Neuropeptide Y and Galanin Binding Sites in Rat and Monkev Lumbar Dorsal Root Ganalia and Spinal Cord and Effect of Peripheral Axotomy

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [¹²⁵I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I–IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae 1–11. [¹²⁵I]NPY binding was most strongly attenuated by NPY_13–36, a Y2 agonist, and partially inhibited by [Leu³¹,Pro³⁴]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY_13–36 completely abolished PYY binding, which was also reduced by [Leu,³¹ Pro³⁴] NPY. However, the increase in PYY binding seen in laminae I–IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu³¹, Pro³⁴] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I–IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord. The present results indicate that after axotomy the synthesis of NPY receptors is increased in rat DRG neurons, especially in large neurons, and is transported to the laminae I–IV of the ipsilateral dorsal horn and into the sciatic nerve. No such up-regulation of the NPY receptor occurred in monkey DRG after axotomy. The Y2 receptor seems to be the main NPY receptor in DRG and the dorsal horn of the rat and monkey spinal cord, but Y1 receptors also exist. The increase in NPY binding sites in laminae I–IV of the dorsal horn after axotomy partly represents Y1 receptors. In contrast to the rat, galanin binding sites could be identified in monkey lumbar DRG. No effect of axotomy on the distribution of galanin binding sites in rat or monkey DRG and dorsal horn was detected, suggesting their presence on local dorsal horn neurons (or central afferents).     

Effect of Peripheral Axotomy on Expression of Neuropeptide Y Receptor mRNA in Rat Lumbar Dorsal Root Ganglia

Using in situ hybridization, the expression of the mRNA for a neuropeptide Y (NPY) receptor, was studied in lumbar (L) 4 and 5 dorsal root ganglia (DRGs) of normal rats and at various intervals after unilateral sciatic nerve transection. Twenty percent of all normal DRG neurons were NPY receptor mRNA-positive, and the majority of these neurons were of the small type, with only a few labelled medium-sized and large neurons. In L5 normal ganglia NPY receptor mRNA colocalized with substance P, calcitonin gene-related peptide and galanin mRNAs in small neurons, but not in medium-sized or large neurons containing these peptides. NPY receptor mRNA was not observed in somatostatin or nitric oxide synthase mRNA-positive neurons. Sciatic nerve transection induced a marked decrease in NPY receptor mRNA levels. However, in parallel there was a transient increase in the number of NPY receptor mRNA-positive small neuron profiles, but the intensity of labelling was mostly very low, although a few strongly labelled, small neuron profiles were also encountered. In addition, axotomy caused a marked increase in the number of NPY receptor mRNA-positive large neuron profiles in the ipsilateral DRGs, and they constituted 15–20% of counted DRG neuron profiles and 45–65% of counted large neuron profiles, 7–28 days after axotomy. In L5 DRGs, ipsilateral to the axotomy, NPY receptor mRNA colocalized with NPY mRNA in many large and some medium-sized neuron profiles, with galanin mRNA in some small, medium-sized and large neuron profiles and with vasoactive intestinal polypeptide mRNA in some small and medium-sized neuron profiles and a few large profiles. Occasionally, NPY receptor mRNA was observed in nitric oxide synthase mRNA-positive small neurons. In the dorsal horn, NPY receptor mRNA-positive small neurons were concentrated in lamina II at L4 and L5 levels, and were scattered in deeper laminae. No marked changes were observed ipsilateral to the axotomy. No NPY receptor mRNA-positive cells were found in the normal rat gracile nucleus, or in this nucleus after axotomy. These results show that a NPY receptor may be a prejunctional receptor in primary afferent neurons and play a role in the modulation of somatosensatory information, both in normal and lesioned primary afferent DRG cells. However, axotomy induced a distinct shift in NPY receptor mRNA expression from small to large neurons, indicating that sensitivity to NPY is switched from one modality to another. Thus, not only several sensory neuropeptides, as shown in previous studies, but at least also one of the peptide receptors change their expression dramatically in response to axotomy, suggesting complex adaptive responses.     

Effect of peripheral nerve lesion and lumbar sympathectomy on peptide regulation in dorsal root ganglia in the NGF-overexpressing mouse

Galanin is a peptide normally expressed at low levels both in sensory and in sympathetic neurons. It is strongly upregulated after peripheral nerve lesions, and it has been proposed that nerve growth factor (NGF) plays a role in this regulation. In the present study the effect of both sciatic nerve transection and lumbar sympathectomy on galanin in lumbar dorsal root ganglia (DRGs) was examined in mice overexpressing NGF (NGFOE) in the skin under the keratin promoter. The superior cervical ganglia (SCG) were also studied. In the DRG pericellular baskets containing tyrosine hydroxylase- (TH) and galanin-like immunoreactivity (LI) were found, mostly in the same fibers. Galanin-positive baskets were also found in the trigeminal ganglia. However, only single neuropeptide Y (NPY)-positive baskets were observed within the DRGs. No marked difference in number of galanin-positive neurons was seen between wild-type and NGFOE mice. After sciatic nerve transection galanin was upregulated in DRG neurons to about the same extent in NGFOE mice as in wild-type mice. Galanin-, but not TH-LIs decreased in the pericellular baskets. After lumbar sympathectomy both galanin- and TH-immunoreactive baskets disappeared, suggesting a sympathetic origin. In the SCG the very low galanin mRNA levels were strongly increased after lesion of the carotid nerves, both in wild-type and in NGFOE mice. However, whereas NPY mRNA levels decreased in the SCG after axotomy in the wild-type mice, there was a distinct increase in the NGFOE mice. Our results show that high NGF levels in skin induce formation of pericellular baskets in DRGs expressing galanin- and TH-LI and that galanin in these baskets is strongly influenced by peripheral axotomy. However, overexpression of NGF did not markedly influence galanin expression in DRG neurons, neither normally nor after nerve lesions. Finally, expression of NPY in sympathetic ganglia is differently regulated in NGFOE compared to wild-type mice.     

Increase of preprotachykinin mRNA and substance P immunoreactivity in spared dorsal root ganglion neurons following partial sciatic nerve injury

Complete sciatic nerve injury reduces substance P (SP) expression in primary sensory neurons of the L4 and L5 dorsal root ganglia (DRG), due to loss of target-derived nerve growth factor (NGF). Partial nerve injury spares a proportion of DRG neurons, whose axons lie in the partially degenerating nerve, and are exposed to elevated NGF levels from Schwann and other endoneurial cells involved in Wallerian degeneration. To test the hypothesis that SP is elevated in spared DRG neurons following partial nerve injury, we compared the effects of complete sciatic nerve transection (CSNT) with those of two types of partial injury, partial sciatic nerve transection (PSNT) and chronic constriction injury (CCI). As expected, a CSNT profoundly decreased SP expression at 4 and 14 days postinjury, but after PSNT and CCI the levels of preprotachykinin (PPT) mRNA, assessed by in situ hybridization, and the SP immunoreactivity (SP-IR) of the L4 and L5 DRGs did not decrease, nor did dorsal horn SP-IR decrease. Using retrograde labelling with fluorogold to identify spared DRG neurons, we found that the proportion of these neurons expressing SP-IR 14 days after injury was much higher than in neurons of normal DRGs. Further, the highest levels of SP-IR in individual neurons were detected in ipsilateral L4 and L5 DRG neurons after PSNT and CCI. We conclude that partial sciatic nerve injury elevates SP levels in spared DRG neurons. This phenomenon might be involved in the development of neuropathic pain, which commonly follows partial nerve injury.     

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.     

Effect of a graded single constriction of the rat sciatic nerve on pain behavior and expression of immunoreactive NPY and NPY Y1 receptor in DRG neurons and spinal cord

In the present study, the rat sciatic nerve was constricted to varying degrees using only one ligature with a very thin polyethylene sheath placed between nerve and ligature thread. Complete nerve transection was studied for comparison. With a 40-80% constriction of the nerve we observed allodynia to a similar extent as in the so-called Bennett model based on four loose ligatures. We also monitored changes in the expression of neuropeptide Y (NPY) and the NPY Y1 receptor (Y1R) in the lumbar 4-5 dorsal root ganglia (DRG) and dorsal horn and found upregulation of NPY and downregulation of the Y1R in DRG neurons after injury. These results indicate that similar peptide and receptor changes occur in this model as after axotomy and in other nerve injury models, although the immunohistochemical and behavioral changes seem to be dependent on the degree of constriction of the nerve. Thus, it seems relevant to monitor the degree of constriction when evaluating pain and other post-injury events. The possibility that some of the changes in NPY-ergic neurotransmission are related to the generation of allodynia is discussed; as well as the possibility to use this mononeuropathic model based on a single ligature nerve constriction (SLNC) as a complementary approach to other widely used pain models.     

A preliminary study on DRGs and spinal cord of a galanin receptor 2-EGFP transgenic mouse

The neuropeptide galanin functions via three G-protein coupled receptors, Gal_1–3-R. Both Gal₁-R and ₂-R are involved in pain signaling at the spinal level. Here a Gal₂-R-EGFP transgenic (TG) mouse was generated and studied in pain tests and by characterizing Gal₂-R expression in both sensory ganglia and spinal cord. After peripheral spared nerve injury, mechanical allodynia developed and was ipsilaterally similar between wild type (WT) and TG mice. A Gal₂-R-EGFP-positive signal was primarily observed in small and medium-sized dorsal root ganglion (DRG) neurons and in spinal interneurons and processes. No significant difference in size distribution of DRG neuronal profiles was found between TG and WT mice. Both percentage and fluorescence intensity of Gal₂-R-EGFP-positive neuronal profiles were overall significantly upregulated in ipsilateral DRGs as compared to contralateral DRGs. There was an ipsilateral reduction in substance P-positive and calcitonin gene-related peptide (CGRP)-positive neuronal profiles, and this reduction was more pronounced in TG as compared to WT mice. Moreover, Gal₂-R-EGFP partly co-localized with three pain-related neuropeptides, CGRP, neuropeptide Y and galanin, both in intact and injured DRGs, and with galanin also in local neurons in the superficial dorsal horn. Taken together, the present results provide novel information on the localization and phenotype of DRG and spinal neurons expressing the second galanin receptor, Gal₂-R, and on phenotypic changes following peripheral nerve injury. Gal2-R may also be involved in autoreceptor signaling.     

Neuronal-glial differential expression of TGF-alpha and its receptor in the dorsal root ganglia in response to sciatic nerve lesion.

Injury to peripheral nerves often results in structural and functional changes in the dorsal root ganglia (DRG). Although the mechanisms underlying these changes remain largely unknown, satellite cell activation and up-regulation of several neurotrophic factors in the DRG occur in response to the nerve lesion, modulating the plasticity of affected neurons. To investigate potential roles of transforming growth factor alpha (TGF-alpha) in these plastic changes in the DRG following a sciatic nerve transection, here we examined the expression in DRGs of TGF-alpha and its receptor (EGF receptor), molecules known to be mitogenic to glia and Schwann cells and to be neurotrophic for some differentiated neurons. In the normal DRGs, TGF-alpha and its receptor are expressed mainly in small neurons and satellite cells surrounding some large or medium-sized neurons as determined by immunohistochemistry and in situ hybridization. In response to sciatic nerve lesion, there was a marked and differential up-regulation of TGF-alpha and EGF receptor expression within DRG, evident as early as 24 h after lesion and lasting for at least 14 days. While the up-regulated TGF-alpha was localized mainly on satellite cells in the ipsilateral and contralateral DRGs, EGF receptor up-regulation was mainly neuronal (with the expression expanding to include all neurons) in the ipsilateral DRGs, but mainly glial in the contralateral DRGs. These changes in TGF-alpha and its receptor expression suggest that TGF-alpha may play a role in the satellite cell proliferation and/or activation as well as in neuronal survival after nerve lesion.     

Effect of axotomy on expression of NPY, galanin, and NPY Y1 and Y2 receptors in dorsal root ganglia and the superior cervical ganglion studied with double-labeling in situ hybridization and immunohistochemistry

Using double-labeling techniques for both in situ hybridization and immunohistochemistry some peptides and peptide receptors were studied quantitatively in a sensory and a sympathetic ganglion after axotomy. In the lumbar 5 dorsal root ganglion (DRG) normally no neuropeptide Y- and only a few galanin-positive cell bodies are seen. Following complete transection of the sciatic nerve around 60% of all neuropeptide Y (NPY) neuron profiles (NPs) were galanin positive (+) and 33-44% of all galanin NPs were NPY(+). A good agreement between immunohistochemistry and in situ hybridization was observed for NPY and galanin. NPY Y1- and Y2-receptor (R) mRNAs were found in around 40% of all NPY mRNA(+) NPs, and more than half of the Y1-R mRNA(+) NPs and two-thirds of the Y2-R mRNA(+) NPs were NPY(+). In addition, more than one-third of the galanin mRNA-containing NPs showed colocalization with NPY receptor mRNAs and up to 70% of the Y2-R mRNA(+) NPs also expressed galanin mRNA. In the control superior cervical ganglion (SCG) 10% of the NPY(+) NPs were Y2-R mRNA(+), and 85% of the Y2-R(+) NPs were NPY mRNA(+), and the corresponding percentages after axotomy were around 35 and 45%, respectively. Following axotomy of the carotid nerves around half of all NPY(+) NPs were galanin(+), and conversely around 50% of all galanin NPs were NPY(+) at the mRNA level, whereas much lower percentages (15 and 9%, respectively) were observed with immunohistochemistry. These results demonstrate that double-labeling procedures are valid tools to quantitatively evaluate coexistence situations in sensory and sympathetic ganglia, showing a high degree of coexistence for NPY and galanin in axotomized neurons both in the lumbar 5 DRG and in the SCG. However, the immunohistochemical analysis in the SCG demonstrated much lower numbers of peptide-positive neurons than seen with in situ hybridization, suggesting that the latter technique is more sensitive. The fact that a considerable number of neurons express NPY together with Y1- and/or Y2-Rs indicates that both receptors may act as autoreceptors, the Y1-R presumably at the level of the cell body and the Y2-R on nerve terminals in the dorsal horn and/or the periphery. The present results also show that in both sensory and sympathetic neurons there is a strong upregulation of the Y2-R after nerve injury, suggesting a possible role in trophic and regenerative events.     

Neuronal–Glial Differential Expression of TGF-α and Its Receptor in the Dorsal Root Ganglia in Response to Sciatic Nerve Lesion

Injury to peripheral nerves often results in structural and functional changes in the dorsal root ganglia (DRG). Although the mechanisms underlying these changes remain largely unknown, satellite cell activation and up-regulation of several neurotrophic factors in the DRG occur in response to the nerve lesion, modulating the plasticity of affected neurons. To investigate potential roles of transforming growth factor α (TGF-α) in these plastic changes in the DRG following a sciatic nerve transection, here we examined the expression in DRGs of TGF-α and its receptor (EGF receptor), molecules known to be mitogenic to glia and Schwann cells and to be neurotrophic for some differentiated neurons. In the normal DRGs, TGF-α and its receptor are expressed mainly in small neurons and satellite cells surrounding some large or medium-sized neurons as determined by immunohistochemistry and in situ hybridization. In response to sciatic nerve lesion, there was a marked and differential up-regulation of TGF-α and EGF receptor expression within DRG, evident as early as 24 h after lesion and lasting for at least 14 days. While the up-regulated TGF-α was localized mainly on satellite cells in the ipsilateral and contralateral DRGs, EGF receptor up-regulation was mainly neuronal (with the expression expanding to include all neurons) in the ipsilateral DRGs, but mainly glial in the contralateral DRGs. These changes in TGF-α and its receptor expression suggest that TGF-α may play a role in the satellite cell proliferation and/or activation as well as in neuronal survival after nerve lesion.     

Nitric oxide synthase-like immunoreactivity in lumbar dorsal root ganglia and spinal cord of rat and monkey and effect of peripheral axotomy

With the immunofluorescence technique, nitric oxide synthase (NOS)-like immunoreactivity (LI) was found in a few medium-sized and small sensory neurons in lumbar (L) 4 and L5 dorsal root ganglia (DRG) of normal rat, and in most of these neurons, NOS-LI coexisted with calcitonin gene-related peptide and sometimes with substance P and galanin. NOS-immunoreactive nerve fibers, terminals and small neurons were also located in the dorsal horn of the segments 4 and 5 of the rat lumbar spinal cord with the highest density in inner lamina II. Many NOS-positive neurons and fibers were seen in the area around the central canal. A sparse network of NOS-immunoreactive nerve fibers was found in the ventral horn. After unilateral sciatic nerve cut in the rat, the number of NOS-positive neurons increased in the ipsilateral L4 and L5 DRGs, mainly in medium and small neurons, but also in some large neurons and very small neurons. NOS-LI could now also be seen in the ipsilateral dorsal roots, and in an increased number of fibers and terminals in both outer and inner lamina II of the ipsilateral dorsal horn. The number of NOS-immunoreactive neurons in lamina II of the ipsilateral dorsal horn was reduced. In the monkey L4 and L5 DRGs, many small neurons were NOS-immunoreactive, but only a few weakly stained nerve fibers and terminals were found in laminae I-IV of the dorsal horn at L4 and L5 lumbar levels. A few NOS-positive neurons were present in lamina X. The number of NOS-immunoreactive neurons was somewhat reduced in DRGs 14 days after peripheral axotomy, but no certain effect was seen in the dorsal horn. These results, together with earlier in situ hybridization studies, demonstrate that axotomy in rat induces a marked upregulation of NOS synthesis in primary sensory neurons, thus suggesting a role for NO in lesioned sensory neurons. In contrast, no such effect was recorded in monkey, perhaps indicating distinct species differences. © 1993 Wiley-Liss, Inc.     

单侧坐骨神经完全结扎术后Ⅰ型囊泡膜谷氨酸转运体在大鼠腰髓、背根神经节和结扎远侧端神经干内表达的变化

目的　 研究单侧坐骨神经结扎对大鼠腰 4～ 5脊髓节段和相应的背根神经节 (DRGs)内VGluT1样免疫阳性反应产物表达的影响以及VGluT1通过轴浆流向外周转运的情况。 方法　 采用免疫组织化学方法观察单侧坐骨神经结扎后不同时间内腰 4～ 5脊髓节段、DRGs和结扎部位的近、远侧端神经干内VGLuT1样免疫阳性反应强度的变化。结果　 (1)坐骨神经结扎后第 1和第 2天 ,VGluT1样免疫阳性产物在结扎的同侧腰 4～ 5脊髓节段和相应节段的DRGs内未检测到明显变化 ;但自术后第 4天开始 ,可观察到VGluT1样免疫阳性产物的表达在上述部位逐渐减弱 ;VGluT1样免疫阳性产物表达的降低在上述部位所出现的时间和程度相平行。 (2 )结扎后第 1天即可观察到VGluT1样免疫阳性产物在坐骨神经结扎部位近侧端的表达有所升高 ,但自术后第 4天开始逐渐降低 ;而VGluT1样免疫阳性产物在坐骨神经结扎部位远侧端的表达自结扎后第 1天起就逐渐降低 ,至第 4周时已完全消失。结论　 (1)DRG神经元合成VGluT1,并通过轴浆流将VGLluT1向中枢突和周围突运输 ,故腰髓内部分VGluT1样阳性末梢起源于DRG神经元 ;(2 )外周神经的损伤很易影响到DRG神经元内VGluT1的合成     

Secretory pathways of neuropeptides in rat lumbar dorsal root ganglion neurons and effects of peripheral axotomy

Using immunocytochemistry combined with confocal and electron microscopy, the secretory pathways related to substance P (SP), calcitonin gene-related peptide (CGRP), galanin (GAL), and neuropeptide Y (NPY) were investigated in neurons in rat lumbar (L) 4 and L5 dorsal root ganglia (DRGs) before and after peripheral axotomy. All four peptides were processed through the regulated secretory pathway in many small neurons in normal DRGs, and CGRP through this pathway also in some large neurons. In many small neurons, two neuropeptides could be sorted into the same or separate large dense-core vesicles (LDCVs). The LDCVs had a significantly larger diameter in small as compared to large DRG neurons. Fourteen days after sciatic nerve cut, the levels of SP- and CGRP-like immunoreactivities (-LIs) and the number of LDCVs containing these peptides were markedly reduced, but SP- and CGRP-LIs were still seen in the regulated pathway. GAL-LI was markedly increased in many small neurons and some large neurons and NPY-LI mainly in large neurons. Both peptides were particularly abundant in the Golgi region. In small neurons, the number of LDCVs containing GAL- or NPY-LI was increased, but did not appear to reach the numbers containing SP- or CGRP-LI in normal DRG neurons. After axotomy, CGRP-LI and GAL-LI were often in separate LDCVs. One type of NPY-positive large neurons showed budding off of LDCVs after axotomy, but also some “scattered” labeling in the cytoplasm. In the second type, NPY-LI was mainly found in multivesicular bodies. In several myelinated nerve fibers a “diffuse” distribution of NPY was seen together with some LDCVs containing NPY-LI. In contrast, in unmyelinated nerve fibers, NPY-, GAL-, SP-, and CGRP-LIs were always observed in LDCVs. Thus, both in normal and axotomized DRG neurons, peptides are processed through the regulated pathway. However, in some large neurons, NPY is, in addition, secreted through the constitutive pathway, perhaps as a consequence of limited sorting mechanisms for NPY, i.e., the plasticity of the secretory mechanisms does not match the rate of peptide synthesis after axotomy. © 1995 Wiley-Liss, Inc.     

Partial sciatic nerve ligation induced more dramatic increase of neuropeptide Y immunoreactive axonal fibers in the gracile nucleus of middle-aged rats than in young adult rats

Neuropeptide changes in primary sensory neurons caused by partial nerve injury are likely involved in the development of neuropathic pain. In this study, using immunocytochemistry, we examined neuropeptide Y (NPY) expression in lumbar dorsal root ganglion (DRG) cells of young adult (2-3 months old) and middle-aged (8-10 months old) rats 4 weeks after partial sciatic nerve ligation (PSNL). Significantly higher NPY immunoreactivity was induced in the injured side DRG neurons, the dorsal horn and the gracile nuclei in middle-aged rats than in young rats. Using combined fluorescent dye tracing and NPY immunostaining, we found in middle-aged rats that 46% injured DRG neurons projected to the gracile nucleus and 45% of injured neurons were also NPY-IR, whereas 42% spared DRG neurons projected to the gracile nucleus and 18% of spared neurons were also NPY-IR. Thus PSNL induces NPY up-regulation in spared as well as injured DRG neurons, both contribute to the increased NPY immunoreactivity in the gracile nucleus in the middle-aged rats. The more dramatic increase of NPY in DRG neurons of middle-aged rats after PSNL shows that the responses to partial nerve injury are age-dependent, that suggests a possible relevance to the higher incidence of neuropathic pain in human middle age.     

NPY-like immunoreactivity in sensory nerve fibers in rat sciatic neuroma.

Neuropeptide Y (NPY)-like immunoreactivity was examined by fluorescence immunohistochemistry in rat sciatic neuromas, L5 dorsal root ganglias (DRGs) and L5 dorsal roots 1-3 weeks after chronic nerve injury. Anterograde tracing demonstrated that a large number of NPY-positive neuroma fibers were sensory. These fibers were mostly large diameter axons, in line with the finding that a majority of NPY-immunoreactive neurons in the DRG were medium- to large-sized neurons which showed immunoreactivity to the neurofilament antibody RT 97. In dorsal roots NPY immunoreactivity was strong after sciatic neuroma formation. Dorsal rhizotomy and ligation, on the other hand, did not induce NPY immunoreactivity at any of the sites examined.     

Chronic nerve compression injury induces a phenotypic switch of neurons within the dorsal root ganglia

Chronic nerve compression (CNC) injury initiates a series of pathological changes within the peripheral nerve at the site of injury. However, to date, little work has been performed to explore neuronal cell body responses to CNC injury. Here we show a preferential upregulation of growth-associated protein-43 (GAP-43) and enhanced Fluoro Ruby uptake by the small-diameter calcitonin gene-related protein (CGRP) and isolectin B4 (IB4)-positive neurons in the L4 and L5 ipsilateral dorsal root ganglion (DRG) 2 weeks and 1 month post injury. Furthermore, L4 and L5 DRGs ipsilateral to CNC injury also demonstrated a marked reduction in neurofilament 200 (NF-200) neurons and an increase in CGRP and IB4 neurons at early time points. All numbers normalized to values comparable to those of control when the DRG was evaluated 6 months post injury. Quantification of glial-derived neurotrophic factor (GDNF) protein revealed an upregulation in L4 and L5 DRG followed by a return to baseline values at later stages following injury. Upregulation of GDNF expression by Schwann cells was also readily apparent with both immunohistochemistry and Western blot analysis of 1 month compressed sciatic nerve specimens. Thus, CNC induces a phenotypic change in the DRG that appears to be temporally associated with increases in GDNF protein expression at and near the site of the compression injury in the nerve.     

坐骨神经非冻结性冷损伤后背根神经节细胞的凋亡

目的观察大鼠坐骨神经在非冻结性低温作用后,腰段脊髓L4-L6背根神经节(dorsal root ganglion,DRG)凋亡神经元的数量以及形态学改变,探讨周围神经非冻结性冷损伤致DRG神经元凋亡的情况。方法雄性Wistar大鼠33只,随机分为1周组、2周组、3周组,每组11只。每只大鼠取任意一侧坐骨神经为实验侧,给予冷损伤(4℃,2h),对照侧坐骨神经同样方法暴露,不给予低温处理。根据坐骨神经和DRG的病理变化,分别取两侧的L4、L5、L6背根神经节于低温结束后1周、2周、3周采用流式细胞仪(Annexin/PI法)和Tunel法对DRG神经元凋亡进行定量和定性检测。结果流式细胞仪(Annexin/PI法)定量测定结果显示,实验侧凋亡率明显高于对照侧。Tunel法检测发现受冷侧的DRG出现典型的Tunel染色阳性的早期凋亡细胞,半定量测定显示实验侧DRG神经元凋亡率明显高于对照侧。两种方法均显示受冷后1周开始凋亡细胞增多,2周时到达高峰,3周时略下降。结论非冻结性低温可以造成坐骨神经对应的L4、L5、L6DRG神经元出现凋亡,凋亡的高峰出现在冷损伤后2周,以早期凋亡为主。凋亡可能是坐骨神经冷损伤的机制之一。     

Effect of Axotomy on Expression of NPY, Galanin, and NPY Y1 and Y2 Receptors in Dorsal Root Ganglia and the Superior Cervical Ganglion Studied with Double-Labeling in Situ Hybridization and Immunohistochemistry

Using double-labeling techniques for both in situ hybridization and immunohistochemistry some peptides and peptide receptors were studied quantitatively in a sensory and a sympathetic ganglion after axotomy. In the lumbar 5 dorsal root ganglion (DRG) normally no neuropeptide Y- and only a few galanin-positive cell bodies are seen. Following complete transection of the sciatic nerve around 60% of all neuropeptide Y (NPY) neuron profiles (NPs) were galanin positive (+) and 33–44% of all galanin NPs were NPY⁺. A good agreement between immunohistochemistry and in situ hybridization was observed for NPY and galanin. NPY Y1- and Y2-receptor (R) mRNAs were found in around 40% of all NPY mRNA⁺ NPs, and more than half of the Y1-R mRNA⁺ NPs and two-thirds of the Y2-R mRNA⁺ NPs were NPY⁺. In addition, more than one-third of the galanin mRNA-containing NPs showed colocalization with NPY receptor mRNAs and up to 70% of the Y2-R mRNA⁺ NPs also expressed galanin mRNA. In the control superior cervical ganglion (SCG) 10% of the NPY⁺ NPs were Y2-R mRNA⁺, and 85% of the Y2-R⁺ NPs were NPY mRNA⁺, and the corresponding percentages after axotomy were around 35 and 45%, respectively. Following axotomy of the carotid nerves around half of all NPY⁺ NPs were galanin⁺, and conversely around 50% of all galanin NPs were NPY⁺ at the mRNA level, whereas much lower percentages (15 and 9%, respectively) were observed with immunohistochemistry. These results demonstrate that double-labeling procedures are valid tools to quantitatively evaluate coexistence situations in sensory and sympathetic ganglia, showing a high degree of coexistence for NPY and galanin in axotomized neurons both in the lumbar 5 DRG and in the SCG. However, the immunohistochemical analysis in the SCG demonstrated much lower numbers of peptide-positive neurons than seen with in situ hybridization, suggesting that the latter technique is more sensitive. The fact that a considerable number of neurons express NPY together with Y1- and/or Y2-Rs indicates that both receptors may act as autoreceptors, the Y1-R presumably at the level of the cell body and the Y2-R on nerve terminals in the dorsal horn and/or the periphery. The present results also show that in both sensory and sympathetic neurons there is a strong upregulation of the Y2-R after nerve injury, suggesting a possible role in trophic and regenerative events.