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Central changes in primary afferent fibers following peripheral nerve lesions
Pergamon
PII:
Neuroscience Vol. 77, No. 4, pp. 1115–1122, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(96)00528-3
CENTRAL CHANGES IN PRIMARY AFFERENT FIBERS FOLLOWING PERIPHERAL NERVE LESIONS R. E. COGGESHALL,*† H. A. LEKAN,* T. P. DOUBELL,‡ A. ALLCHORNE‡ and C. J. WOOLF‡ *Department of Anatomy and Neurosciences, and Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, TX 77555-1069, U.S.A. ‡Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, U.K. Abstract––Cutting or crushing rat sciatic nerve does not significantly reduce the number of central myelinated sensory axons in the dorsal roots entering the fourth and fifth lumbar segments even over very extended periods of time. Unmyelinated axons were reduced by 250%, but only long after sciatic nerve lesions (four to eight months), and reinnervation of the peripheral target did not rescue these axons. This indicates that a peripheral nerve lesion sets up a slowly developing but major shift towards large afferent fiber domination of primary afferent input into the spinal cord. In addition, since myelinated axons are never lost, this is good evidence that the cells that give rise to these fibers are also not lost. If this is the case, this would indicate that adult primary sensory neurons with myelinated axons do not depend on peripheral target innervation for survival. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: primary afferents, transection, dorsal root axons, nociception.
Cell death is an integral component of the development of the nervous system.49 This death is usually ascribed to competition for a limited supply of targetproduced neurotrophins,19,30,31,49 and this construct is often called the neurotrophic theory.48 In support, peripheral nerve lesions in young animals,57,59 null mutations of the neurotrophins nerve growth factor (NGF),17 brain-derived neurotrophic factor (BDNF)16,42 and neurotrophin-3,23,25 and mutations of the neurotrophin receptors for trkA,61 trkB37 and trkC,36 are reported to result in substantial dorsal root ganglion (DRG) cell loss. In the adult, the peripheral target still produces neurotrophins,7 which maintain, after retrograde transport to the cell body,22 aspects of the differentiated cell phenotype.41 Disrupting contact with the target rapidly changes the phenotype, with the downor up-regulation of neuropeptides,35 cytoskeletal proteins68 and growth-associated proteins.11,34 Some of these changes can be prevented by neurotrophin replacement.18,26,28,67,72 Adult cell survival is also thought to depend on peripheral neurotrophin production and retrograde transport, as evidenced by reports of DRG cell loss after adult nerve lesions, combined with the observations that administration of trophic factors such as NGF prevents this loss.18,26,28,67,72 †To whom correspondence should be addressed. Abbreviations: BDNF, brain-derived growth factor; DRG, dorsal root ganglion; L4, fourth lumbar; L5, fifth lumbar; NGF, nerve growth factor; PB, phosphate buffer.
A question about studies on cell loss, however, relates to the use of profile counts, because calibrations demonstrate that such counts may lead to biased estimates of cell numbers.13,14,16,50,52 Because of this and also because we can ask whether a particular type of dorsal root axon is lost, we reexamined the issue of loss after adult peripheral nerve section by counting dorsal root axons following sciatic nerve lesions. Our reasoning is that if transection results in substantial DRG cell loss, this should be reflected in a fall-off in numbers of the fourth and fifth lumbar (L4 and L5, respectively) dorsal root axons, and if a particular population of DRG cells is preferentially vulnerable, this should be reflected in the loss of a particular type of axon. Loss of sensory axons and presumably neurons after nerve injury has implications for changes in sensitivity to primary afferent stimuli73 and may justify neurotrophin replacement therapy clinically.45 Thus, a re-examination of the central changes in primary afferent neurons that follow peripheral axotomy is pertinent. EXPERIMENTAL PROCEDURES
Adult Sprague–Dawley rats (UCL Biological Services) of either sex weighing 125–200 g had, under halothane (2–4%) anesthesia, either the left sciatic nerve cut (ligated and then transected) or crushed (smooth-tipped forceps for 1 min, until translucent) at mid-thigh levels. The animals recovered well and the protocol was approved by the Animal Care and Use Committee of University College London and met the standards approved by the International Association for the
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R. E. Coggeshall et al. Table 1. Axon numbers in L4 and L5 dorsal roots after sciatic nerve lesions in adult rats L4
Naive (young) Naive (old) Naive (total) 2-week cut 4-week cut 8-week cut 16-week cut 32-week cut 8-week crush 16-week crush 32-week crush
L5
n
MY
UN
n
MY
UN
7 4 11 5 3 4 4 7 3 3 6
5092&151 4790&96 4982&109 4965&297 5195&566 4583&211 4606&258 4617&130 4651&83 5001&1126 4819&162
8834&564 8898&881 8857&454 8890&611 9492&816 9859&1108 8195&1456 5167&329 7126&42 5814&778 4366&420
6 4 10 6 4 4 4 8 4 3 7
4900&209 5434&324 5114&190 4357&185 4773&156 4239&209 4370&501 4979&212 4440&250 4977&100 4540&250
11326&807 9648&617 10654&586 9459&491 10366&1337 10796&785 7370&723 6618&709 9536&317 9258&991 6134&587
Results given are the mean number of axons&S.E.M. in each group. MY, myelinated axons; UN, unmyelinated axons; n, number of animals.
Study of Pain. At various times following surgery (Table 1), the animals were terminally anesthetized with sodium pentabarbitone (lithobarb, 1.6 g/kg) and perfused with 3% paraformaldehyde, 3% glutaraldehyde and 0.1% picric acid in 0.1 M phosphate buffer (PB; pH 7.4). The L4 and L5 dorsal roots were removed, soaked in fresh fixative for one to three days, rinsed in PB, placed in 2% osmium tetroxide in PB for 2 h, rinsed, stained en bloc with 1% uranyl acetate in 70% ethyl alcohol for 1 h, dehydrated in ascending concentrations of ethyl alcohol and embedded in a mixture of Epon and Araldite. Cross-sections of each root were taken, photographed on a Philips 300 electron microscope and montages were done at approximately #750. Each montage was divided into four-inch squares with a random orientation, and myelinated axons were counted in every other square. Total myelinated axon numbers (MY) were estimated by multiplying the counts by 2. In two montages all myelinated fibers were counted, and the above estimates in the same roots were within 2% of the complete counts. An orthogonal pattern of eight to 12 dots was then placed randomly on each montage and these dots were used as the centers of higher power pictures (2#5000). Myelinated and unmyelinated axons were counted in these pictures, which contained approximately 20% of the axons in each root. Exclusion and inclusion lines were used for the counts from each micrograph. Total unmyelinated axon numbers (UN) were estimated by multiplying the ratio of unmyelinated to myelinated axons (un/my) in the high-power pictures from each root by total myelinated axon numbers: UN=MY·(un/my). In two cases all unmyelinated fibers were counted and the above estimates on the same roots were within 3% of the complete counts. Two sets of naive (normal) animals were done. The first group consisted of seven ‘‘young’’ naive animals weighing the same as the experimental animals at the time of surgery (125–200 g). The second set consisted of four ‘‘old’’ naive animals aged 11 months (§400 g) at the time of killing. The roots of these animals were counted as above. Statistical analyses were done utilizing one-way analysis of variance with P¦0.01 as a measure of significance. RESULTS
A photomontage of a typical L4 dorsal root is shown in Fig. 1. Note the numerous myelinated axons, identified by their electron-dense myelin sheaths. Unmyelinated axons, which cannot be clearly seen at this low magnification, are located in the interstices between the myelinated axons. At
higher powers (Fig. 2), both myelinated and unmyelinated axons can be clearly identified and counted. The axon counts from young and old naives are essentially the same (Table 1), and no statistically significant differences between the groups could be demonstrated. Thus, there is no drop in axon number as a function of age. Accordingly, these values were combined and used as the control values to compare with the results of surgery. The axon counts from the L4 and L5 roots at various times following either cut (ligation plus transection) or crush of the sciatic nerve and the control values are presented in tabular form in Table 1 and graphically in Fig. 3. Note that myelinated fiber numbers do not change significantly no matter what the surgery or time of survival. Also note that unmyelinated fiber numbers trend down at 16 weeks post-lesion and are decreased by approximately 50% from the naive animals in both L4 and L5 at 32+ weeks post-lesion following either cut or crush of the sciatic. These decreases at 32+ weeks for cut and crush and for both segments are all statistically significant. A final point is that the graphs from the crush as compared to the cut animals are essentially indistinguishable.
DISCUSSION
This study has generated a number of unexpected findings. Firstly, peripheral nerve section fails to produce any significant reduction in the total number of central dorsal root axons for at least 16 weeks after injury. Secondly, we presume that neurons with myelinated axons do not die at all, since the numbers of these fibers do not change even at eight to nine months post-lesion. Finally, a subpopulation of central unmyelinated axons does disappear after nerve lesion, but only after a long delay, independently of whether the injured nerve is permitted to reinnervate the peripheral target (crush) or not (ligation and section).
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Fig. 1. A montage of a normal L4 dorsal root from an adult rat. The large clear round or oval structures are blood vessels emptied by the perfusion (arrowhead). The predominant structures seen at this magnification are the myelinated axons (arrows), which are distinguished by the presence of the electron-dense myelin sheath. Scale bar=50 µm.
A first question is why previous studies report a loss of DRG neurons by 30–60 days following the lesions,5,9,21,33,46,54,55,56,58,65,77 when we fail to detect any dorsal root axon loss until 2224 days? One possible reason relates to the use of profile counts for estimating cell numbers. The difficulty is that profiles of neurons (a profile is what is seen in a histological section) rather than the neurons themselves are counted. Since numbers of profiles depend on many variables besides cell numbers,13,15 profile counts, even with assumption-based correction procedures,1,39 often lead to biased estimates of cell numbers.14,50,52 Since we find no post-lesion axon loss during the time when large numbers of cells are reported to be lost, we feel it reasonable to consider the possibility that the counts in previous studies are the result of changes in cell size and shape rather than cell numbers. This is a major reason why we turned to dorsal root counts. Since each cross-sectioned axonal profile in the root represents a single axon (in contrast to cells which are subdivided into several profiles during sectioning), axon counts will produce unbiased estimates of axon numbers. It is interesting that our estimates of axon numbers for the L4 and L5
dorsal roots are close to the total number of DRG cells as estimated by optical disector techniques.63 This would seem to indicate that each DRG cell in these segments gives rise to one central axon, which has recently been shown.64 A second possibility to explain the differences between our results and earlier cell counts is that cells are lost, but there is concomitant sprouting of dorsal root axons from surviving cells so that total axon numbers do not drop until at least 16 weeks after injury. To our knowledge, there has been little suggestion of afferent fiber sprouting specifically in dorsal roots following peripheral nerve lesions, but there is a considerable literature indicating that nerve lesions result in central primary afferent sprouting,75 and it is certainly possible that this sprouting could begin by axons dividing in the dorsal root or ganglion. A third possibility is that axons survive in the roots even though the cells that give rise to these axons die. This we regard as very unlikely, however, because dorsal rhizotomy results in the death of all dorsal axons in the stump of the root separated from the ganglion.
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Fig. 2. A higher power picture of a normal L4 rat dorsal root. Note the myelinated axons (arrows) of various sizes, each with its characteristic sheath. Also visible at this magnification are the more numerous unmyelinated axons (arrowheads), which are small, electron-lucent, round or oval profiles occupying troughs of the Schwann cell cytoplasm. Scale bar=5 µm.
To determine whether previous cell counts are in error or dorsal root axons sprout after peripheral axotomy, it will be necessary to count DRG cells by stereological techniques and in the same animals determine the numbers of dorsal root axons. Preliminary data indicate that DRG cells are not lost by four weeks post-lesion but that considerable numbers are gone by 32 weeks post-lesion. A second question is why only unmyelinated axons are lost in the dorsal root after peripheral nerve injury? This may reflect different neurotrophin sensitivities of subpopulations of primary sensory neurons. In the adult, primary sensory neurons with myelinated axons predominantly express trkB and trkC, with a smaller group (218%) also expressing trkA.6,44,76 These axons, and presumably the cells that give rise to them, remain viable after peripheral nerve lesions. By contrast, approximately 50% of the small DRG cells that give rise to unmyelinated axons express trkA, with the other 50% failing to express any currently known trk receptor.6,76 Since central axon loss after nerve injury is restricted to unmyelinated axons, it would be interesting to determine if either trkA or the non-trk-expressing cells are lost. A problem, though, is that trk receptors change after axotomy.24,66 Also, if all trkA-expressing cells die after a nerve lesion, one would have expected some
loss of myelinated fibers, unless they survive because they also co-express some other trk receptor. An ancillary question is what proportion of cells with unmyelinated axons in the sciatic die when their peripheral axons are transected? As determined by retrograde labeling, the proportions of L4 and L5 DRG cells that project through the sciatic nerve range from 50% to 70%.10,21,33,62 There are questions about completeness of labeling,43,51 but if we accept the proportions as approximately correct, the numbers of cells that give rise to sciatic nerve unmyelinated axons that eventually die after nerve lesions must be high. Indeed, since the total axon count in dorsal roots L4 and L5 is 229,000, with a 2:1 ratio of unmyelinated to myelinated axons, and since 50% of the unmyelinated axons are lost after axotomy, the loss may represent the majority of cells with unmyelinated axons in the sciatic nerve. That small cells, which are presumed to give rise to unmyelinated sensory axons, preferentially die is an old idea.9,43,53 What we now add is that: (i) it is only the unmyelinated axons that are lost, (ii) the proportion of dying axons is high, (iii) it takes a very long time for the death of the axons, and (iv) reinnervation of the target does not prevent the axon loss. A third question is what prevents cell death for sensory neurons with myelinated axons and presumably some cells with unmyelinated axons after they are deprived of contact with peripheral targets? This may reflect either non-target-dependent neurotrophin production or non-dependence on neurotrophins for survival. One source of NGF and BDNF after nerve crush is cells in the distal stump.32,47 The problem with this as an explanation for the lack of cell death is that no difference was found between transection and crush animals, in that transected sensory axons will not be exposed to the neurotrophin-rich distal stump and crushed axons will. Alternative sources of neurotrophins may be the central target, in this case the spinal cord, or cells within the DRG itself. Trophins in the spinal cord have not been reported to be involved in cell survival in adults, in contrast to newborns.76 There are several reports of neurotrophin production in DRG neurons, however, particularly BDNF in trkA-expressing cells,70 with some increase in NGF after nerve injury.60 Furthermore, an autocrine release of BDNF has been shown to contribute to the capacity of some adult DRG neurons to survive in culture in the absence of exogenous growth factors.2 Overall, however, the findings speak strongly against the idea that retrograde peripheral neurotrophin transport has anything to do with survival of DRG cells with central myelinated axons following peripheral nerve lesions in adult rats. This conclusion is buttressed by previous observations that peripheral axotomy leads to few changes in dorsal roots in adult cats58 and little Marchi degeneration in the dorsal columns in adult rats.4 For cells with unmyelinated axons, interruption of retrograde neurotrophin flow is associated with profound cell
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Fig. 3. Bar graphs representing means&S.E.M. of myelinated and unmyelinated axon counts for the L4 and L5 dorsal roots at various times following cut or crush of the sciatic nerve. The naive counts were obtained by combining the ‘‘young’’ and ‘‘old’’ naive counts as mentioned in the text. Counts for cut and crush were compared against the naive data for each group. Statistical significance of P¦0.01 (asterisk) is indicated.
death, but because of the long delay, whether the interruption is causally related to this cell death is not known. Questions from the above are why is the unmyelinated axon loss so slow, and why did it occur both after nerve section and nerve crush? At present we do not know, but it may reflect either rapid sprouting from surviving DRG cells followed by a slow resolution or a slow program of lesion-induced changes
which are not prevented by subsequent reinnervation. Alternatively, since not all fibers successfully reinnervate peripheral targets after nerve crush, it may be that those that fail to reinnervate eventually die. Peripheral axotomy causes changes in chemical phenotype, membrane excitability and structure of primary sensory cell bodies.20,35,71 Peripheral but not central axotomy also results in the up-regulation of growth-associated genes,12 which may contribute to
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the central sprouting of myelinated afferent fibers into lamina II.40,74 Additionally, peripheral nerve section results in degenerative changes in the central terminals of myelinated fibers in the deep dorsal horn,3,5,27,29,69 which have been interpreted as a transganglionic degeneration reflecting post-axotomy DRG cell loss.3 However, since we do not see loss of myelinated axons in the roots when transganglionic degenerative changes are maximal, these changes presumably reflect ‘‘transganglionic atrophy’’38 or ‘‘dying back’’ of central axon terminals8 rather than actual transganglionic degeneration. CONCLUSION
The loss of approximately 50% of C-fibers in dorsal roots without a notable loss of myelinated
axons after peripheral nerve injury means that sensory input changes substantially, with a slowly developing but major shift towards domination by myelinated fibers. These findings are in keeping with the generation of A-fiber-mediated pain after nerve injury.45,73 In addition, the reduction in C-fibers may also contribute to trophic changes in the target as a result of a reduction in the efferent neurogenic inflammatory action subserved by these fibers and the sensory function they subserve. Acknowledgements—We wish to thank Gena Krannig for preparation of tissue, sectioning and counting of axons. We also thank Lyn Schilling for preparing the manuscript. This work is supported by a Bristol-Myers Squibb unrestricted Pain Research Grant, the Medical Research Council of Great Britain, and by NIH grants NS 10161, NS 11255 and NS 07185.
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R. E. Coggeshall et al. Richardson P. M., Verge Issa V. M. K. and Riopelle R. J. (1986) Distribution of neuronal receptors for nerve growth factor in the rat. J. Neurosci. 6, 2312–2321. Risling M., Aldskogius H. and Hildebrand C. (1983) Effects of sciatic nerve crush on the L7 spinal roots and dorsal root ganglia in kittens. Expl Neurol. 79, 176–187. Risling M., Aldskogius H., Hildebrand C. and Remahl S. (1983) Effects of sciatic nerve resection on L7 spinal roots and dorsal root ganglia in adult cats. Expl Neurol. 82, 568–580. Schmalbruch H. (1987) Loss of sensory neurons after sciatic nerve section in the rat. Anat. Rec. 219, 323–329. Sebert M. E. and Shooter E. M. (1993) Expression of mRNA for neurotrophic factors and their receptors in the rat dorsal root ganglion and sciatic nerve following nerve injury. J. Neurosci. Res. 36, 357–367. Smeyne R. J., Klein R., Schnapp A., Long S. K., Bryant S., Lewin A., Lira S. A. and Barbacid M. (1994) Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 386, 246–249. Swett J. E., Torigoe Y., Elie V. R., Bourassa C. M. and Miller P. G. (1991) Sensory axons in the rat sciatic nerve. Expl Neurol. 114, 82–103. Tandrup T. (1993) A method for unbiased and efficient estimation of number and mean volume of specified neuron subtypes in rat dorsal root ganglion. J. comp. Neurol. 329, 269–276. Tandrup T. (1995) Are neurons in the dorsal root ganglion pseudounipolar? A comparison of the number of neurons and number of myelinated and unmyelinated fibres in the dorsal root. J. comp. Neurol. 357, 341–347. Tessler A., Himes B. T., Krieger N. R., Murray M. and Goldberger M. E. (1985) Sciatic nerve transection produces death of dorsal root ganglion cells and reversible loss of substance P in spinal cord. Brain Res. 332, 209–218. Verge V. M. K., Merlio J. P., Grondin J., Ernfors P., Persson H., Riopelle R. J., Ho¨kfelt T. and Richardson P. M. (1992) Colocalization of NGF binding sites, trk mRNA and low affinity NGF receptor mRNA in primary sensory neurons: response to injury and infusion of NGF. J. Neurosci. 12, 4011–4022. Verge V. M. K., Richardson P. M., Wiesenfeld-Hallin Z. and Ho¨kfelt T. (1995) Differential influence of nerve growth factor on neuropeptide expression in vivo: a novel role in peptide suppression in adult sensory neurons. J. Neurosci. 15, 2081–2096. Verge V. M. K., Tetzlaff W., Bisby M. A. and Richardson P. M. (1990) Influence of nerve growth factor on neurofilament gene expression in mature primary sensory neurons. J. Neurosci. 10, 2018–2025. Westrum L. E., Canfield R. C. and Black R. G. (1976) Transganglionic degeneration in the spinal trigeminal nucleus following removal of tooth pulps in adult cats. Brain Res. 101, 137–140. Wetmore C. and Olson L. (1995) Neuronal and nonneuronal expression of neurotrophins and their receptors in sensory and sympathetic ganglia suggest new intercellular trophic interactions. J. comp. Neurol. 353, 143–159. Willis W. D. and Coggeshall R. E. (1991) Sensory Mechanisms of the Spinal Cord, 2nd edn. Plenum, New York. Wong J. and Oblinger M. M. (1991) NGF rescues substance P expression but not neurofilament or tubulin gene expression in axotomized sensory neurons. J. Neurosci. 11, 543–552. Woolf C. J. and Doubell T. P. (1994) The pathophysiology of chronic pain-increased sensitivity to low threshold Aâfibre inputs. Curr. Opin. Neurobiol. 4, 525–534. Woolf C. J., Shortland P. and Coggeshall R. E. (1992) Peripheral nerve injury triggers central sprouting of myelinated afferents. Nature 355, 75–78. Woolf C. J., Shortland P., Reynolds M., Ridings J., Doubell T. and Coggeshall R. E. (1995) Reorganization of central terminals of myelinated primary afferents in the rat dorsal horn following peripheral axotomy. J. comp. Neurol. 359, 1–14. Wright D. E. and Snider W. D. (1995) Neurotrophin receptor mRNA expression defines distinct populations of neurons in rat dorsal root ganglia. J. comp. Neurol. 351, 329–338. Ygge J. and Aldskogius H. (1984) Intracostal nerve transection and its effect on the dorsal root ganglion, a quantitative study on thoracic ganglion cell numbers and sizes in the rat. Expl Brain Res. 55, 402–408. (Accepted 26 September 1996)
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Neuroscience Vol. 77, No. 4, pp. 1115–1122, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(96)00528-3
CENTRAL CHANGES IN PRIMARY AFFERENT FIBERS FOLLOWING PERIPHERAL NERVE LESIONS R. E. COGGESHALL,*† H. A. LEKAN,* T. P. DOUBELL,‡ A. ALLCHORNE‡ and C. J. WOOLF‡ *Department of Anatomy and Neurosciences, and Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, TX 77555-1069, U.S.A. ‡Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, U.K. Abstract––Cutting or crushing rat sciatic nerve does not significantly reduce the number of central myelinated sensory axons in the dorsal roots entering the fourth and fifth lumbar segments even over very extended periods of time. Unmyelinated axons were reduced by 250%, but only long after sciatic nerve lesions (four to eight months), and reinnervation of the peripheral target did not rescue these axons. This indicates that a peripheral nerve lesion sets up a slowly developing but major shift towards large afferent fiber domination of primary afferent input into the spinal cord. In addition, since myelinated axons are never lost, this is good evidence that the cells that give rise to these fibers are also not lost. If this is the case, this would indicate that adult primary sensory neurons with myelinated axons do not depend on peripheral target innervation for survival. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: primary afferents, transection, dorsal root axons, nociception.
Cell death is an integral component of the development of the nervous system.49 This death is usually ascribed to competition for a limited supply of targetproduced neurotrophins,19,30,31,49 and this construct is often called the neurotrophic theory.48 In support, peripheral nerve lesions in young animals,57,59 null mutations of the neurotrophins nerve growth factor (NGF),17 brain-derived neurotrophic factor (BDNF)16,42 and neurotrophin-3,23,25 and mutations of the neurotrophin receptors for trkA,61 trkB37 and trkC,36 are reported to result in substantial dorsal root ganglion (DRG) cell loss. In the adult, the peripheral target still produces neurotrophins,7 which maintain, after retrograde transport to the cell body,22 aspects of the differentiated cell phenotype.41 Disrupting contact with the target rapidly changes the phenotype, with the downor up-regulation of neuropeptides,35 cytoskeletal proteins68 and growth-associated proteins.11,34 Some of these changes can be prevented by neurotrophin replacement.18,26,28,67,72 Adult cell survival is also thought to depend on peripheral neurotrophin production and retrograde transport, as evidenced by reports of DRG cell loss after adult nerve lesions, combined with the observations that administration of trophic factors such as NGF prevents this loss.18,26,28,67,72 †To whom correspondence should be addressed. Abbreviations: BDNF, brain-derived growth factor; DRG, dorsal root ganglion; L4, fourth lumbar; L5, fifth lumbar; NGF, nerve growth factor; PB, phosphate buffer.
A question about studies on cell loss, however, relates to the use of profile counts, because calibrations demonstrate that such counts may lead to biased estimates of cell numbers.13,14,16,50,52 Because of this and also because we can ask whether a particular type of dorsal root axon is lost, we reexamined the issue of loss after adult peripheral nerve section by counting dorsal root axons following sciatic nerve lesions. Our reasoning is that if transection results in substantial DRG cell loss, this should be reflected in a fall-off in numbers of the fourth and fifth lumbar (L4 and L5, respectively) dorsal root axons, and if a particular population of DRG cells is preferentially vulnerable, this should be reflected in the loss of a particular type of axon. Loss of sensory axons and presumably neurons after nerve injury has implications for changes in sensitivity to primary afferent stimuli73 and may justify neurotrophin replacement therapy clinically.45 Thus, a re-examination of the central changes in primary afferent neurons that follow peripheral axotomy is pertinent. EXPERIMENTAL PROCEDURES
Adult Sprague–Dawley rats (UCL Biological Services) of either sex weighing 125–200 g had, under halothane (2–4%) anesthesia, either the left sciatic nerve cut (ligated and then transected) or crushed (smooth-tipped forceps for 1 min, until translucent) at mid-thigh levels. The animals recovered well and the protocol was approved by the Animal Care and Use Committee of University College London and met the standards approved by the International Association for the
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R. E. Coggeshall et al. Table 1. Axon numbers in L4 and L5 dorsal roots after sciatic nerve lesions in adult rats L4
Naive (young) Naive (old) Naive (total) 2-week cut 4-week cut 8-week cut 16-week cut 32-week cut 8-week crush 16-week crush 32-week crush
L5
n
MY
UN
n
MY
UN
7 4 11 5 3 4 4 7 3 3 6
5092&151 4790&96 4982&109 4965&297 5195&566 4583&211 4606&258 4617&130 4651&83 5001&1126 4819&162
8834&564 8898&881 8857&454 8890&611 9492&816 9859&1108 8195&1456 5167&329 7126&42 5814&778 4366&420
6 4 10 6 4 4 4 8 4 3 7
4900&209 5434&324 5114&190 4357&185 4773&156 4239&209 4370&501 4979&212 4440&250 4977&100 4540&250
11326&807 9648&617 10654&586 9459&491 10366&1337 10796&785 7370&723 6618&709 9536&317 9258&991 6134&587
Results given are the mean number of axons&S.E.M. in each group. MY, myelinated axons; UN, unmyelinated axons; n, number of animals.
Study of Pain. At various times following surgery (Table 1), the animals were terminally anesthetized with sodium pentabarbitone (lithobarb, 1.6 g/kg) and perfused with 3% paraformaldehyde, 3% glutaraldehyde and 0.1% picric acid in 0.1 M phosphate buffer (PB; pH 7.4). The L4 and L5 dorsal roots were removed, soaked in fresh fixative for one to three days, rinsed in PB, placed in 2% osmium tetroxide in PB for 2 h, rinsed, stained en bloc with 1% uranyl acetate in 70% ethyl alcohol for 1 h, dehydrated in ascending concentrations of ethyl alcohol and embedded in a mixture of Epon and Araldite. Cross-sections of each root were taken, photographed on a Philips 300 electron microscope and montages were done at approximately #750. Each montage was divided into four-inch squares with a random orientation, and myelinated axons were counted in every other square. Total myelinated axon numbers (MY) were estimated by multiplying the counts by 2. In two montages all myelinated fibers were counted, and the above estimates in the same roots were within 2% of the complete counts. An orthogonal pattern of eight to 12 dots was then placed randomly on each montage and these dots were used as the centers of higher power pictures (2#5000). Myelinated and unmyelinated axons were counted in these pictures, which contained approximately 20% of the axons in each root. Exclusion and inclusion lines were used for the counts from each micrograph. Total unmyelinated axon numbers (UN) were estimated by multiplying the ratio of unmyelinated to myelinated axons (un/my) in the high-power pictures from each root by total myelinated axon numbers: UN=MY·(un/my). In two cases all unmyelinated fibers were counted and the above estimates on the same roots were within 3% of the complete counts. Two sets of naive (normal) animals were done. The first group consisted of seven ‘‘young’’ naive animals weighing the same as the experimental animals at the time of surgery (125–200 g). The second set consisted of four ‘‘old’’ naive animals aged 11 months (§400 g) at the time of killing. The roots of these animals were counted as above. Statistical analyses were done utilizing one-way analysis of variance with P¦0.01 as a measure of significance. RESULTS
A photomontage of a typical L4 dorsal root is shown in Fig. 1. Note the numerous myelinated axons, identified by their electron-dense myelin sheaths. Unmyelinated axons, which cannot be clearly seen at this low magnification, are located in the interstices between the myelinated axons. At
higher powers (Fig. 2), both myelinated and unmyelinated axons can be clearly identified and counted. The axon counts from young and old naives are essentially the same (Table 1), and no statistically significant differences between the groups could be demonstrated. Thus, there is no drop in axon number as a function of age. Accordingly, these values were combined and used as the control values to compare with the results of surgery. The axon counts from the L4 and L5 roots at various times following either cut (ligation plus transection) or crush of the sciatic nerve and the control values are presented in tabular form in Table 1 and graphically in Fig. 3. Note that myelinated fiber numbers do not change significantly no matter what the surgery or time of survival. Also note that unmyelinated fiber numbers trend down at 16 weeks post-lesion and are decreased by approximately 50% from the naive animals in both L4 and L5 at 32+ weeks post-lesion following either cut or crush of the sciatic. These decreases at 32+ weeks for cut and crush and for both segments are all statistically significant. A final point is that the graphs from the crush as compared to the cut animals are essentially indistinguishable.
DISCUSSION
This study has generated a number of unexpected findings. Firstly, peripheral nerve section fails to produce any significant reduction in the total number of central dorsal root axons for at least 16 weeks after injury. Secondly, we presume that neurons with myelinated axons do not die at all, since the numbers of these fibers do not change even at eight to nine months post-lesion. Finally, a subpopulation of central unmyelinated axons does disappear after nerve lesion, but only after a long delay, independently of whether the injured nerve is permitted to reinnervate the peripheral target (crush) or not (ligation and section).
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Fig. 1. A montage of a normal L4 dorsal root from an adult rat. The large clear round or oval structures are blood vessels emptied by the perfusion (arrowhead). The predominant structures seen at this magnification are the myelinated axons (arrows), which are distinguished by the presence of the electron-dense myelin sheath. Scale bar=50 µm.
A first question is why previous studies report a loss of DRG neurons by 30–60 days following the lesions,5,9,21,33,46,54,55,56,58,65,77 when we fail to detect any dorsal root axon loss until 2224 days? One possible reason relates to the use of profile counts for estimating cell numbers. The difficulty is that profiles of neurons (a profile is what is seen in a histological section) rather than the neurons themselves are counted. Since numbers of profiles depend on many variables besides cell numbers,13,15 profile counts, even with assumption-based correction procedures,1,39 often lead to biased estimates of cell numbers.14,50,52 Since we find no post-lesion axon loss during the time when large numbers of cells are reported to be lost, we feel it reasonable to consider the possibility that the counts in previous studies are the result of changes in cell size and shape rather than cell numbers. This is a major reason why we turned to dorsal root counts. Since each cross-sectioned axonal profile in the root represents a single axon (in contrast to cells which are subdivided into several profiles during sectioning), axon counts will produce unbiased estimates of axon numbers. It is interesting that our estimates of axon numbers for the L4 and L5
dorsal roots are close to the total number of DRG cells as estimated by optical disector techniques.63 This would seem to indicate that each DRG cell in these segments gives rise to one central axon, which has recently been shown.64 A second possibility to explain the differences between our results and earlier cell counts is that cells are lost, but there is concomitant sprouting of dorsal root axons from surviving cells so that total axon numbers do not drop until at least 16 weeks after injury. To our knowledge, there has been little suggestion of afferent fiber sprouting specifically in dorsal roots following peripheral nerve lesions, but there is a considerable literature indicating that nerve lesions result in central primary afferent sprouting,75 and it is certainly possible that this sprouting could begin by axons dividing in the dorsal root or ganglion. A third possibility is that axons survive in the roots even though the cells that give rise to these axons die. This we regard as very unlikely, however, because dorsal rhizotomy results in the death of all dorsal axons in the stump of the root separated from the ganglion.
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Fig. 2. A higher power picture of a normal L4 rat dorsal root. Note the myelinated axons (arrows) of various sizes, each with its characteristic sheath. Also visible at this magnification are the more numerous unmyelinated axons (arrowheads), which are small, electron-lucent, round or oval profiles occupying troughs of the Schwann cell cytoplasm. Scale bar=5 µm.
To determine whether previous cell counts are in error or dorsal root axons sprout after peripheral axotomy, it will be necessary to count DRG cells by stereological techniques and in the same animals determine the numbers of dorsal root axons. Preliminary data indicate that DRG cells are not lost by four weeks post-lesion but that considerable numbers are gone by 32 weeks post-lesion. A second question is why only unmyelinated axons are lost in the dorsal root after peripheral nerve injury? This may reflect different neurotrophin sensitivities of subpopulations of primary sensory neurons. In the adult, primary sensory neurons with myelinated axons predominantly express trkB and trkC, with a smaller group (218%) also expressing trkA.6,44,76 These axons, and presumably the cells that give rise to them, remain viable after peripheral nerve lesions. By contrast, approximately 50% of the small DRG cells that give rise to unmyelinated axons express trkA, with the other 50% failing to express any currently known trk receptor.6,76 Since central axon loss after nerve injury is restricted to unmyelinated axons, it would be interesting to determine if either trkA or the non-trk-expressing cells are lost. A problem, though, is that trk receptors change after axotomy.24,66 Also, if all trkA-expressing cells die after a nerve lesion, one would have expected some
loss of myelinated fibers, unless they survive because they also co-express some other trk receptor. An ancillary question is what proportion of cells with unmyelinated axons in the sciatic die when their peripheral axons are transected? As determined by retrograde labeling, the proportions of L4 and L5 DRG cells that project through the sciatic nerve range from 50% to 70%.10,21,33,62 There are questions about completeness of labeling,43,51 but if we accept the proportions as approximately correct, the numbers of cells that give rise to sciatic nerve unmyelinated axons that eventually die after nerve lesions must be high. Indeed, since the total axon count in dorsal roots L4 and L5 is 229,000, with a 2:1 ratio of unmyelinated to myelinated axons, and since 50% of the unmyelinated axons are lost after axotomy, the loss may represent the majority of cells with unmyelinated axons in the sciatic nerve. That small cells, which are presumed to give rise to unmyelinated sensory axons, preferentially die is an old idea.9,43,53 What we now add is that: (i) it is only the unmyelinated axons that are lost, (ii) the proportion of dying axons is high, (iii) it takes a very long time for the death of the axons, and (iv) reinnervation of the target does not prevent the axon loss. A third question is what prevents cell death for sensory neurons with myelinated axons and presumably some cells with unmyelinated axons after they are deprived of contact with peripheral targets? This may reflect either non-target-dependent neurotrophin production or non-dependence on neurotrophins for survival. One source of NGF and BDNF after nerve crush is cells in the distal stump.32,47 The problem with this as an explanation for the lack of cell death is that no difference was found between transection and crush animals, in that transected sensory axons will not be exposed to the neurotrophin-rich distal stump and crushed axons will. Alternative sources of neurotrophins may be the central target, in this case the spinal cord, or cells within the DRG itself. Trophins in the spinal cord have not been reported to be involved in cell survival in adults, in contrast to newborns.76 There are several reports of neurotrophin production in DRG neurons, however, particularly BDNF in trkA-expressing cells,70 with some increase in NGF after nerve injury.60 Furthermore, an autocrine release of BDNF has been shown to contribute to the capacity of some adult DRG neurons to survive in culture in the absence of exogenous growth factors.2 Overall, however, the findings speak strongly against the idea that retrograde peripheral neurotrophin transport has anything to do with survival of DRG cells with central myelinated axons following peripheral nerve lesions in adult rats. This conclusion is buttressed by previous observations that peripheral axotomy leads to few changes in dorsal roots in adult cats58 and little Marchi degeneration in the dorsal columns in adult rats.4 For cells with unmyelinated axons, interruption of retrograde neurotrophin flow is associated with profound cell
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Fig. 3. Bar graphs representing means&S.E.M. of myelinated and unmyelinated axon counts for the L4 and L5 dorsal roots at various times following cut or crush of the sciatic nerve. The naive counts were obtained by combining the ‘‘young’’ and ‘‘old’’ naive counts as mentioned in the text. Counts for cut and crush were compared against the naive data for each group. Statistical significance of P¦0.01 (asterisk) is indicated.
death, but because of the long delay, whether the interruption is causally related to this cell death is not known. Questions from the above are why is the unmyelinated axon loss so slow, and why did it occur both after nerve section and nerve crush? At present we do not know, but it may reflect either rapid sprouting from surviving DRG cells followed by a slow resolution or a slow program of lesion-induced changes
which are not prevented by subsequent reinnervation. Alternatively, since not all fibers successfully reinnervate peripheral targets after nerve crush, it may be that those that fail to reinnervate eventually die. Peripheral axotomy causes changes in chemical phenotype, membrane excitability and structure of primary sensory cell bodies.20,35,71 Peripheral but not central axotomy also results in the up-regulation of growth-associated genes,12 which may contribute to
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the central sprouting of myelinated afferent fibers into lamina II.40,74 Additionally, peripheral nerve section results in degenerative changes in the central terminals of myelinated fibers in the deep dorsal horn,3,5,27,29,69 which have been interpreted as a transganglionic degeneration reflecting post-axotomy DRG cell loss.3 However, since we do not see loss of myelinated axons in the roots when transganglionic degenerative changes are maximal, these changes presumably reflect ‘‘transganglionic atrophy’’38 or ‘‘dying back’’ of central axon terminals8 rather than actual transganglionic degeneration. CONCLUSION
The loss of approximately 50% of C-fibers in dorsal roots without a notable loss of myelinated
axons after peripheral nerve injury means that sensory input changes substantially, with a slowly developing but major shift towards domination by myelinated fibers. These findings are in keeping with the generation of A-fiber-mediated pain after nerve injury.45,73 In addition, the reduction in C-fibers may also contribute to trophic changes in the target as a result of a reduction in the efferent neurogenic inflammatory action subserved by these fibers and the sensory function they subserve. Acknowledgements—We wish to thank Gena Krannig for preparation of tissue, sectioning and counting of axons. We also thank Lyn Schilling for preparing the manuscript. This work is supported by a Bristol-Myers Squibb unrestricted Pain Research Grant, the Medical Research Council of Great Britain, and by NIH grants NS 10161, NS 11255 and NS 07185.
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