Neurotrophins from dorsal root ganglia trigger allodynia after spinal nerve injury in rats

Injury to peripheral nerves often results in chronic pain which is difficult to relieve. The mechanism underlying the pain syndrome remains largely unknown. In previous studies we showed that neurotrophins are up-regulated in satellite cells around sensory neurons following sciatic nerve lesion. In the present study, we have examined whether the neurotrophins in the dorsal root ganglia play any role in allodynia after nerve injury. Antibodies to different neurotrophins, directly delivered to injured dorsal root ganglia, significantly reduced (with different time sequences) the percentage of foot withdrawal responses evoked by von Frey hairs. The antibodies to nerve growth factor acted during the early phase but antibodies to neurotrophin-3 and brain-derived neurotrophic factor were effective during the later phase. Exogenous nerve growth factor or brain-derived neurotrophic factor, but not neurotrophin-3, directly delivered to intact dorsal root ganglia, trigger a persistent mechanical allodynia. Our results showed that neurotrophins within the dorsal root ganglia after peripheral nerve lesion are involved in the generation of allodynia at different stages. These studies provide the first evidence that ganglia-derived neurotrophins are a source of nociceptive stimuli for neuropathic pain after peripheral nerve injury.     

Dynamic expression of Slit1–3 and Robo1–2 in the mouse peripheral nervous system after injury

The Slit family of axon guidance cues act as repulsive molecules for precise axon pathfinding and neuronal migration during nervous system development through interactions with specific Robo receptors.Although we previously reported that Slit1–3 and their receptors Robo1 and Robo2 are highly expressed in the adult mouse peripheral nervous system,how this expression changes after injury has not been well studied.Herein,we constructed a peripheral nerve injury mouse model by transecting the right sciatic nerve.At 14 days after injury,quantitative real-time polymerase chain reaction was used to detect mRNA expression of Slit1–3 and Robo1–2 in L4–5 spinal cord and dorsal root ganglia,as well as the sciatic nerve.Immunohistochemical analysis was performed to examine Slit1–3,Robo1–2,neurofilament heavy chain,F4/80,and vimentin in L4–5 spinal cord,L4 dorsal root ganglia,and the sciatic nerve.Co-expression of Slit1–3 and Robo1–2 in L4 dorsal root ganglia was detected by in situ hybridization.In addition,Slit1–3 and Robo1–2 protein expression in L4–5 spinal cord,L4 dorsal root ganglia,and sciatic nerve were detected by western blot assay.The results showed no significant changes of Slit1–3 or Robo1–2 mRNA expression in the spinal cord within 14 days after injury.In the dorsal root ganglion,Slit1–3 and Robo1–2 mRNA expression were initially downregulated within 4 days after injury;however,Robo1–2 mRNA expression returned to the control level,while Slit1–3 mRNA expression remained upregulated during regeneration from 4–14 days after injury.In the sciatic nerve,Slit1–3 and their receptors Robo1–2 were all expressed in the proximal nerve stump;however,Slit1,Slit2,and Robo2 were barely detectable in the nerve bridge and distal nerve stump within 14 days after injury.Slit3 was highly ex-pressed in macrophages surrounding the nerve bridge and slightly downregulated in the distal nerve stump within 14 days after injury.Robo1 was upregulated in vimentin-positive cells and migrating Schwann cells inside the nerve bridge.Robo1 was also upregulated in Schwann cells of the distal nerve stump within 14 days after injury.Our findings indicate that Slit3 is the major ligand expressed in the nerve bridge and distal nerve stump during peripheral nerve regeneration,and Slit3/Robo signaling could play a key role in peripheral nerve repair after injury.This study was approved by Plymouth University Animal Welfare Ethical Review Board (approval No.30/3203) on April 12,2014.     

Substance P and calcitonin gene-related peptide expression in dorsal root ganglia in sciatic nerve injury rats

The neuropeptides, substance P and calcitonin gene-related peptide, have been shown to be involved in pain transmission and repair of sciatic nerve injury. A model of sciatic nerve defect was prepared by dissecting the sciatic nerve at the middle, left femur in female Sprague Dawley rats. The two ends of the nerve were encased in a silica gel tube. L5 dorsal root ganglia were harvested 7, 14 and 28 days post sciatic nerve injury for immunohistochemical staining. Results showed that substance P and cal- citonin gene-related peptide expression increased significantly in dorsal root ganglion of rats with sci- atic nerve injury. This increase peaked at 7 days, declined at 14 days, and reduced to normal levels by 28 days post injury. The findings indicate that the neuropeptides, substance P and calcitonin gene- related peptide, mainly increased in the early stages after sciatic nerve injury.     

Macrophages contribute to the maintenance of stable regenerating neurites following peripheral nerve injury

Normal adult uninjured nerve is unable to support axonal regeneration. We have studied the mechanisms underlying the regeneration of peripheral nerve by culturing adult mouse dorsal root ganglia (DRG) explants on unfixed, longitudinal cryosections of either the uninjured sciatic nerve or the distal segment of the transected sciatic nerve. We found that, initially, DRG grew vigorously on cryosections of both uninjured and postinjury sciatic nerves. However, the neurites began to degenerate shortly after contact with the uninjured nerve, whereas those growing on postinjury nerve substrate remained healthy for up to 9 days in culture. This ability to support stable outgrowth peaked at 8 days, gradually decreased by 10 days, and disappeared by 20 days after injury. Macrophages appeared in the distal segment by 4 days postinjury and had infiltrated its entire length by 8 days. Uninjured nerve cryosections could be rendered supportive of stable outgrowth by preincubation with macrophage-conditioned medium or by brief trypsinization. The activity of the macrophage-conditioned medium was augmented upon activation of macrophages. Together these findings suggest that the environment of the sciatic nerve undergoes a transformation during Wallerian degeneration such that it becomes transiently supportive of the stable outgrowth of neurites. This transformation may be mediated by a proteolytic activity, generated by activated macrophages, that removes a putative "degeneration signal" protein normally present in the adult nerve and thus contributes to the maintenance of stable regenerating neurites.     

Upregulation of brain-derived neurotrophic factor in the sensory pathway by selective motor nerve injury in adult rats

Selective motor nerve injury by lumbar 5 ventral root transection (L5 VRT) induces neuropathic pain, but the underlying mechanisms remain unknown. Previously, increased expression and secretion of brain-derived neurotrophic factor (BDNF) had been implicated in injury-induced neuropathic pain in the sensory system. In this study, as a step to examine potential roles of BDNF in L5 VRT-induced neuropathic pain, we investigated BDNF gene and protein expression in adult rats with L5 VRT. L5 VRT induced a dramatic upregulation of BDNF mRNA in intact sensory neurons in the ipsilateral L5 dorsal root ganglia (DRG), in non-neuronal cells in the ipsilateral sciatic nerve, and in motoneurons in the ipsilateral spinal cord. L5 VRT also induced de novo synthesis of BDNF mRNA in spinal dorsal horn neurons and in glial cells in the white matter of the ipsilateral spinal cord. Consistent with the mRNA expression pattern, BDNF protein was also mainly upregulated in all populations of sensory neurons in the ipsilateral L5 DRG and in spinal neurons and glia. Quantitative analysis by ELISA showed that the BDNF content in the DRG and sciatic nerve peaked on day 1 and remained elevated 14 days after L5 VRT. These results suggest that increased BDNF expression in intact primary sensory neurons and spinal cord may be an important factor in the induction of neuropathic pain without axotomy of sensory neurons.     

Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons

Most current studies quantify axon regeneration by immunostaining regeneration-associated proteins,representing indirect measurement of axon lengths from both sensory neurons in the dorsal root ganglia and motor neurons in the spinal cord.Our recently developed method of in vivo electroporation of plasmid DNA encoding for enhanced green fluorescent protein into adult sensory neurons in the dorsal root ganglia provides a way to directly and specifically measure regenerating sensory axon lengths in whole-mount nerves.A mouse model of sciatic nerve compression was established by squeezing the sciatic nerve with tweezers.Plasmid DNA carrying enhanced green fluorescent protein was transfected by ipsilateral dorsal root ganglion electroporation 2 or 3 days before injury.Fluorescence distribution of dorsal root or sciatic nerve was observed by confocal microscopy.At 12 and 18 hours,and 1,2,3,4,5,and 6 days of injury,lengths of regenerated axons after sciatic nerve compression were measured using green fluorescence images.Apoptosis-related protein caspase-3 expression in dorsal root ganglia was determined by western blot assay.We found that in vivo electroporation did not affect caspase-3 expression in dorsal root ganglia.Dorsal root ganglia and sciatic nerves were successfully removed and subjected to a rapid tissue clearing technique.Neuronal soma in dorsal root ganglia expressing enhanced green fluorescent protein or fluorescent dye-labeled microRNAs were imaged after tissue clearing.The results facilitate direct time course analysis of peripheral nerve axon regeneration.This study was approved by the Institutional Animal Care and Use Committee of Guilin Medical University,China(approval No.GLMC201503010)on March 7,2014.     

Preconditioning selective ventral root injury promotes plasticity of ascending sensory neurons in the injured spinal cord of adult rats – possible roles of brain-derived neurotrophic factor, TrkB and p75 neurotrophin receptor

Preconditioning sciatic nerve injury enhances axonal regeneration of ascending sensory neurons after spinal cord injury. A key question is whether direct injury of sensory nerves is necessary for the enhanced regeneration. The lumbar 5 ventral root transection (L5 VRT) model, a model of selective motor nerve injury, provides a useful tool to address this question. Here we examined the effects of a preconditioning L5 VRT on the regeneration after a subsequent dorsal column transection (DCT) in adult Sprague–Dawley rats. We found that L5 VRT 1 week before DCT increased the number of Fast Blue (FB)-labeled neurons in the L5 dorsal root ganglia (DRG) and promoted sprouting/regenerating axons to grow into the glial scar. L5 VRT also induced a dramatic upregulation of expression of brain-derived neurotrophic factor (BDNF) in the preconditioned DRG and in the injured spinal cord. Moreover, almost all of the FB-labeled sprouting/regenerating neurons expressed BDNF, and approximately 55% of these neurons were surrounded by p75 neurotrophin receptor-positive glial cells. This combined injury led to an increase in the number of BDNF- and TrkB-immunoreactive nerve fibers in the dorsal column caudal to the lesion site. Taken together, these findings demonstrate that L5 VRT promotes sprouting/regeneration of ascending sensory neurons, indicating that sensory axotomy may not be essential for the plasticity of injured dorsal column axons. Thus, the sensory neurons could be preprimed in the regenerative milieu of Wallerian degeneration and neuroinflammation, which might alter the expression of neurotrophic factors and their receptors, facilitating sprouting/regeneration of ascending sensory neurons.     

miR-30c promotes Schwann cell remyelination following peripheral nerve injury

Differential expression of miRNAs occurs in injured proximal nerve stumps and includes miRNAs that are firstly down-regulated and then gradually up-regulated following nerve injury. These miRNAs might be related to a Schwann cell phenotypic switch. miR-30c, as a member of this group, was further investigated in the current study. Sprague-Dawley rats underwent sciatic nerve transection and proximal nerve stumps were collected at 1, 4, 7, 14, 21, and 28 days post injury for analysis. Following sciatic nerve injury, miR-30c was down-regulated, reaching a minimum on day 4, and was then upregulated to normal levels. Schwann cells were isolated from neonatal rat sciatic nerve stumps, then transfected with miR-30c agomir and co-cultured in vitro with dorsal root ganglia. The enhanced expression of miR-30c robustly increased the amount of myelin-associated protein in the co-cultured dorsal root ganglia and Schwann cells. We then modeled sciatic nerve crush injury in vivo in Sprague-Dawley rats and tested the effect of perineural injection of miR-30c agomir on myelin sheath regeneration. Fourteen days after surgery, sciatic nerve stumps were harvested and subjected to immunohistochemistry, western blot analysis, and transmission electron microscopy. The direct injection of miR-30c stimulated the formation of myelin sheath, thus contributing to peripheral nerve regeneration. Overall, our findings indicate that miR-30c can promote Schwann cell myelination fol-lowing peripheral nerve injury. The functional study of miR-30c will benefit the discovery of new therapeutic targets and the development of new treatment strategies for peripheral nerve regeneration.     

重组腺病毒载体AxCA-BDNF基因转染修复坐骨神经损伤*☆

背景：如何促进周围神经损伤修复与再生一直是基础与临床研究的热点。基因治疗有可能成为今后解决该问题的主要手段之一。 目的：观察携带小鼠脑源性神经营养因子(brain-derived neurotrophic factor,BDNF) cDNA表达片段的重组腺病毒载体AxCA-BDNF转染大鼠损伤坐骨神经后BDNF的表达,以及脊髓前角运动神经元的存活和神经生长情况。 方法：切除成年Wistar大鼠股中部10 mm长的坐骨神经,AxCA-BDNF转染组、BDNF组和对照组分别用硅胶管内置AxCA-BDNF原液,BDNF溶液或空白病毒稀释液桥接坐骨神经两断端。术后3,7,14 d,1,2,4个月应用原位杂交和免疫组织化学方法检测损伤坐骨神经及相应脊髓节段BDNF mRNA和蛋白的表达,并观察损伤坐骨神经的组织学及超微结构改变,再生的神经元及有髓神经纤维数目和髓鞘厚度。 结果与结论：术后3,7,14 d及1个月时,AxCA-BDNF转染组损伤坐骨神经近、远端神经干及脊髓(L3~6)中BDNF mRNA和蛋白水平明显高于BDNF组和对照组(P < 0.01)。光、电镜病理组织学检查和图像分析证实,BDNF基因转染后,脊髓前角运动神经元存活数量、新生神经纤维数目及其髓鞘厚度、神经联接的再形成均明显优于对照组(P < 0.01)。说明经腺病毒介导转染的BDNF基因可在大鼠坐骨神经内有效表达,并通过轴突逆行转运到了相应的脊髓神经元,不仅能促进损伤神经纤维再生,也能保护损伤的脊髓神经元。 关键词：坐骨神经损伤;重组腺病毒;脑源性神经营养因子;基因转染;免疫组织化学;原位分子杂交;神经再生     

Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms

Human umbilical cord-derived mesenchymal stem cells(h UCMSCs) represent a promising young-state stem cell source for cell-based therapy. h UCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the paracrine effects of h UCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that h UCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with h UCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These findings suggest that paracrine action may be a key mechanism underlying the effects of h UCMSCs in peripheral nerve repair.     

Role of neurotrophic factors in enhancing linear axonal growth of ganglionic sensory neurons in vitro

Neurotrophins play a major role in the regulation of neuronal growth such as neurite sprouting or regeneration in response to nerve injuries. The role of nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor in maintaining the survival of peripheral neurons remains poorly understood. In regenerative medicine, different modalities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. This study was to investigate the influence of nerve growth factor, neurotrophin-3 and brain-derived neurotrophic factor on the growth of neurites using two in vitro models of dorsal root ganglia explants and dorsal root ganglia-derived primary cell dissociated cultures. Quantitative data showed that the total neurite length and tortuosity were differently influenced by trophic factors. Nerve growth factor and, indirectly, brain-derived neurotrophic factor stimulate the tortuous growth of sensory fibers and the formation of cell clusters. Neurotrophin-3, however, enhances neurite growth in terms of length and linearity allowing for a more organized and directed axonal elongation towards a peripheral target compared to the other growth factors. These findings could be of considerable importance for any clinical application of neurotrophic factors in peripheral nerve regeneration. Ethical approval was obtained from the Regione Piemonte Animal Ethics Committee ASLTO1(file # 864/2016-PR) on September 14, 2016.     

Trophic influence of the distal nerve segment on GABAA receptor expression in axotomized adult sensory neurons

The depolarizing action of gamma-aminobutyric acid (GABA), or the GABAA receptor agonist muscimol, on rat dorsal root (L4 and L5) fibers is attenuated following transection, but not crush, of the sciatic nerve. Following discrete nerve crush, axons actively regenerate and contact both the distal nerve segment and the peripheral target tissues. The aim of the present study was to distinguish between these two regions as possible sources of trophic support for retrograde maintenance of dorsal root GABA receptor sensitivity. A surgical procedure was employed to permit a delimited segment of axonal regeneration while prohibiting reestablishment of end organ innervation; the sciatic nerve was crushed and a ligature was placed 3 cm distal to the crush site. Under these conditions, the injury-induced decrement in the dorsal root GABA response, observed between 12 and 21 postoperative days, was significantly attenuated relative to that of ligated nerves, in which regeneration into the distal stump does not occur. The data suggest that nerve transection by ligation restricts trophic support for maintenance of GABA receptor expression in dorsal root ganglion (DRG) neurons. Furthermore, during regeneration the denervated distal nerve segment assumes a neurotrophic role in the maintenance of dorsal root GABA sensitivity, consistent with the hypothesis that growth factors derived from reactive Schwann cells may positively regulate the expression of receptors on axotomized sensory neurons.     

Trophic influence of the distal nerve segment on GABAA receptor expression in axotomized adult sensory neurons

The depolarizing action of γ-aminobutyric acid (GABA), or the GABA_A receptor agonist muscimol, on rat dorsal root (L4 and L5) fibers is attenuated following transection, but not crush, of the sciatic nerve (15). Following discrete nerve crush, axons actively regenerate and contact both the distal nerve segment and the peripheral target tissues. The aim of the present study was to distinguish between these two regions as possible sources of trophic support for retrograde maintenance of dorsal root GABA receptor sensitivity. A surgical procedure was employed to permit a delimited segment of axonal regeneration while prohibiting reestablishment of end organ innervation; the sciatic nerve was crushed and a ligature was placed 3 cm distal to the crush site. Under these conditions, the injury-induced decrement in the dorsal root GABA response, observed between 12 and 21 postoperative days, was significantly attenuated relative to that of ligated nerves, in which regeneration into the distal stump does not occur. The data suggest that nerve transection by ligation restricts trophic support for maintenance of GABA receptor expression in dorsal root ganglion (DRG) neurons. Furthermore, during regeneration the denervated distal nerve segment assumes a neurotrophic role in the maintenance of dorsal root GABA sensitivity, consistent with the hypothesis that growth factors derived from reactive Schwann cells may positively regulate the expression of receptors on axotomized sensory neurons.     

Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury

The transient receptor potential cation channel subfamily V member 1(TRPV1) provides the sensation of pain(nociception). However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517(300 mg/kg), a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immunofluorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clusters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve.     

Galanin and its receptor system promote the repair of injured sciatic nerves in diabetic rats

Various studies have reported that galanin can promote axonal regeneration of dorsal root ganglion neuronsin vitro and inhibit neuropathic pain. However, little is known about its effects on diabetic peripheral neuropathy, andin vivo experimental data are lacking. We hypothesized that repeated applications of exogenous galanin over an extended time frame may also repair nerve damage in diabetic peripheral neuropathy, and relieve pain in vivo. We found that neuropathic pain occurred in streptozotocin-induced diabetic rats and was more severe after sciatic nerve pinch injury at 14 and 28 days than in diabetic sham-operated rats. Treatment with exogenous galanin alleviated the neuropathic pain and promoted sciatic nerve regeneration more effectively in diabetic rats than in non-diabetic rats after sciatic nerve pinch injury. This was accompanied by changes in the levels of endogenous galanin, and its receptors galanin receptor 1 and galanin receptor 2 in the dorsal root ganglia and the spinal dorsal horn when compared with nerve pinch normal rats. Our results show that application of exogenous galanin daily for 28 days can promote the regeneration of injured sciatic nerves, and alleviate neuropathic pain in diabetic rats.     

Intrinsic versus extrinsic factors in determining the regeneration of the central processes of rat dorsal root ganglion neurons: The influence of a peripheral nerve graft

The relative contribution of intrinsic growth capacity versus extrinsic growth-promoting factors in determining the capacity of transected dorsal root axons to regenerate long distances was studied. L4 dorsal root axons regenerating into 4-cm peripheral nerve grafts on transected dorsal roots were counted. Few dorsal root myelinated axons regenerated to the distal end of the grafts by 10 weeks unless the sciatic nerve was also crushed. Regeneration of unmyelinated axons was also increased by peripheral lesions. Crush or transection of the dorsal roots without grafting did not alter GAP-43 mRNA expression in L4 dorsal root ganglion (DRG) cells. Grafting a peripheral nerve onto the cut end of an L4 dorsal root doubled the number of DRG cells expressing high levels of GAP-43 mRNA after a delay of several weeks. Peripheral nerve crush at the time of nerve grafting resulted in a very rapid rise in GAP-43 mRNA expression, which then declined to a steady level, twice that of controls, by 7 weeks. Thus, the rapid increase in the number of DRG neurons expressing high levels of GAP-43 mRNA after peripheral but not central axotomy correlates with the regeneration of central axons through nerve grafts. Because GAP-43 mRNA is slowly upregulated in a subpopulation of sensory neurons in response to exposure of their central axons to a peripheral nerve environment, environments favourable for axonal growth may act by increasing the intrinsic growth response of neurons. Lack of intrinsic growth capacity may contribute to the failure of dorsal root axons to regenerate into the spinal cord. © 1996 Wiley-Liss, Inc.     

Nitric oxide synthase and growth-associated protein are coexpressed in primary sensory neurons after peripheral injury

A possible role for nitric oxide in growth and regeneration of dorsal root ganglion (DRG) afferents has been explored in lesion experiments by comparing immunocytochemistry for nitric oxide synthase (NOS) with that for the growth-associated phosphoprotein 43 (GAP-43). Sciatic nerve ligature induced a progressive increase in the number of small DRG cell profiles immunopositive for NOS between 2 days and 4 weeks of survival. In the proximal stump of the ligature, NOS-immunopositive fibers began to appear 2 days after injury and their growth cones were especially evident after 7 days. NOS-immunopositive fibers appeared past (i.e., distal to) the ligature at 14 days of survival and extended for at least 6 mm in either direction 4 weeks after the lesion. Dorsal root ligature alone at L4–L5 did not result in expression of NOS in DRG neurons or in the appearence of NOS-immunopositive fibers. In rats with dorsal root ligature and nerve ligature, the results were similar to those with nerve ligature only. DRG cell profiles immunopositive for GAP-43 kept increasing from 2 days to 4 weeks after sciatic nerve ligature and included small neurons initially and large neurons subsequently. Numerous axons became GAP-43 immunopositive on both sides of the ligature from 2 days after injury. In double-labeled material, about 80% of DRG cell profiles immunopositive for NOS were also immunopositive for GAP-43. The two antigens co-occurred in peripheral nerve axons proximal to the ligature starting at about 7 days and distal to it at about 2 weeks after ligature. Thus, in response to nerve lesion, nitric oxide may not only provide an injury signal to the central nervous system but may also contribute to the growth and regeneration of injured axons. J. Comp. Neurol. 404:64–74, 1999. © 1999 Wiley-Liss, Inc.