CCK－8和NDAP对大鼠脑和脊髓组织c－fos表达的影响

为探讨八肽胆囊收缩素（ＣＣｋ－８）和阿片肽相互作用的分子机理,利用抗体免疫沉淀技术研究了ＣＣＫ－８与ＮＤＡＰ（ｋ阿片受体激动剂）对大鼠脑（去皮层和小脑）和脊髓背柱组织Ｆｏｓ蛋白的影响。结果表明,０．１μｍｏｌ／ＬＣＣＫ－８可显著刺激脑和脊髓组织中Ｆｏｓ蛋白增加（分别是对照组的３．８倍和３．６倍）。相同浓度的ＮＤＡＰ对Ｆｏｓ蛋白的生成亦有一定的诱导作用,分别是对照组的２．７倍和２．６倍。ＣＣＫ－８和ＮＤＡＰ共同处理组织,Ｆｏｓ蛋白生成水平相似（脑）或高于（脊髓）ＣＣＫ~－８单独诱导的水平。结果表明,ＣＣＫ－８和ＮＤＡＰ均可直接诱导大鼠脑和脊髓组织ｃ－ｆｏｓ的表达,它们对ｃ－ｆｏｓ表达的相互作用在脑和脊髓中呈现不同的模式。     

强啡肽A和CCK—8对大鼠脊髓突触小体摄取^45Ca的影响

为了探讨血管紧张素Ⅱ(AⅡ)和八肽胆囊收缩素(CCK-8)这两种肽的抗阿片作用机理,本实验中观察了三种阿片类物质(吗啡、强啡肽和 DPDPE)和两种抗阿片物质(AⅡ和 CCK-8)对大鼠脊髓突触小体摄取~(45) Ca 的影响。结果表明:(1)在脊髓腹柱突触小体上,10nmol/L—1μmol/L 的吗啡、强啡肽 A(Dyn A)和 DPDPE 对~(45)Ca 摄取均有较弱的抑制作用;(2)CCK-8在浓度高达lμmol/L 时对~(45)Ca 摄取有较弱的抑制作用;(3)AⅡ在浓度高达lμmol/L时也不影响腹柱突触小体摄取~(45)Ca;(4)在背柱的突触小体制备中,上述阿片物质中 Dyn A 对~(45)Ca 摄取有较强的抑制作用,并被 k 受体阻断剂 nor-BNI 所阻断。10和100nmol/L 的 CCK-8能翻转lμmol/L Dyn A 对~(45)Ca 摄取的抑制作用;(5)A Ⅱ不能翻转Dyn A 的抑制作用。以上结果提示,CCK-8阻断 Dyn A 抑制脊髓背柱突触小体摄取 Ca~(2+)的作用可能是其行为学中抗阿片作用的机理之一。AⅡ对脊髓 Ca~(2+)摄取和 Dyn A 抑制脊髓 Ca~(2+)摄取的作用皆无影响,与行为学中观察到的 AⅡ在脊髓内不能对抗阿片镇痛的现象一致,进一步说明 CCK-8和AⅡ拮抗阿片类物质对神经末梢 Ca~(2+)摄取的影响可能是其抗阿片作用的重要机理之一。     

Time Course of Immediate Early Gene Protein Expression in the Spinal Cord following Conditioning Stimulation of the Sciatic Nerve in Rats

Long-term potentiation induced by conditioning electrical stimulation of afferent fibers is a widely studied form of synaptic plasticity in the brain and the spinal cord. In the spinal cord dorsal horn, long-term potentiation is induced by a series of high-frequency trains applied to primary afferent fibers. Conditioning stimulation (CS) of sciatic nerve primary afferent fibers also induces expression of immediate early gene proteins in the lumbar spinal cord. However, the time course of immediate early gene expression and the rostral-caudal distribution of expression in the spinal cord have not been systematically studied. Here, we examined the effects of sciatic nerve conditioning stimulation (10 stimulus trains, 0.5 ms stimuli, 7.2 mA, 100 Hz, train duration 2 s, 8 s intervals between trains) on cellular expression of immediate early genes, Arc, c-Fos and Zif268, in anesthetized rats. Immunohistochemical analysis was performed on sagittal sections obtained from Th13- L5 segments of the spinal cord at 1, 2, 3, 6 and 12 h post-CS. Strikingly, all immediate early genes exhibited a monophasic increase in expression with peak increases detected in dorsal horn neurons at 2 hours post-CS. Regional analysis showed peak increases at the location between the L3 and L4 spinal segments. Both Arc, c-Fos and Zif268 remained significantly elevated at 2 hours, followed by a sharp decrease in immediate early gene expression between 2 and 3 hours post-CS. Colocalization analysis performed at 2 hours post-CS showed that all c-Fos and Zif268 neurons were positive for Arc, while 30% and 43% of Arc positive neurons were positive for c-Fos and Zif268, respectively. The present study identifies the spinal cord level and time course of immediate early gene (IEGP) expression of relevance for analysis of IEGPs function in neuronal plasticity and nociception.     

八肽胆囊收缩素对抗阿片肽的中枢性降血压作用

过去的工作已经证明八肽胆囊收缩素(CCK-8)能够对抗阿片肽的镇痛作用,本工作探讨CCK-8是否能够对抗阿片肽的心血管抑制作用。给戊巴比妥钠麻醉大鼠脊髓蛛网膜下腔(ith)注射 CCK-8可以对抗 ith 注射 mu(μ)型阿片受体激动剂[NMePhe~3,D-Pro~4]Morphiceptin(PL017)(5μg)、delta(δ)型受体激动剂[D-Ala~2,D-Leu~5]Enkephalin(DADLE)(25μg)和 Kappa(K)型受体激动剂[N-Me Tyr,N-Me Arg~7,D-Leu~8]Dynorphin 1-8 ethyla-mide(66A-078)(1μg)引起的降低血压和减慢心率作用。在 MAP 的表现上,CCK-8的拮抗作用(10μg及以下剂量)具有量-效关系,并可被 CCK 受体阻断剂丙谷胺(Proglumide)(100μg)翻转。在 HR 的表现上,上述剂量的 CCK-8也显示了一定的拮抗作用,但量-效关系不如 MAP 表现得明显。单纯将 CCK-8或 Proglumide ith 注射,可见大剂量(50μg)CCK-8可以引起明显的降血压作用和短时的降心率作用,小剂量(0.05μg)CCK-8则表现出明显的降心率作用;ith 注射 Proglumide 100μg,30 min 后也表现出减慢心率的作用。以上结果提示:在脊髓水平,一定剂量范围内的 CCK-8能够对抗阿片肽的心血管抑制效应,此对抗作用是通过 CCK 受体实现的。本工作的结果支持关于 CCK-8是一种抗阿片物质的设想。     

Differential bile-pancreatic secretory effects of CCK-58 and CCK-8

In this work, we 1) synthesized rat CCK-58, 2) determined the amounts and forms of rat CCK in whole blood after stimulation of its release by casein, 3) determined the potency of CCK-8 and CCK-58 peptides to displace labeled CCK-8 from CCK(A) and CCK(B) receptors transfected into Chinese hamster ovary (CHO) cells, and 4) examined the biological actions of CCK-8 and rat CCK-58 in an anesthetized rat model. CCK-58 was the only detected endocrine form of CCK in rat blood. Synthetic rat CCK-58 was less potent than CCK-8 for displacing the label from CCK(A) and CCK(B) receptors in transfected CHO cells. However, rat CCK-58 was more potent than CCK-8 for stimulation of pancreatic protein secretion in the anesthetized rat. In addition, CCK-58 but not CCK-8 stimulated fluid secretion in this anesthetized rat model. These data suggest that regions outside the COOH terminus of rat CCK-58 influence the expression of CCK biological activity. The presence of only CCK-58 in the circulation and the fact that its biological activity differs from CCK-8 suggests that CCK-58 deserves scrutiny in other physiological models of CCK activity.     

Spinal cord stimulation improves survival in ischemic skin flaps: an experimental study of the possible mediation by calcitonin gene-related peptide

Currently, spinal cord stimulation is used to treat ischemia and ischemic pain, with the best results observed in vasospastic cases. It was earlier demonstrated that spinal cord stimulation may attenuate experimentally induced vasospasm in an island flap in the rat. The present study was designed to investigate whether preemptive spinal cord stimulation could increase long-term flap survival and to explore the neurohumoral mediation of the effect. A total of 56 rats were implanted with chronic spinal cord stimulation systems. Three days later, a groin flap based on the superficial epigastric vessels was harvested, and the single feeding artery was occluded by a detachable microvascular clip. After 12 hours, the clip was removed. Flap survival was evaluated after 7 days. Immediately before flap surgery, two groups of animals received 30 minutes of stimulation using current clinical parameters and with stimulation amplitudes of 70 (n = 10) or 90 percent (n = 8) of that evoking muscular contractions. The outcomes in these groups were compared with those in two control groups (n = 20; n = 10). In one group, an additional calcitonin gene-receptor peptide (CGRP) antagonist was intravenously injected before stimulation (n = 8). In the control groups without stimulation, virtually all flaps necrotized. In treated groups, flap survival was 60 percent at the lower intensity and almost 90 percent at the higher one. The administration of a CGRP antagonist before treatment reduced its efficacy to below 40 percent survival. The differences between the untreated and treated groups were significant. The decrease in survival after CGRP-receptor block was significant in one of two tests. Preemptive spinal cord stimulation increases survival of skin flaps with critical ischemia. The effects are dependent on the stimulation intensity and are possibly mediated by the release of CGRP in the periphery.     

Cryptic organisation within an apparently irregular rostrocaudal distribution of interneurons in the embryonic zebrafish spinal cord

The molecules and mechanisms involved in patterning the dorsoventral axis of the developing vertebrate spinal cord have been investigated extensively and many are well known. Conversely, knowledge of mechanisms patterning cellular distributions along the rostrocaudal axis is relatively more restricted. Much is known about the rostrocaudal distribution of motoneurons and spinal cord cells derived from neural crest but there is little known about the rostrocaudal patterning of most of the other spinal cord neurons. Here we report data from our analyses of the distribution of dorsal longitudinal ascending (DoLA) interneurons in the developing zebrafish spinal cord. We show that, although apparently distributed irregularly, these cells have cryptic organisation. We present a novel cell-labelling technique that reveals that DoLA interneurons migrate rostrally along the dorsal longitudinal fasciculus of the spinal cord during development. This cell-labelling strategy may be useful for in vivo analysis of factors controlling neuron migration in the central nervous system. Additionally, we show that DoLA interneurons persist in the developing spinal cord for longer than previously reported. These findings illustrate the need to investigate factors and mechanisms that determine “irregular” patterns of cell distribution, particularly in the central nervous system but also in other tissues of developing embryos.     

反复电针对慢性痛的累加治疗作用及其机制研究

本研究从基础和临床两方面观察了反复电针对慢性痛的累加治疗作用,并结合疼痛患者及慢性痛动物模型中几种神经肽的放射免疫测定及相应受体拮抗剂的药理学研究结果,探讨了产生累加效应的可能机制。结果表明,在临床脊髓损伤性痉挛患者,１００Ｈｚ穴位体表电刺激有效地缓解痉挛并有累加效应;在临床慢性痛患者,２／１５Ｈｚ变频ＴＥＮＳ刺激有效地治疗疼痛并具有累加效应。在关节炎模型大鼠,电针刺激能产生明显的镇痛并具有累加效     

Interactions between spinal cord stimulation and activity blockade in the regulation of synaptogenesis and motoneuron survival in the chick embryo

The present study investigated the effects of spinal cord stimulation, neuromuscular blockade, or a combination of the two on neuromuscular development both during and after the period of naturally occurring motoneuron death in the chick embryo. Electrical stimulation of the spinal cord was without effect on motoneuron survival, synaptogenesis, or muscle properties. By contrast, activity blockade rescued motoneurons from cell death and altered synaptogenesis. A combination of spinal cord stimulation and activity blockade resulted in a marked increase in motoneuron death, and also altered synaptogenesis similar to that seen with activity blockade alone. Perturbation of normal nerve–muscle interactions by activity blockade may increase the vulnerability of developing motoneurons to excessive excitatory afferent input (spinal cord stimulation) resulting in excitotoxic-induced cell death. © 1993 John Wiley & Sons, Inc.     

Cortical potentials evoked by epidural stimulation of the cervical and thoracic spinal cord in man

Scalp somatosensory evoked potentials (SEPs) were recorded after electrical stimulation of the spinal cord in humans. Stimulating electrodes were placed at different vertebral levels of the epidural space over the midline of the posterior aspect of the spinal cord. The wave form of the response differed according to the level of the stimulating epidural electrodes. Cervical stimulation elicited an SEP very similar to that produced by stimulation of upper extremity nerves, e.g., bilateral median nerve SEP, but with a shorter latency. Epidural stimulation of the lower thoracic cord elicited an SEP similar to that produced by stimulation of lower extremity nerves. The results of upper thoracic stimulation appeared as a mixed upper and lower extremity type of SEP. The overall amplitudes of SEPs elicited by the epidural stimulation were higher than SEPs elicited by peripheral nerve stimulation. In 4 patients the CV along the spinal cord was calculated from the difference in latencies of the cortical responses to stimulation at two different vertebral levels. The CVs were in the range of 45–65 m/sec. The method was shown to be promising for future study of spinal cord dysfunctions.     

Noninvasive method to control the human spinal locomotor systems

The mechanism of interactions between receptor activation in the musculoskeletal system and stimulation of the spinal cord in the regulation of locomotor behavior was studied in healthy subjects. Afferent stimulation was tested for effect on the patterns of stepping movements induced by percutaneous stimulation of the spinal cord. A combination of percutaneous spinal cord stimulation and vibratory stimulation was shown to increase the amplitude of leg movements. It was demonstrated that vibratory stimulation of limb muscles at a frequency of less than 30 Hz can be used to control involuntary movements elicited by noninvasive stimulation of the spinal cord.     

Calcitonin gene-related peptide 8-37 inhibits the evoked discharge frequency of wide dynamic range neurons in dorsal horn of the spinal cord in rats.

The present study was performed to explore the effect of calcitonin gene-related peptide 8-37 (CGRP8-37) on the electrical stimulation-evoked discharge frequency of wide dynamic range (WDR) neurons in the dorsal horn of the spinal cord in rats. The discharge frequencies of WDR neurons were evoked by transdermic electrical stimulation applied on the ipsilateral hindpaw. CGRP8-37 was applied directly on the dorsal surface of the L3 to L5 spinal cord. After the administration of 3 nmol of CGRP8-37, the evoked discharge frequency of WDR neurons decreased significantly, an effect lasting more than 30 min. The results indicate that CGRP receptors play an important role in the transmission of presumed nociceptive information in the dorsal horn of the spinal cord.     

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.     

Glial tumor necrosis factor alpha (TNFα) generates metaplastic inhibition of spinal learning

Injury-induced overexpression of tumor necrosis factor alpha (TNFα) in the spinal cord can induce chronic neuroinflammation and excitotoxicity that ultimately undermines functional recovery. Here we investigate how TNFα might also act to upset spinal function by modulating spinal plasticity. Using a model of instrumental learning in the injured spinal cord, we have previously shown that peripheral intermittent stimulation can produce a plastic change in spinal plasticity (metaplasticity), resulting in the prolonged inhibition of spinal learning. We hypothesized that spinal metaplasticity may be mediated by TNFα. We found that intermittent stimulation increased protein levels in the spinal cord. Using intrathecal pharmacological manipulations, we showed TNFα to be both necessary and sufficient for the long-term inhibition of a spinal instrumental learning task. These effects were found to be dependent on glial production of TNFα and involved downstream alterations in calcium-permeable AMPA receptors. These findings suggest a crucial role for glial TNFα in undermining spinal learning, and demonstrate the therapeutic potential of inhibiting TNFα activity to rescue and restore adaptive spinal plasticity to the injured spinal cord. TNFα modulation represents a novel therapeutic target for improving rehabilitation after spinal cord injury.     

Role of spinal voltage-dependent calcium channel alpha 2 delta-1 subunit in the expression of a neuropathic pain-like state in mice

The present study was undertaken to investigate the role of spinal voltage-dependent calcium channel alpha(2)delta-1 subunit in the expression of a neuropathic pain-like state induced by partial sciatic nerve ligation in mice. In cultured spinal neurons, gabapentin (GBP), which displays the inhibitory effect of alpha(2)delta-1 subunit, suppressed the extracellular Ca(2+) influx induced by KCl, whereas it failed to inhibit the intracellular Ca(2+) release induced by inositol-1,4,5-triphosphate. Seven days after sciatic nerve ligation, the protein level of alpha(2)delta-1 subunit in the ipsilateral spinal cord was clearly increased compared to that observed in sham-operated mice. In addition, the mRNA level of alpha(2)delta-1 subunit was significantly increased in the dorsal root ganglion, but not in the spinal cord, of nerve-ligated mice. Under these conditions, a marked decrease in the latency of paw-withdrawal against a thermal stimulation and tactile stimulation, induced by sciatic nerve ligation was abolished by repeated intrathecal (i.t.) treatment with GBP. Additionally, the persistent reduction in the nociceptive threshold by i.t. treatment with GBP at the early stage of the neuropathic pain-like state was maintained for 7 days even after GBP withdrawal. It is of interest to note that a single i.t. post-injection of GBP showed a marked and transient inhibitory effect on the developed neuropathic pain-like state, whereas repeated i.t. post-treatment with GBP produced a persistent inhibitory effect during the treatment. In conclusion, we propose here that the neuropathic pain-like state with sciatic nerve ligation is associated with the increased level of the alpha(2)delta-1 subunit of Ca(2+) channels at the sensory nerve terminal in the spinal dorsal horn of mice. Furthermore, the present data provide evidence that the neuropathic pain may be effectively controlled by repeated treatment with GBP at the early stage.     

Spinal astrocytes contribute to the circadian oscillation of glutamine synthase, cyclooxygenase-1 and clock genes in the lumbar spinal cord of mice

Spinal astrocytes have key roles in the regulation of pain transmission. However, the relationship between astrocytes and the circadian system in the spinal cord remains poorly defined. In the current study, the circadian variations in the expression of several clock genes in the lumbar spinal cord of mice were examined by using real-time PCR. The expression of Period1, Period2 and Cryptochrome1 showed significant circadian oscillations, each gene peaking in the early evening. The expression of Bmal1 mRNA also exhibited a circadian pattern, peaking from around midnight to early morning. The mRNA levels of Cryptochrome2 were slightly, but not significantly altered. Molecules related to pain transmission were also investigated. The mRNA expression of glutamine synthase (GS), and cyclooxygenases (COXs), known to be involved in various spinal sensory functions, showed rhythmicity with a peak in the early evening, although the expression of the neurokinin-1 receptor, subunits of the N-methyl-d-aspartate receptor, and glutamate transporters did not change. In addition, we found that protein levels of GS and COX-1 were also high at midnight compared with midday. Furthermore, we examined the effect of intrathecal fluorocitrate (100pmol), an inhibitor of astrocytic metabolism, on the expression of oscillating genes in lumbar spinal cord. Fluorocitrate significantly suppressed astrocyte function. Furthermore, the circadian oscillation of clock gene expression and GS and COX-1 expression were suppressed. Together, these results suggest that a significant circadian rhythmicity of the expression of clock genes is present in the spinal cord and that the components of the circadian clock timed by astrocytes might contribute to spinal functions, including nociceptive processes.     

Locomotion induced by spinal cord stimulation in the neonate rat in vitro.

The present studies employed the neonate rat brain stem-spinal cord preparation to determine whether electrical stimulation of the lumbosacral enlargement (LE) of the spinal cord itself can be used to elicit locomotion, and whether or not such stimulation persists in inducing locomotion following midthoracic spinal cord transection or hindlimb deafferentation. Results suggest that (1) stimulation of the dorsal columns or ventral funiculus of the LE is effective in inducing airstepping in the neonatal rat brain stem-spinal cord limb-attached preparation; (2) central disconnection by midthoracic spinal cord transection does not alter LE-stimulation-induced airstepping and may lead to an increase in stepping frequency if suprathreshold stimulation is used; and (3) dorsal root section also leads to an increase in the frequency of suprathreshold LE-stimulation-induced locomotion, but there is not further increase in frequency if a spinal cord transection is performed in addition to dorsal rhizotomy.     

Sensorimotor regulation of movements: Novel strategies for the recovery of mobility

A series of observations have provided important insight into properties of the spinal as well as supraspinal circuitries that control posture and movement. We have demonstrated that spinal rats can regain full weight-bearing standing and stepping over a range of speeds and directions with the aid of electrically enabling motor control (eEmc), pharmacological modulation (fEmc), and training [1, 2]. Also, we have reported that voluntary control movements of individual joints and limbs can be regained after complete paralysis in humans [3, 4]. However, the ability to generate significant levels of voluntary weight-bearing stepping with or without epidural spinal cord stimulation remains limited. Herein we introduce a novel method of painless transcutaneous electrical enabling motor control (pcEmc) and sensory enabling motor control (sEmc) strategy to neuromodulate the physiological state of the spinal cord. We have found that a combination of a novel non-invasive transcutaneous spinal cord stimulation and sensory-motor stimulation of leg mechanoreceptors can modulate the spinal locomotor circuitry to state that enables voluntary rhythmic locomotor movements.