0.05 % sodium borohydride solution for 1 h. The sections were incubated overnight with mouse monoclonal antibodies to phosphorylated neurofilament (clone 2 F l l , Dako, Copenhagen, Denmark), diluted 1:40 in PBS/BSA, rinsed with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG (1:200, Dako, Copenhagen, Denmark) for 1 h. The sections were then mounted in 90 % glycerol in veronal-buffered saline containing 10 % 1,4-diazabicyclo (2.2.2) octane [17]. For electron microscopic examination, sections were stained with NGFR antibodies and embedded in Epon as described. Ultrathin sections were contrasted with lead citrate for i min and viewed in an electron microscope (Zeiss EM 109, Oberkochen, Germany).
30-Results
3
g
20-
Frequency distributions of myelinated-fibre diameters in v e n t r a l a n d d o r s a l r o o t s
,o_ r 5
10
15 diameter (JJm)
Fig. 1. Myelinated-fibre-frequencydistributions in ventral roots of a control case (upper) and two cases of amyotrophic lateral sclerosis (ALS; middle and lower). In ALS, minor changes in the frequency distribution are seen in one group (middle), marked changes with a shift to fibres with small diameters are seen in the other case
(lower)
A l l t h e r o o t s o b t a i n e d f r o m t h e A L S p a t i e n t s s h o w e d an i n c r e a s e in s m a l l - f i b r e p e r c e n t a g e s a n d a d e c r e a s e in l a r g e - f i b r e p e r c e n t a g e s (Table 1, Fig. 1). Two g r o u p s w e r e d i s c e r n e d ; o n e s h o w e d m a r k e d c h a n g e s in t h e f r e q u e n c y d i s t r i b u t i o n a n d an i n c r e a s e in t h e fibre d e n s i t y (Fig. 1, l o w e r ) , w h i l s t t h e o t h e r s h o w e d m i n o r c h a n g e s in t h e f r e q u e n c y d i s t r i b u t i o n a n d a d e c r e a s e in t h e fibre d e n s i t y (Fig. 1, m i d d l e ) . I n t h e d o r s a l r o o t s , t h e f i b r e d e n s i t y was d e c r e a s e d in all A L S cases, w i t h f r e q u e n c y d i s t r i b u t i o n s s h o w i n g no d i f f e r e n c e s w h e n c o m p a r e d w i t h c o n t r o l s (Fig. 2).
651 Table 1. Morphometric quantification of ventral and dorsal roots in ALS and controls Ventral roots % of fibres - 7 ~ t m
fibre density (1/mm2)
Dorsal roots % of fibres 7 am
fibre density (1/mm2)
Control-1 Control-2 Control-3
13.9 23.5 21.8
86.1 76.5 78.2
5490 6739 4638
74.8 75.8 69.7
25.2 24.2 30.3
13998 14951 13933
ALS-1 ALS-2 ALS-3 ALS-4
92.6 92.7 36.8 32.4
7.4 7.3 63.2 67.6
15588 14103 2189 2602
73.1 69.2 66.3 75.6
26.9 30.8 33.7 24.4
7172 12085 11732 11060
30-
o- 20--
10--
rll 5
10
5
10
15 diameter (jJm)
30-
g
&
20-
10-
ri 15
diameter (IJm) Fig. 2. Myelinated-fibre-frequency distributions in dorsal roots of a control case (upper) and a case of ALS (lower). Myelinated fibres are similarly distributed
N G F R i m m u n o s t a i n i n g in the peripheral nerves I n all cases, N G F R i m m u n o s t a i n i n g was visible in the o u t e r layers o f the vessel walls a n d in the perineurial sheaths (Fig. 3). I n the A L S material, N G F R i m m u n o s taining was also seen inside the fascicles; the i m m u n o s taining h a d a s p o t t e d aspect and an irregular distribution p a t t e r n . I n s o m e areas within the fascicles, a dense p a t t e r n o f N G F R i m m u n o s t a i n i n g was seen, whilst in o t h e r areas little or no staining was detectable (Fig. 3). D o u b l e labelling with antibodies to p h o s p h o r y l a t e d n e u r o f i l a m e n t , r e v e a l e d a n o r m a l o c c u r r e n c e of axons in areas with little or no N G F R i m m u n o s t a i n i n g . I n areas
Fig. 3. Nerve growth factor receptor (NGFR) immunoreactivity in a sciatic nerve in ALS. Immunostaining with a spotted aspect and irregular distribution is seen within the fascicles. There is also immunostaining in the outer layers of vessel walls (arrows) and in the perineurial sheaths. Bar = 50 ~tm
with a dense N G F R i m m u n o s t a i n i n g p a t t e r n , however, p h o s p h o r y l a t e d n e u r o f i l a m e n t i m m u n o l a b e l l i n g was i n f r e q u e n t (Fig. 4). E l e c t r o n m i c r o s c o p y revealed N G F R i m m u n o s t a i n ing l o c a t e d at the m e m b r a n e s and in the cytoplasms of cells containing a basal lamina, indicative of S c h w a n n cells.
652
Fig. 4. A Phosphorylated neurofilament immunostaining in a sciatic nerve fascicle in ALS. B N G F R immunostaining of the same area in the same section. Many NGFR-positive spots are seen in
areas with little or no phosphorylated neurofilament immunostaining and vice versa. Bar = 25 ,am
NGFR immunostaining in the ventral roots In the ventral roots, NGFR immunoreactivity was present in all cases in the outer layer of the vessel walls and in the connective tissue surrounding the fascicles (Fig. 5). In ALS, NGFR immunostaining was also seen within the fascicles and had the same spotted aspect as in the peripheral nerves (Fig. 6a). In addition, in the ALS cases positive staining of elongated cells extending from the perifascicular connective tissue into the nerve root fascicles was also evident (Fig. 6c). These cells had a spindle-like nucleus and electron microscopy showed that they were not covered by basal laminas.
]i"
~S
~7
Fig. 5. N G F R immunostaining in a control ventral root. Immunolabelling is seen in the connective tissue surrounding the fascicles but is absent within the fascicles. Bar = 50 ~tm
653
Fig. 6A-C. NGFR immunostaining in nerve roots of a case of ALS. A Ventral root. Throughout the whole of the fascicles immunolabelling with a spotted aspect is present. Positive labelling of the perifascicular connective tissue can also be seen. B Dorsal
N G F R immunostaining in the dorsal roots N G F R immunostaining was seen in all cases in the vessel walls and in the perifascicular connective tissue, exactly as was seen in the ventral roots. In A L S cases, N G F R labelling was also positive within the fascicles, but the distribution was less dense than in the ventral roots (Fig. 6 b). Again, positively stained elongated cells were seen, but were less numerous here than in the ventral roots.
N G F R immunostaining in the spinal cord In all cases, N G F R immunoreactivity was detected in the substantia gelatinosa of the dorsal horns. In the r e m a i n d e r of the spinal cord N G F R immunostaining was absent b o t h in A L S and in controls. Neither m o t o r neurons nor glial cells revealed any immunoreactivity (Fig. 7). T h e transitions between the spinal cords and the ventral and dorsal roots were also studied. N G F R immunostaining was negative in areas with central
root. Immunolabelling is present, but is seen much less frequently than in the ventral root. C Detail of elongated cells in a ventral root extending from the perifascicular connective tissue into the nerve root fascicle. Bars A, B = 50 gm; C = 10 am
myelin. In contrast, areas with peripheral myelin showed N G F R immunolabelling c o m p a r a b l e to the nerve roots m o r e distally as described above.
Discussion The two groups apparent in the ventral roots in the A L S material using frequency distributions and fibre densities, have previously b e e n described and discussed [10, 22, 28, 29, 34]. In contrast to prior observations [18], the decrease in fibre densities in the dorsal roots of the A L S cases was distributed equally over all fibre diameters. O u r results indicate that d e n e r v a t e d Schwann cells in A L S do express N G F R . A disorder in the functioning of Schwann cells in A L S has previously b e e n suggested [10]. A r g u m e n t s in favour of Schwann cell involvement are the presence of m a n y fibres with a variety of internodal lengths [10], a relatively high frequency of segmental demyelination in the sural aerves [5] and a decrease in the m o t o r nerve conduction velocity, not
654
Fig. 7. A Ventral horn of the spinal cord of a case of ALS, immunostained for NGFR. Immunolabelling is seen in a vessel wall, but is absent from motor neuron cell bodies and glial cells. B Same area of the same section counterstained with hematoxylin,
showing the presence of motor neuron cell bodies and nonneuronal cells. C Positive NGFR immunostaining in the ventral root in the same section. Bar = 100 ~tm
only in the distal parts but also in the proximal parts of the peripheral nerves (discussed by H a n y u et al. [10]). Our results, however, do not support the theory of a primary Schwann cell disorder in ALS, as there is no apparent difference between the expression of N G F R by denervated Schwann cells in ALS and by Schwann cells in experimentally injured nerves [30]. The results of our double-labelling studies with antibodies against phosphorylated neurofilament and N G F R suggest that N G F R immunostaining may be used as an indicator of axonal degeneration, because positive N G F R immunostaining correlates with the absence of intact axons. Positive N G F R immunoreactivity in dorsal roots points to axonal loss. This may explain the decreased fibre density in the dorsal roots and supports the notion that ALS is not a pure m o t o r syndrome [6, 18]. In view of the frequency distributions mentioned earlier, we must conclude that the axonal loss in the dorsal roots is not confined to large axons, but is distributed equally over all fibre densities. N G F R immunostaining in the substantia gelatinosa of the dorsal horns confirms previous observations by Yip and Johnson [38] in rats. These authors demonstrated that N G F R immunoreactivity in the substantia gelatinosa was absent following dorsal root section, suggesting localisation of N G F R in the central processes of sensory nerve fibres.
In contrast to Schwann cells, glial cells in the spinal cord revealed no N G F R immunostaining. Experimentally, N G F R have been detected in several astrocytic cell lines in tissue cultures and in retinal Mtiller cells, a modified astrocyte type [21, 26], but N G F R immunostaining on the glial cells of animal spinal cords has never been detected. N G F R immunostaining is present in developing m o t o r neurons [25, 36]. N G F R m R N A and N G F R have also been detected in m o t o r neurons during regeneration of their damaged axons [7, 35]. One might, therefore, expect to find N G F R in neurons forming collateral axonal sprouts. Collateral sprouting is a feature of ALS [3, 32] and expression of N G F R in m o t o r neurons in the spinal cord of ALS patients is, therefore, conceivable. N G F R immunostaining, however, was absent in the m o t o r neurons in the ventral horns. T h e r e are several possible explanations for this phenomenon. Firstly, the hypothesis that m o t o r neurons forming collateral sprouts show increased N G F R expression may be wrong. Regeneration as is seen following crush or transection of axons does not occur in ALS. This may explain the difference between our results and the findings of Wood et al. [35]. Secondly, the sensitivity of our detection m e t h o d may be too low to be able to demonstrate N G F R in the spinal m o t o r neurons. Thirdly, the absence of N G F R in m o t o r neurons may be the
655 expression of a defect, characteristic for ALS. A d d i t i o n al e x p e r i m e n t s are r e q u i r e d to c o n f i r m or disprove this latter hypothesis. A c c o r d i n g to S c h a t t e m a n et al. [26], two factors c o n t r i b u t e to the p r e s e n c e o f N G F R in vessel walls. Firstly, N G F R i m m u n o s t a i n i n g has b e e n o b s e r v e d on vascular pericytes and secondly, s y m p a t h e t i c nerve fibres i n n e r v a t i n g t h e vessel walls m a y also carry N G F R . Perineurial N G F R i m m u n o l a b e l l i n g has b e e n described by C h e s a et al. [4]. T h e f u n c t i o n o f N G F R on perineurial cells is at present unclear. Obviously, the perifascicular cells in the ventral and dorsal roots are c o m p a r a b l e to those in the perineurial sheaths as far as N G F R expression is c o n c e r n e d . T h e N G F R - p o s i t i v e e l o n g a t e d cells e x t e n d i n g f r o m the perifascicular c o n n e c t i v e tissue into the fascicles of the nerve roots in A L S are p r o b a b l y fibroblasts. Fibroblasts in tissue culture do n o t show N G F R i m m u n o r e a c tivity [37, 39]; n e i t h e r have fibroblasts in the distal s e g m e n t of a t r a n s e c t e d nerve b e e n r e p o r t e d as showing any N G F R immunoreactivity. N G F m R N A , however, has b e e n d e t e c t e d in fibroblast-like cells in the distal s e g m e n t o f e x p e r i m e n t a l l y transected nerves [13] and this m a y indicate a functional role for s o m e specialised fibroblasts in the r e g e n e r a t i o n o f nerve fibres. M a c r o p h a g e s in lesioned peripheral nerves m a y also be e l o n g a t e d and c o m e to r e s e m b l e fibroblasts [23]. T h e cells in o u r material, however, were longer and less stellate t h a n the m a c r o p h a g e s described by P e r r y et al. [23].
11.
12. 13.
14.
15. 16. 17.
18.
19. 20. 21.
References 22. 1. Adams JC (1981) Heavy metal intensification of DAB-based HRP reaction products. J Histochem Cytochem 29: 775-780 2. Banerjee SR Snyder SH, Cuatrecasas R Green LA (1973) Binding of nerve growth factor in superior cervical ganglia. Proc Natl Acad Sci USA 70:2519-2523 3. Bonduelle M (1975) Amyotrophic lateral sclerosis. Handb Clin Neurol 22:281-338 4. Chesa PG, Rettig WJ, Thomson TM, Old L J, Melamed MR (1988) Immunohistochemical analysis of nerve growth factor receptor expression in normal and malignant human tissues. J Histochem Cytochem 36:383-389 5. Dayan AD, Graveson GS, Illis LS, Robinson PK (1969) Schwann cell damage in motoneuron disease. Neurology 19: 242-246 6. Dyck PJ, Stevens JC, Mulder DW, Espinosa RE (1975) Frequency of nerve fiber degeneration of peripheral motor and sensory neurons in amyotrophic lateral sclerosis: morphometry of deep and superficial peroneal nerves. Neurology 25: 781-787 7. Ernfors E Henschen A, Olson L, Persson H (1989) Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons. Neuron 2:1605-1613 8. Frazier WA, Boyd LE Bradshaw RA (1974) Properties of specific binding of lZSI-nerve growth factor to responsive peripheral neurons. J Biol Chem 249:5513-5519 9. Greene LA, Shooter EM (1980) The nerve growth factor: biochemistry, synthesis, and mechanism of action. Annu Rev Neurosci 3:353-402 10. Hanyu N. Oguchi K, Yanagisawa N, Tsukagoshi H (1982) Degeneration and regeneration of ventral root motor fibers in
23.
24.
25. 26. 27. 28.
29.
30.
amyotrophic lateral sclerosis. Morphometric studies of cervical ventral roots. J Neurol Sci 55:99-115 Hefti E Hartikka J, Salvatierra A,Weiner WJ, Mash DC (1986) Localization of nerve growth factor receptors in cholinergic neurons of the human basal forebrain. Neurosci Lett 69: 37-41 Herrup K, Shooter EM (1973) Properties of the B-nerve growth factor receptor of avian dorsal root ganglia. Proc Natl Acad Sci USA 70:3884-3888 Heumann R, Korsching S, Bandtlow C, Thoenen H (1987) Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection. J Cell Biol 104: 1623-1631 Hilderink PH, Veldman H, Jennekens FGI (1989) A simple method for immunostaining of axons in plastic embedded material. Proceedings of the 30th Dutch Federation meeting. Dutch Foundation Federation of Medical Scientific Societies, Maastricht. Abstr no 169 Johnson EM, Rich KM, Yip HK (1986) The role of NGF in sensory neurons in vivo. Trends Neurosci 9:33-37 Johnson EM,TaniuchiM, DiStefanoPS (1988) Expression and possible function of nerve growth factor receptors in Schwann cells. Trends Neurosci 11:299-304 Johnson GD, Davidson RS, McNamee KC, Russell G, Goodwin D, Holborow EJ (1982) Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy. J Immuuol Methods 55:231-242 KawamuraY, Dyck PJ, Masatake S, Okazaki H,Tateishi J, Doi H (1981) Morphometric comparison of the vulnerability of peripheral motor and sensory neurons in amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 40:(;67-675 Levi-Montalcini R (1987) The Nerve Growth Factor 35 years later. Science 237:1154-1162 Levi-Montalcini R, Angeletti P (1968) Nerve growth factor. Physiol Rev 48:534-569 Lindsay RM (1979) Adult rat brain astrocytes support survival of both NGF-dependent and NGF-insensitive neurones. Nature 282:80-82 Ohnishi Y, Makifuchi T, Ikuta F (1980) Morphometric investigation of the myelinated fibers of the anterior spinal roots in amyotrophic lateral sclerosis and Shy-Drager syndrome. Clin Neurol 20:809-815 Perry VH, Brown MC, Gordon S (1987) The macrophage response to central and peripheral nerve injury. A possible role for macrophages in regeneration. J Exp Med 165: 1218-1223 Raivich G, Kreutzberg GW (1987) Expression of growth factor receptors in injured nervous tissue. I. Axotomy leads to ashift in the cellular distribution of specific t3-nerve growth factor binding in the injured and regenerating PNS. J Neurocytol 16:689-700 Raivich G, Zimmermann A, Sutter A (1985) The spatial and temporal pattern of [3NGF receptor expression in the developing chick embryo. EMBO J 4:637-644 Schatteman GC, Gibbs L, Lanahan AA, Claude P, Bothwell M (1988) Expression of NGF receptor in the developing and adult primate central nervous system. J Neurosci 8:860-873 Snider WD, Johnson EM (1989) Neurotrophic molecules. Ann Neurol 26:489-506 Sobue G, Matsuoka Y, Mukai E, Takayanagi T, Sobue I, Hashizume Y (1981) Spinal and cranial motor nerve roots in amyotrophic lateral sclerosis and X-linked recessive bulbospihal muscular atrophy: morphometric and teased fiber study. Acta Neuropathol (Berl) 55:227-235 Sobue G, MatsuokaY, Mukai E,Takayanagi T, Sobue t (1981) Pathology of myelinated fibers in cervical and lumbar ventral spinal roots in amyotrophic lateral sclerosis. J Neurol Sci 50: 413-421 Taniuchi M, Clark HB, Johnson EM (1986) Induction of nerve growth factor receptor in Schwann cells after axotomy. Proc Natl Acad Sci USA 83:4094-4098
656 31. Taniuchi M, Clark HB, Schweitzer JB, Johnson EM (1988) Expression of nerve growth factor receptors by Schwann cells of axotomized peripheral nerves: ultrastmctural location, suppression by axonal contact, and binding properties. J Neurosci 8:664-681 32. Telerman-Toppet N, CoOrs C (1978) Motor innervation and fiber-type pattern in amyotrophic lateral sclerosis and in Charcot-Marie-Tooth disease. Muscle Nerve 1:133-139 33. Thoenen H, Barde YA (1980) Physiology of nerve growth factor. Physiol Rev 60:1284-1335 34. Wohlfart G, Swank PL (1941) Pathology of amyotrophic lateral sclerosis, fiber analysis of the ventral roots and pyramidal tracts of the spinal cord. Arch Neurol Psychiatry 46: 783 -799 35. Wood SJ, Pritchard J, Sofroniew MV (1990) Re-expression of nerve growth factor receptor after axonal injury recapitulates a
36. 37. 38. 39.
developmental event in motor neurons: differential regulation when regeneration is allowed or prevented. Eur J Neurosci 2: 650-657 Yah Q, Johnson EM (1988) An immunohistochemical study of the nerve growth factor receptor in developing rats. J Neurosci 8:3481-3498 Yasuda T, Sobue G, Mokuno K, Kreider B, Pleasure D (1987) Cultured rat Schwann cells express low affinity receptors for nerve growth factor. Brain Res 436:113-119 Yip HK, Johnson EM (1987) Nerve growth factor receptors in rat spinal cord: an autoradiographic and immunohistochemical study. Neuroscience 22:267-279 Zimmermann A, Sutter A (1983) B-Nerve growth factor (~NGF) receptors on glial cells. Cell-cell interaction between neurones and Schwann cells in cultures of chick sensory ganglia. EMBO J 2:879-885
