- Email: [email protected]
Morphological changes in spinal motor neurons giving rise to long-term regenerated sciatic nerve axons
Brain Research, 463 (1988) 69-77 Elsevier
69
BRE 14003
Morphological changes in spinal motor neurons giving rise to long-term regenerated sciatic nerve axons C.M. Bowe 1, C.H.
Y u I a n d S.G. W a x m a n 2'3
~Section of Neurobiology and Department of Pediatrics, Brown University, Providence, RI (U.S.A.)and eDepartment of Neurology, Yale University, New Haven, CT (U.S.A.) and 3PVA/EPVA Neuroscience and Regeneration Research Center, VA Hospital, West Haven, CT (U.S.A.)
(Accepted 11 May 1988) Key words: Axon reaction; Chromatolysis; Motor neuron; Nerve injury; Regeneration; Perikaryal response
Morphological properties of rat spinal motor neurons were examined 14-16 months following unilateral sciatic nerve crush and compared to the properties observed in neurons contralateral to injury and in cord segments from age-matched control rats. Regenerated and control motor neurons were identified by retrograde labelling with HRP applied to sciatic nerves distal to the site of crush or at a comparable location in control nerves. Many of the experimental motor neurons were enlarged and had thickened dendritic processess compared to the finer dendrites seen in control cells. Mean cell area ipsilateral to the crush lesions was larger than mean control cell area (P-value < 0.001) despite representation of all control cell areas in the regenerated population. These data suggest that persistent or continued morphological abnormalities occur in mammalian motor neurons following simple sciatic crush injury when examined at extended times beyond the period of axonal regeneration and clinical recovery.
INTRODUCTION The acute sequence of morphological, biochemical and physiological changes in the region of the cell body following axotomy have been extensively reviewed 3A2'23'26. Within hours to days of axonal injury, perikarya of injured fibers exhibit a response (classically termed the chromolytic response) in association with nuclear eccentricity, nucleolar enlargement and cellular swelling. The severity and duration of the acute perikaryal reaction vary depending on age, the species examined, and the lesion model employed 9'26'3436A0"42. In general, the most dramatic and sustained changes are observed following transection and ligation 3'9'27'44. Maximal cell loss, typically occurring within 1-5 weeks of injury, is significantly influenced by the axons' ability to re-establish connection with peripheral targets 9'27"41. Normal morphological properties have been reported by 4 - 6 months in ceils giving rise to fully regenerated axons 2'34'41. Few studies have examined perikaryal
morphology beyond the period during which axonal regeneration is 'completed' and only a limited number have investigated late-occurring changes using retrograde labelling techniques to specifically identify cells of origin of regenerated fibers. Even when peripheral nervous system axons can re-establish continuity with peripheral targets and functional recovery is observed, persistent abnormalities of both physiological and morphological properties of the regenerated fibers are observed beyond one year following nerve crush injury 6A9'2°'22'35. The present study of rat spinal motor neurons was performed to assess whether there are morphological differences, compared to normal, in cell bodies giving rise to long-term regenerated sciatic axons. The results of this examination of regenerated neurons provide evidence for alterations of cell morphology, size and possibly retrograde transport of horseradish peroxidase (HRP) observed beyond one year following nerve injury. Preliminary results of this study were presented in abstract form s .
Correspondence: C.M. Bowe, Section of Neurobiology, Box G, Brown University, Providence, RI 02912, U.S.A.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
70 MATERIALS AND METHODS Four anesthetized (xylazine 10 mg/kg + ketamine 75 mg/kg) Wistar rats underwent unilateral sciatic crush lesions at 4 (2 rats) or 10 (2 rats) weeks of age. The left sciatic nerve was exposed at the level of the sciatic notch and fine dissecting forceps were used to apply uniform pressure for 20 s across the width of the nerve, proximal to the origin of the nerve to the biceps femoris muscle. Experimental and two agematched control animals were similarly housed and maintained for 12-15 months following surgery. Observation of the animals during the recovery period indicated functional recovery of the affected hindpaw by 6-8 weeks following nerve crush. The size of the ipsilateral (reinnervated) and contralateral control limbs was symmetrical. Anesthetized experimental and control animals underwent bilateral transection of sciatic nerves at 14-16 months of age at a site 5-10 mm distal to the site described for sciatic crush or at a comparable location in contralateral and control nerves. A 15-20 mm segment of sciatic nerve, distal to the site of transection was excised for physiological investigation. Proximal sciatic nerve stumps were gently suctioned into a short length of polyethylene tubing which was subsequently filled with 40% HRP VI (Sigma) mixed in Kreb's solution. The distal end of the tubing was sealed with bone wax, the surgical incision was sutured, and the animals were permitted to survive for a period of 48 h prior to sacrifice for histological studies. At the conclusion of the survival period, animals were heavily anesthetized with pentobarbital (50 mg/kg) and perfused via a left ventricular catheter with normal saline containing 50% sodium nitrite until blood cleared from vessels. Animals were then perfused with 700-800 ml of 1% paraformaldehyde, 1% glutaraldehyde, 3% sucrose in 0.1 M PO4 buffer at pH 7.4. At the completion of fixation, the posterior T12-S 1 vertebrae were removed. Left and right L 4, L 5 and L 6 dorsal root ganglia were identified under visualization with a dissecting microscope and the associated ventral and dorsal roots were followed to their point of insertion in the lumbar cord. Spinal cord segments L2_3, L4_6 and S1-2 were removed en bloc and placed in a fixative solution containing 30% sucrose for 1 h prior to transfer to diluted POa buffer.
On the following day, 50 ~tm cord sections were cut on a freezing microtome, rinsed in diluted PO 4 buffer and preincubated in ammonium molybdate and tetramethylbenzidine according to a protocol previously described 29. Sections were gently agitated during incubation with 1.8% H2O 2 for several hours. A portion of the sections was counterstained with neutral red. Sections were examined under light microscopy for the presence and location of HRP labelled motor neurons and were photographed at x310 magnification. Somatic areas were determined for all labelled motor neurons identified in photomicrographs of L4_6 cord segments from 3 lesioned and two control animals. Area was calculated by multiplying the product of two perpendicular radii for each labelled cell 5. Measurements through somatic projections were avoided. A total of 888 cells were evaluated (578 ipsilateral to the sciatic crush, 93 contralateral and 217 from control animals). The clustered distribution and intensity of HRP reaction observed in motor neurons ipsilateral to the lesion (discussed below) may have facilitated their recognition and may account for the increased number of labelled cells identified on the lesioned side. Similarly, the number of dispersed, smaller diameter, motor neurons may have been underestimated in field counts of control specimens. However, by including all labelled cell areas in our calculations, we avoided biasing our cell samples toward the apparent larger cell population in experimental sections, t-Tests were performed between group data derived from individual control and lesioned animals, as well as between summed control and experimental cell groups. RESULTS Segments of sciatic nerve 5-10 mm distal to the crush site were removed for physiological examination at the time that long-term regenerated nerves were transected and exposed to HRP. The physiological properties of these regenerated nerves have been evaluated and will be presented in another paper (in preparation7). Though recordings indicated that fibers had regenerated beyond the site of crush and into the area used for HRP application, sensitivity of regenerated nerves to potassium channel blocking
71
4
l ~!iiil)~]!i~i;~!i~ii!ii!ill
A
C
i~ii? ¸ :;iiiil
!!i!i!!!!!!ili~i!i!i!i!i~!iii!i!i
J
B:
I
Fig. 1. Photomicrographs of lumbar cord sections taken from a 14-month-old rat following left sciatic crush lesion at 10 weeks of age. Sciatic nerves were transected and exposed to H R P for 48 h prior to sacrifice. Cross-sections contralateral to the nerve crush (A and C) are presented with sections from the same cord level ipsilateral to the lesion (B and D, respectively). Labelled motor neurons on the control side were uniformly distributed within the region of sciatic representation. Labelled regenerated cells were more densely clustered and appeared enlarged. T h o u g h individual neurons on both the ipsilateral and contralateral sides varied in the intensity of H R P labelling, in general the regenerated neurons were more densely labelled. Scale bar = 100/~m.
72
P
A
b
C
--
D /
Fig. 2. Photomicrographs of control (A and C) and regenerated (B and D) motor neurons at higher magnifications. A: a section from a contralateral cord section illustrating a large control cell with fine dendritic processes characteristic of normal spinal motor neurons. B: a section ipsilateral to the sciatic crush including small diameter regenerated cells with multiple thickened dendrites (arrows). C and D: fields taken from cord segments contralateral (C) and ipsilateral (D) to the lesion. Thickened and tortuous dendritic processes were often seen in regenerated motor neurons (arrow) but were not observed in contralateral or control cord segments. Scale bar A, B - 50 ktm; C, D = 25/~m.
73 agents was m a r k e d l y increased c o m p a r e d to that observed in contralateral and age-matched control nerves. E x p e r i m e n t a l and control m o t o r neurons examined in cord segments proximal and distal to the L4_ 6 region did not exhibit H R P reaction product. However, within the L4_ 6 segments, localization of the cells of origin of regenerated fibers differed from that observed on the contralateral control sides or in comparable segments from control animals. Paired sections of lumbar cord taken at the same level from the control (Fig. 1A, C) and the lesioned (Fig. 1B, D, respectively) sides are presented in Fig. 1. Intensely labelled nests of large cells were observed in the crushed side (Fig. 1B, D) c o m p a r e d to the m o r e dispersed appearance of labelled m o t o r neurons in the contralateral side (Fig. 1A, C). The less dense pattern of distribution was also observed in sections from control animals. A l t h o u g h large cells were frequently seen on the lesioned side, smaller cells were also present in the same fields. Labelled m o t o r neurons observed contralateral to the side of crush are presented at higher magnification in Fig. 2 A illustrating the characteristic dendritic processes which were typically observed on the nonlesioned side, as well as in cord segments from control animals. In contrast, many of the labelled m o t o r neurons on the lesioned side, even those with smaller somatic areas (Fig. 2B), had thicker dendritic processes (arrows). This difference was frequently observed, even when the largest control cells (Fig. 2C) were c o m p a r e d to large regenerated m o t o r neurons (Fig. 2D arrow). R e g e n e r a t e d m o t o r neurons of all sizes were generally m o r e densely labelled than contralateral control cells; lateralized differences in H R P - r e a c t i o n product were not noted in control animals. Eccentric nuclei were not observed in regenerated m o t o r neurons; enlarged r e g e n e r a t e d cells with thickened dendritic processes were present in fields containing more normal appearing labelled cells. Examination of cord cross-sections suggested that labelled m o t o r neurons ipsilateral to the crush lesions were generally larger than labelled cells on the contralateral side or from control animals. M e a n cell area was calculated for summed control (698 Bm 2 + 19.32 S . E . M . ) and regenerated cell populations (908 # m 2 + 16.47 S . E . M . ) t-Tests p e r f o r m e d b e t w e e n individual control groups (one contralateral cell group
and two control cell groups) and between 3 regenerated cell groups showed no significant differences in cell area within control and regenerated categories. H o w e v e r , differences between summed control and lesioned cell areas were significant at a P-value of <0.001. The distributions of cell areas for control and experimental m o t o r neurons are p r e s e n t e d in Fig. 3, representing cell areas in 200 ~tm 2 bins. Although all area bins seen in the control cell population were r e p r e s e n t e d in the lesioned cell group, regenerated cell areas were generally larger than those of control cells. M o r e o v e r , cell areas >2000 ~tm 2 were only seen in the r e g e n e r a t e d cell population. DISCUSSION The observation of p e r i k a r y a l enlargement and
2S,
CONTROL
20
PERCENT TOTAL CELLS
Is.
,oF s
o
I 200
|
AREA (p2)
2s
PERCENT CELLS
REGENERATED
IS,
10,
5
0 2~
I 600
I 10G0
1400
I i ~'T--1 I ImO 2200
AREA (~2)
Fig. 3. The frequency distribution of control (n 310) and regenerated (n = 578) cell areas segregated into 200~um2 bins. All control area bins are represented in the regenerated population, but a rightward shift of cell areas distribution is noted. There is an increase in absolute area Values of the largest regenerated cells. =
74 thickened dendritic processes in long-term regenerated motor neurons following sciatic nerve crush was unexpected in view of the numerous reports which suggest restoration of normal perikaryal properties following more severe nerve injuries. Previous examinations of the perikaryal response to axonal injury have focused on immediate morphological alterations and the time course of return of normal properties in surviving cell bodies during the period of axonal regeneration 2,26.34,41,42. Though the duration and severity of cell body abnormalities vary for different species and lesion models, in general, properties of cells surviving axonal injury are reported to be comparable to those of control neurons within several months of nerve crush or transection. Inasmuch as our examinations were limited to a period 14-16 months following injury, we cannot differentiate between persistent and progressive changes. The apparent discrepancy between our observation of appreciable morphological changes in longterm regenerated motor neurons and those of previous investigators who have reported normal perikaryal characteristics several months after axonal injury may have resulted from our use of retrograde labelling with HRP. This technique allowed us to distinguish cells of origin of regenerated fibers from non-regenerated or unlesioned cells in the same regions. Morphological differences between lesioned and control motor neurons may have been accentuated by the increased density of HRP-reaction product consistently observed in the regenerated cells. Quantified differences in retrograde labelling between experimental and control cells are not provided by the present study but the observation of differences raises the possibility that altered uptake or
tralateral entorhinal cortex ~4. It is possible that the increased labelling we observed in long-term regenerated motor neurons resulted from the greater number of large cells present in the experimental cell population or from a relatively more intense, acute perikaryal reaction in regenerated vs control cells following nerve transection and HRP exposure 48 h prior to sacrifice. This latter explanation seems unlikely since other features of the immediate perikaryal reaction, i.e. nuclear eccentricity, were not evident. Increases in cell area of up to 160% have been reported for cells examined during the period of maximal chromatolysis 1'2'4'5'27'44. Examinations extended beyond the time of acute perikaryal reaction in motor neurons suggest that cell areas of neurons ipsilateral to lesions return to control values within 1-6 months of crush or axotomy. Barron et al. 5 reported that regenerated and control cervical motor neuron areas were comparable at 30 days following brachial plexotomy. However, at 60, 75 and 90 days post-injury, cell areas ipsilateral to the lesion were larger. Histograms of cell area distribution, 90 days following injury, indicated a shift toward larger cell area in the regenerated vs contralateral control population. Cavanaugh 9 attributed her observation of enlarged mean cell volume in axotomized dorsal root ganglion cells to the selective loss of smaller cell populations. In the present study, frequency distributions indicate that in addition to a reduced proportion of small cells in the regenerated cell population, absolute area values were larger than controls. It is interesting, in this regard, that similar patterns of perikaryal enlargement have been reported in regenerated frog optic nerve, associated with massive cell loss 37, and during active sprouting in both rat entorhinal cortex ~4 and red nucleus 31.
retrograde transport of HRP occurs in long-term regenerated neurons. Increased deposition of HRPreaction product has been described in acutely injured axons 18'29. Halperin and LaVail Is proposed that although delayed uptake of H R P immediately occurs following optic nerve injury, increased accumulation of HRP is subsequently observed in lesioned neurons due to an increased rate of uptake, transport or both or a decrease of the rate of degradation. Both increased cell size and enhanced transport of HRP have been reported for entorhinal neurons during active sprouting into acutely denervated con-
Although the regenerative capacity of the mammalian peripheral nervous system has been emphasized, it is now well established that a number of physiological and morphological abnormalities persist in mammalian axons for many months after peripheral nerve transection despite evidence of successful regrowth and functional recovery. Decreased conduction velocity, initially observed at the time of perikaryal changes and axonal thinning 1°-~2"23, persists in association with shortened internodal distances and the slow acquisition of normal myelin thickness 1°'2°. Similar abnormalities are observed even following le-
75 sions that result in optimal regeneration 17. Several investigators have demonstrated alteration of ionic channel conductances beyond one year following simple nerve crush injury 6'22'35. Hildebrand et a1.19'2° have suggested that altered physiological properties of regenerated axons may, in part, be secondary to the chronic disruption of paranodal myelin sheaths and altered nodal localization along the axonal membrane that is observed following nerve crush in adult rats. The long-term effect of chronic axonal abnormalities on perikaryal properties has not been previously examined. Chronic neuronal alteration following nerve injury is not restricted to regenerated axons but has been reported in reinnervated motor units as well. Examination of motor unit properties at extended periods after initial injury indicate that motor unit number is established within 2 months of injury; motor unit tension returns to normal over a more protracted time c o u r s e 15'16. Gordon and Stein 16 concluded that motor unit tension is maintained, despite loss of some motor axons, by hypertrophy of muscle fibers. Increased axonal diameters have been described following chronic induction of muscle hypertrophy 13'43, particularly in slow, oxidative and fatigue-resistant muscle fibers. Cavanaugh 9 observed a 32% increase in somatic volume of regenerated spinal ganglion cells when peripheral sensory fields were increased for regenerated sensory fibers. The changes we observed in cell area and dendritic morphology may relate to alterations in the size of muscle fibers or receptive fields innervated by regenerated axons or by increased terminal arborization after regeneration. Physiological properties of injured perikarya are dramatically altered during the period of maximal chromatolysis following n e r v e injury 11'12'23'32'41. Somatodendritic excitability is compromised and conduction of impulses through the initial axonal segment is markedly impaired. The association of these physiological changes with observed dendritic retraction 32 and loss of somatic and proximal dendritic excitatory synapses 38'39 has been emphasized. However, Mendell et al. 2s, in a chronically axotomized motor neuron lesion model, reported a dissociation in the timing of initial physiological changes, first observed 6 days post-transection, and the subsequent motor neuron deafferentation occurring 60 days following injury. Further support that initial changes in motor
neuron physiological properties occur independently of presynaptic alterations is provided by Kuno et al. 24 who reported the absence of physiological abnormalities in a motor neurons examined 29-46 days following dorsal root sections. Although most authors suggest that observed abnormalities are reversible when regeneration is successful, data from single cell recordings in long-term regenerated cell bodies are not available. Several lines of evidence indicate that response to injury may differ in the timing of initiation, intensity, and duration for different classes of neurons 15A6'21' 27,30,34,36. Stein and colleagues 15'21'3°, employing a lesion model in which transected mixed nerves were chronically prevented from regenerating with peripheral targets, have shown that fiber degeneration and cell loss differ for motor and sensory neurons. When initially evaluated at 45 days following transection and ligation, fibers from both cell populations exhibited a similar rate of atrophy. However, in physiological examinations at extended periods following injury (145 and 252 days), degeneration progressed at a faster rate and for a longer duration in fast conducting afferent fibers compared to a motor neurons. A differential response to nerve injury in motor and sensory axons could result in a distinctive sequence of alterations in pre- and post-synaptic activity at the motor neuron level. Initial changes in perikaryal size, dendritic arborization and synaptic contacts with la afferent fibers have been described in chromatolytic motor neurons acutely following peripheral transection and crush. Purves and Hadley 33, sequentially examining the dendritic morphology of normal adult rat superior cervical ganglion cells, reported remodelling of postsynaptic elements including extension, retraction and formation of dendrites, during observation periods of up to 3 weeks. If such plasticity is a generalized characteristic of mammalian neurons, dendritic morphology of regenerated motor neurons may be influenced by delayed and/or progressive changes in sensory input. These data emphasize the need for further investigation of cell bodies giving rise to long-term regenerated axons, including further study of long-term neuronal cell body changes following a variety of peripheral nerve lesion models, in order to more clearly elucidate underlying mechanisms. Particular attention
76 s h o u l d be g i v e n to the t i m e c o u r s e of t h e s e p e r i k a r y a l
ACKNOWLEDGEMENTS
c h a n g e s , the b i o c h e m i c a l e v e n t s that u n d e r l i e t h e m and the possibility that d i f f e r e n t p o p u l a t i o n s of n e u -
This w o r k was s u p p o r t e d by T h e R h o d e Island
rons m a y vary in their l o n g - t e r m r e s p o n s e to axonal
F o u n d a t i o n and, in part, by T h e D a n i e l H e u m a n n
injury.
F u n d and the M e d i c a l R e s e a r c h Service, V e t e r a n s Administration.
REFERENCES 1 Aldskogius, H., Barron, K.D. and Regal, R., Axon reaction in dorsal motor vagal and hypoglossal neurons of the adult rat, J. Comp. Neurol., 193 (1980)165-178. 2 Barr, M.L. and Hamilton, J.D., A quantitative study of certain morphological changes in spinal motor neurons during axon reaction, J. Comp. Neurol., 89 (1948) 93-124. 3 Barron, K.D., Comparative observations on the cytologic reactions of central and peripheral nerve cells to axotomy. In C.C. Kao and R.P. Bunge (Eds.), Spinal Cord Reconstruction, Raven, New York, 1982, pp. 7-40. 4 Barron, K.D., Chiang, T.Y., Daniels, A.C. and Doolin, P.F., Subcellular accompaniments of axon reaction in cervical motoneurons of the cat. In H.M. Zimmerman (Ed.), Progress in Neuropathology, Vol. i, Grune and Stratton, New York, 1971, pp. 255-280. 5 Barron, K.D., Cova, J., Scheibly, M.E. and Kohberger, R., Morphometric measurements and RNA content of axotomized feline cervical motoneurons, J. Neurocytol., 11 (1982) 707- 720. 6 Bowe, C.M., Hildebrand, C., Kocsis, J.D. and Waxman, S.G., Physiological properties of regenerated rat sciatic nerve following lesions at different postnatal ages, Dev. Brain Res., 34 (1986) 123-131. 7 Bowe, C.M. and Yu, C.H., Sensitivity of regenerated sciatic nerve to 4-aminopyridine and tetraethylammonium following injury at birth and late maturation, Soc. Neurosci. Abstr., 13 (1987) 1211. 8 Bowe, C.M. and Yu, C.H., Perikaryal changes in spinal motor neurons giving rise to long-term regenerated axons, Soc. Neurosci. Abstr., 14 (1988) in press. 9 Cavanaugh, M.W., Quantitative effects of the peripheral innervation area on nerves and spinal ganglion cells, J. Comp. Neurol., 94 (1951) 181-219. 10 Cragg, B.G. and Thomas, P.K., The conduction velocity of regenerated peripheral nerve fibers, J. Physiol. (Lond.), 171 (1964) 164-175. 11 Czeh, G., Kudo, N. and Kuno, M., Membrane properties and conduction velocity in sensory neurones following central or peripheral axotomy, J. Physiol. (Lond.), 270 (1977) 165-180. 12 Eccles, J.C., Libet, B. and Young, R.R., The behavior of chromatolysed motoneurones studied by intracellular recording, J. Physiol. (Lond.), 143 (1958) 11-40. 13 Edds, M.V., Hypertrophy of nerve fibers to functionally overloaded muscles, J. Comp. Neurol., 93 (1950) 259-275. 14 Goldschmidt, R.B. and Steward, O., Time course of increases in retrograde labelling and increases in cell size of entorhinal cortex neurons sprouting in response to unilateral entorhinal lesions, J. Comp. Neurol., 189 (1980) 359-379. 15 Gordon, T., Hoffer, J.A., Jhamandas, J. and Stein, R.B.,
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Long-term effects of axotomy on neural activity during cat locomotion, J. Physiol. (Lond.), 303 (1980) 243-263. Gordon, T. and Stein, R.B., Time course and extent of recovery in reinnervated motor units of cat triceps surae muscles, J. Physiol. (Lond.), 323 (1982) 307-323. Haftek, J. and Thomas, P.K., Electron-microscope observations on the effects of localized crush injuries on the connective tissues of peripheral nerve, J. Anat., 103 (1968) 233-243. Halperin, J.J. and LaVail, J.H., A study of the dynamics of retrograde transport and accumulation of horseradish peroxidase in injured neurons, Brain Research, 100 (1975) 253-269. Hildebrand, C., Kocsis, J.D., Berglund, S. and Waxman, S.G., Remodelling of internodes in regenerated rat sciatic nerve, Brain Research, 358 (1985) 163-170. Hildebrand, C., Mustafa, G.Y., Bowe, C.M., Berglund, S. and Kocsis, J.D., Nodal spacing along regenerated axons following crush lesion of the developing rat sciatic nerve, Dev. Brain Res., 32 (1986) 147-154. Hoffer, J.A., Stein, R.B. and Gordon, T., Differential atrophy of sensory and motor fibers following section of cat peripheral nerves, Brain Research, 178 (1979) 347-361. Kocsis, J.D., Waxman, S.G., Hildebrand, C. and Ruiz, J.A., Regenerating mammalian nerve fibers: changes in action potential waveform and firing characteristics following blockage of potassium conductance, Proc. R. Soc. Ser. B., 217 (1982) 277-287. Kuno, M. and Llinas, R., Alterations of synaptic action in chromatolysed motoneurones of the cat, J. Physiol. (Lond.), 210 (1970) 823-838. Kuno, M., Miyata, Y. and Munoz-Martinez, E.J., Differential reaction of fast and slow alpha motor neurons to axotomy, J. Physiol. (Lond.), 240 (1974) 725-739. Lavelle, A., Levels of maturation and reactions to injury during neuronal development, Progr. Brain Res., 40 (1973) 161-160. Lieberman, A.R., The axon reaction: a review of the principal features of perikaryal responses to axon injury, Int. Rev. Neurobiol., 14 (1971) 49-124. Lieberman, A.R., Some factors affecting retrograde neuronal responses to axonal lesions. In R. Bellairs and E.G. Grays, (Eds.), Essays on the Nervous System, Clarendon, Oxford, 1974, pp. 71-105. Mendell, L.M., Munson, J.B. and Scott, J.G., Alterations of synapses on axotomized motoneurons, J. Physiol. (Lond.), 255 (1976) 67-79. Mesulam, M.-M., Principles of horseradish peroxidase neuro-chemistry and their application for tracing neural pathways. In M.-M. Mesulam (Ed.), Tracing Neural Connections with Horseradish Peroxidase, Wiley, New York, 1982, pp. 1-151. Milner, T.E. and Stein, R.B., The effects of axotomy on
77
31
32
33
34 35
36
37
the conduction of action potentials in peripheral sensory and motor fibres, J. Neurol. Neurosurg. Psychiat., 44 (1981) 485-496. Prendergast, J. and Stelzner, D.J., Changes in the magnocellular portion of the red nucleus following thoracic hemisection in the neonatal and adult rat, J. Comp. Neurol., 166 (1976) 163-172. Purves, D., Functional and structural changes in mammalian sympathetic neurons following interruption of their axons, J. Physiol. (Lond.), 252 (1975) 429-463. Purves, D. and Hadley, R.D., Changes in the dendritic branching of adult mammalian neurones revealed by repeated imaging in situ, Nature (Lond.), 315 (1985) 404-406. Ranson, S.W., Alterations in spinal ganglion cells following neurotomy, J. Neurol. Psychol., 19 (1909) 125-152. Ritchie, J.M., Sodium and potassium channels in regenerating and developing mammalian myelinated nerves, Proc. R. Soc. Set. B., 215 (1982) 273-287. Romanes, G.J., Motor localization and the effects of nerve iniury on the ventral horn cells of the spinal cord, J. Anat., 80 (1946) 117-131. Stelzner, D.J. and Strauss, J.A., Increase in ganglion cell size after optic nerve regeneration in the frog, Rana pi-
piens, Exp. Neurol., 100 (1988) 210-215. 38 Summer, B.E.H., A quantitative analysis of the response of presynaptic boutons to postsynaptic motor neuron axotomy, Exp. Neurol., 46 (1975) 605-615. 39 Summer, B.E.H. and Watson, W.E., Retraction and expansion of the dendritic tree of motor neurons in adult rats in vivo, Nature (Lond.), 233 (1971) 273-275. 40 Torvik, A., Central chromatolysis and the axon reaction: a reappraisal, Neuropath. Appl. Neurobiol., 2 (1976) 423-432. 41 Torvik, A. and Skjorlen, F., Electron microscopic observations on nerve cell regeneration and degeneration after axon lesions, Acta Neuropath., 17 (1971) 248-264. 42 Torvik, A. and Soreide, A.J., Nerve cell regeneration after axonal lesions in newborn rabbits. Light and electron microscopic study, J. Neuropathol. Exp. Neurol., 31 (1972) 683-695. 43 Walsh, J.V., Burke, R.E., Rymer, W.Z. and Tsairis, P., Effect of compensatory hypertrophy studied in individual motor units in medial gastrocnemius muscle of the cat, J. Neurophysiol., 41 (1978)496-508. 44 Watson, W.E., Observations on the nucleolar and total cell body nucleic acid of injured nerve cells, J. Physiol. (Lond.), 196 (1968) 655-676.
69
BRE 14003
Morphological changes in spinal motor neurons giving rise to long-term regenerated sciatic nerve axons C.M. Bowe 1, C.H.
Y u I a n d S.G. W a x m a n 2'3
~Section of Neurobiology and Department of Pediatrics, Brown University, Providence, RI (U.S.A.)and eDepartment of Neurology, Yale University, New Haven, CT (U.S.A.) and 3PVA/EPVA Neuroscience and Regeneration Research Center, VA Hospital, West Haven, CT (U.S.A.)
(Accepted 11 May 1988) Key words: Axon reaction; Chromatolysis; Motor neuron; Nerve injury; Regeneration; Perikaryal response
Morphological properties of rat spinal motor neurons were examined 14-16 months following unilateral sciatic nerve crush and compared to the properties observed in neurons contralateral to injury and in cord segments from age-matched control rats. Regenerated and control motor neurons were identified by retrograde labelling with HRP applied to sciatic nerves distal to the site of crush or at a comparable location in control nerves. Many of the experimental motor neurons were enlarged and had thickened dendritic processess compared to the finer dendrites seen in control cells. Mean cell area ipsilateral to the crush lesions was larger than mean control cell area (P-value < 0.001) despite representation of all control cell areas in the regenerated population. These data suggest that persistent or continued morphological abnormalities occur in mammalian motor neurons following simple sciatic crush injury when examined at extended times beyond the period of axonal regeneration and clinical recovery.
INTRODUCTION The acute sequence of morphological, biochemical and physiological changes in the region of the cell body following axotomy have been extensively reviewed 3A2'23'26. Within hours to days of axonal injury, perikarya of injured fibers exhibit a response (classically termed the chromolytic response) in association with nuclear eccentricity, nucleolar enlargement and cellular swelling. The severity and duration of the acute perikaryal reaction vary depending on age, the species examined, and the lesion model employed 9'26'3436A0"42. In general, the most dramatic and sustained changes are observed following transection and ligation 3'9'27'44. Maximal cell loss, typically occurring within 1-5 weeks of injury, is significantly influenced by the axons' ability to re-establish connection with peripheral targets 9'27"41. Normal morphological properties have been reported by 4 - 6 months in ceils giving rise to fully regenerated axons 2'34'41. Few studies have examined perikaryal
morphology beyond the period during which axonal regeneration is 'completed' and only a limited number have investigated late-occurring changes using retrograde labelling techniques to specifically identify cells of origin of regenerated fibers. Even when peripheral nervous system axons can re-establish continuity with peripheral targets and functional recovery is observed, persistent abnormalities of both physiological and morphological properties of the regenerated fibers are observed beyond one year following nerve crush injury 6A9'2°'22'35. The present study of rat spinal motor neurons was performed to assess whether there are morphological differences, compared to normal, in cell bodies giving rise to long-term regenerated sciatic axons. The results of this examination of regenerated neurons provide evidence for alterations of cell morphology, size and possibly retrograde transport of horseradish peroxidase (HRP) observed beyond one year following nerve injury. Preliminary results of this study were presented in abstract form s .
Correspondence: C.M. Bowe, Section of Neurobiology, Box G, Brown University, Providence, RI 02912, U.S.A.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
70 MATERIALS AND METHODS Four anesthetized (xylazine 10 mg/kg + ketamine 75 mg/kg) Wistar rats underwent unilateral sciatic crush lesions at 4 (2 rats) or 10 (2 rats) weeks of age. The left sciatic nerve was exposed at the level of the sciatic notch and fine dissecting forceps were used to apply uniform pressure for 20 s across the width of the nerve, proximal to the origin of the nerve to the biceps femoris muscle. Experimental and two agematched control animals were similarly housed and maintained for 12-15 months following surgery. Observation of the animals during the recovery period indicated functional recovery of the affected hindpaw by 6-8 weeks following nerve crush. The size of the ipsilateral (reinnervated) and contralateral control limbs was symmetrical. Anesthetized experimental and control animals underwent bilateral transection of sciatic nerves at 14-16 months of age at a site 5-10 mm distal to the site described for sciatic crush or at a comparable location in contralateral and control nerves. A 15-20 mm segment of sciatic nerve, distal to the site of transection was excised for physiological investigation. Proximal sciatic nerve stumps were gently suctioned into a short length of polyethylene tubing which was subsequently filled with 40% HRP VI (Sigma) mixed in Kreb's solution. The distal end of the tubing was sealed with bone wax, the surgical incision was sutured, and the animals were permitted to survive for a period of 48 h prior to sacrifice for histological studies. At the conclusion of the survival period, animals were heavily anesthetized with pentobarbital (50 mg/kg) and perfused via a left ventricular catheter with normal saline containing 50% sodium nitrite until blood cleared from vessels. Animals were then perfused with 700-800 ml of 1% paraformaldehyde, 1% glutaraldehyde, 3% sucrose in 0.1 M PO4 buffer at pH 7.4. At the completion of fixation, the posterior T12-S 1 vertebrae were removed. Left and right L 4, L 5 and L 6 dorsal root ganglia were identified under visualization with a dissecting microscope and the associated ventral and dorsal roots were followed to their point of insertion in the lumbar cord. Spinal cord segments L2_3, L4_6 and S1-2 were removed en bloc and placed in a fixative solution containing 30% sucrose for 1 h prior to transfer to diluted POa buffer.
On the following day, 50 ~tm cord sections were cut on a freezing microtome, rinsed in diluted PO 4 buffer and preincubated in ammonium molybdate and tetramethylbenzidine according to a protocol previously described 29. Sections were gently agitated during incubation with 1.8% H2O 2 for several hours. A portion of the sections was counterstained with neutral red. Sections were examined under light microscopy for the presence and location of HRP labelled motor neurons and were photographed at x310 magnification. Somatic areas were determined for all labelled motor neurons identified in photomicrographs of L4_6 cord segments from 3 lesioned and two control animals. Area was calculated by multiplying the product of two perpendicular radii for each labelled cell 5. Measurements through somatic projections were avoided. A total of 888 cells were evaluated (578 ipsilateral to the sciatic crush, 93 contralateral and 217 from control animals). The clustered distribution and intensity of HRP reaction observed in motor neurons ipsilateral to the lesion (discussed below) may have facilitated their recognition and may account for the increased number of labelled cells identified on the lesioned side. Similarly, the number of dispersed, smaller diameter, motor neurons may have been underestimated in field counts of control specimens. However, by including all labelled cell areas in our calculations, we avoided biasing our cell samples toward the apparent larger cell population in experimental sections, t-Tests were performed between group data derived from individual control and lesioned animals, as well as between summed control and experimental cell groups. RESULTS Segments of sciatic nerve 5-10 mm distal to the crush site were removed for physiological examination at the time that long-term regenerated nerves were transected and exposed to HRP. The physiological properties of these regenerated nerves have been evaluated and will be presented in another paper (in preparation7). Though recordings indicated that fibers had regenerated beyond the site of crush and into the area used for HRP application, sensitivity of regenerated nerves to potassium channel blocking
71
4
l ~!iiil)~]!i~i;~!i~ii!ii!ill
A
C
i~ii? ¸ :;iiiil
!!i!i!!!!!!ili~i!i!i!i!i~!iii!i!i
J
B:
I
Fig. 1. Photomicrographs of lumbar cord sections taken from a 14-month-old rat following left sciatic crush lesion at 10 weeks of age. Sciatic nerves were transected and exposed to H R P for 48 h prior to sacrifice. Cross-sections contralateral to the nerve crush (A and C) are presented with sections from the same cord level ipsilateral to the lesion (B and D, respectively). Labelled motor neurons on the control side were uniformly distributed within the region of sciatic representation. Labelled regenerated cells were more densely clustered and appeared enlarged. T h o u g h individual neurons on both the ipsilateral and contralateral sides varied in the intensity of H R P labelling, in general the regenerated neurons were more densely labelled. Scale bar = 100/~m.
72
P
A
b
C
--
D /
Fig. 2. Photomicrographs of control (A and C) and regenerated (B and D) motor neurons at higher magnifications. A: a section from a contralateral cord section illustrating a large control cell with fine dendritic processes characteristic of normal spinal motor neurons. B: a section ipsilateral to the sciatic crush including small diameter regenerated cells with multiple thickened dendrites (arrows). C and D: fields taken from cord segments contralateral (C) and ipsilateral (D) to the lesion. Thickened and tortuous dendritic processes were often seen in regenerated motor neurons (arrow) but were not observed in contralateral or control cord segments. Scale bar A, B - 50 ktm; C, D = 25/~m.
73 agents was m a r k e d l y increased c o m p a r e d to that observed in contralateral and age-matched control nerves. E x p e r i m e n t a l and control m o t o r neurons examined in cord segments proximal and distal to the L4_ 6 region did not exhibit H R P reaction product. However, within the L4_ 6 segments, localization of the cells of origin of regenerated fibers differed from that observed on the contralateral control sides or in comparable segments from control animals. Paired sections of lumbar cord taken at the same level from the control (Fig. 1A, C) and the lesioned (Fig. 1B, D, respectively) sides are presented in Fig. 1. Intensely labelled nests of large cells were observed in the crushed side (Fig. 1B, D) c o m p a r e d to the m o r e dispersed appearance of labelled m o t o r neurons in the contralateral side (Fig. 1A, C). The less dense pattern of distribution was also observed in sections from control animals. A l t h o u g h large cells were frequently seen on the lesioned side, smaller cells were also present in the same fields. Labelled m o t o r neurons observed contralateral to the side of crush are presented at higher magnification in Fig. 2 A illustrating the characteristic dendritic processes which were typically observed on the nonlesioned side, as well as in cord segments from control animals. In contrast, many of the labelled m o t o r neurons on the lesioned side, even those with smaller somatic areas (Fig. 2B), had thicker dendritic processes (arrows). This difference was frequently observed, even when the largest control cells (Fig. 2C) were c o m p a r e d to large regenerated m o t o r neurons (Fig. 2D arrow). R e g e n e r a t e d m o t o r neurons of all sizes were generally m o r e densely labelled than contralateral control cells; lateralized differences in H R P - r e a c t i o n product were not noted in control animals. Eccentric nuclei were not observed in regenerated m o t o r neurons; enlarged r e g e n e r a t e d cells with thickened dendritic processes were present in fields containing more normal appearing labelled cells. Examination of cord cross-sections suggested that labelled m o t o r neurons ipsilateral to the crush lesions were generally larger than labelled cells on the contralateral side or from control animals. M e a n cell area was calculated for summed control (698 Bm 2 + 19.32 S . E . M . ) and regenerated cell populations (908 # m 2 + 16.47 S . E . M . ) t-Tests p e r f o r m e d b e t w e e n individual control groups (one contralateral cell group
and two control cell groups) and between 3 regenerated cell groups showed no significant differences in cell area within control and regenerated categories. H o w e v e r , differences between summed control and lesioned cell areas were significant at a P-value of <0.001. The distributions of cell areas for control and experimental m o t o r neurons are p r e s e n t e d in Fig. 3, representing cell areas in 200 ~tm 2 bins. Although all area bins seen in the control cell population were r e p r e s e n t e d in the lesioned cell group, regenerated cell areas were generally larger than those of control cells. M o r e o v e r , cell areas >2000 ~tm 2 were only seen in the r e g e n e r a t e d cell population. DISCUSSION The observation of p e r i k a r y a l enlargement and
2S,
CONTROL
20
PERCENT TOTAL CELLS
Is.
,oF s
o
I 200
|
AREA (p2)
2s
PERCENT CELLS
REGENERATED
IS,
10,
5
0 2~
I 600
I 10G0
1400
I i ~'T--1 I ImO 2200
AREA (~2)
Fig. 3. The frequency distribution of control (n 310) and regenerated (n = 578) cell areas segregated into 200~um2 bins. All control area bins are represented in the regenerated population, but a rightward shift of cell areas distribution is noted. There is an increase in absolute area Values of the largest regenerated cells. =
74 thickened dendritic processes in long-term regenerated motor neurons following sciatic nerve crush was unexpected in view of the numerous reports which suggest restoration of normal perikaryal properties following more severe nerve injuries. Previous examinations of the perikaryal response to axonal injury have focused on immediate morphological alterations and the time course of return of normal properties in surviving cell bodies during the period of axonal regeneration 2,26.34,41,42. Though the duration and severity of cell body abnormalities vary for different species and lesion models, in general, properties of cells surviving axonal injury are reported to be comparable to those of control neurons within several months of nerve crush or transection. Inasmuch as our examinations were limited to a period 14-16 months following injury, we cannot differentiate between persistent and progressive changes. The apparent discrepancy between our observation of appreciable morphological changes in longterm regenerated motor neurons and those of previous investigators who have reported normal perikaryal characteristics several months after axonal injury may have resulted from our use of retrograde labelling with HRP. This technique allowed us to distinguish cells of origin of regenerated fibers from non-regenerated or unlesioned cells in the same regions. Morphological differences between lesioned and control motor neurons may have been accentuated by the increased density of HRP-reaction product consistently observed in the regenerated cells. Quantified differences in retrograde labelling between experimental and control cells are not provided by the present study but the observation of differences raises the possibility that altered uptake or
tralateral entorhinal cortex ~4. It is possible that the increased labelling we observed in long-term regenerated motor neurons resulted from the greater number of large cells present in the experimental cell population or from a relatively more intense, acute perikaryal reaction in regenerated vs control cells following nerve transection and HRP exposure 48 h prior to sacrifice. This latter explanation seems unlikely since other features of the immediate perikaryal reaction, i.e. nuclear eccentricity, were not evident. Increases in cell area of up to 160% have been reported for cells examined during the period of maximal chromatolysis 1'2'4'5'27'44. Examinations extended beyond the time of acute perikaryal reaction in motor neurons suggest that cell areas of neurons ipsilateral to lesions return to control values within 1-6 months of crush or axotomy. Barron et al. 5 reported that regenerated and control cervical motor neuron areas were comparable at 30 days following brachial plexotomy. However, at 60, 75 and 90 days post-injury, cell areas ipsilateral to the lesion were larger. Histograms of cell area distribution, 90 days following injury, indicated a shift toward larger cell area in the regenerated vs contralateral control population. Cavanaugh 9 attributed her observation of enlarged mean cell volume in axotomized dorsal root ganglion cells to the selective loss of smaller cell populations. In the present study, frequency distributions indicate that in addition to a reduced proportion of small cells in the regenerated cell population, absolute area values were larger than controls. It is interesting, in this regard, that similar patterns of perikaryal enlargement have been reported in regenerated frog optic nerve, associated with massive cell loss 37, and during active sprouting in both rat entorhinal cortex ~4 and red nucleus 31.
retrograde transport of HRP occurs in long-term regenerated neurons. Increased deposition of HRPreaction product has been described in acutely injured axons 18'29. Halperin and LaVail Is proposed that although delayed uptake of H R P immediately occurs following optic nerve injury, increased accumulation of HRP is subsequently observed in lesioned neurons due to an increased rate of uptake, transport or both or a decrease of the rate of degradation. Both increased cell size and enhanced transport of HRP have been reported for entorhinal neurons during active sprouting into acutely denervated con-
Although the regenerative capacity of the mammalian peripheral nervous system has been emphasized, it is now well established that a number of physiological and morphological abnormalities persist in mammalian axons for many months after peripheral nerve transection despite evidence of successful regrowth and functional recovery. Decreased conduction velocity, initially observed at the time of perikaryal changes and axonal thinning 1°-~2"23, persists in association with shortened internodal distances and the slow acquisition of normal myelin thickness 1°'2°. Similar abnormalities are observed even following le-
75 sions that result in optimal regeneration 17. Several investigators have demonstrated alteration of ionic channel conductances beyond one year following simple nerve crush injury 6'22'35. Hildebrand et a1.19'2° have suggested that altered physiological properties of regenerated axons may, in part, be secondary to the chronic disruption of paranodal myelin sheaths and altered nodal localization along the axonal membrane that is observed following nerve crush in adult rats. The long-term effect of chronic axonal abnormalities on perikaryal properties has not been previously examined. Chronic neuronal alteration following nerve injury is not restricted to regenerated axons but has been reported in reinnervated motor units as well. Examination of motor unit properties at extended periods after initial injury indicate that motor unit number is established within 2 months of injury; motor unit tension returns to normal over a more protracted time c o u r s e 15'16. Gordon and Stein 16 concluded that motor unit tension is maintained, despite loss of some motor axons, by hypertrophy of muscle fibers. Increased axonal diameters have been described following chronic induction of muscle hypertrophy 13'43, particularly in slow, oxidative and fatigue-resistant muscle fibers. Cavanaugh 9 observed a 32% increase in somatic volume of regenerated spinal ganglion cells when peripheral sensory fields were increased for regenerated sensory fibers. The changes we observed in cell area and dendritic morphology may relate to alterations in the size of muscle fibers or receptive fields innervated by regenerated axons or by increased terminal arborization after regeneration. Physiological properties of injured perikarya are dramatically altered during the period of maximal chromatolysis following n e r v e injury 11'12'23'32'41. Somatodendritic excitability is compromised and conduction of impulses through the initial axonal segment is markedly impaired. The association of these physiological changes with observed dendritic retraction 32 and loss of somatic and proximal dendritic excitatory synapses 38'39 has been emphasized. However, Mendell et al. 2s, in a chronically axotomized motor neuron lesion model, reported a dissociation in the timing of initial physiological changes, first observed 6 days post-transection, and the subsequent motor neuron deafferentation occurring 60 days following injury. Further support that initial changes in motor
neuron physiological properties occur independently of presynaptic alterations is provided by Kuno et al. 24 who reported the absence of physiological abnormalities in a motor neurons examined 29-46 days following dorsal root sections. Although most authors suggest that observed abnormalities are reversible when regeneration is successful, data from single cell recordings in long-term regenerated cell bodies are not available. Several lines of evidence indicate that response to injury may differ in the timing of initiation, intensity, and duration for different classes of neurons 15A6'21' 27,30,34,36. Stein and colleagues 15'21'3°, employing a lesion model in which transected mixed nerves were chronically prevented from regenerating with peripheral targets, have shown that fiber degeneration and cell loss differ for motor and sensory neurons. When initially evaluated at 45 days following transection and ligation, fibers from both cell populations exhibited a similar rate of atrophy. However, in physiological examinations at extended periods following injury (145 and 252 days), degeneration progressed at a faster rate and for a longer duration in fast conducting afferent fibers compared to a motor neurons. A differential response to nerve injury in motor and sensory axons could result in a distinctive sequence of alterations in pre- and post-synaptic activity at the motor neuron level. Initial changes in perikaryal size, dendritic arborization and synaptic contacts with la afferent fibers have been described in chromatolytic motor neurons acutely following peripheral transection and crush. Purves and Hadley 33, sequentially examining the dendritic morphology of normal adult rat superior cervical ganglion cells, reported remodelling of postsynaptic elements including extension, retraction and formation of dendrites, during observation periods of up to 3 weeks. If such plasticity is a generalized characteristic of mammalian neurons, dendritic morphology of regenerated motor neurons may be influenced by delayed and/or progressive changes in sensory input. These data emphasize the need for further investigation of cell bodies giving rise to long-term regenerated axons, including further study of long-term neuronal cell body changes following a variety of peripheral nerve lesion models, in order to more clearly elucidate underlying mechanisms. Particular attention
76 s h o u l d be g i v e n to the t i m e c o u r s e of t h e s e p e r i k a r y a l
ACKNOWLEDGEMENTS
c h a n g e s , the b i o c h e m i c a l e v e n t s that u n d e r l i e t h e m and the possibility that d i f f e r e n t p o p u l a t i o n s of n e u -
This w o r k was s u p p o r t e d by T h e R h o d e Island
rons m a y vary in their l o n g - t e r m r e s p o n s e to axonal
F o u n d a t i o n and, in part, by T h e D a n i e l H e u m a n n
injury.
F u n d and the M e d i c a l R e s e a r c h Service, V e t e r a n s Administration.
REFERENCES 1 Aldskogius, H., Barron, K.D. and Regal, R., Axon reaction in dorsal motor vagal and hypoglossal neurons of the adult rat, J. Comp. Neurol., 193 (1980)165-178. 2 Barr, M.L. and Hamilton, J.D., A quantitative study of certain morphological changes in spinal motor neurons during axon reaction, J. Comp. Neurol., 89 (1948) 93-124. 3 Barron, K.D., Comparative observations on the cytologic reactions of central and peripheral nerve cells to axotomy. In C.C. Kao and R.P. Bunge (Eds.), Spinal Cord Reconstruction, Raven, New York, 1982, pp. 7-40. 4 Barron, K.D., Chiang, T.Y., Daniels, A.C. and Doolin, P.F., Subcellular accompaniments of axon reaction in cervical motoneurons of the cat. In H.M. Zimmerman (Ed.), Progress in Neuropathology, Vol. i, Grune and Stratton, New York, 1971, pp. 255-280. 5 Barron, K.D., Cova, J., Scheibly, M.E. and Kohberger, R., Morphometric measurements and RNA content of axotomized feline cervical motoneurons, J. Neurocytol., 11 (1982) 707- 720. 6 Bowe, C.M., Hildebrand, C., Kocsis, J.D. and Waxman, S.G., Physiological properties of regenerated rat sciatic nerve following lesions at different postnatal ages, Dev. Brain Res., 34 (1986) 123-131. 7 Bowe, C.M. and Yu, C.H., Sensitivity of regenerated sciatic nerve to 4-aminopyridine and tetraethylammonium following injury at birth and late maturation, Soc. Neurosci. Abstr., 13 (1987) 1211. 8 Bowe, C.M. and Yu, C.H., Perikaryal changes in spinal motor neurons giving rise to long-term regenerated axons, Soc. Neurosci. Abstr., 14 (1988) in press. 9 Cavanaugh, M.W., Quantitative effects of the peripheral innervation area on nerves and spinal ganglion cells, J. Comp. Neurol., 94 (1951) 181-219. 10 Cragg, B.G. and Thomas, P.K., The conduction velocity of regenerated peripheral nerve fibers, J. Physiol. (Lond.), 171 (1964) 164-175. 11 Czeh, G., Kudo, N. and Kuno, M., Membrane properties and conduction velocity in sensory neurones following central or peripheral axotomy, J. Physiol. (Lond.), 270 (1977) 165-180. 12 Eccles, J.C., Libet, B. and Young, R.R., The behavior of chromatolysed motoneurones studied by intracellular recording, J. Physiol. (Lond.), 143 (1958) 11-40. 13 Edds, M.V., Hypertrophy of nerve fibers to functionally overloaded muscles, J. Comp. Neurol., 93 (1950) 259-275. 14 Goldschmidt, R.B. and Steward, O., Time course of increases in retrograde labelling and increases in cell size of entorhinal cortex neurons sprouting in response to unilateral entorhinal lesions, J. Comp. Neurol., 189 (1980) 359-379. 15 Gordon, T., Hoffer, J.A., Jhamandas, J. and Stein, R.B.,
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Long-term effects of axotomy on neural activity during cat locomotion, J. Physiol. (Lond.), 303 (1980) 243-263. Gordon, T. and Stein, R.B., Time course and extent of recovery in reinnervated motor units of cat triceps surae muscles, J. Physiol. (Lond.), 323 (1982) 307-323. Haftek, J. and Thomas, P.K., Electron-microscope observations on the effects of localized crush injuries on the connective tissues of peripheral nerve, J. Anat., 103 (1968) 233-243. Halperin, J.J. and LaVail, J.H., A study of the dynamics of retrograde transport and accumulation of horseradish peroxidase in injured neurons, Brain Research, 100 (1975) 253-269. Hildebrand, C., Kocsis, J.D., Berglund, S. and Waxman, S.G., Remodelling of internodes in regenerated rat sciatic nerve, Brain Research, 358 (1985) 163-170. Hildebrand, C., Mustafa, G.Y., Bowe, C.M., Berglund, S. and Kocsis, J.D., Nodal spacing along regenerated axons following crush lesion of the developing rat sciatic nerve, Dev. Brain Res., 32 (1986) 147-154. Hoffer, J.A., Stein, R.B. and Gordon, T., Differential atrophy of sensory and motor fibers following section of cat peripheral nerves, Brain Research, 178 (1979) 347-361. Kocsis, J.D., Waxman, S.G., Hildebrand, C. and Ruiz, J.A., Regenerating mammalian nerve fibers: changes in action potential waveform and firing characteristics following blockage of potassium conductance, Proc. R. Soc. Ser. B., 217 (1982) 277-287. Kuno, M. and Llinas, R., Alterations of synaptic action in chromatolysed motoneurones of the cat, J. Physiol. (Lond.), 210 (1970) 823-838. Kuno, M., Miyata, Y. and Munoz-Martinez, E.J., Differential reaction of fast and slow alpha motor neurons to axotomy, J. Physiol. (Lond.), 240 (1974) 725-739. Lavelle, A., Levels of maturation and reactions to injury during neuronal development, Progr. Brain Res., 40 (1973) 161-160. Lieberman, A.R., The axon reaction: a review of the principal features of perikaryal responses to axon injury, Int. Rev. Neurobiol., 14 (1971) 49-124. Lieberman, A.R., Some factors affecting retrograde neuronal responses to axonal lesions. In R. Bellairs and E.G. Grays, (Eds.), Essays on the Nervous System, Clarendon, Oxford, 1974, pp. 71-105. Mendell, L.M., Munson, J.B. and Scott, J.G., Alterations of synapses on axotomized motoneurons, J. Physiol. (Lond.), 255 (1976) 67-79. Mesulam, M.-M., Principles of horseradish peroxidase neuro-chemistry and their application for tracing neural pathways. In M.-M. Mesulam (Ed.), Tracing Neural Connections with Horseradish Peroxidase, Wiley, New York, 1982, pp. 1-151. Milner, T.E. and Stein, R.B., The effects of axotomy on
77
31
32
33
34 35
36
37
the conduction of action potentials in peripheral sensory and motor fibres, J. Neurol. Neurosurg. Psychiat., 44 (1981) 485-496. Prendergast, J. and Stelzner, D.J., Changes in the magnocellular portion of the red nucleus following thoracic hemisection in the neonatal and adult rat, J. Comp. Neurol., 166 (1976) 163-172. Purves, D., Functional and structural changes in mammalian sympathetic neurons following interruption of their axons, J. Physiol. (Lond.), 252 (1975) 429-463. Purves, D. and Hadley, R.D., Changes in the dendritic branching of adult mammalian neurones revealed by repeated imaging in situ, Nature (Lond.), 315 (1985) 404-406. Ranson, S.W., Alterations in spinal ganglion cells following neurotomy, J. Neurol. Psychol., 19 (1909) 125-152. Ritchie, J.M., Sodium and potassium channels in regenerating and developing mammalian myelinated nerves, Proc. R. Soc. Set. B., 215 (1982) 273-287. Romanes, G.J., Motor localization and the effects of nerve iniury on the ventral horn cells of the spinal cord, J. Anat., 80 (1946) 117-131. Stelzner, D.J. and Strauss, J.A., Increase in ganglion cell size after optic nerve regeneration in the frog, Rana pi-
piens, Exp. Neurol., 100 (1988) 210-215. 38 Summer, B.E.H., A quantitative analysis of the response of presynaptic boutons to postsynaptic motor neuron axotomy, Exp. Neurol., 46 (1975) 605-615. 39 Summer, B.E.H. and Watson, W.E., Retraction and expansion of the dendritic tree of motor neurons in adult rats in vivo, Nature (Lond.), 233 (1971) 273-275. 40 Torvik, A., Central chromatolysis and the axon reaction: a reappraisal, Neuropath. Appl. Neurobiol., 2 (1976) 423-432. 41 Torvik, A. and Skjorlen, F., Electron microscopic observations on nerve cell regeneration and degeneration after axon lesions, Acta Neuropath., 17 (1971) 248-264. 42 Torvik, A. and Soreide, A.J., Nerve cell regeneration after axonal lesions in newborn rabbits. Light and electron microscopic study, J. Neuropathol. Exp. Neurol., 31 (1972) 683-695. 43 Walsh, J.V., Burke, R.E., Rymer, W.Z. and Tsairis, P., Effect of compensatory hypertrophy studied in individual motor units in medial gastrocnemius muscle of the cat, J. Neurophysiol., 41 (1978)496-508. 44 Watson, W.E., Observations on the nucleolar and total cell body nucleic acid of injured nerve cells, J. Physiol. (Lond.), 196 (1968) 655-676.