Neurogenesis in adult rat dorsal root ganglia

Nerve cells in mammalian species, including primary sensory neurons in the dorsal root ganglia (DRGs), are thought to be generated pre- or perinatally. The only known exceptions are olfactory receptor cells and some cortical microneurons. We now report results of experiments in which the number of neurons in the L4 and L5 DRGs of normal adult rats was counted from serial 10-micrometers paraffin sections stained with cresyl violet. Contrary to expectations, we found that there is a gradual increase in the number of DRG neurons as the animals age. The neuronal population nearly doubles over the adult life of the animal.     

Expression patterns of erythropoietin and its receptor in the developing spinal cord and dorsal root ganglia

Recombinant human erythropoietin (EPO) is neuroprotective in animal models of adult spinal cord injury, and reduces apoptosis in adult dorsal root ganglia after spinal nerve crush. The present work demonstrates that spinal cord and dorsal root ganglia share dynamic expression patterns of EPO and its receptor (EPOR) during development. C57Bl mice from embryonic days (E) 8 (E8) to E19 were studied. In spinal cord and dorsal root ganglia, EPOR expression in all precursor cells preceded the expression of EPO in subsets of neurons. On E11, EPO-immunoreactive spinal motoneurons and ganglionic sensory neurons resided adjacent to EPOR-expressing radial glial cells and satellite cells, respectively. From E12 onwards, EPOR-immunoreactivity decreased in radial glial cells and, transiently, in satellite cells. Simultaneously, large-scale apoptosis of motoneurons and sensory neurons started, and subsets of neurons were labelled by antibodies against EPOR. Viable neurons expressed EPO and EPOR. Up to E12.5, apoptotic cells were EPOR-immunopositive, but variably EPO-immunonegative or EPO-immunopositive. Thereafter, EPO-immunonegative and EPOR-immunopositive apoptotic cells predominated. Our findings suggest that EPO-mediated neuron-glial and, later, neuron–neuronal interactions promote the differentiation and/or the survival of subsets of neurons and glial cells in central as well as in peripheral parts of the embryonic nervous system. Correspondingly, expression of phospho-Akt-1/protein-kinase B extensively overlapped expression sites of EPO and EPOR, but was absent from apoptotic cells. Identified other sites of EPO and/or EPOR expression include radial glial cells that transform to astrocytes, cells of the floor plate and notochord as well as neural crest-derived boundary cap cells at motor exit points and cells of the primary sympathetic chain.     

Binding patterns of lectins with GalNAc specificity in the mouse dorsal root ganglia and spinal cord

To localize membrane glycoconjugates in neurons of the mouse spinal cord and dorsal root ganglia (DRG), cryostat sections of newborn (P0), 7 day-old (P7), P14, P21 and P31 animals were stained with ten FITC-conjugated plant lectins, the majority of them recognizing N-acetyl-D-galactosamine (GalNAc) terminal sugar residues. In the dorsal root ganglia of P0 animals, the different lectins showed distinct patterns of labeling in either cells of the nervous system, including neurons, or other structures such as nerves or blood vessels. Moreover, some of these lectins showed important changes in their pattern of labeling during postnatal development. This was especially relevant for lectins that label a subpopulation of small-sized cells that have been previously identified as the nociceptive cells of the DRG. Enzymatic digestion of sections with neuraminidase removes sialic acid from the carbohydrate chains of glycoconjugates thus exposing novel sugar residues. When this treatment was applied to DRG sections from postnatal animals the pattern of lectin staining was either changed or eliminated and heterogeneous subsets of glycoconjugates normally masked by this sugar were exposed. In the spinal cord of PO animals, none of the lectins labeled cells in the central gray matter. However, after the enzymatic digestion of sections with neuraminidase, spinal cord motoneurons and some other cells were labeled by two of the lectins suggesting that GalNAc residues present in these cells are normally masked by terminal sialic acid. Altogether, these results show important changes in the temporal and spatial expression of glycoconjugates that may be relevant for the postnatal development of the CNS and PNS of mice.     

Expression of ATP receptors in the rat dorsal root ganglion and spinal cord

Extracellular purine nucleotides and nucleosides play important roles in the nervous system, e.g., neurotransmission, neuromodulation, chemoattraction and acute inflammation. Extracellular nucleotides act through ATP receptors (P2 receptors). P2 receptors are classified into two families: the P2X receptors are ionotropic ligand-gated ion channels and the P2Y receptors are metabotropic G-protein-coupled receptors. Currently, seven P2X receptors (P2X1–7) and eight P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) are recognized. In the sensory nervous system, ATP is suggested to be one of first mediators of tissue damage, which activates primary afferents. Nerve injury often leads to neuropathic pain, such as mechanical allodynia and painful responses to normally innocuous stimuli. Peripheral nerve injury induces the upregulation of molecules in activated microglia in the spinal cord. Microglia in the spinal cord may play an important role in the development and maintenance of neuropathic pain. A prominent signaling pathway in the development of neuropathic pain involves ATP acting on microglial purinergic receptors. This review focuses on the expression of P2X and P2Y receptors mRNAs in the pain transmission pathway, i.e., in the dorsal root ganglion (DRG) and spinal cord. Furthermore, we suggest that the multiple microglial P2Y receptors activated by peripheral nerve injury may play a key role in the development of neuropathic pain.     

Differential galanin upregulation in dorsal root ganglia and spinal cord after graded single ligature nerve constriction of the rat sciatic nerve

Single ligature nerve constriction (SLNC) is a newly developed animal model for the study of neuropathic pain. SLNC of the rat sciatic nerve induces pain-related behaviors, as well as changes in the expression of neuropeptide tyrosine and the Y(1) receptor in lumbar dorsal root ganglia (DRGs) and spinal cord. In the present study, we have analyzed the expression of another neuropeptide, galanin, in lumbar DRGs and spinal cord after different degrees of constriction of the rat sciatic nerve. The nerve was ligated and reduced to 10-30, 40-80 or 90% of its original diameter (light, medium or strong SLNCs). At different times after injury (7, 14, 30, 60 days), lumbar 4 and 5 DRGs and the corresponding levels of the spinal cord were dissected out and processed for galanin-immunohistochemistry. In DRGs, SLNC induced a gradual increase in the number of galanin-immunoreactive (IR) neurons, in direct correlation with the degree of constriction. Thus, after light SLNC, a modest upregulation of galanin was observed, mainly in small-sized neurons. However, following medium or strong SLNCs, there was a more drastic increase in the number of galanin-IR neurons, involving also medium and large-sized cells. The highest numbers of galanin-IR neurons were detected 14 days after injury. In the dorsal horn of the spinal cord, medium and strong SLNCs induced a marked ipsilateral increase in galanin-like immunoreactivity in laminae I-II. These results show that galanin expression in DRGs and spinal cord is differentially regulated by different degrees of nerve constriction and further support its modulatory role on neuropathic pain.     

The immunocytochemical distribution of seven peptides in the spinal cord and dorsal root ganglia of horse and pig

Summary The distribution of calcitonin gene-related peptide (CGRP), enkephalin, galanin, neuropeptide Y (NPY), somatostatin, tachykinins and vasoactive intestinal polypeptide (VIP) was compared in cervical, thoracic, lumbar and sacral segmental levels of spinal cord and dorsal root ganglia of horse and pig.In both species, immunoreactivity for the peptides under study was observed at all segmental levels of the spinal cord. Peptide-immunoreactive fibres were generally concentrated in laminae I–III, the region around the central canal, and in the autonomic nuclei. A general increase in the number of immunoreactive nerve fibres was noted in the lumbosacral segments of the spinal cord, which was particularly exaggerated in the case of VIP immunoreactivity. In the horse, some CGRP-, somatostatin- or tachykinin-immunoreactive cell bodies were present in the dorsal horn. In the pig, cells immunoreactive for somatostatin, enkephalin or NPY were noted in a similar location.In the ventral horn most motoneurones were CGRP-immunoreactive in both species. However, in pig many other cell types were CGRP-immunoreactive not only in the ventral horn, but also in laminae V–VI of the dorsal horn.With the exception of enkephalin and NPY immunoreactivity, which was not seen in pig dorsal root ganglia, all peptides studied were localised to neuronal cell bodies and/or fibres in the dorsal root ganglia. In both species, immunolabelled cell bodies were observed in ganglia from cervical, thoracic, lumbar and sacral levels, with the exception of VIP-immunoreactive cells that were detected only in the lumbosacral ganglia. Numerous CGRP- and tachykinin-immunoreactive cell bodies were visualised in both species, while the cells immunolabelled with other peptide antisera were much lower in number.In both species, immunostaining of serial sections revealed that a subset of CGRP-immunoreactive cells co-expressed tachykinin, galanin or somatostatin immunoreactivity. In the horse some enkephalin-immunoreactive cells were also CGRP positive and occasionally combinations of three peptides, e.g. CGRP, tachykinin and galanin or CGRP, tachykinin and enkephalin were identified.The results obtained suggest that the overall pattern of distribution of peptide immunoreactivities is in general agreement with that so far described in other mammals, although some species variations have been observed, particularly regarding the presence of immunoreactive cell bodies in the dorsal horn of the spinal cord.     

神经营养因子-3对大鼠脊髓半横断后背根神经节c-Jun表达的影响

目的：研究神经营养因子-3(NT-3)对脊髓半横断后背根神经节c-Jun表达的影响,探索NT-3促进脊髓修复的作用机制。方法：将实验动物分为：对照组,损伤组和NT-3注射组,应用荧光免疫组化法结合激光扫描共聚焦显微镜,观察各组背根神经节c-Jun的表达,并计数细胞核完整的神经元数目。结果：脊髓损伤后,背根神经节的细胞内c-Jun的表达上调;NT-3注射组脊髓损伤侧背根神经节神经元的c-Jun表达明显上调,背根神经节内细胞核呈完整状态的神经元数量明显增多。结论：(1)c-Jun在轴突损伤后表达上调。(2)NT-3对轴突损伤后的神经元有保护作用。(3)NT-3可能通过使c-Jun表达上调而发挥其促神经再生作用。     

Expression of peripherin in solid transplants of foetal spinal cord and dorsal root ganglia grafted to the injured cervical spinal cord of adult rats

The expression of the neuronal type III intermediate filament protein peripherin was studied in E14 spinal cord fragments and E15 dorsal root ganglia 1–30 weeks after their transplantation to the injured cervical spinal cord of the adult rat. In the dorsal root ganglion transplants, the surviving neurons generally appeared as a rather healthy looking population of small strongly immunoreactive cells which are very similar to the small dorsal root ganglion neurons of adult control rats. In the spinal cord transplants, there were only a few peripherin-immunoreactive neurons, morphologically close to the motoneurons or to the preganglionic sympathetic neurons of adult rats. In both types of transplants, peripherin expression of the immunoreactive neurons was apparently correlated with the previously established ability of these transplanted neurons for extensive axonal growth into a co-grafted peripheral nerve.     

Expression of brain-derived neurotrophic factor in rat dorsal root ganglia, spinal cord and gracile nuclei in experimental models of neuropathic pain.

Chronic constriction injury of the sciatic nerve and lumbar L5 and L6 spinal nerve ligation provide animal models for pain syndromes accompanying peripheral nerve injury and disease. In the present study, we evaluated changes in brain-derived neurotrophic factor (BDNF) immunoreactivity in the rat L4 and L5 dorsal root ganglia (DRG) and areas where afferents from the DRG terminates (the L4/5 spinal cord and gracile nuclei) in these experimental models of neuropathic pain. Chronic constriction injury induced significant increase in the percentage of small, medium and large BDNF-immunoreactive neurons in the ipsilateral L4 and L5 DRG. Following spinal nerve ligation, the percentage of large BDNF-immunoreactive neurons increased significantly, and that of small BDNF-immunoreactive neurons decreased markedly in the ipsilateral L5 DRG, while that of BDNF-immunoreactive L4 DRG neurons of all sizes showed marked increase. Both chronic constriction injury and spinal nerve ligation induced significant increase in the number of BDNF-immunoreactive axonal fibers in the superficial and deeper laminae of the L4/5 dorsal horn and the gracile nuclei on the ipsilateral side.Considering that BDNF may modulate nociceptive sensory inputs and that injection of antiserum to BDNF significantly reduces the sympathetic sprouting in the DRG and allodynic response following sciatic nerve injury, our results also may suggest that endogenous BDNF plays an important role in the induction of neuropathic pain after chronic constriction injury and spinal nerve ligation. In addition, the increase of BDNF in L4 DRG may contribute to evoked pain which is known to be mediated by input from intact afferent from L4 DRG following L5 and L6 spinal nerve ligation.     

Distribution of neurons expressing calcitonin gene-related peptide mRNAs in the brain stem, spinal cord and dorsal root ganglia of rat and guinea-pig

In situ hybridization histochemistry was used to localize calcitonin gene-related peptide mRNAs in spinal cord, brain stem and dorsal root ganglion neurons of the rat and guinea-pig. A 32P-labeled 23-base-long (23mer) oligodeoxyribonucleotide (oligomer) complementary to calcitonin gene-related peptide mRNA sequences encoding residues 23-30 of calcitonin gene-related peptide was used primarily as a probe (CGRP I probe). A 32mer complementary to mRNA sequences for residues 10-20 of calcitonin gene-related peptide (CGRP II probe) was also used as a positive control for specificity of the 23mer for calcitonin gene-related peptide mRNA. In both the guinea-pig and rat calcitonin gene-related peptide mRNA was localized specifically to neurons of the dorsal root ganglion, to spinal motoneurons and to motoneurons of the hypoglossal, facial and accessory facial motor nuclei. Differences in the distribution of calcitonin gene-related peptide mRNA between the rat and guinea-pig included a higher proportion of rat dorsal root ganglion neurons containing calcitonin gene-related peptide mRNA and the localization of calcitonin gene-related peptide mRNA to motoneurons of the ambiguus motor nucleus, parabrachial and peripeduncular nucleus of the rat but not the guinea-pig. In the guinea-pig, in contrast, calcitonin gene-related peptide mRNA was localized also to motoneurons of the abducens, trigeminal, trochlear and oculomotor nerves. The neuronal groups in the intact rat found here to contain calcitonin gene-related mRNA have also been shown previously to contain calcitonin gene-related peptide immunoreactivity in colchicine-treated rats. Colchicine-treated rats, however, have been found to contain additional groups of calcitonin gene-related peptide immunoreactive neurons which, in the intact rats used in the present study, showed no detectable hybridization with the calcitonin gene-related peptide probe.     

Quantitative autoradiographic localisation of [125I]endothelin-1 binding sites in spinal cord and dorsal root ganglia of the rat.

The autoradiographic distribution of [125I] endothelin (ET)-1 binding sites was studied in the spinal cord and dorsal root ganglia of developing and adult rat. In the spinal cord, high density of [125I]ET-1 binding sites were diffusely distributed throughout the grey matter whereas in the ganglia discrete silver grains were localised primarily on the satellite cells. A variation in the density of binding sites was evident, particularly in the spinal cord, during development. These data, in conjunction with other reports, suggest a possible neuromodulatory role for ET-1 in spinal cord and dorsal root ganglia of the rat.     

Distribution of substance P-like immunoreactivity in the spinal cord and dorsal root ganglia of the human foetus and infant

Using the indirect immunofluorescence method, the distribution of substance P-like-immunoreactivity was studied in spinal cord and dorsal root ganglia of 25 human foetuses ranging from 12 to 29 weeks of gestational age. The spinal cord and dorsal root ganglia of three infants (1 day-, 2 and 4 month-old) were also investigated as a post-natal reference. On the whole, the substance P distribution patterns seen in infants were already visible throughout most of foetal life. The highest density of substance P-like-immunoreactive fibres was localized over the superficial layers of the dorsal grey horn. Punctiform immunofluorescence was often found over the white matter especially in the funiculi dorsalis et lateralis. In the ventral horn, substance P immunoreactive fibres were few and far between in the grey matter and were only detected from foetal stage 16 weeks. In addition, longitudino-frontal sections through the dorsal regions revealed repetitive arrangements of substance P-like-immunoreactive fibres along the whole spinal cord. In dorsal root ganglia only a few immunoreactive cells were observed. These findings demonstrate the wide and early occurrence of substance P-like-immunoreactivity in the human foetus spinal cord and dorsal root ganglia. They suggest that the development of the substance P neuronal system begins early in ontogenesis and is regionally differentiated.     

Changes in PDGF expression in spared dorsal root ganglia and associated spinal dorsal horns in cats subjected to partial dorsal root ganglionectomy

Changes in the platelet derived growth factor (PDGF) in the spared dorsal root ganglia (DRG) and associated spinal dorsal horns were evaluated in cats subjected to unilateral removal of L1-L5 and L7-S2 DRG, sparing the L6 DRG. The number of PDGF immunopositive neurons and protein expression decreased significantly in the spared DRG and associated dorsal horns of the L3 and L6 cord segments at 3 days post-operation (dpo). It bottomed to the lowest level at 7 dpo in the DRG, then returned to the control level at 14 dpo; while in the L6 dorsal horn, it rapidly increased at 7 dpo and exceeded the control level at 14 dpo. This showed a significant upregulation in the spared DRG and associated spinal dorsal horns, especially in the L6 cord segment following a transient decrease. Meanwhile, a significant upregulation of PDGF mRNA was also seen in L6 DRG and L3 and L6 dorsal horns at 3 dpo. The upregulation of the endogenous PDGF in the said structures indicated a potential role of this factor in spinal cord plasticity after partial dorsal root ganglia removal in cats.     

Regeneration of dorsal root axons into experimentally altered glial environments in the rat spinal cord

Exposure of the lumbar spinal cord of rats to X-rays 3 days after birth results in changes in the composition of central glia. Shortly after irradiation, there is both retardation of central myelin formation and a loss of integrity of the astrocyte-derived glia limitans on the dorsal surface of the cord. Subsequently, Schwann cells invade, undergo division and myelinate axons in the dorsal funiculi in the irradiated region of the cord, creating there an environment similar to that of peripheral nerve. The present study was undertaken to compare the ability of lesioned dorsal root axons to grow back into the altered glial environments that exist within the spinal cord after irradiation. This regrowth was assessed by injecting Fluoro-Gold into the spinal cord and subsequently examining neurons in the dorsal root ganglia (DRG) for the presence of this label. Numbers of retrogradely labeled neurons were counted in the DRG in both injured and contralateral non-injured sides. Non-irradiated control rats had almost no labeled DRG neurons on the injured side, whereas Fluoro-Gold labeled neurons were observed in substantial numbers in the DRG on the injured side of irradiated rats. There was a definite trend in the data, indicating that the longer the interval between irradiation and root injury, the greater the number of labeled neurons. Since the Fluoro-Gold labeling technique does not allow for visualization of the labeled axons within the spinal cord, a few animals were used to assess anterograde labeling with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP/HRP) from the dorsal root into the spinal cord. HRP-filled regenerating axons were visualized in dorsal white and gray matter of the irradiated spinal cord. Such axons were not present in the non-irradiated spinal cords. Radiation-induced changes in glial populations are discussed, particularly with regard to the temporal sequence of these changes and their possible relationship to the conversion of a normally non-permissive environment into one conducive to axonal regrowth.     

Bilateral dorsal root potentials in the lower sacral spinal cord

Summary Bilateral dorsal root potentials (DRPs) evoked in the S3 dorsal roots by stimulation of the S2 and L6 dorsal roots and the cutaneous afferents entering the spinal cord in the lumbar segments have been studied in spinal cats. Stimulation of all these afferents produces DRPs which have the same amplitude on both sides of the spinal cord. During longlasting repetitive stimulation the negativity of the ipsilateral dorsal root is maintained only when this stimulation is applied to the neighbouring dorsal root. Depression of the testing DRPs produced by preceding single volleys or repetitive stimulation is only slightly larger on the contralateral side of the cord. The difference between depression of the DRPs on both sides of the cord is significantly smaller in the S3 than in the L7 segment. Following conditioning tetanization both ipsi- and contralateral DRPs undergo depression. The pattern of bilateral DRPs in the S3 segment significantly differs from that observed in the L7 segment and these differences correspond to the already known distinct arrangement of the substantia gelatinosa in the two parts of the cord.     

Oscillatory interaction between dorsal root excitability and dorsal root potentials in the spinal cord of the turtle.

The response to dorsal root stimulation, at one to two times threshold, was investigated in the isolated cervical enlargement of the turtle spinal cord. At frequencies near 10 Hz the synaptic response in motoneurons and the cord dorsum potential, after an initial lag time, oscillated in amplitude with a period of more than 1 s. The mono- and polysynaptyic postsynaptic response in motoneurons, the pre- and postsynaptic component of the cord dorsum potential and the dorsal root potential oscillated in synchrony. These oscillations were only observed with stimulus frequencies in the range 9-11 Hz. The oscillating response could only be evoked from stimulus sites to which dorsal root potentials were conducted from the spinal cord (2-3 mm). At more distant stimulus sites cyclic variations in amplitude of the cord dorsum potential and the synaptic response in motoneurons were not observed. During an oscillating spinal response to a stimulus train in one dorsal root filament, the response evoked by a stimulus in another short filament (2-3 mm) from the same root varied in amplitude with the induced oscillation. The spinal response to a stimulus in a longer filament (i.e. more than 3 mm) did not oscillate. It is argued that the oscillating responses described rely on interactions between distributed elements rather than on unit oscillators. We also show that primary afferent transmission is unaffected by the substantial variations in dorsal root potentials during oscillations.     

Somatostatin is increased in the dorsal root ganglia of adjuvant-inflamed rat

To determine whether biosynthesis of somatostatin is enhanced in the primary sensory neurons by inflammatory pain, we examined the effects of adjuvant inoculation on the content of immunoreactive somatostatin, mainly composed of somatostatin-14 and somatostatin-28, in the dorsal root ganglia and the spinal cord of the rat. The adjuvant inoculation, which produced long-lasting inflammation and hyperalgesia, increased the content of immunoreactive somatostatin, especially somatostatin-14, in the dorsal root ganglia at L4-L6 levels with no change in the dorsal and ventral horns of lumbar enlargement. Such an increase was enhanced by an intrathecal injection of colchicine (0.2 mg) that inhibits axonal flow of somatostatin. Chronic administration of the anti-inflammatory analgesic, sodium diclofenac (3 mg.kg-1.d-1), abolished an adjuvant-induced increase in the content of immunoreactive somatostatin in the dorsal root ganglia. These results suggest that the turnover (biosynthesis and axonal flow) of somatostatin in the primary sensory neurons is enhanced in the presence of persisting inflammatory pain, and support the idea that somatostatin-containing primary afferents are involved in the transmission of pain in the spinal dorsal horn.     

Immunocytochemistry of B-50 (GAP-43) in the spinal cord and in dorsal root ganglia of the adult cat

Summary The distribution of the neural-specific growth associated protein B-50 (GAP-43), which persists in the mature spinal cord and dorsal root ganglia, has been studied by light and electron microscopic immunohistochemistry in the cat. Throughout the spinal cord, B-50 immunoreactivity was seen confined to the neuropil, whereas neuronal cell bodies were unreactive. The most conspicuous immunostaining was observed in the dorsal horn, where it gradually decreased from superficial laminae (I–II) toward more ventral laminae (III–V), and in the central portion of the intermediate gray (mainly lamina X). In these regions, the labelling was localized within unmyelinated, small diameter nerve fibres and axon terminals. In the rest of the intermediate zone (laminae VI–VIII), B-50 immunoreactivity was virtually absent. The intermediolateral nucleus in the thoracic and cranial lumbar cord showed a circumscribed intense B-50 immunoreactivity brought about by the labelling of many axon terminals on preganglionic sympathetic neurons. In motor nuclei of the ventral horn (lamina IX), low levels of B-50 immunoreactivity were present in a few axon terminals on dendritic and somal profiles of motoneurons. In dorsal root ganglia, B-50 immunoreactivity was mainly localized in the cell bodies of small and medium-sized sensory neurons. The selective distribution of persisting B-50 immunoreactivity in the mature cat throughout sensory, motor, and autonomie areas of the spinal cord and in dorsal root ganglia suggests that B-50-positive systems retain in adult life the capacity for structural and functional plasticity.