Effects of peripheral nerve injury on delta opioid receptor (DOR) immunoreactivity in the rat spinal cord

Morphine and other opioids have direct analgesic actions in the spinal cord and chronic spinal administration of opioid agonists is used clinically in patients suffering from severe, chronic pain. Neuropathic pain resulting from peripheral nerve injury is often less sensitive to opioid therapy than other forms of chronic pain in both humans and animal models. Changes in spinal mu-opioid receptor (MOR) expression have been demonstrated in animal models of neuropathic pain. However, these changes alone fail to account for the attenuation of opioid activity. Reduced expression of delta-opioid receptors (DOR) following peripheral nerve injury has been reported but most of these reports are limited to subjective observation. The magnitude and consistency of these changes is therefore unclear. In addition, previous studies did not evaluate the effects of nerve injury on behavioral measures to confirm induction of aberrant pain symptoms. We therefore performed quantitative image analysis to evaluate the effect of peripheral nerve injury on DOR-immunoreactivity in spinal cord sections from rats previously characterized for sensory responsiveness. We observed statistically significant decreases ipsilateral to nerve injury in all three models tested: sciatic nerve transection, chronic constriction injury of the sciatic nerve and L5/L6 spinal nerve ligation. These results suggest that decreases in the expression of DOR are a common feature of peripheral nerve injury.     

Increased phosphorylated-mu-opioid receptor immunoreactivity in the mouse spinal cord following sciatic nerve ligation

The present study was designed to determine whether a state of neuropathic pain induced by sciatic nerve ligation could alter phosphorylated-mu-opioid receptor-like immunoreactivity in the superficial dorsal horn of the mouse spinal cord. Mice with sciatic nerve ligation exhibited a significant suppression of the morphine-induced antinociception. Under this condition, phosphorylated-mu-opioid receptor-like immunoreactivity was clearly increased on the ipsilateral side in the superficial laminae of the L5 lumbar spinal dorsal horn in nerve-ligated mice. These findings suggest that the phosphorylation of the mu-opioid receptor in the spinal cord under a neuropathic pain-like state may, at least in part, contribute to the reduction in the antinociceptive effect produced by morphine in the mouse.     

Changes in beta-2 adrenergic receptor and AMP-activated protein kinase alpha-2 subunit in the rat vestibular nerve after labyrinthectomy

In the present study, to elucidate the role of vestibular ganglion (VG) after the unilateral labyrinthine damage, we examined quantitative changes in mRNA expression of beta-adrenergic receptors (bARs) and AMP-activated protein kinase alpha catalytic subunits (aAMPKs) in VG after unilateral labyrinthectomy (UL) in rats. Using the real-time PCR method, beta2 AR mRNA expression in bilateral VG and AMPK alpha2 mRNA expression in the ipsilateral VG were significantly up-regulated with the maximum increase at the postoperative 7 day and 1 day, respectively. The up-regulation of beta2 AR in bilateral VG was long-lasting until 28 days after UL and that of AMPK alpha2 in the ipsilateral VG was just transient within 7 days after UL. These mRNA changes were supported by immunohistochemical data. According to previous reports, both of bARs and aAMPKs could regulate mitochondrial uncoupling protein (UCP) mRNA expression in several kinds of tissues and therefore might have thermogenic neurotransmission and antioxidant neuroprotective roles in neuronal tissues. UL requires not only long-lasting response of VG for central vestibular neuro-plasticity around 2-4 weeks but rapid response of VG against apoptosis of peripheral vestibular epithelia-neuronal synapses. The present findings suggest that beta2 AR in bilateral VG and AMPK alpha2 in the ipsilateral VG might play important signaling roles after the unilateral labyrinthine damage.     

Light and electron microscopic localization of alpha-1 adrenergic receptor immunoreactivity in the rat striatum and ventral midbrain

Electrophysiological and pharmacological studies have demonstrated that alpha-1 adrenergic receptor (α1AR) activation facilitates dopamine (DA) transmission in the striatum and ventral midbrain. However, because little is known about the localization of α1ARs in dopaminergic regions, the substrate(s) and mechanism(s) underlying this facilitation of DA signaling are poorly understood. To address this issue, we used light and electron microscopy immunoperoxidase labeling to examine the cellular and ultrastructural distribution of α1ARs in the caudate putamen, nucleus accumbens, ventral tegmental area, and substantia nigra in the rat. Analysis at the light microscopic level revealed α1AR immunoreactivity mainly in neuropil, with occasional staining in cell bodies. At the electron microscopic level, α1AR immunoreactivity was found primarily in presynaptic elements, with scarce postsynaptic labeling. Unmyelinated axons and about 30–50% terminals forming asymmetric synapses contained the majority of presynaptic labeling in the striatum and midbrain, while in the midbrain a subset of terminals forming symmetric synapses also displayed immunoreactivity. Postsynaptic labeling was scarce in both striatal and ventral midbrain regions. On the other hand, only 3–6% of spines displayed α1AR immunoreactivity in the caudate putamen and nucleus accumbens. These data suggest that the facilitation of dopaminergic transmission by α1ARs in the mesostriatal system is probably achieved primarily by pre-synaptic regulation of glutamate and GABA release.     

Nitric oxide synthase immunoreactivity in rat spinal cord.

Immunoreactivity to nitric oxide synthase (NOS-IR) and choline acetyltransferase (ChAT-IR) was detected in the adult rat spinal cord using the avidin-biotin-peroxidase technique. Intensely stained NOS-positive neurons with cell processes were observed in the intermediolateral cell column of the thoracic and sacral segments and around the central canal of all segments. These areas also contained ChAT-IR neurons. A number of small- to medium-sized NOS-IR cells were noted in the superficial and deeper laminae throughout the entire cord. NOS-IR was not detected in the ventral horn motoneurons, which were, however, ChAT-IR. The results indicate that NOS-IR is present in autonomic preganglionic neurons and in selected neurons in the dorsal horn and lamina X, but appears to be absent in motoneurons.     

大鼠脊髓损伤后不同时间点5-HT2A受体免疫反应性的变化

目的:研究大鼠脊髓骶段(第2骶节)横断伤后5-羟色胺2A受体免疫反应(5-HT2AR-IR)的变化.方法:大鼠随机分为7组,即2h、8h和1d、2d、7d、30 d、60 d组,每组又随机分为手术组和假手术组.手术组在大鼠第2骶髓节段完全横断,假手术组只去除椎板,保留硬脊膜的完整,采用免疫荧光组织化学方法检测脊髓损伤下段5-HT2A-IR的变化.结果:2、8h手术组脊髓前角运动神经元区5-HT 2A-IR密度与假手术组比较无明显不同,1d后与假手术组比较开始增高,5-HT2A-IR密度随时间延长呈逐渐增加的趋势,直到手术后60 d达到最高水平.结论:慢性脊髓骶段完全横断后脊髓前角运动神经元的5-HT2A受体上调可能是5-羟色胺去神经支配后超敏感反应和痉挛发生的潜在机制.     

Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the rat brain and spinal cord

A complete mapping study on the 1,25-dihydroxyvitamin D3 receptor immunoreactivity within the rat central nervous system was performed with a monoclonal and a polyclonal antibody. Specific immunostaining was observed within both nuclear and cytoplasmic compartments of a variety of cells in the cerebellum, mesopontine area, diencephalon, cortex, spinal cord, and limbic system. Both monoclonal and polyclonal antibodies provided similar staining patterns. The monoclonal antibody stained distinct domains within the nuclei of all and the cytoplasm of specific neuronal cell types, like motor neurons, Purkinje cells, and pyramidal cells of the cortex more clearly than the polyclonal antibody. The expression of vitamin D3 receptor in the rat central nervous system was confirmed by in situ hybridisation. The widespread distribution of vitamin D3 receptor in distinct portions of the sensory, motor, and limbic brain systems suggests multiple functional properties of 1,25-dihydroxyvitamin D3 in the central nervous system.     

Increase of galanin-like immunoreactivity in rat hypothalamic arcuate neurons after peripheral nerve injury

Galanin and galanin receptors are widely distributed within the central nervous system, and may play important roles in pain signaling and modulation. In the present study, we examined the galanin immunoreactivity (IR) in the hypothalamus and the amygdala following peripheral nerve injury. Four weeks after the operation, the ipsilateral mechanical threshold in the spared nerve injury (SNI) group (0.87 +/- 0.33 g) was significantly lower than that in the sham group (12.53 +/- 3.41 g; P < 0.05). In the SNI group, the number of galanin-IR neurons per section in the arcuate nucleus (Arc) of the hypothalamus was 10.2 +/- 1.7, significantly higher than that in the sham group (5.6 +/- 1.0; P < 0.05). These data suggest that the galanin-ergic neurons in the Arc may be involved in the functional modulation of descending pain modulation system following peripheral nerve injury.     

Metastin-like immunoreactivity in the rat medulla oblongata and spinal cord

Metastin, the product of metastasis suppressor gene KiSS-1, is proposed to be the natural ligand for the G-protein-coupled receptor GPR54, known also as AXOR12. This immunohistochemical study, using a rabbit polyclonal antiserum against the human metastin fragment (45-54)-NH(2), showed that in rats metastin-like immunoreactivity (MTS-LI) was present in neurons of the nucleus of the solitary tract and caudoventrolateral reticular nucleus, and in cell processes of the spinal trigeminal tract and lateral reticular nucleus. MTS-LI was confined mainly to neurons and fibers at or caudal to the area postrema. In the spinal cord, MTS-LI cell processes formed a dense plexus in superficial layers I and II of the dorsal horn. The pattern of distribution of MTS-LI in the medulla and spinal cord suggests that this novel peptide may participate in autonomic and sensory neural signaling.     

Increased nerve growth factor receptor mRNA in contused rat spinal cord

Peripheral nerve injury induces the expression of nerve growth factor receptor (NGFR). To determine whether a similar induction results from injury of the spinal cord, NGFR mRNA content was determined using Northern blot hybridization analysis of total RNA from spinal cords of rats contused in the mid-thoracic region. By four days after contusion NGFR mRNA was significantly increased in the thoracic segments that included the injury site. The induction was maximal at 7 days, about 5- to 7-fold the level of uninjured controls, and remained 4 times higher than controls at 14 and 28 days after injury. These results suggest that axotomy in the CNS may also trigger the molecular mechanism(s) leading to up-regulation of NGFR expression.     

大鼠坐骨神经损伤早期NF-κB和Bcl-2在脊髓运动神经元的诱导激活

目的：探讨大鼠坐骨神经损伤早期NF—KB和Bcl-2在脊髓中的表达变化。方法：健康成年sD大鼠48只，随机分为正常组、假手术组和右侧坐骨神经压榨组，动物存活不同时问（6，12，24和72h）获取L。脊髓段，用免疫组织化学方法检测脊髓内NF—KB和Bcl-2的表达变化。结果：（1）正常L4-6脊髓内，NF—KB和Bcl-2免疫阳性产物主要位于前角运动神经元胞浆内，NF—KB活化少，Bcl-2表达低；（2）损伤后双侧脊髓中NF—κB免疫阳性产物则主要位于胞核内，呈活化状态，损伤侧活化起始时问（6h）早于损伤对照侧（12h），且6、12、24hNF—κB活化细胞数明显高于对照侧（P〈0．05），72h双侧比较无明显差异（P〉0．05）。（3）损伤后6h损伤侧脊髓中Bcl-2免疫阳性反应即增强，12h达到高峰，24h下降，72h又增高；其中6、12、72h损伤侧Bcl-2免疫阳性反应灰度值与对照侧和正常组相比差别具有统计学意义（P〈0．05），而24h损伤侧与对照侧和正常组比较无统计学差异（P〉0．05）。结论：坐骨神经损伤后脊髓前角细胞中NF—κB的活化，激活了其下游抗凋亡基因Bcl-2的转录，两者可能在损伤早期参与抑制细胞凋亡，促进细胞存活和神经再生。     

Effects of spinal cord lesions and rhizotomies on cholinergic and opiate receptor binding sites in rat spinal cord

Changes in the distribution of [3H]quinuclidinylbenzilate ([3H]QNB), [3H] alpha-bungaro-toxin ([3H]alpha-Btx) and [3H]etorphine binding sites were studied autoradiographically, and cholinacetyltransferase (ChAT) activity radioenzymatically, in the C6-7 segments of rats 1-20 days after combined dorsal and ventral C3-8 rhizotomies and spinal cord lesions at C3. After dorsal and ventral rhizotomies the number of [3H]QNB, [3H]alpha-Btx and [3H]etorphine binding sites were reduced ipsilaterally in the dorsal horn and those of [3H]QNB and [3H]alpha-Btx in the ventral horn. In the ventral horn ChAT activity was significantly reduced. After a unilateral spinal cord lesion at C3, ChAT activity was reduced in the ipsilateral ventral horn at C6-7 caudal to the lesions, whereas no change in receptor binding sites was observed.     

腓肠神经移植修复大鼠陈旧性脊髓损伤的作用

目的:探讨利用自体周围神经组织移植修复大鼠陈旧性脊髓损伤病理机制,为临床应用提供实验依据。方法:利用改良Allens撞击方法建立脊髓打击损伤模型,12周后,将大鼠分为2组,实验组切取后肢腓肠神经,利用显微外科技术去除神经外膜,将其修剪成小段,游离移植于脊髓损伤处,对照组不作处理。分别于术后2、4、12周,在光镜及电镜下观察脊髓损伤段及移植周围神经再生情况。术后4、8及12周分别对两组动物进行脊髓诱发电位检查。结果:对照组脊髓变性,可见瘢痕和空洞,实验组术后12周,损伤区脊髓与周围神经融合良好。脊髓诱发电位检查神经移植组优于对照组。结论:周围神经组织游离移植修复大鼠陈旧性脊髓损伤后,存活良好,为再生轴突跨越损伤段脊髓提供通道。     

周围神经阻滞麻醉对脊髓生物学的影响

背景：周围神经阻滞麻醉后是怎样产生功能抑制的,对脊髓和大脑中枢有何影响,是怎样调节周围神经的,中枢在解剖形态上有何改变,目前尚不明确。 目的：观察周围神经阻滞麻醉对脊髓神经细胞生物学的影响。 方法：将60只新西兰大白兔随机分为坐骨神经阻滞麻醉组、蛛网膜下腔阻滞麻醉组和硬膜外腔阻滞麻醉组,每组20只;每组再分实验组和对照组,每组10只。坐骨神经阻滞麻醉组的实验组在股骨头与坐骨结节连线中点注射利多卡因,蛛网膜下腔阻滞麻醉组的实验组在蛛网膜下腔注射布比卡因,硬膜外腔阻滞麻醉组的实验组在硬膜外腔注射利多卡因,各组的对照均在相同部位注射生理盐水。 结果与结论：坐骨神经阻滞麻醉、硬膜外腔阻滞麻醉或蛛网膜下腔阻滞麻醉30 min后,均有相应节段脊髓灰质后角Ⅲ、Ⅳ板层小圆细胞、前角Ⅸ板层外侧大多角细胞的尼氏体减少,核偏向一端,c-Fos蛋白表达减弱或无表达,提示脊髓相应节段的神经细胞功能受到抑制;另外蛛网膜下腔阻滞麻醉后,脊髓软脊膜有分层或断裂现象。     

Localization of N-type Ca2+ channels in the rat spinal cord following chronic constrictive nerve injury

Previous studies have shown that spinal L-type, N-type, and P-type Ca²⁺-channel blockers are effective in modulating pain behavior caused nerve injury. In the present work, using the loose ligation of the sciatic nerve model, we characterized the time course of the appearance of tactile and cold allodynia and the corresponding spinal expression of the N-type Ca²⁺ channel α_1B-subunit after nerve ligation. Within 1 week after ligation, the majority of rats developed a unilateral sensitivity to mechanical stimulation (von Frey filaments), as well as sensitivity to cold, which persisted for 30 days. Immunocytochemical analysis of the spinal cord in sham-operated animals for the α_1B-subunit showed a smooth, moderate staining pattern in the superficial laminae I–II, as well as in ventral α-motoneurons. In nerve-ligated animals, an intense, dot-like immunoreactivity in the ipsilateral dorsal horn was observed from 5–20 days after nerve ligation. The most prominent α_1B-subunit upregulation was found in the outer as well as the inner part of lamina II (II_o, II_i), extending from the medial toward the lateral region of the L4 and L5 spinal segments. The behavioral changes which developed after chronic constriction injury directly correlated with the α_1B-subunit upregulation in the corresponding spinal cord segments. These data suggest that upregulation of the spinal α_1B-subunit may play an important role in the initiation and maintenance of pain state after peripheral nerve injury. Electronic Publication     

Characterization of nestin expression in the spinal cord of GFP transgenic mice after peripheral nerve injury

Many studies have shown that activation and increase in the number of astrocytes and microglia in the spinal cord participate in the initiation and maintenance of neuropathic pain, but little attention has been paid to the responses of neural progenitor cells to peripheral nerve injury. Nestin, a class VI intermediate filament protein, is expressed both in neuronal and glial progenitors as well as in their common precursors; and nestin-positive cells appear in the brain and spinal cord following various forms of damage to these regions. To clarify the responses of neural progenitor cells to nerve injury, we applied L5 spinal nerve transection (L5-SNT) to nestin-promoter GFP (pNestin-GFP) transgenic mice to narrow the target to them. While pNestin-GFP expression was strongly retained in the ependyma lining the central canal of the transgenic spinal cord even in adulthood, it was markedly reduced in the dorsal horn during postnatal development by day 7. Increases in pNestin-GFP expression and labeling by the proliferation marker 5-bromodeoxyuridine were broadly found in the dorsal horn of adult mice on day 3 after L5-SNT. On the other hand, the activation and increase in number of microglia and astrocytes are restricted to the superficial layer of the dorsal horn, the central terminal of injured primary afferent fibers. Purinergic P2X agonist α, β-MeATP increased [Ca²⁺]i in nestin-positive cells in the superficial layer ipsilateral to nerve injury and P2 receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (PPADS) blocked the expression and elongation of pNestin-GFP fibers in the slice culture of the spinal cord. These results with pNestin-GFP transgenic mice demonstrate that nestin-positive cells proliferate in the dorsal horn in response to peripheral nerve injury and suggest that ATP may contribute to the expression of nestin and activation of neural progenitor cells after nerve injury.     

Morphometric analysis in the rat spinal cord following peripheral nerve transection with the use of MAP2 immunohistochemistry]

The physiological function of the spinal cord is regulated by two different kinds of information, namely, afferent inputs from the primary sensory fibers and descending inputs from the brain. The former conveys the sensory information from the peripheral region via the dorsal root ganglia to spinal interneurons or directly to motor neurons and brings about the voluntary and/or involuntary muscle movement. In the present study, the time-related changes in the dendrocytoarchitecture of the rat spinal cord following the transection of afferent and efferent connections from/to the peripheral organs were observed by using immunohistochemistry for microtubule-associated protein 2 (MAP2) and Nissl staining. To investigate the changes quantitatively, morphometric analyses were performed on the section of gray matter of tissue samples stained with MAP2-antisera. One to 24 weeks after hemitransection of the dorsal and ventral roots, a remarkable reduction in the area and progressive changes in the shape of the gray matter were seen in both the anterior and posterior horns on the lesioned side. These changes were found to be due to the reduction of the width of the anterior and posterior horns on the lesioned side rather than of the height. At the later postoperative stage, atrophy of the Fasciculus gracilis and disappearance of motoneurons on the lesioned side were recognized in Nissl stained tissue samples.     

Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin

The temporal course of changes in peptide expression in the dorsal root ganglia L4 and L5 and in the dorsal horn of the spinal cord has been studied in rats subjected to a sciatic nerve transection at a mid-thigh level following different survival times. Galanin-, substance P-, vasoactive intestinal polypeptide-, peptide histidine-isoleucine- and calcitonin gene-related peptide-like immunoreactivities have been studied both by immunohistochemistry and radioimmunoassay. Galanin messenger ribonucleic acid has also been studied by in situ hybridization in the dorsal root ganglia of normal and lesioned animals. In addition, a group of animals with a sciatic nerve crush was studied to compare possible differences in peptide expression after both types of lesions. The results show that the transection induces an increase in the number of cell bodies expressing galanin-like immunoreactivity in the ganglia, and that the galanin levels rise about 120-fold after three and 14 days of survival. This increase reflected increased synthesis of the peptide, since there was a rise in the galanin messenger ribonucleic acid already at 24 h post-lesion, which was maintained for at least 60 days. In the spinal cord there was an increase of staining in the midportion of the outer layers of the dorsal horn that corresponded to fibers thought to arise from cells of the dorsal root ganglia affected by the transection. Also a depletion of substance P-like and an increase in vasoactive intestinal polypeptide- and peptide histidine-isoleucine-like immunoreactivities in the dorsal root ganglia were confirmed. These changes were shown to be rapidly detectable and were paralleled by similar changes in the dorsal horn of the spinal cord. For calcitonin gene-related peptide the immunohistochemistry was inconclusive, and the radioimmunoassay showed no detectable changes. After nerve crush a transient increase in the number of galanin immunoreactive neurons was observed, as well as a decrease in the number of neurons showing substance P-like immunoreactivity. These changes were most noticeable between six and 14 days of survival. After this, peptide expression seemed to return slowly to normal, that is by day 45 post-crush only a few cells showed galanin-like, and many sensory neurons expressed substance P-like immunoreactivity. The results demonstrate that when primary sensory neurons are peripherally lesioned they respond in a complex manner, altering their normal production of peptides by increasing or decreasing their synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in the axonal membrane potential and Ca2+ concentration associated with peripheral nerve grafting after spinal cord injury

We performed peripheral nerve allografting in rats with spinal cord injury, and measured motor function and axonal membrane potential as well as Ca(2+) concentration of the nerve grafting spinal cord area by using a behavior observation system and a confocal laser-scanning microscope, respectively. In our experiments, we produced a model of peripheral nerve grafting after spinal cord injury by peripheral nerve allografting (sciatic nerve) in rats with spinal cord injury (thoracic cord hemisection). The group with spinal cord injury that underwent peripheral nerve grafting showed improvement in motor function, a significant increase in the axonal action potential, and a slight increase in the Ca(2+) concentration compared with the group that did not undergo nerve grafting.