Molecular Brain Research, 15 (1992) 291-297
291
© 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00 BRES 70483
Induction of nerve growth factor receptor (p75 NGFr)m R N A within hypoglossal motoneurons following axonal injury Robert C. Hayes a, Ronald G. Wiley b and David M. Armstrong
a
a Department of Anatomy and Cell Biology /FGIN, Georgetown University, Washington, DC 20007 (USA) and b Departments of Neurology and Pharmacology, VA Medical Center, Nashville, TN 37212 (USA)
(Accepted 19 May 1992)
Key words: Nerve growth factor receptor; Motoneuron; Transection; Axonal crush; Vincristine; Regeneration; In situ hybridization
The hypoglossal nerve is a useful model system for analysis of gene expression in injured motoneurons. In particular, we sought to determine whether the increased appearance of the low affinity nerve growth factor receptor (p75NGFr)observed immunocytochemically following nerve injury can be directly correlated to increased levels of the p75 NGFr mRNA. The present study also examined the relative effects of nerve crush versus nerve transection on the expression of p75 NGFr mRNA. In sham-operated or intact animals, p75 NGFr mRNA is detected rarely and then only at levels slightly higher than background. Following unilateral transection or crush of the rat hypoglossal nerve, the levels of p75 NGFrmRNA increase in a time dependent fashion that parallels the appearance of the protein as reported previously. Moreover, this increase in p75 NGFr mRNA following transection is dependent on a signal from the injured site, since blockage of axonal transport with vincristine also blocks the increased p75 rqGFrmRNA levels. When comparing the effect of nerve crush to nerve transection, we observed that the intensity of the response was greater in the crush paradigm versus that observed following transection. The duration of the response following nerve crush was shorter than that observed following transection of the nerve. The increase in p75 NGFr mRNA after crush was most robust 4 days postlesion and appeared more robust primarily due to a 90-150% increased number of motoneurons expressing p75NGFr mRNA when compared to nerve transection. These data suggest that nerve crush is more effective than nerve transection in eliciting increased p75 NGF~mRNA levels.
INTRODUCTION F o r several r e a s o n s , h y p o g l o s s a l m o t o n e u r o n s r e p r e sent a useful m o d e l for studying h o w injury m o d i f i e s the expression of various gene products. In particular, t h e h y p o g l o s s a l n u c l e u s has a well c h a r a c t e r i z e d disc r e t e n u c l e a r o r g a n i z a t i o n with a p u r e s o m a t o m o t o n e u r o n p r o j e c t i o n t h a t is r e a d i l y accessible to m a n i p u l a t i o n with v a r i o u s agents. Previously, it h a d b e e n r e p o r t e d t h a t t r a n s e c t i n g o r c r u s h i n g t h e hyp o g l o s s a l n e r v e p r o d u c e s a fall in c h o l i n e a c e t y l t r a n s f e r a s e (CHAT) i m m u n o r e a c t i v i t y in h y p o g l o s s a l m o t o r neurons and causes the concomitant appearance of n e r v e g r o w t h f a c t o r r e c e p t o r (p75 NGFr) p r o t e i n t't2'29. T h e f u n c t i o n a l significance o f this l a t t e r o b s e r v a t i o n is u n c l e a r l a r g e l y b e c a u s e it h a s b e e n g e n e r a l l y a s s u m e d t h a t a d u l t m o t o n e u r o n s a r e n o t r e s p o n s i v e to N G F u n d e r n o r m a l c o n d i t i o n s 26. H o w e v e r , s o m e p75 NGFrpositive i m m u n o r e a c t i v i t y has b e e n o b s e r v e d in t h e rat
h y p o g l o s s a l n u c l e u s following i n t r a c e r e b r o v e n t r i c u l a r i n j e c t i o n o f colchicine 18. M o r e o v e r , c r u s h i n g t h e sciatic n e r v e r e s u l t e d in e x p r e s s i o n o f p75 NGFr m R N A within spinal c o r d m o t o n e u r o n s 5. I n t e r e s t i n g l y , sciatic n e r v e damage induces expression of these same NGF receptors within S c h w a n n cells in t h e vicinity o f t h e i n j u r e d site 8'24'25. E x p r e s s i o n o f p75 NGFr by S c h w a n n cells is s u p p r e s s e d as t h e r e g e n e r a t i n g axons r e i n s t a t e c o n t a c t with the S c h w a n n cell surface s u p p o r t i n g t h e view t h a t p75 NGFr m a y well p a r t i c i p a t e in s o m e functions i m p o r t a n t to m o t o n e u r o n r e g e n e r a t i o n . R e c e n t e x p e r i m e n t s in which we o b s e r v e d t h a t p75 yGFr i m m u n o r e a c t i v i t y can b e i n h i b i t e d following a d m i n i s t r a t i o n o f vincristine (a m i c r o t u b u l e i n h i b i t o r t h a t blocks fast axonal transp o r t ) at a l o c a t i o n b e t w e e n t h e i n j u r e d site a n d t h e cell b o d y suggest t h a t a signal m a y o r i g i n a t e f r o m t h e i n j u r e d site itself a n d be r e t r o g r a d e l y t r a n s p o r t e d 16. In o r d e r to test w h e t h e r such a signal l e a d s to i n c r e a s e d p75 NGFr m R N A levels within m o t o n e u r o n s following
Correspondence: R.C. Hayes, Department of Anatomy and Cell Biology/FGIN, Washington, DC 20007, USA.
292 injury, we investigated the affects of nerve transection c o m p a r e d to nerve crush on p75 Nc;v~ m R N A levels in rat hypoglossal m o t o n e u r o n s using in situ hybridization histochemistry. MATERIALS AND METHODS
Aninlals attd surgical procedures In the present study, we used tissue sections from the same animals as in our immunocytochemical study i. Details of the surgical procedures used to injure the XIIth nerve were previously described ~'>. Sham-oper-
ated animals were closed after exposing the nerve. W o u n d s were closed with Michel w o u n d clips and animals returned to cages for one of several time periods. At least two animals each were allowed to survive for periods of 1, 4, 9, 30 and 90 days following unilateral transection of the hypoglossal nerve and 1, 4, 9, 12 and 31 days following unilateral nerve crush. After the appropriate survival time, the rats were anesthetized (i.p.) and perfused with fresh 4% formaldeh y d e / 0 . 1 M sodium phosphate buffer (PB). Following perfusion, brains were removed and the brainstems sectioned at 40 p,m with a sliding microtome. All tissue
ld
30d
B
£
4d
90d
C
F
Fig. 1. Autoradiograms from [)SS]p75NGFrriboprobe hybridized to tissue sections following unilateral transection of the rat hypoglossal nerve: (A) sham control: (B) I day; (C) 4 days; (D) 9 days; (E) 30 days; and (F) 90 days posllesion.
293 sections were stored in a cryoprotectant solution as previously described ~. It is our experience that tissue sections can be stored in cryoprotectant solution at - 2 0 ° C for many months without any effect on the quality of the in situ hybridization signal. For the vincristine studies, 50 /xg vincristinc sulfate was applied proximal to the transected nerve followed by a 7 day survival period as described by Moix et al. j6
In situ hybridization histochemistry In situ hybridization histochemistry was carried out after permeabilizing 'free-floating' tissue sections sequentially for 2 × 3 min in 0.1 M glycine/PB; 15 min in 0,3% Triton X-100/PB; 30 min at 37°C in 1 / z g / m l
proteinase K; and then postfixing 5 min in fresh 4% f o r m a l d e h y d e / P B . Tissue sections were rinsed with PB following each step. Throughout these experiments riboprobes were labeled with [35S]CTP. The p75 NG~r plasmid was kindly provided by Dr. C.R. Buck 4. After linearization with EcoRl, a 270-nucleotide antisense probe was transcribed with T3 R N A polymerase (BRL) against a portion of the 5' coding region of the rat p75 NGFr gene (bases 430-700) 19. Prehybridization was carried out for at least 1 h at 55°C in 1.67 × SSPE (1 × = 0.18 M N a C I - 1 0 mM NaPO4, pH 7.4-1 mM EDTA), 50% deionized formamide, 1 × Denhardt's, 25 mM DTT, 10% PEG-8000, 0.1% SDS, 100 /xg/ml d e n a t u r e d / s h e a r e d herring sperm DNA. Tissue was
9d
ib
D 12d
E
Fig. 2. Autoradiograms from [35Slp75NGFrriboprobe hybridized to tissue sections following unilateral crush of the rat hypoglossal nerve: (A) sham control; (B) 1 day; (C) 4 days; (D) 9 days; (E) 12 days: and (F) 31 days postlesion.
m
295 TABLE I Number of hypoglossal neurons expressing p75 NGFr m R N A 4 days postlesion
The values represent the mean number of cells per tissue section. For each animal 4 tissue sections were examined. The tissue sections were examined under the light microscope and within each section the number of cells expressing p75 NGFr mRNA were manually counted. The hypoglossal nucleus was divided into two zones (i.e., dorsal and ventral) in order to ascertain whether any regional differences existed. Standard deviations are included. Region
Dorsal Ventral
Transection
Crush
A l,g. %
Animal I
Animal 2
Animal 3
Animal 4
increase
11+5 23 _+7
8:t:2 18-+9
21_+3 39 + 9
26+8 39 ± 5
153% 91%
hybridized for 16-18 h at 55°C in prehybridization solution to which 10/zl of denatured probe (4 min at 95°C) was added to obtain a total volume equal to 0.25 ml (8-10 x 10 6 cpm per section). Following hybridization, tissue sections were washed at 55°C using 2 × SSC (1 × = 0.15 M NaC1-0.015 M sodium citrate, p H 7.0) + 10 mM D T T for 40 min; 25 i z g / m l RNAse A in 0.5 M N a C I / T E for 40 min; 2 x SSC + 5 mM DT-I" twice for 20 min; and 0.1 × SSC + 5 mM D D T twice for 20 min each.
1495 /.~m2. Only those grain clusters in which the number of silver grains per unit area were at least 2-fold above background values were used. Background levels were defined as the mean of five separate calculations within a region surrounding each the hypoglossal nuclei. Importantly, in this experiment the crushed and transected rats were processed in parallel in order to control for any variability in the in situ hybridization procedure itself. RESULTS
Data analysis and quantitation
Tissue sections were mounted onto glass slides and exposed to Dupont Cronex 4 X-ray film for 6 days. After developing the film, slides were dehydrated through graded alcohol, air dried and dipped in Kodak NTB-2 photographic emulsion diluted 1 : 1 with d H 2 0 . The slides were developed 18 days later in D19 and fixed in F-5 fixative, counterstained for Nissl substance, dehydrated through graded alcohols, coverslipped with D P X mountant and examined under the light microscope using dark field optics. The number of grain clusters were counted manually for each of the various time points. In order to more quantitatively assay the effect of the two experimental paradigms, we selected tissue sections from the 4 day survival group and carried out grain counts using an IBAS imaging system. The imaging system was attached to a Zeiss microscope. All counts were carried out under bright field optics using a 40 × objective, as described by WeissWunder and Chesselet 27. In this study we determined the density of silver grains within a predefined circle of
In sham-operated or non-operated control rats, little if any evidence of p75 N~Fr m R N A was observed in the autoradiograms (Figs. 1 and 2). However, as early as 1 day following transection, p75 NGFr message levels began to increase in the hypoglossal nucleus ipsilateral to the lesion. The amount of p75 NGFr m R N A continued to rise and reached maximum levels 4 days postlesion and thereafter maintained these levels throughout the first month. By 90 days posttransection, p75 N~Fr message content had returned to basal levels. In contrast, crushing the nerve (Fig. 2) resulted in a more robust elevation of p75 NGFr m R N A levels than that observed following transection of the nerve, even though the duration of the response was briefer. Following nerve crush, p75 N~vr m R N A levels peaked approximately 4 days following crush, decreasing steadily at 9 and 12 days survival, and returning back to basal levels by 31 days. In order to assess further the response of motoneurons to nerve injury, the mounted tissue sections were
TABLE II Mean grain counts within hypoglossal neurons 4 days postlesion
The mean number of grains counted in a defined area of 1495 ~m 2 were determined using the IBAS imaging system(using a 40× objective). For each animal a total of 4 tissue sections were assayed. Standard deviations are included. Region
Dorsal Ventral
Transection
Crush
Avg. %
Animal 1
Animal 2
Animal 3
Animal 4
increase
111 + 48 148+ 64
86 _+40 95 + 59
145 + 95 184+ 119
116+ 56 156+ 82
32% 40%
296 dipped in photographic emulsion, developed, and examined under dark field optics (Fig. 3). Specifically, we focused on the 4 day time point since it correlated with a peak response in both transected and crush paradigms. Initial observations suggested that the number of cells showing a positive signal were increased following nerve crush when compared to transected nerve. Moreover, the intensity of silver grains within these individual clusters appeared to be greater following nerve crush. After further assaying cell number using the IBAS imaging system, the data indicated that the more robust response observed 4 days following nerve crush (Figs. 2 and 3) when compared to nerve transection (Figs. 1 and 3) was largely due to a 90-150% increased recruitment of cells expressing p75 NGvr mRNA (Table I). Furthermore, a 30% to 40% increase in overall grain density per unit area suggested that the more robust response following nerve crush may also be attributed to increased amount of message per cell (Table II). In order to address whether a signal originating from the injured site accounts for the increased p75 NGF~ mRNA levels, vincristine sulfate (50 /~g) was applied proximal to the transected nerve stump and animals were sacrificed 7 days postlesion. The increased signal normally seen 7 days following nerve transection was not present when axonal transport was blocked. In fact, vincristine treatment of the transected nerve gave results indistinguishable from non-operated control animals (data not shown). DISCUSSION The current study demonstrates that: (1) injury to the rat hypoglossal nerve results in an increase in the steady state levels of p75 Ncvr message; and, (2) nerve crush results in a shorter, yet more robust response than transecting:the nerve. Furthermore, a time dependent increase in p75 NGFr m R N A is demonstrated in a manner analogous to that previously shown immunocytochemically using the 192 IgG antibody 1. This antibody has been identified by others as recognizing the low affinity N G F receptor (i.e. p75NGFr) 6'15. These results also indicate that the increased protein levels reported previously are correlated with increased levels of p75 NGF~ message rather than a buildup of non-transported p75 NGFr protein. Whether the protein is derived entirely from de novo synthesis is not entirely clear, since very low levels of protein and associated mRNA could occasionally be observed in sham-operated and non-operated control rats. However, the vincristine study presented here and that by Moix et al. ~6 suggest de novo protein synthesis of p75 NGvr likely predominates.
The present findings indicating that nerve crush results in a more robust yet briefer elevation of p75 NGFr mRNA levels is largely in agreement with the report of Saika et al. 21, who observed a comparable effect following crush and transection of rat facial nerve using a rat oligonucleotide p75 NGFr probe. In their study and the present one, the response was maximum at approximately 4 days postlesion and was attributed to an increased number of cells expressing p75 NGEr mRNA in the crushed versus transected paradigm. However, the present study also suggests that the more robust response may in part be attributed to an increase in the number of p75 NGrr m R N A molecules per cell following nerve crush. However, these latter measurements may severely underestimate the differences in grain counts between crushed and transected motoneurons since in the crushed paradigm the larger number of silver grains were often juxtaposed to one another thus resulting in closely packed grains being imaged as a single grain 2'28. Despite this potential shortcoming, the data supports the notion that crushing the nerve is a more effective stimulus in eliciting increased p75 NGFr m R N A levels. The difference in duration of the response of motoneurons following nerve crush and transection may in part reflect the ability of crushed motoneurons to reinnervate their target, as demonstrated previously by Wood et al. 29 in their studies with hypoglossal motoneurons and again by Saika et al. 21 with facial motoneurons. It is of interest, though, that target reinnervation likely is not a prerequisite for long term hypoglossal motoneuron survival, as demonstrated by Snider and Thanedar 23. They reported that the majority of hypoglossal motoneurons survive complete nerve transection for periods up to 1 year, consistent also with our previously reported observations 1. Therefore, the decreased number of motoneurons expressing the p75 NcFr message is likely not the result of fewer motoneurons surviving nerve transection. In our hands, crushing the nerve is more injurious to the surrounding perineurium, including Schwann cells, when compared to a surgically precise transection. In an elegant series of studies, nonneuronal cells surrounding an injured site have been shown to express both NGF and p75 NGFr5"7`8'24,25.It is believed that both proteins may be involved in the regeneration of the injured peripheral nerve since expression of both molecules is inhibited following contact of the Schwann cells with the growing axon 7'8'24'25. Previous studies 9 have demonstrated that N G F infusion can increase the in vivo expression of p75 NcFr mRNA, thus raising the possibility that the injury provoked induction of NGF by Schwann ceils may in an analogous fashion cause an
297 i n d u c t i o n of p75 NGFr m R N A within hypoglossal mot o n e u r o n s . This possibility is c o n s i s t e n t with the vincristine d a t a n o t e d in this study a n d a r e c e n t study in which we suggested that the de novo expression of p75 N°Fr is likely in r e s p o n s e to a signal g e n e r a t e d at the site of local n e r v e injury a n d which is c o m m u n i cated d o w n s t r e a m to the cell soma x6. However, induction of p75 NGFr does not imply N G F participates directly in a physiological response, b u t may act t h r o u g h some o t h e r Signaling m e c h a n i s m . I n any case, what this signal is a n d how it affects p75 NGFr expression is currently u n k n o w n . W h e t h e r the low affinity N G F receptor p r o b e used in these studies implies trophic s u p p o r t (e.g. N G F 6, B D N F 2°, NT-5 3, C D F 14, C N T F 17'22) is necessary for r e g e n e r a t i o n of hypoglossal m o t o n e u r o n s can n o t be d e t e r m i n e d u n t i l similar studies are carried out with probes specific to o t h e r genes recently isolated, such as the different (i.e. high affinity-confiring) trk g e n e s !°'tl'13. REFERENCES 1 Armstrong, D.M., Brady, R., Hersh, L.B., Hayes, R.C. and Wiley, R.G., Expression of choline acetyltransferase and nerve growth factor receptor within hypoglossal motoneurons following nerve injury, J. Comp. Neurol., 304 (1991) 596-607. 2 Beckman, W.C., Evaluation of systems for quantitation of autoradiograms, J. Histochem. Cytochem., 29 (1981) 212-215. 3 Berkemeier, L.R., Winslow, J.W., Kaplan, D.R., Nikolics, K., Goeddel, D.V~ and Rosenthal, A., Neurotrophin-5: a novel neurotrophic factor that activates trk and trkB, Neuron, 7 (1991) 857-866. 4 Buck, C.R., Martinez, H.J., Chao, M.V. and Black, I.B., Differential expression of the nerve growth factor receptor gene in multiple brain regions, Dev. Brain Res., 44 (1988) 259-268. 5 Enfors, P., Henschen, A., Olson, L. and Persson, H., Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons, Neuron, 2 (1989) 1605-1613. 6 Hempstead~ B.L.; Martin-Zanca, D., Kaplan, D.R., Parada, L.F. and Chap, M.V., High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor, Nature, 35~0(1991) 678-682. 7 Heumann, R., Korsching, S., Bandtlow, C. and Thoenen, H., Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection, J. Cell Biol., 104 (1987) 1623-1631. 8 Heumann, R., Lindholm, D., Bandtlow, C., Meyer, M., Radeke, M.J., Misko, T.P., Shooter, E. and Thoenen, H., Differential regulation of mRNA encoding nerve growth factor and its receptor in rat sciatic nerve during development, degeneration, and regulation: role of macrophages, Proc. Natl. Acad. Sci. USA, 84 (1987b) 8735-8739. 9 Higgins, G.A., Koh, S., Chen, K.S. and Gage, F.H., NGF induction of NGF-receptor gene expression and cholinergic neuronal hypertrophy in the basal forebrain of the adult rat, Neuron, 3 (1989) 247-256. 10 Klein, R., Parada, L.F., Coulier, F. and Barbacid, M., trkB, a
novel tyrosine protein kinase receptor expressed during mouse neural development, EMBO J., 8 (1989) 3701-3709. 11 Lamballe, F., Klein, R. and Barbacid, M., trkC, a new member of the trk family of tyrosine protein kinases, is a receptor for neurotrophin-3, Cell, 66 (1991) 967-979. 12 Lams, B.E., Isacson, O. and Sofroniew, M.V., Loss of transmitter-associated enzyme following axotomy does not indicate death of brainstem cholinergic neurons, Brain Res., 475 (1988) 401-406. 13 Martin-Zanca, D., Oskam, R., Mitra, G., Copeland, T. and Barbacid, M., Molecular and biochemical characterization of the human trk proto-oncogene, Mol. Cell. Biol., 9 (1989) 24-33. 14 McManaman, J.L., Oppenheim, R.W., Prevette, D. and Marchetti, D., Rescue of motoneurons from cell death by a pui'ified skeletal muscle polypeptide: effects of the ChAT development factor CDF, Neuron, 4 (1990) 891-898. 15 Meakin, S.O. and Shooter, E.M., Molecular investigations on the high affinity nerve growth factor receptor, Neuron, 66 (1991) 152-163. 16 Moix, L.J., Greeson, D.M., Armstrong, D.M. and Wiley, R.G., Separate signals mediate hypoglossal motor neurons response to axonal injury, Brain Res., in press. 17 Oppenheim, R.W., Prevette, D., Qin-Wei, Y., Collins, F. and MacDonald, J., Control of embryonic motoneuron survival in vivo by neurotrophic factor, Science, 251 (1991) 1616-1618. 18 Pioro, E.P. and Cuello, A.C., Distribution of nerve growth factor receptor-like immunoreactivity in the adult rat central nervous system. Effect of colchicine and correlation with the cholinergic system--II. Brainstem, cerebellum and spinal cord, Neuroscience, 34 (1990) 89-110. 19 Promega Protocols and Application GuMe, 2nd edn., 1991. 20 Rodriguez-Taber, A., Dechant, G. and Barde, Y.-A., Binding of brain-derived neurotrophic factor to the nerve growth factor receptor, Neuron, 4 (1990) 487-492. 2l Saika, T., Senba, E., Noguchi, K., Sato, M., Yoshida, S., Kubo, T., Matsunaga, T. and Tohyama, S., Effects of nerve crush and transection on mRNA levels for nerve growth factor receptor in the rat facial motoneurons, Mol. Brain Res., 9 (1991) 157-160. 22 Sendtner, M., Kreutzberg, G.W. and Thoenen, H., Ciliary neurotrophic factor prevents the degeneration of motor neurons after axotomy, Nature, 345 (1990) 440-441. 23 Snider, W.D. and Thanedar, S., Target dependence of hypoglossal motor neurons during development and in maturity, J. Comp. Neurol., 279 (1989) 489-498. 24 Taniuchi, M. and Johnson, E.M., Induction of nerve growth factor receptor in Schwann cells after axotomy, Proc. Natl. Acad. Sci. USA, 83 (1986) 4094-4098. 25 Taniuchi, M., Clark, H.B., Schweitzer, J.B. and Johnson, E.M., Expression of nerve growth factor receptors by Schwann cells of axotomized peripheral nerves: ultrastructural location, suppression by axonal contact and binding properties, J. Neurosci., 8 (1989) 664-681. 26 Yip, H.K. and Johnson, E.M., Nerve growth factor receptors in rat spinal cord: an autoradiographic and immunohistochemical study, Neuroscience, 22 (1987) 267-279. 27 Weiss-Wunder, L.T. and Chesselet, M.-F., Subpopulations of mesencephalic dopaminergic neurons express different levels of tyrosine hydroxylase messenger RNA, J. Comp. Neurol., 303 (1991) 478-488. 28 Verge, V.M.K., Riopelle, R.J. and Richardson, P.M., Nerve growth factor receptors on normal and injured sensory neurons, J. Neurosci., 9 (1989) 914-922. 29 Wood, S.J., Pritchard, J. and Sofroniew, M.V., Re-expression of nerve growth factor receptor after axonal injury recapitulates a developmental event in motor neurons: differential regulation when regeneration is allowed or prevented, Fur. J. Neurosci., 2 (1990) 650-657.
291
© 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00 BRES 70483
Induction of nerve growth factor receptor (p75 NGFr)m R N A within hypoglossal motoneurons following axonal injury Robert C. Hayes a, Ronald G. Wiley b and David M. Armstrong
a
a Department of Anatomy and Cell Biology /FGIN, Georgetown University, Washington, DC 20007 (USA) and b Departments of Neurology and Pharmacology, VA Medical Center, Nashville, TN 37212 (USA)
(Accepted 19 May 1992)
Key words: Nerve growth factor receptor; Motoneuron; Transection; Axonal crush; Vincristine; Regeneration; In situ hybridization
The hypoglossal nerve is a useful model system for analysis of gene expression in injured motoneurons. In particular, we sought to determine whether the increased appearance of the low affinity nerve growth factor receptor (p75NGFr)observed immunocytochemically following nerve injury can be directly correlated to increased levels of the p75 NGFr mRNA. The present study also examined the relative effects of nerve crush versus nerve transection on the expression of p75 NGFr mRNA. In sham-operated or intact animals, p75 NGFr mRNA is detected rarely and then only at levels slightly higher than background. Following unilateral transection or crush of the rat hypoglossal nerve, the levels of p75 NGFrmRNA increase in a time dependent fashion that parallels the appearance of the protein as reported previously. Moreover, this increase in p75 NGFr mRNA following transection is dependent on a signal from the injured site, since blockage of axonal transport with vincristine also blocks the increased p75 rqGFrmRNA levels. When comparing the effect of nerve crush to nerve transection, we observed that the intensity of the response was greater in the crush paradigm versus that observed following transection. The duration of the response following nerve crush was shorter than that observed following transection of the nerve. The increase in p75 NGFr mRNA after crush was most robust 4 days postlesion and appeared more robust primarily due to a 90-150% increased number of motoneurons expressing p75NGFr mRNA when compared to nerve transection. These data suggest that nerve crush is more effective than nerve transection in eliciting increased p75 NGF~mRNA levels.
INTRODUCTION F o r several r e a s o n s , h y p o g l o s s a l m o t o n e u r o n s r e p r e sent a useful m o d e l for studying h o w injury m o d i f i e s the expression of various gene products. In particular, t h e h y p o g l o s s a l n u c l e u s has a well c h a r a c t e r i z e d disc r e t e n u c l e a r o r g a n i z a t i o n with a p u r e s o m a t o m o t o n e u r o n p r o j e c t i o n t h a t is r e a d i l y accessible to m a n i p u l a t i o n with v a r i o u s agents. Previously, it h a d b e e n r e p o r t e d t h a t t r a n s e c t i n g o r c r u s h i n g t h e hyp o g l o s s a l n e r v e p r o d u c e s a fall in c h o l i n e a c e t y l t r a n s f e r a s e (CHAT) i m m u n o r e a c t i v i t y in h y p o g l o s s a l m o t o r neurons and causes the concomitant appearance of n e r v e g r o w t h f a c t o r r e c e p t o r (p75 NGFr) p r o t e i n t't2'29. T h e f u n c t i o n a l significance o f this l a t t e r o b s e r v a t i o n is u n c l e a r l a r g e l y b e c a u s e it h a s b e e n g e n e r a l l y a s s u m e d t h a t a d u l t m o t o n e u r o n s a r e n o t r e s p o n s i v e to N G F u n d e r n o r m a l c o n d i t i o n s 26. H o w e v e r , s o m e p75 NGFrpositive i m m u n o r e a c t i v i t y has b e e n o b s e r v e d in t h e rat
h y p o g l o s s a l n u c l e u s following i n t r a c e r e b r o v e n t r i c u l a r i n j e c t i o n o f colchicine 18. M o r e o v e r , c r u s h i n g t h e sciatic n e r v e r e s u l t e d in e x p r e s s i o n o f p75 NGFr m R N A within spinal c o r d m o t o n e u r o n s 5. I n t e r e s t i n g l y , sciatic n e r v e damage induces expression of these same NGF receptors within S c h w a n n cells in t h e vicinity o f t h e i n j u r e d site 8'24'25. E x p r e s s i o n o f p75 NGFr by S c h w a n n cells is s u p p r e s s e d as t h e r e g e n e r a t i n g axons r e i n s t a t e c o n t a c t with the S c h w a n n cell surface s u p p o r t i n g t h e view t h a t p75 NGFr m a y well p a r t i c i p a t e in s o m e functions i m p o r t a n t to m o t o n e u r o n r e g e n e r a t i o n . R e c e n t e x p e r i m e n t s in which we o b s e r v e d t h a t p75 yGFr i m m u n o r e a c t i v i t y can b e i n h i b i t e d following a d m i n i s t r a t i o n o f vincristine (a m i c r o t u b u l e i n h i b i t o r t h a t blocks fast axonal transp o r t ) at a l o c a t i o n b e t w e e n t h e i n j u r e d site a n d t h e cell b o d y suggest t h a t a signal m a y o r i g i n a t e f r o m t h e i n j u r e d site itself a n d be r e t r o g r a d e l y t r a n s p o r t e d 16. In o r d e r to test w h e t h e r such a signal l e a d s to i n c r e a s e d p75 NGFr m R N A levels within m o t o n e u r o n s following
Correspondence: R.C. Hayes, Department of Anatomy and Cell Biology/FGIN, Washington, DC 20007, USA.
292 injury, we investigated the affects of nerve transection c o m p a r e d to nerve crush on p75 Nc;v~ m R N A levels in rat hypoglossal m o t o n e u r o n s using in situ hybridization histochemistry. MATERIALS AND METHODS
Aninlals attd surgical procedures In the present study, we used tissue sections from the same animals as in our immunocytochemical study i. Details of the surgical procedures used to injure the XIIth nerve were previously described ~'>. Sham-oper-
ated animals were closed after exposing the nerve. W o u n d s were closed with Michel w o u n d clips and animals returned to cages for one of several time periods. At least two animals each were allowed to survive for periods of 1, 4, 9, 30 and 90 days following unilateral transection of the hypoglossal nerve and 1, 4, 9, 12 and 31 days following unilateral nerve crush. After the appropriate survival time, the rats were anesthetized (i.p.) and perfused with fresh 4% formaldeh y d e / 0 . 1 M sodium phosphate buffer (PB). Following perfusion, brains were removed and the brainstems sectioned at 40 p,m with a sliding microtome. All tissue
ld
30d
B
£
4d
90d
C
F
Fig. 1. Autoradiograms from [)SS]p75NGFrriboprobe hybridized to tissue sections following unilateral transection of the rat hypoglossal nerve: (A) sham control: (B) I day; (C) 4 days; (D) 9 days; (E) 30 days; and (F) 90 days posllesion.
293 sections were stored in a cryoprotectant solution as previously described ~. It is our experience that tissue sections can be stored in cryoprotectant solution at - 2 0 ° C for many months without any effect on the quality of the in situ hybridization signal. For the vincristine studies, 50 /xg vincristinc sulfate was applied proximal to the transected nerve followed by a 7 day survival period as described by Moix et al. j6
In situ hybridization histochemistry In situ hybridization histochemistry was carried out after permeabilizing 'free-floating' tissue sections sequentially for 2 × 3 min in 0.1 M glycine/PB; 15 min in 0,3% Triton X-100/PB; 30 min at 37°C in 1 / z g / m l
proteinase K; and then postfixing 5 min in fresh 4% f o r m a l d e h y d e / P B . Tissue sections were rinsed with PB following each step. Throughout these experiments riboprobes were labeled with [35S]CTP. The p75 NG~r plasmid was kindly provided by Dr. C.R. Buck 4. After linearization with EcoRl, a 270-nucleotide antisense probe was transcribed with T3 R N A polymerase (BRL) against a portion of the 5' coding region of the rat p75 NGFr gene (bases 430-700) 19. Prehybridization was carried out for at least 1 h at 55°C in 1.67 × SSPE (1 × = 0.18 M N a C I - 1 0 mM NaPO4, pH 7.4-1 mM EDTA), 50% deionized formamide, 1 × Denhardt's, 25 mM DTT, 10% PEG-8000, 0.1% SDS, 100 /xg/ml d e n a t u r e d / s h e a r e d herring sperm DNA. Tissue was
9d
ib
D 12d
E
Fig. 2. Autoradiograms from [35Slp75NGFrriboprobe hybridized to tissue sections following unilateral crush of the rat hypoglossal nerve: (A) sham control; (B) 1 day; (C) 4 days; (D) 9 days; (E) 12 days: and (F) 31 days postlesion.
m
295 TABLE I Number of hypoglossal neurons expressing p75 NGFr m R N A 4 days postlesion
The values represent the mean number of cells per tissue section. For each animal 4 tissue sections were examined. The tissue sections were examined under the light microscope and within each section the number of cells expressing p75 NGFr mRNA were manually counted. The hypoglossal nucleus was divided into two zones (i.e., dorsal and ventral) in order to ascertain whether any regional differences existed. Standard deviations are included. Region
Dorsal Ventral
Transection
Crush
A l,g. %
Animal I
Animal 2
Animal 3
Animal 4
increase
11+5 23 _+7
8:t:2 18-+9
21_+3 39 + 9
26+8 39 ± 5
153% 91%
hybridized for 16-18 h at 55°C in prehybridization solution to which 10/zl of denatured probe (4 min at 95°C) was added to obtain a total volume equal to 0.25 ml (8-10 x 10 6 cpm per section). Following hybridization, tissue sections were washed at 55°C using 2 × SSC (1 × = 0.15 M NaC1-0.015 M sodium citrate, p H 7.0) + 10 mM D T T for 40 min; 25 i z g / m l RNAse A in 0.5 M N a C I / T E for 40 min; 2 x SSC + 5 mM DT-I" twice for 20 min; and 0.1 × SSC + 5 mM D D T twice for 20 min each.
1495 /.~m2. Only those grain clusters in which the number of silver grains per unit area were at least 2-fold above background values were used. Background levels were defined as the mean of five separate calculations within a region surrounding each the hypoglossal nuclei. Importantly, in this experiment the crushed and transected rats were processed in parallel in order to control for any variability in the in situ hybridization procedure itself. RESULTS
Data analysis and quantitation
Tissue sections were mounted onto glass slides and exposed to Dupont Cronex 4 X-ray film for 6 days. After developing the film, slides were dehydrated through graded alcohol, air dried and dipped in Kodak NTB-2 photographic emulsion diluted 1 : 1 with d H 2 0 . The slides were developed 18 days later in D19 and fixed in F-5 fixative, counterstained for Nissl substance, dehydrated through graded alcohols, coverslipped with D P X mountant and examined under the light microscope using dark field optics. The number of grain clusters were counted manually for each of the various time points. In order to more quantitatively assay the effect of the two experimental paradigms, we selected tissue sections from the 4 day survival group and carried out grain counts using an IBAS imaging system. The imaging system was attached to a Zeiss microscope. All counts were carried out under bright field optics using a 40 × objective, as described by WeissWunder and Chesselet 27. In this study we determined the density of silver grains within a predefined circle of
In sham-operated or non-operated control rats, little if any evidence of p75 N~Fr m R N A was observed in the autoradiograms (Figs. 1 and 2). However, as early as 1 day following transection, p75 NGFr message levels began to increase in the hypoglossal nucleus ipsilateral to the lesion. The amount of p75 NGFr m R N A continued to rise and reached maximum levels 4 days postlesion and thereafter maintained these levels throughout the first month. By 90 days posttransection, p75 N~Fr message content had returned to basal levels. In contrast, crushing the nerve (Fig. 2) resulted in a more robust elevation of p75 NGFr m R N A levels than that observed following transection of the nerve, even though the duration of the response was briefer. Following nerve crush, p75 N~vr m R N A levels peaked approximately 4 days following crush, decreasing steadily at 9 and 12 days survival, and returning back to basal levels by 31 days. In order to assess further the response of motoneurons to nerve injury, the mounted tissue sections were
TABLE II Mean grain counts within hypoglossal neurons 4 days postlesion
The mean number of grains counted in a defined area of 1495 ~m 2 were determined using the IBAS imaging system(using a 40× objective). For each animal a total of 4 tissue sections were assayed. Standard deviations are included. Region
Dorsal Ventral
Transection
Crush
Avg. %
Animal 1
Animal 2
Animal 3
Animal 4
increase
111 + 48 148+ 64
86 _+40 95 + 59
145 + 95 184+ 119
116+ 56 156+ 82
32% 40%
296 dipped in photographic emulsion, developed, and examined under dark field optics (Fig. 3). Specifically, we focused on the 4 day time point since it correlated with a peak response in both transected and crush paradigms. Initial observations suggested that the number of cells showing a positive signal were increased following nerve crush when compared to transected nerve. Moreover, the intensity of silver grains within these individual clusters appeared to be greater following nerve crush. After further assaying cell number using the IBAS imaging system, the data indicated that the more robust response observed 4 days following nerve crush (Figs. 2 and 3) when compared to nerve transection (Figs. 1 and 3) was largely due to a 90-150% increased recruitment of cells expressing p75 NGvr mRNA (Table I). Furthermore, a 30% to 40% increase in overall grain density per unit area suggested that the more robust response following nerve crush may also be attributed to increased amount of message per cell (Table II). In order to address whether a signal originating from the injured site accounts for the increased p75 NGF~ mRNA levels, vincristine sulfate (50 /~g) was applied proximal to the transected nerve stump and animals were sacrificed 7 days postlesion. The increased signal normally seen 7 days following nerve transection was not present when axonal transport was blocked. In fact, vincristine treatment of the transected nerve gave results indistinguishable from non-operated control animals (data not shown). DISCUSSION The current study demonstrates that: (1) injury to the rat hypoglossal nerve results in an increase in the steady state levels of p75 Ncvr message; and, (2) nerve crush results in a shorter, yet more robust response than transecting:the nerve. Furthermore, a time dependent increase in p75 NGFr m R N A is demonstrated in a manner analogous to that previously shown immunocytochemically using the 192 IgG antibody 1. This antibody has been identified by others as recognizing the low affinity N G F receptor (i.e. p75NGFr) 6'15. These results also indicate that the increased protein levels reported previously are correlated with increased levels of p75 NGF~ message rather than a buildup of non-transported p75 NGFr protein. Whether the protein is derived entirely from de novo synthesis is not entirely clear, since very low levels of protein and associated mRNA could occasionally be observed in sham-operated and non-operated control rats. However, the vincristine study presented here and that by Moix et al. ~6 suggest de novo protein synthesis of p75 NGvr likely predominates.
The present findings indicating that nerve crush results in a more robust yet briefer elevation of p75 NGFr mRNA levels is largely in agreement with the report of Saika et al. 21, who observed a comparable effect following crush and transection of rat facial nerve using a rat oligonucleotide p75 NGFr probe. In their study and the present one, the response was maximum at approximately 4 days postlesion and was attributed to an increased number of cells expressing p75 NGEr mRNA in the crushed versus transected paradigm. However, the present study also suggests that the more robust response may in part be attributed to an increase in the number of p75 NGrr m R N A molecules per cell following nerve crush. However, these latter measurements may severely underestimate the differences in grain counts between crushed and transected motoneurons since in the crushed paradigm the larger number of silver grains were often juxtaposed to one another thus resulting in closely packed grains being imaged as a single grain 2'28. Despite this potential shortcoming, the data supports the notion that crushing the nerve is a more effective stimulus in eliciting increased p75 NGFr m R N A levels. The difference in duration of the response of motoneurons following nerve crush and transection may in part reflect the ability of crushed motoneurons to reinnervate their target, as demonstrated previously by Wood et al. 29 in their studies with hypoglossal motoneurons and again by Saika et al. 21 with facial motoneurons. It is of interest, though, that target reinnervation likely is not a prerequisite for long term hypoglossal motoneuron survival, as demonstrated by Snider and Thanedar 23. They reported that the majority of hypoglossal motoneurons survive complete nerve transection for periods up to 1 year, consistent also with our previously reported observations 1. Therefore, the decreased number of motoneurons expressing the p75 NcFr message is likely not the result of fewer motoneurons surviving nerve transection. In our hands, crushing the nerve is more injurious to the surrounding perineurium, including Schwann cells, when compared to a surgically precise transection. In an elegant series of studies, nonneuronal cells surrounding an injured site have been shown to express both NGF and p75 NGFr5"7`8'24,25.It is believed that both proteins may be involved in the regeneration of the injured peripheral nerve since expression of both molecules is inhibited following contact of the Schwann cells with the growing axon 7'8'24'25. Previous studies 9 have demonstrated that N G F infusion can increase the in vivo expression of p75 NcFr mRNA, thus raising the possibility that the injury provoked induction of NGF by Schwann ceils may in an analogous fashion cause an
297 i n d u c t i o n of p75 NGFr m R N A within hypoglossal mot o n e u r o n s . This possibility is c o n s i s t e n t with the vincristine d a t a n o t e d in this study a n d a r e c e n t study in which we suggested that the de novo expression of p75 N°Fr is likely in r e s p o n s e to a signal g e n e r a t e d at the site of local n e r v e injury a n d which is c o m m u n i cated d o w n s t r e a m to the cell soma x6. However, induction of p75 NGFr does not imply N G F participates directly in a physiological response, b u t may act t h r o u g h some o t h e r Signaling m e c h a n i s m . I n any case, what this signal is a n d how it affects p75 NGFr expression is currently u n k n o w n . W h e t h e r the low affinity N G F receptor p r o b e used in these studies implies trophic s u p p o r t (e.g. N G F 6, B D N F 2°, NT-5 3, C D F 14, C N T F 17'22) is necessary for r e g e n e r a t i o n of hypoglossal m o t o n e u r o n s can n o t be d e t e r m i n e d u n t i l similar studies are carried out with probes specific to o t h e r genes recently isolated, such as the different (i.e. high affinity-confiring) trk g e n e s !°'tl'13. REFERENCES 1 Armstrong, D.M., Brady, R., Hersh, L.B., Hayes, R.C. and Wiley, R.G., Expression of choline acetyltransferase and nerve growth factor receptor within hypoglossal motoneurons following nerve injury, J. Comp. Neurol., 304 (1991) 596-607. 2 Beckman, W.C., Evaluation of systems for quantitation of autoradiograms, J. Histochem. Cytochem., 29 (1981) 212-215. 3 Berkemeier, L.R., Winslow, J.W., Kaplan, D.R., Nikolics, K., Goeddel, D.V~ and Rosenthal, A., Neurotrophin-5: a novel neurotrophic factor that activates trk and trkB, Neuron, 7 (1991) 857-866. 4 Buck, C.R., Martinez, H.J., Chao, M.V. and Black, I.B., Differential expression of the nerve growth factor receptor gene in multiple brain regions, Dev. Brain Res., 44 (1988) 259-268. 5 Enfors, P., Henschen, A., Olson, L. and Persson, H., Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons, Neuron, 2 (1989) 1605-1613. 6 Hempstead~ B.L.; Martin-Zanca, D., Kaplan, D.R., Parada, L.F. and Chap, M.V., High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor, Nature, 35~0(1991) 678-682. 7 Heumann, R., Korsching, S., Bandtlow, C. and Thoenen, H., Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection, J. Cell Biol., 104 (1987) 1623-1631. 8 Heumann, R., Lindholm, D., Bandtlow, C., Meyer, M., Radeke, M.J., Misko, T.P., Shooter, E. and Thoenen, H., Differential regulation of mRNA encoding nerve growth factor and its receptor in rat sciatic nerve during development, degeneration, and regulation: role of macrophages, Proc. Natl. Acad. Sci. USA, 84 (1987b) 8735-8739. 9 Higgins, G.A., Koh, S., Chen, K.S. and Gage, F.H., NGF induction of NGF-receptor gene expression and cholinergic neuronal hypertrophy in the basal forebrain of the adult rat, Neuron, 3 (1989) 247-256. 10 Klein, R., Parada, L.F., Coulier, F. and Barbacid, M., trkB, a
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