A single re-implanted ventral root exerts neurotropic effects over multiple spinal cord segments in the adult rat

Spinal cord injuries, particularly traumatic injuries to the conus medullaris and cauda equina, are typically complex and involve multiple segmental levels. Implantation of avulsed ventral roots into the spinal cord as a repair strategy has been shown to be neuroprotective and promote axonal regeneration by spinal cord neurons into an implanted root. However, it is not well known over what distance in the spinal cord an implanted ventral root can exert its neurotropic effect. Here, we investigated whether an avulsed L6 ventral root acutely implanted into the rat spinal cord after a four level (L5–S2) unilateral ventral root avulsion injury may exert neurotropic effects on autonomic and motor neurons over multiple spinal cord segments at 6 weeks postoperatively. Using retrograde labeling techniques and stereological quantification methods, we demonstrate that autonomic and motor neurons from all four lesioned spinal cord segments, spanning more than an 8 mm rostro-caudal distance, reinnervated the one implanted root. The rostro-caudal distribution suggested a gradient of neurotropism, where the axotomized neurons closest to the implanted site had the highest probability of root reinnervation. These results suggest that implantation of a single ventral root may provide neurotropic effects to injured neurons at the site of lesion as well as in the adjacent spinal cord segments. Our findings may be of translational research interest for the development of surgical repair strategies after multi-level conus medullaris and cauda equina injuries, in which fewer ventral roots than spinal cord segments may be available for implantation.     

Minocycline protects motor but not autonomic neurons after cauda equina injury

Conus medullaris/cauda equina injuries typically result in loss of bladder, bowel, and sexual functions, partly as a consequence of autonomic and motor neuron death. To mimic these injuries, we previously developed a rodent lumbosacral ventral root avulsion (VRA) injury model, where both autonomic and motor neurons progressively die over several weeks. Here, we investigate whether minocycline, an antibiotic with putative neuroprotective effects, may rescue degenerating autonomic and motor neurons after VRA injury. Adult female rats underwent lumbosacral VRA injuries followed by a 2-week treatment with either minocycline or vehicle injected intraperitoneally. The sacral segment of the spinal cord was studied immunohistochemically using choline acetyltransferase (ChAT) and activated caspase-3 at 4 weeks post-operatively. Minocycline increased the survival of motoneurons but not preganglionic parasympathetic neurons (PPNs). Further investigations demonstrated that a larger proportion of motoneurons expressed activated caspase-3 compared to PPNs after VRA injury and indicated an association with minocycline’s differential neuroprotective effect. Our findings suggest that minocycline may protect degenerating motoneurons and expand the therapeutic window of opportunity for surgical repair of proximal root lesions affecting spinal motoneurons.     

Reimplantation of avulsed lumbosacral ventral roots in the rat ameliorates injury-induced degeneration of primary afferent axon collaterals in the spinal dorsal columns

Injuries to the cauda equina/conus medullaris portion of the spinal cord can result in motor, sensory, and autonomic dysfunction, and neuropathic pain. In rats, unilateral avulsion of the motor efferents from the lumbosacral spinal cord results in at-level allodynia, along with a corresponding glial and inflammatory response in the dorsal horn of the spinal cord segments immediately rostral to the lesion. Here, we investigated the fate of intramedullary primary sensory projections following a motor efferent lesion. The lumbosacral (L6 and S1) ventral roots were unilaterally avulsed from the rat spinal cord (VRA; n=9). A second experimental group had the avulsed roots acutely reimplanted into the lateral funiculus (Imp; n=5), as this neural repair strategy is neuroprotective, and promotes the functional reinnervation of peripheral targets. A laminectomy-only group served as controls (Lam; n=7). At 8 weeks post-lesion, immunohistochemical examination showed a 42% reduction (P<0.001) in the number of RT97-positive axons in the ascending tracts of the dorsal funiculus of the L4-5 spinal segment in VRA rats. Evidence for degenerating myelin was also present. Reimplantation of the avulsed roots ameliorated axon and myelin degeneration. Axons in the descending dorsal corticospinal tract were unaffected in all groups, suggesting a specificity of this lesion for spinal primary sensory afferents. These results show for the first time that a lesion restricted to motor roots can induce the degeneration of intramedullary sensory afferents. Importantly, reimplantation of the lesioned motor roots ameliorated sensory axon degeneration. These data further support the therapeutic potential for reimplantation of avulsed ventral roots following trauma to the cauda equina/conus medullaris.     

An electron microscopic analysis of the left phrenic nerve in the rat

In this electron microscopic study, the axonal categories in the left phrenic nerve at its entrance to the diaphragm have been determined. At a level 3 mm rostral to the diaphragm, the left phrenic nerve contains approximately 700 axons: 57% are myelinated and 43% are unmyelinated. The dorsal root ganglion cells give rise to 31% of the myelinated axons and the ventral root contributes 69%. Of the unmyelinated axons, the dorsal root ganglion cell contributes 59%, the cervical sympathetic chain 24%, and 17% course through the ventral roots. These ventral root unmyelinated axons are presumably preganglionic efferents since the proximal stump of the ventral root showed no decrease in unmyelinated axons after ventral rhizotomy.     

Motoneurons reinnervate skeletal muscle after ventral root implantation into the spinal cord of the cat

By use of intracellular recording and staining with horseradish peroxidase it was found that alpha and probably also gamma motoneurons were able to reinnervate ventral root implants after an avulsion of ventral roots at the spinal cord surface in the cat. The reinnervation of the implant was achieved after an initial growth of new axons in central nervous system tissue. Reinnervating neurons could be excited or inhibited by segmental reflex activity and their axons could conduct nerve impulses. The character of muscle twitch responses elicited by electrical stimulation of implanted roots strongly indicated that denervated muscles were reinnervated by new motor axons via the implant.     

马尾神经受压大鼠脊髓圆锥神经元的形态、凋亡及脑源性神经营养因子 mRNA的表达变化

目的 观察大鼠马尾神经受压后脊髓圆锥神经元的形态变化,检测脊髓神经元凋亡数量及脑源性神经营养因子(BDNF)mRNA的表达,以及探讨上述变化的可能机制. 方法 将90只成年SD大鼠分为马尾受压模型组、假手术组与正常对照组,分别于造模术后30min、2h、4h、8h、1d、3d、1周、2周、3周取样.采用光镜与透射电镜观察马尾受压后脊髓圆锥神经细胞的形态变化;采用原位缺口末端标记(TUNEL)技术检测细胞凋亡;原位杂交法测定BDNF mRNA阳性细胞的数量.计算单位面积内的阳性细胞数,单因素方差分析比较马尾受压后不同时间组与对照组之间的差异. 结果 马尾神经受压可导致脊髓圆锥神经元形态结构出现明显伤害性改变.TUNEL染色与BDNF mRNA原位杂交显示,阳性细胞分别于制模术后8h、4h起较对照组有明显增多,并于术后3d达高峰,术后3周上述阳性细胞的数量仍明显高于对照组. 结论 马尾神经受压后,可导致相应脊髓圆锥神经细胞结构的明显变化,其凋亡数量明显增加,说明马尾受损可导致中枢神经元的不可逆损伤,这可能是马尾综合征逾期手术效果不理想的重要原因之一.在马尾严重受压后,神经元及胶质细胞的BDNFmRNA的表达明显增加,可能对神经细胞的保护及修复起一定作用.     

Stretch receptor-associated expression of alpha 3 isoform of the Na+, K+-ATPase in rat peripheral nervous system

Expression of the neuronal alpha(3) isoform of the Na(+),K(+)-ATPase (alpha(3) Na(+),K(+)-ATPase) was studied in the rat peripheral nervous system using histological and immunohistochemical techniques. Non-uniform expression of the alpha(3) Na(+),K(+)-ATPase was observed in L5 ventral and dorsal roots, dorsal root ganglion, sciatic nerve and its branches into skeletal muscle. The alpha(3) Na(+),K(+)-ATPase was not detected in nerve fibers in skin, saphenous and sural nerves. In dorsal root ganglion 12+/-2% of neurons were immunopositive for alpha(3) Na(+),K(+)-ATPase and all these neurons were large primary afferents that were not labeled by Griffonia simplicifolia isolectin B4 (marker of small primary sensory neurons). In dorsal and ventral roots 27+/-3% and 40+/-3%, respectively, of myelinated axons displayed immunoreactivity for alpha(3) Na(+),K(+)-ATPase. In contrast to the dorsal roots, strong immunoreactivity in ventral roots was observed only in myelinated axons of small caliber, presumably gamma-efferents. In the mixed sciatic nerve alpha(3) Na(+),K(+)-ATPase was detected in 26+/-5% of myelinated axons (both small and large caliber). In extensor hallicus proprius and lumbricales hind limb muscles alpha(3) Na(+),K(+)-ATPase was detected in some intramuscular axons and axonal terminals on intrafusal muscle fibers in the spindle equatorial and polar regions (regions of afferent and efferent innervation of the muscle stretch receptor, respectively). No alpha(3) Na(+),K(+)-ATPase was found in association with innervation of extrafusal muscle fibers or in tendon-muscle fusion regions. These data demonstrate non-uniform expression of the alpha(3) isoform of the Na(+),K(+)-ATPase in rat peripheral nervous system and suggest that alpha(3) Na(+),K(+)-ATPase is specifically expressed in afferent and efferent axons innervating skeletal muscle stretch receptors.     

犬马尾与骶神经根的解剖学观察

目的：为从马尾和骶神经根途径开展犬的神经泌尿学研究提供解剖依据。方法：对3只犬灌后进行解剖,并对22只犬进行术中观察,总结马尾、骶神经根的解剖特征。结果：犬的盆底器官由S1～S2脊髓节段和神经根支配;脊髓圆锥延续较长,达L6椎体下缘,而马尾神经较短;髓神经前后根出硬膜后,有各自独立的硬膜囊包绕直至后根神经节处,长1～1.5cm。结论：犬马尾和骶神经根的解剖特征与人类不同。     

Reactive changes in dorsal roots and dorsal root ganglia after C7 dorsal rhizotomy and ventral root avulsion/replantation in rabbits

Current surgical treatment of spinal root injuries aims at reconnecting ventral roots to the spinal cord while severed dorsal roots are generally left untreated. Reactive changes in dorsal root ganglia (DRGs) and in injured dorsal roots after such complex lesions have not been analysed in detail. We studied dorsal root remnants and lesioned DRGs 6 months after C7 dorsal rhizotomy, ventral root avulsion and immediate ventral root replantation in adult rabbits. Replanted ventral roots were fixed to the spinal cord with fibrin glue only or with glue containing ciliary neurotrophic factor and/or brain-derived neurotrophic factor. Varying degrees of degeneration were observed in the deafferented dorsal spinal cord in all experimental groups. In cases with well-preserved morphology, small myelinated axons extended into central tissue protrusions at the dorsal root entry zone, suggesting sprouting of spinal neuron processes into the central dorsal root remnant. In lesioned DRGs, the density of neurons and myelinated axons was not significantly altered, but a slight decrease in the relative frequency of large neurons and an increase of small myelinated axons was noted (significant for axons). Unexpectedly, differences in the degree of these changes were found between control and neurotrophic factor-treated animals. Central axons of DRG neurons formed dorsal root stumps of considerable length which were attached to fibrous tissue surrounding the replanted ventral root. In cases where gaps were apparent in dorsal root sheaths, a subgroup of dorsal root axons entered this fibrous tissue. Continuity of sensory axons with the spinal cord was never observed. Some axons coursed ventrally in the direction of the spinal nerve. Although the animal model does not fully represent the situation in human plexus injuries, the present findings provide a basis for devising further experimental approaches in the treatment of combined motor/sensory root lesions.     

Fibre composition of the ventral roots L7 and S1 in the owl monkey (Aotus trivirgatus)

Summary The ventral roots L7 and S1 of the owl monkeyAotus trivirgatus, were examined by electron microscopy. On average, these roots contain 2950 and 1837 myelinated axons respectively. In both roots the myelinated axons have bimodal size distributions, but the S1 root contains more small myelinated axons. Both roots contain a substantial proportion of unmyelinated axon profiles (UAP). In the L7 root the proportion of UAP decreases as the spinal cord is approached, from 19% distally to 5% in the juxtamedullary rootlets. Unmyelinated and very small myelinated CNS-type axons have not been observed in the L7 transitional region. The average S1 root contains some 40% unmyelinated axons at all examined proximo-distal levels. Unmyelinated/ very small myelinated axons are easily found on the CNS side of the S1 transitional region, in direct relation to motoraxon bundles. Bundles of unmyelinated and small myelinated axons occur in the ventral pia mater of both segments. The unmyelinated axons in the L7 root of the owl monkey appear to be arranged like those in the feline L7 ventral root, possibly representing afferents. It is likely that most unmyelinated and small myelinated axons in the ventral root S1 are autonomic efferents.     

Regenerating supernumerary axons are cholinergic and emerge from both autonomic and motor neurons in the rat spinal cord

Multipolar neurons in the mammalian nervous system normally exhibit one axon and several dendrites. However, in response to an axonal injury, adult motoneurons may regenerate supernumerary axons. Supernumerary axons emerge from the cell body or dendritic trees in addition to the stem motor axon. It is not known whether these regenerating axons contain neurotransmitters for synaptic transmission at their terminals. Here, using immunohistochemistry for choline acetyltransferase, an enzyme that synthesizes acetylcholine, we demonstrate the emergence of cholinergic supernumerary axons at 6 weeks after a unilateral L5-S2 ventral root avulsion and acute implantation of the avulsed L6 ventral root into the adult rat spinal cord. Light microscopic serial reconstruction of choline acetyltransferase immunoreactive arbors shows that these supernumerary axons originate from both autonomic and motor neurons. The supernumerary axons emerge from the cell body or dendrites, exhibit an abnormal projection pattern within the intramedullary gray and white matters, make frequent abrupt turns in direction, and form bouton-like swellings as well as growth cone-like terminals. Double labeling immunohistochemistry studies show that the choline acetyltransferase immunoreactive supernumerary axons co-localized with two proteins associated with axonal growth and elongation, growth-associated protein 43 and p75, the low affinity neurotrophic factor receptor. Our findings suggest that regenerating supernumerary axons selectively transport and store choline acetyltransferase, supporting the notion that supernumerary axons may develop functional and active synaptic transmission. Therefore, regenerating supernumerary axons may contribute to the plasticity in neural circuits following injury in the adult nervous system.     

腹腔镜辅助下腰椎骨折前路减压内固定术

目的探讨在腹腔镜辅助下，经腹膜后腰肌间隙人路的小切口对腰椎爆裂性骨折椎管减压、植骨、内固定手术的可行性。方法13例腰椎爆裂性骨折，在腹腔镜辅助下经腹膜后腰肌间隙入路，应用腹腔镜、自制手术器械，进行椎体爆裂性骨折并脊髓圆锥、马尾神经压迫症病例的椎管减压、植骨、内固定手术。结果全部病例切口Ⅰ期愈合，椎管内骨块清除彻底。椎管减压充分，植骨块融合良好，内固定器无松动；1例L4椎体爆裂性骨折、马尾神经受压病例同时还伴有T6骨折、完全性截瘫，术后脊髓功能未恢复；4例圆锥、马尾神经受压，术后功能基本恢复。结论腹腔镜辅助下经腹膜后腰肌间隙人路的小切口腰椎手术，可以完成椎体爆裂性骨折并脊髓圆锥、马尾神经压迫症的椎管减压、植骨、内固定术。     

脊神经前后根的应用显微解剖学研究

目的　探讨脊神经前后根神经小束的分束标准和数目 ,为临床手术提供可靠的依据和新的手术标准。方法　在 15例 (30侧 )成人脊柱标本上 ,对L1～S2节段的脊神经前后根进行形态学观察和显微测量。结果　脊髓圆锥部脊神经根是由根丝逐步汇合而成。应用显微外科技术 ,后根一般可分为 10～ 18小束 ,前根一般分为 6～ 11小束 ,其小束的直径是基本一致 ,数值较为恒定。结论　在高选择性手术 ,脊神经前后根的分束标准应尽量细 ,这样有利于准确切断引起痉挛的Ia类神经纤维 ,一般前根达到 6～ 11小束 ,后根达到 10～18小束     

Re-utilization of Schwann cells during ingrowth of ventral root afferents in perinatal kittens

Ventral roots in all mammalian species, including humans, contain significant numbers of unmyelinated axons, many of them afferents transmitting nociceptive signals from receptive fields in skin, viscera, muscles and joints. Observations in cats indicate that these afferents do not enter the spinal cord via the ventral root, but rather turn distally and enter the dorsal root. Some unmyelinated axons are postganglionic autonomic efferents that innervate blood vessels of the root and the pia mater. In the feline L7 segment, a substantial proportion of unmyelinated axons are not detectable until late in perinatal development. The mechanisms inducing this late ingrowth, and the recruitment of Schwann cells (indispensable, at this stage, for axonal survival and sustenance), are unknown. We have counted axons and Schwann cells in both ends of the L7 ventral root in young kittens and made the following observations. (1) The total number of axons detectable in the root increased throughout the range of investigated ages. (2) The number of myelinated axons was similar in the root's proximal and distal ends. The increased number of unmyelinated axons with age is thus due to increased numbers of small unmyelinated axons. (3) The number of separated large probably promyelin axons was about the same in the proximal and distal ends of the root. (4) Schwann cells appeared to undergo redistribution, from myelinated to unmyelinated axons. (5) During redistribution of Schwann cells they first appear as aberrant Schwann cells and then become endoneurial X-cells temporarily free of axonal contact. We hypothesize that unmyelinated axons invade the ventral root from its distal end, that this ingrowth is particularly intense during the first postnatal month and that disengaged Schwann cells, eliminated from myelinated motoneuron axons, provide the ingrowing axons with structural and trophic support.     

Mechanisms underlying recurrent inhibition in the sacral parasympathetic outflow to the urinary bladder.

1. In cats with the sacral dorsal roots cut on one side electrical stimulation (15-40 c/s) of the central end of the transected ipsilateral pelvic nerve depressed spontaneous bladder contractions. The depression was abolished by transecting the ipsilateral sacral ventral roots. 2. Electrical stimulation of acutely or chronically transected ('deafferented') sacral ventral roots depressed spontaneous bladder contractions and the firing of sacral parasympathetic preganglionic neurones innervating the bladder. The depression of neuronal firing occurred ipsilateral and contralateral to the point of stimulation, but only occurred with stimulation of sacral roots containing preganglionic axons and only with stimulation of sacral roots containing preganglionic axons and only at intensities of stimulation (0-7-4V) above the threshold for activation of these axons. 3. The inhibitory responses were not abolished by strychnine administered by micro-electrophoresis to preganglionic neurones, but were blocked by the intravenous administration of strychnine. 4. The firing of preganglionic neurones elicited by micro-electrophoretic administration of an excitant amino acid (DL-homocysteic acid) was not depressed by stimulation of the ventral roots. 5. It is concluded that the inhibition of the sacral outflow to the bladder by stimulation of sacral ventral roots is related to antidromic activation of vesical preganglionic axons. Collaterals of these axons must excite inhibitory interneurones which in turn depress transmission at a site on the micturition reflex pathway prior to the preganglionic neurones.     

Delayed riluzole treatment is able to rescue injured rat spinal motoneurons

The effect of delayed 2-amino-6-trifluoromethoxy-benzothiazole (riluzole) treatment on injured motoneurons was studied. The L4 ventral root of adult rats was avulsed and reimplanted into the spinal cord. Immediately after the operation or with a delay of 5, 10, 14 or 16 days animals were treated with riluzole (n=5 in each group) while another four animals remained untreated. Three months after the operation the fluorescent dye Fast Blue was applied to the proximal end of the cut ventral ramus of the L4 spinal nerve to retrogradely label reinnervating neurons. Three days later the spinal cords were processed for counting the retrogradely labeled cells and choline acetyltransferase immunohistochemistry was performed to reveal the cholinergic cells in the spinal cords. In untreated animals there were 20.4+/-1.6 (+/-S.E.M.) retrogradely labeled neurons while in animals treated with riluzole immediately or 5 and 10 days after ventral root avulsion the number of labeled motoneurons ranged between 763+/-36 and 815+/-50 (S.E.M.). Riluzole treatment starting at 14 and 16 days after injury resulted in significantly lower number of reinnervating motoneurons (67+/-4 and 52+/-3 S.E.M., respectively). Thus, riluzole dramatically enhanced the survival and reinnervating capacity of injured motoneurons not only when treatment started immediately after injury but also in cases when riluzole treatment was delayed for up to 10 days. These results suggest that motoneurons destined to die after ventral root avulsion are programmed to survive for some time after injury and riluzole is able to rescue them during this period of time.     

GAP-43 expression in the developing rat lumbar spinal cord.

The expression of the growth-associated protein GAP-43, detected by immunocytochemistry, has been studied in the developing rat lumbar spinal cord over the period E11 (embryonic day 11), when GAP-43 first appears in the spinal cord, to P29 (postnatal day 29) by which time very little remains. Early GAP-43 expression in the fetal cord (E11-14) is restricted to dorsal root ganglia, motoneurons, dorsal and ventral roots and laterally positioned and contralateral projection neurons and axons. Most of the gray matter is free of stain. The intensity of GAP-43 staining increases markedly as axonal growth increases, allowing clear visualization of the developmental pathways taken by different groups of axons. Later in fetal life (E14-19), as these axons find their targets and new pathways begin to grow, the pattern of GAP-43 expression changes. During the period, GAP-43 staining in dorsal root ganglia, motoneurons, and dorsal and ventral roots decreases, whereas axons within the gray matter begin to express the protein and staining in white matter tracts increases. At E17-P2 there is intense GAP-43 labelling of dorsal horn neurons with axons projecting into the dorsolateral funiculus and GAP-43 is also expressed in axon collaterals growing into the gray matter from lateral and ventral white matter tracts. At E19-P2, GAP-43 is concentrated in axons of substantia gelatinosa. Overall levels decline in the postnatal period, except for late GAP-43 expression in the corticospinal tract, and by P29 only this tract remains stained.     

Complement activation after lumbosacral ventral root avulsion injury

A lumbosacral ventral root avulsion (VRA) injury results in a pronounced loss of motoneurons, in part due to apoptosis. Caspase inhibitors may rescue motoneurons after a VRA in neonatal rats, but this treatment approach has been unsuccessful to protect motoneurons subjected to the same injury in adult rats. Other mechanisms may contribute to the retrograde motoneuron death encountered in adult animals. Here, we study whether the complement system, a part of the innate immune system, contributes to motoneuron death after a lumbosacral VRA. Adult Sprague-Dawley rats underwent a unilateral L5-S2 VRA injury. At 10 days postoperatively, quantitative immunohistochemical studies demonstrated that the lytic membrane attack complex (MAC) targeted approximately 38% of axotomized motoneurons. The MAC inhibitor Clusterin was concurrently expressed at significantly higher levels in astrocytes and de novo in 30% of the remaining motoneurons. Our data suggest that complement activation and necrosis contribute to motoneuron death after lumbosacral VRA injuries. We speculate that inhibition of MAC may constitute a potential neuroprotective strategy following cauda equina injuries.     

Glial reactions in a rodent cauda equina injury and repair model

In the adult rat, an avulsion injury of lumbosacral ventral roots results in a progressive and pronounced loss of the axotomized motoneurons. A subsequent acute implantation of an avulsed ventral root into the spinal cord has neuroprotective effects. However, it has not been known whether a surgical implantation of an avulsed ventral root into the spinal cord for neural repair purposes affects intramedullary glial and microglial reactions. Here, adult female Sprague-Dawley rats underwent a unilateral L5-S2 ventral root avulsion injury with or without acute implantation of the L6 ventral root into the spinal cord. At 4 weeks postoperatively, immunohistochemistry using primary antibodies to GFAP (astrocytes), Ox-42 (microglia), and ED-1 (macrophages) was performed at the L6 spinal cord segment, and quantified using densitometry. Our results show that a lumbosacral ventral root avulsion injury induces an activation of astrocytes, microglia, and macrophages in the ventral horn. Interestingly, an acute implantation of an avulsed root into the white matter does not significantly affect the activation of glial cells or macrophages in the ventral horn. We speculate that neuroprotective and axonal growth promoting benefits of the combined glial and microglial/ macrophage responses may outweigh their potential negative effects, as previous studies have shown that implantation of avulsed roots is a successful strategy in promoting reinnervation of peripheral targets.