0270.6474/84/0407-1736$02.00/O The Journal of Neuroscience Copyright 0 Society for Neuroscience Vol. 4, No. 7, pp. 1736-1744 Printed in U.S.A. July 1984 A CONDITIONING LESION OF THE PERIPHERAL AXONS OF DORSAL ROOT GANGLION CELLS ACCELERATES REGENERATION OF ONLY THEIR PERIPHERAL AXONS’ MONICA M. ORLINGER” AND RAYMOND J. LASEK Department of Developmental Genetics and Anatomy, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 Received March 15,1983; Revised December 19, 1983; Accepted January 20,1984 Abstract Axotomy of the peripheral axon of dorsal root ganglion (DRG) cells is known to result in chromatolysis and changes in protein synthesis in DRG cells. We investigated whether a stimulus produced by peripheral branch axotomy would affect the regenerative properties of both the central and peripheral axon of the DRG cell equally. To examine this question, a conditioning crush lesion was made distally on the sciatic nerve 2 weeks prior to a testing lesion of either the dorsal root or peripheral branch axon near the DRG. Fast axonal transport of radioactive proteins was used to assess regeneration of DRG axons. In the adult rat, leading peripheral branch axons normally regenerate at a rate of 4.4 mm/day. If a conditioning lesion of the sciatic nerve is made 2 weeks before the test lesion, the rate of peripheral branch axonal regeneration increases by 25% to 5.5 mm/day. This effect is not limited to the fastest growing axons in the nerve since a population of more slowly growing axons also exhibits accelerated outgrowth in response to a prior peripheral axotomy. In contrast to this, the fastest growing central branch axons of DRG cells, which normally regenerate at a rate of 2.5 mm/day, are not significantly affected by a prior peripheral axotomy. A population of more slowly growing axons in the dorsal root also does not exhibit accelerated outgrowth in response to a peripheral conditioning lesion. The results of these experiments indicate that changes in the DRG neuron’s metabolism induced by prior axotomy of its peripheral axon do not affect the regenerative properties of both axons equally. This raises the possibility that accelerated axonal outgrowth in only one axonal branch results from a differentially regulated supply of proteins to the two axons by the DRG cell body. The peripheral extensions of neurons, such as den- sensory receptors. A number of asymmetries in the sup- drites and axons, are readily distinguished by structural, ply of proteins from the DRG cell body to the two axons functional, and biochemical criteria. The selective rout- exist. For example, the amount of rapidly transported ing of materials from the cell body into particular neu- protein (Lasek, 1968; Ochs, 1972; Ochs et al., 1978), the rites may play a central role in establishing such regional amount of cytoskeletal protein carried in slow axonal differences in different parts of a neuron. Studies of transport, and the rate of slow transport are greater in axonal transport in dorsal root ganglion (DRG) cells the peripheral axon than in the central axon (Lasek, have provided support for this suggestion. The DRG cell 1968; Komiya and Kurokawa, 1978; Mori et al., 1979; is well suited to the study of differential routing since it Lasek et al., 1983; Wujek and Lasek, 1983). has two long, functionally distinct axons that branch Differences in axonal transport are of importance be- from a short stem axon. One branch courses centrally cause they may determine several different structural through the dorsal root to synapse on central neurons, and functional features of the peripheral and central and the other proceeds peripherally to terminate on DRG axon. For example, the diameter of the peripheral axon is larger than that of the central axon (Suh et al., 1984). This structural difference, in large part, results ’ This research was supported by National Institutes of Health Grant from the differential amounts of slowly transported cy- NS-14900-03 to R. J. L. and National Institutes of Health Traineeship NS-07118-04 to M. M. 0. We wish to thank Dr. Irvine McQuarrie for toskeletal polymers entering the two branches and the helpful discussions and suggestions and Ms. Shirley Ricketts and Ms. differential rates of transport of these elements in each Vivian Pocek for excellent technical assistance throughout the work. axonal branch (Lasek et al., 1983). Axonal transport of * To whom correspondence should be addressed. the cytoskeleton is clearly involved in axonal regenera- 1736 The Journal of Neuroscience Regeneration of DRG Axons after a Conditioning Lesion 1737 tion and may determine the rate of axonal elongation the L5 dorsal root at 8 to 10 mm from the L5 DRG (Fig. (Lasek, 1981; Lasek and Hoffman, 1976; Lasek et al., 1). The L5 spinal nerve was exposed at the L4-L5 spinal 1981; McQuarrie, 1983). A notable functional difference nerve junction outside of the vertebral column; the L5 between the two axonal branches of the DRG cell is that dorsal root was exposed at t,he level of the L4 DRG by a the rate of regeneration of peripheral branch axons is partial laminectomy. The lesion consisted of crushing about twice that of dorsal root axons (Komiya, 1981; the nerve twice with a #5 Dumont jeweler’s forceps for Wujek and Lasek, 1983). The axonal regeneration rate 15 set, a method that completely transects all axons of correlates with the rate of slow transport, particularly of the nerve (McQuarrie et al., 1977). At the end of surgery, slow component b (SCb), in each axonal branch (Komiya overlying muscles, fascia, and skin were closed with silk and Kurokawa, 1978; Komiya, 1981; Wujek and Lasek, suture. 1983). These observations indicate that a differential Conditioning lesion. Some animals received a condi- supply of proteins to the two branches of the DRG cell tioning lesion 2 weeks prior to the test lesion (Fig. 1). may affect the functional properties of these axons. The conditioning lesion consisted of crushing the sciatic An interesting question arises when considering the nerve in the midthigh (50 to 60 mm from the L5 DRG) maintenance of two quite distinct axons by the DRG cell with #5 Dumont jeweler’s forceps twice for 15 sec. Two body: Would a stimulus which alters the metabolism of weeks later, a test lesion was made as described above. the neuron change the regenerative properties of its two A 2-week conditioning interval was used since this inter- axons equally? In this paper we address this question by va! has been shown to be optimal for various neuronal stimulating a chromatolytic response in the DRG cell in systems studied to date (Grafstein and McQuarrie, 1978: order to produce changes in protein synthesis in the cell Forman et al., 1981). body. Chromatolysis can be induced in mammalian This paradigm provided four groups of animals: one DRG cells by cutting the peripheral, but not the central, group had only a test crush of the peripheral branch of axon (reviewed in Cragg, 1970; Lieberman, 1971). This the DRG (spinal nerve); one group had only a test crush results in changes in the levels of cytoskeletal protein of the central branch (dorsal root); a third group sus- synthesis (Perry and Wilson, 1981; Hall, 1982). The tained a conditioning lesion of the peripheral branch effects of these cell body changes on the peripheral and (sciatic nerve) 2 weeks prior to a test crush of the central axons were assessedby measuring the regenera- peripheral branch (spinal nerve); and a fourth group tive capacity of the axons 2 weeks after inducing the sustained a conditioning lesion of the sciatic nerve 2 chromatolytic response. This experimental approach, a weeks prior to a test crush of the dorsal root (Fig. 1). conditioning lesion paradigm, which entails inducing a neuron to regenerate with one lesion and testing the effects of this lesion with a subsequent axotomy, is known to result in an acceleration of the rate of axonal regeneration in several systems. (McQuarrie and Graf- stein, 1973, 1981; McQuarrie et al., 1977; Grafstein and McQuarrie, 1978; Forman et al., 1980, 1981; McQuarrie, CONDITIONING 1981, 1984). The effects of a conditioning lesion on LESION subsequent axonal regeneration have been related to changes in the synthesis and export of proteins from the cell body into the axon. For example, changes in the synthesis and transport of slowly transported cytoskele- tal proteins have been correlated with increases in the rate of axonal elongation (reviewed in Grafstein and McQuarrie, 1978; Forman et al., 1981; Lasek et al., 1981; McQuarrie and Grafstein, 1982; McQuarrie, 1984). We report that a peripheral conditioning lesion in- d I creases the rate of axonal elongation of only peripheral I axons of DRG cells. This observation suggests that I I changes in protein synthesis during the chromatolytic ITEST LESION B response to axotomy are differentially transferred to the two axons of the DRG cell, and it provides support for 3 ,iT:iys the hypothesis that the cytoskeletal networks in different regions of the neuron can be selectively regulated. .lO Materials and Methods AXONAL OUTGROWTH ASSESSED Test lesion. The experiments utilized young adult male Figure 1. Schematic showing sites of lesions in the experi- Sprague-Dawley rats (200 to 300 gm). For all surgical ments. The conditioning lesion was a crush of the sciatic nerve procedures, animals were anesthetized with a mixture of in the midthigh (50 to 60 mm from the L5 DRG). Test lesions sodium pentobarbital (27 mg/kg) and chloral hydrate were done either alone or 2 weeks after a conditioning lesion. (128 mg/kg). Regeneration of DRG axons was assessed Test lesions were crushes of either the L5 spinal nerve (test after an axotomizing test lesion.