Increased uptake and transport of cholera toxin B-subunit in dorsal root ganglion neurons after peripheral axotomy: Possible implications for sensory sprouting

In the present study we show that, in contrast to the rat, injection of cholera toxin B-subunit (CTB) into the intact sciatic nerve of Macaca mulatta monkey gives rise to labelling of a sparse network of fibers in laminae I–II of spinal cord and of some mainly small dorsal root ganglion (DRG) neurons. Twenty days after sciatic nerve cut, the percentage of CTB-positive lumbar 5 (L5) DRG neuron profiles increased from 11% to 73% of all profiles. In the spinal cord, a marked increase in CTB labelling was seen in laminae I, II, and the dorsal part of lamina III. In the rat L5 DRGs, 18 days after sciatic nerve cut, the percentage of CTB- and CTB conjugated to horseradish peroxidase (HRP)-labelled neuron profiles increased from 45% to 81%, and from 54% to 87% of all neuron profiles, respectively. Cell size measurements in the rat showed that most of the CTB-positive neuron profiles were small in size after axotomy, whereas most were large in intact DRGs. In the rat spinal dorsal horn, a dense network of CTB-positive fibers covered the whole dorsal horn on the axotomized side, whereas CTB-labelled fibers were mainly seen in laminae III and deeper laminae on the contralateral side. A marked increase in CTB-positive fibers was also seen in the gracile nucleus. The present study shows that in both monkey and rat DRGs, a subpopulation of mainly small neurons acquires the capacity to take up CTB/CTB-HRP after axotomy, a capacity normally not associated with these DRG neurons. These neurons may transganglionically transport CTB and CTB-HRP. Thus, after peripheral axotomy, CTB and CTB-HRP are markers not only for large but also for small DRG neurons and, thus, possibly also for both myelinated and unmyelinated primary afferents in the spinal dorsal horn. These findings may lead to a reevaluation of the concept of sprouting, considered to take place in the dorsal horn after peripheral nerve injury. J. Comp. Neurol. 404:143–158, 1999. © 1999 Wiley-Liss, Inc.     

Axotomy induces preprotachykinin gene expression in a subpopulation of dorsal root ganglion neurons

The distribution of dorsal root ganglion (DRG) cell sizes that show changes in preprotachykinin (PPT) gene expression and substance P (SP) levels following axotomy was examined using RNA blot analysis, in situ hybridization histochemistry, and immunocytochemistry. PPT mRNA was induced in medium-sized (1,000–2,000 μm²) and large-sized (>2,000 μm²) cells in the DRG after axotomy. There was a 165% increase in the number of labeled cells after sciatic transection and a 260% increase after spinal nerve transection which results in axotomy of all the cells in the ganglion. The further increase after spinal nerve transection suggests that the induction occurred in axotomized neurons. PPT mRNA label was also present in a reduced number of small (<1,000 μm²) cells after axotomy. SP immunoreactivity was also induced in medium-and large-sized cells and reduced in small-sized cells. Our findings suggest that the expression of the PPT gene and SP is differentially regulated in different subpopulations of DRG neurons after axotomy and is consistent with the hypothesis that tachykinins may be important in both sensory transmission and regeneration. © 1994 Wiley-Liss, Inc.     

Dorsal root ganglion neurons up-regulate the expression of laminin-associated integrins after peripheral but not central axotomy

The favorable prognosis of regeneration in the peripheral nervous system after axonal lesions is generally regarded as dependent on the Schwann cell basal lamina. Laminins, a heterotrimeric group of basal lamina molecules, have been suggested to be among the factors playing this supportive role. For neurons to utilize laminin as a substrate for growth, an expression of laminin binding receptors, integrins, is necessary. In this study, we have examined the expression of laminin binding integrin subunits in dorsal root ganglion (DRG) neurons after transection to either their peripherally projecting axons, as in the sciatic nerve, followed by regeneration, or the centrally projecting axons in dorsal roots, followed by no or weak regenerative activity. In uninjured DRG, immunohistochemical staining revealed a few neurons expressing integrin subunit alpha6, whereas integrin subunits alpha7 and foremost beta1 were expressed in a majority of neurons. After an injury to the sciatic nerve, mRNAs encoding all three integrins were up-regulated in DRG neurons. By anterograde tracing, immunoreactivity for all studied integrins was also found in association with growing axons after a sciatic nerve crush lesion in vivo. In contrast, mRNA levels remained constant in DRG neurons after a dorsal root injury. Together with previous findings, this suggests that integrin subunits alpha6, alpha7, and beta1 have an important role in the regenerative response following nerve injury and that the lack of regenerative capacity following dorsal root injury could in part be explained by the absence of response in integrin regulation.     

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.     

周围神经损伤后脊神经节感觉神经元胞体形态学的变化

目的 研究周围神经损伤后脊神经节感觉神经元胞体形态学的变化以探讨其主要死广性质。方法 切断并原位吻合大鼠右侧坐骨神经，左侧不作任何处理，作为对照；于术后不同时间取L4-L6脊神经节作光镜和电镜观察，观察脊神经节感觉神经元胞体形态的变化。结果 光镜下，损伤的脊神经节感觉神经元胞体染色质浓染；电镜下，细胞膜内陷，分割细胞内容物成凋亡小体；而对侧脊神经节感觉神经元胞体均一、无变化。结论 大鼠坐骨神经损伤后，脊神经节感觉神经元有死亡，其胞体的形态学变化符合细胞凋亡特征。     

The effect of peripheral nerve injury on dorsal root potentials and on transmission of afferent signals into the spinal cord

The sciatic nerve adults rats was either cut and ligated or was crushed on one side. The response of the spinal cord to stimulation of the proximal part of the injured nerve was examined at various times after the lesion and compared to the effects of stimulating the intact nerve on the other side. During the first 10 days after nerve section the following measures were not affected: (i) the size of the input volley (compound action potential, CAP, measured on a dorsal root that carried sciatic nerve afferents (L5); (ii) the volley running in the dorsal columns; (iii) the dorsal root potential (DRP) evoked on neighbouring dorsal roots which do not contain sciatic afferents (L2 and L3); (iv) the post-synaptic volleys ascending in the spinal cord. However, by the fourth day after nerve section, there was a decrease of the DRP evoked on the ipsilateral L5 dorsal root by stimulation of the cut nerve. By 10 days this DRP had decreased by 50%. There was also a decrease in the DRP on the L5 root evoked by stimulation of the contralateral intact nerve. Crush lesions of the sciatic nerve did not produce DRP charge. Beginning 10–20 days after nerve cut, there was a decrease in the amplitude of the afferent CAP and of all the measures of central response to the afferent volley. We discuss the possibility that the loss of the DRP may be associated with a disinhibition which results in novel receptive fields which we observe in cord cells deafferented by the peripheral nerve section. The decrease of DRP and the appearance of novel receptive fields do not occur if the peripheral nerve is crushed rather than cut.     

Neurite outgrowth and GAP-43 mRNA expression in cultured adult rat dorsal root ganglion neurons: Effects of NGF or prior peripheral axotomy

Adult dorsal root ganglion (DRG) cells are capable of neurite outgrowth in vivo and in vitro after axotomy. We have investigated, in cultured adult rat DRG cells, the relative influence of nerve growth factor (NGF) or a prior peripheral nerve lesion on the capacity of these neurons to produce neurites. Since there is evidence suggesting that the growth-associated protein GAP-43 may play a crucial role in axon elongation during development and regeneration, we have also compared the effect of these treatments on GAP-43 mRNA expression. NGF increased the early neurite outgrowth in a subpopulation of DRG cells. This effect was substantially less, however, than that resulting from preaxotomy, which initiated an early and profuse neurite outgrowth in almost all cells. No difference in the expression of GAP-43 mRNA was found between neurons grown in the presence or absence of NGF over 1 week of culture, in spite of the increased growth produced by NGF. In contrast, cultures of neurons that had been preaxotomized showed substantial increase in GAP-43 mRNA and NGF had, as expected, a significant effect on substance P mRNA levels. Two forms of growth may be present in adult DRG neurons: an NGF-independent, peripheral nerve injury-provoked growth associated with substantial GAP-43 upregulation, and an NGF-dependent growth that may underlie branching or sprouting of NGF-sensitive neurons, but which is not associated with increased levels of GAP-43 mRNA. © 1994 Wiley-Liss, Inc.     

Retinoic acid responsive gene product,midkine, has neurotrophic functions for mouse spinal cord and dorsal root ganglion neurons in culture

Midkine (MK) is the product of a retinoic acid responsive gene and is a member of a new family of heparin-binding growth factors. Neurotrophic effects of MK were examined using cultured spinal cord and dorsal root ganglion (DRG) neurons derived from fetal mouse. MK, which was added to the culture medium at concentrations of 1–100 ng/ml, promoted survival of both types of neurons approximately 5-fold after 7 days in culture. For spinal cord neurons, the increased survival was reflected in an increase of choline acetyltransferase activity. MK also promoted neurite extension in spinal cord (2-fold) and DRG (1.7-fold) neurons. The survival-promoting activity of MK to these neurons was comparable to that of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF). In spite of its significant effects on fetal neurons, MK was ineffective in sustaining survival of DRG neurons derived from postnatal mice. From these results, we conclude that MK is a neurotrophic factor to embryonic spinal cord and DRG neurons, and we propose that MK plays a significant role in embryogenesis of the nervous system. © 1993 Wiley-Liss, Inc.     

Distribution and expression of tissue inhibitors of metalloproteinase in dorsal root entry zone and dorsal column after dorsal root injury

To understand whether tissue inhibitors of metalloproteinase (TIMPs) contribute to the failure of regenerating sensory axons to enter the spinal cord, we used in situ hybridization and immunocytochemistry to examine the expression of TIMP1, TIMP2, and TIMP3 in the dorsal root, dorsal root entry zone (DREZ), and dorsal column after dorsal root injury in adult rats. We found that the three TIMPs and their mRNAs were up-regulated in a time-, region-, and cell-type-specific manner. Strong up-regulation of all three TIMPs was seen in the injured dorsal roots. TIMP2 was also significantly up-regulated in the DREZ and degenerating dorsal column, where TIMP1 and TIMP3 showed only moderate up-regulation. Most cells up-regulating the TIMPs in the DREZ and degenerating dorsal column were reactive astrocytes, but TIMP2 was also up-regulated by microglia/macrophages, especially at long postoperative survival times. These results suggest that TIMPs may be involved in controlling tissue remodelling following dorsal root injury and that manipulation of the expression of TIMPs may provide a means of promoting axonal regeneration into and within the injured spinal cord.     

Enhanced adenoviral gene delivery to motor and dorsal root ganglion neurons following injection into demyelinated peripheral nerves

Injection of viral vectors into peripheral nerves may transfer specific genes into their dorsal root ganglion (DRG) neurons and motoneurons. However, myelin sheaths of peripheral axons block the entry of viral particles into nerves. We studied whether mild, transient peripheral nerve demyelination prior to intraneural viral vector injection would enhance gene transfer to target DRG neurons and motoneurons. The right sciatic nerve of C57BL/6 mice was focally demyelinated with 1% lysolecithin, and the left sciatic nerve was similarly injected with saline (control). Five days after demyelination, 0.5 μl of Ad5‐GFP was injected into both sciatic nerves at the site of previous injection. The effectiveness of gene transfer was evaluated by counting GFP⁺ neurons in the DRGs and ventral horns. After peripheral nerve demyelination, there was a fivefold increase in the number of infected DRG neurons and almost a 15‐fold increase in the number of infected motoneurons compared with the control, nondemyelinated side. Focal demyelination reduced the myelin sheath barrier, allowing greater virus–axon contact. Increased CXADR expression on the demyelinated axons facilitated axoplasmic viral entry. No animals sustained any prolonged neurological deficits. Increased gene delivery into DRG neurons and motoneurons may provide effective treatment for amyotrophic lateral sclerosis, pain, and spinal cord injury. © 2010 Wiley‐Liss, Inc.     

Effects of postnatal anti-NGF on the development of CGRP-IR neurons in the dorsal root ganglion

Experiments were undertaken to examine anatomical correlates of physiological effects of rabbit sera raised against nerve growth factor (anti-NGF) on nociceptive afferents. This antiserum has been shown to deplete the population of A-δ high threshold mechanoreceptors and to reduce neurogenic vasodilatation. Because numerous studies implicate calcitonin gene related peptide (CGRP)- containing sensory neurons in these effects, immunocytochemical and anatomical techniques were used to examine the normal development of CGRP-immunoreactive (-IR) neurons in the dorsal root ganglion (DRG) of rats from 13 days to 19 weeks of age, and to compare this to the development in rats treated neonatally (postnatal days 2-14) with anti-NGF. In controls the rate of increase in the mean diameter of CGRP-IR cells was substantially greater between 13 days and 5 weeks of age than it was between 5 weeks and 19 weeks, in contrast to CGRP-negative neurons whose rate of growth remained relatively constant. Anti-NGF had no significant effect on growth rate, but rats treated with anti-NGF exhibited a reduced proportion of CGRP-IR neurons at 5 weeks. This deficit was reversed by 19 weeks unlike the physiological changes. These results indicate independent regulation of CGRP expression and nociceptor physiology by NGF. J. Comp. Neurol. 392: 489–498, 1998. © 1998 Wiley-Liss, Inc.     

Primary neurons maintain their central axonal arbors in the spinal dorsal horn following peripheral nerve injury: an anatomical analysis using transganglionic transport of horseradish peroxidase

This study in adult cats demonstrates that primary neurons of all sizes survive following the transection and capping with a polyethylene tube of their peripheral processes in the superficial radial nerve. The central axonal arbors of these injured primary neurons remain intact and maintain their normal topographic position across laminae I–VI of the cervical (C6–C8) dorsal horn. In addition, they maintain their synaptic vesicles, some of their synaptic connections and their ability to transport horseradish peroxidase transganglionically.     

Transganglionic regulation of central terminals of dorsal root ganglion cells by nerve growth factor (NGF)

Blockade of axonal transport or transection of the rat sciatic nerve results in transganglionic degenerative atrophy (TDA) of nerve terminals containing fluoride-resistant acid phosphatase (FRAP) in the Rolando substance of the spinal cord. Application of vinblastine (9 micrograms) in a cuff around the sciatic nerve of adult rats blocked the retrograde transport of [125I]NGF in sensory fibers; this amount of vinblastine is identical to the threshold amount that induces TDA. Conversely, application of NGF to the proximal stump of the transected sciatic nerve prevented or delayed the occurrence of TDA as reflected by the maintenance of FRAP in the upper dorsal horn, that otherwise would inevitably disappear following the peripheral nerve lesion. These results suggest that endogenous NGF transported retrogradely in peripheral sensory fibers of the adult rat under normal conditions may be responsible for the regulation of the structural and functional integrity of the central terminals of these FRAP-containing primary sensory neurons and that TDA may be the consequence of the failure of NGF to reach the perikarya of these neurons.     

Functional repair after dorsal root rhizotomy using nerve conduits and neurotrophic molecules

Functional recovery after large excision of dorsal roots is absent because of both the limited regeneration capacity of the transected root, and the inability of regenerating sensory fibers to traverse the dorsal root entry zone. In this study, bioresorbable guidance conduits were used to repair 6-mm dorsal root lesion gaps in rats, while neurotrophin-encoding adenoviruses were used to elicit regeneration into the spinal cord. Polyester conduits with or without microfilament bundles were implanted between the transected ends of lumbar dorsal roots. Four weeks later, adenoviruses encoding NGF or GFP were injected into the spinal cord along the entry zone of the damaged dorsal roots. Eight weeks after injury, nerve regeneration was observed through both types of implants, but those containing microfilaments supported more robust regeneration of calcitonin gene-related peptide (CGRP)-positive nociceptive axons. NGF overexpression induced extensive regeneration of CGRP(+) fibers into the spinal cord from implants showing nerve repair. Animals that received conduits containing microfilaments combined with spinal NGF virus injections showed the greatest recovery in nociceptive function, approaching a normal level by 7-8 weeks. This recovery was reversed by recutting the dorsal root through the centre of the conduit, demonstrating that regeneration through the implant, and not sprouting of intact spinal fibers, restored sensory function. This study demonstrates that a combination of PNS guidance conduits and CNS neurotrophin therapy can promote regeneration and restoration of sensory function after severe dorsal root injury.     

Regulation of chondroitin sulphate proteoglycan and reactive gliosis after spinal cord transection: effects of peripheral nerve graft and fibroblast growth factor 1

M‐J. Lee, C. J. Chen, W‐C. Huang, M‐C. Huang, W‐C. Chang, H‐S. Kuo, M‐J. Tsai, Y‐L. Lin and H. Cheng (2011) Neuropathology and Applied Neurobiology 37, 585–599 Regulation of chondroitin sulphate proteoglycan and reactive gliosis after spinal cord transection: effects of peripheral nerve graft and fibroblast growth factor 1 Aims: The combined treatment of peripheral nerve (PN) graft and fibroblast growth factor (FGF)‐1 for spinal cord injury produces functional recovery, but how it affects injury events is still unknown. This project studied the effect of PN graft and FGF‐1 on white matter degeneration following spinal cord injury. Methods: Rats were divided into four groups: (i) complete spinal cord transection and T8 segment removed; the remaining three groups underwent transection followed by (ii) PN grafting; (iii) supply of exogenous FGF‐1; and (iv) PN grafting plus FGF‐1 treatment. Chondroitin sulphate proteoglycan (CSPG) deposition, astrocytes and macrophage activation, cavity size, and calcitonin gene‐related peptide and synaptophysin immunoreactivity were compared. Results: Peripheral nerve grafting increased CSPG levels compared to transection surgery alone. This CSPG was associated with the proximity to the PN graft. FGF‐1 reduced CSPG deposition in grafted animals regardless of the proximity to the graft. The CSPG reduction was accompanied by reduced GFAP expression and macrophage activation. The amount of CSPG with dissociated glycosaminoglycan did not differ between groups. FGF‐1 in Schwann cell–astrocyte coculture did not reduce CSPG deposition. Furthermore, the PN graft increased the calcitonin gene‐related peptide immunoreactivity and altered the distribution of synaptophysin‐positive axons. Conclusion: Peripheral nerve graft supported sensory re‐innervation and partial protection of the grey matter, but up‐regulated CSPG in the graft–stump junction compared to non‐grafted rats. The reduction of CSPG was caused by FGF‐1–PN synergy, and did not involve dissociation of CSPG or the suppression of a general immune response.     

Co-localized but target-unrelated expression of vasoactive intestinal polypeptide and galanin in rat dorsal root ganglion neurons after peripheral nerve crush injury

Expression of vasoactive intestinal polypeptide (VIP) and galanin in dorsal root ganglion (DRG) neurons is known to be induced by peripheral nerve injury. We investigated (1) whether VIP and galanin were co-expressed by DRG neurons and (2) whether such neurons innervated specified peripheral targets (visceral, cutaneous or muscular). An antibody to the 200 kDa neurofilament subunit (NF200) was used as a marker for large type-A cells in the DRG. VIP and galanin were respectively observed in 22% and 67% of DRG neurons at the L5 spinal level after crushing of the sciatic nerve. Most VIP-containing neurons were small type-B cells (about 90%) and approximately 95% of VIP-containing neurons also showed galanin-like immunoreactivity. Galanin was expressed by both large type-A and small type-B cells. Immunocytochemistry combined with a retrograde tracer revealed that about 70-80% of the small type-B cells in each sensory division displayed VIP-like immunoreactivity, and that most of the tracer-labeled neurons also expressed galanin. These findings suggest that the expression VIP and/or galanin in response to peripheral nerve crush injury is a property common to visceral, cutaneous and muscular sensory neurons.     

Brain-derived neurotrophic factor expression in dorsal root ganglion neurons in response to reanastomosis of the distal stoma after nerve grafting

Studies have shown that retreatment of the distal stoma after nerve grafting can stimulate nerve regeneration. The present study attempted to verify the effects of reanastomosis of the distal stoma, after nerve grafting, on nerve regeneration by assessing brain-derived neurotrophic factor expression in 2-month-old rats. Results showed that brain-derived neurotrophic factor expression in L 2-4 dorsal root ganglia began to increase 3 days after autologous nerve grafting post sciatic nerve injury, peaked at 14 days, decreased at 28 days, and reached similar levels to the sham-surgery group at 56 days. Brain-derived neurotrophic factor expression in L 2-4 dorsal root ganglia began to increase 3 days after reanastomosis of the distal stoma, 59 days after autologous nerve grafting post sciatic nerve injury, significantly increased at 63 days, peaked at 70 days, and gradually decreased thereafter, but remained higher compared with the sham-surgery group up to 112 days. The results of this study indicate that reanastomosis of the distal stoma after orthotopic nerve grafting stimulated brain-derived neurotrophic factor expression in L 2-4 dorsal root ganglia.