Direct evidence for the link between monoaminergic descending pathways and motor activity:: II. A study with microdialysis probes implanted in the ventral horn of the spinal cord

In order to define precisely the relation between descending monoaminergic systems and the motor system, we measured in the ventral horn of spinal cord of adult rats the variations of extracellular concentrations of 5-HT, 5-HIAA, DA and MHPG. Measurements were performed during rest, endurance running on a treadmill, and a post-exercise period, with microdialysis probes implanted permanently for 45 days. We found a slight decrease in both 5-HT and 5-HIAA during locomotion with a more marked decrease during the post-exercise period compared to the mean of rest values. In contrast, the concentration of DA and MHPG increased slightly during the exercise and decreased thereafter. These results, when compared with those of a previous study, which measured monoamines in the spinal cord white matter [C. Gerin, D. Bécquet, A. Privat, Direct evidence for the link between monoaminergic descending pathways and motor activity: I. A study with microdialysis probes implanted in the ventral funiculus of the spinal cord, Brain Res. 704 (1995) 191–201], highlight the complex regulation of the release of monoamines that occurs in the ventral horn.     

Astrocyte-conditioned medium attenuates glutamate-induced apoptotic cell death in primary cultured spinal cord neurons of rats

Abstract

Objectives:

To better understand the neuroprotective role of astrocytes in spinal cord injury (SCI), we investigated whether astrocyte-conditioned medium (ACM) can attenuate glutamate-induced apoptotic cell death in primary cultured spinal cord neurons.

Methods:

Spinal cord neurons were pretreated with ACM for 24?hours. Subsequently, they were exposed to glutamate (125?μM) for 1?hour. The neurons were then incubated for 24?hours. Following that, measurements assessing cell viability and lactate dehydrogenase (LDH) release were performed. Apoptosis was confirmed through cell morphology using Hoechst 33342 staining and terminal deoxynucleotidyl transferase dUTP-mediated nicked end labeling (TUNEL) assay. Assessment for expression of apoptotic enzymes, including Caspase-3, Bcl-2 and Bax, was performed using Western Blot Analysis.

Results:

Astrocyte-conditioned medium pretreatment of neurons showed both an increase in spinal cord neuron viability and a decrease in LDH release in a dose-dependent pattern. Moreover, pretreatment seems to attenuate glutamate-induced apoptotic cell death, antagonise glutamate-induced up-regulation of Caspase-3 expression and downregulate Bcl-2/Bax protein expression ratio.

Conclusions:

By attenuating glutamate-induced apoptotic cell death in primary cultured spinal cord neurons of rats, ACM seems to provide a neuroprotective effect by regulating apoptosis-related protein expression. Our results provide an experimental basis for clinical applications and potential therapeutic use of ACM in SCI.     

Early axon and dendritic outgrowth of spinal accessory motor neurons studied with DiI in fixed tissues

We have utilized lateral diffusion of DiI in fixed tissues (Godement et al., '87: Development 101: 697-713) to study early axon and dendritic outgrowth of spinal accessory motor neurons in embryonic rats. Crystals were placed in the central canal of the cervical spinal cord near the ventral commissure in order to label growing accessory axons anterogradely and on the spinal accessory nerve to label somata and dendrites retrogradely. Animals were studied on E11-E13. We show here that it is possible to stain axonal and dendritic processes from the earliest stages of motor neuron differentiation by using DiI. Our results demonstrate that, unlike axons of other cervical motor neurons, accessory axons traverse the lateral region of the embryonic cord, which consists of neuroepithelial endfeet. Thus an affinity for neuroepithelial endfeet could partially explain their unusual intraspinal trajectory. We also show that morphology of the spinal accessory growth cones differs according to position along the accessory nerve pathway. Finally, we show that accessory motor neuron axons are in the region of their target precursors prior to the initiation of dendritic arborization. Use of DiI in fixed tissue allows study of process outgrowth in mammalian spinal cord with detail previously obtainable only in nonmammalian vertebrates.     

Enhancement of BDNF and activated-ERK immunoreactivity in spinal motor neurons after peripheral administration of BDNF

Brain-derived neurotrophic factor (BDNF) shows neurotrophic effects on adult motor neurons when given systemically, But it is unknown whether systemically administered BDNF is transported to central cell bodies to affect them directly. Here we used immunohistochemistry to investigate the transport of peripherally injected BDNF to spinal motor neurons and the subsequent activation of a signaling pathway. We first injected BDNF into the flexor digitorum brevis (FDB) and analyzed the motor nucleus that projects to the FDB for BDNF immunoreactivity (BDNF-ir) and phosphorylated extracellular signal-regulated kinase (ERK) 1/2 immunoreactivity (pERK1/2-ir). Both immunoreactivities were observed in the motor neuron cell bodies. Next, BDNF was injected subcutaneously (s.c.) into rats with a unilaterally axotomized sciatic nerve. pERK1/2-ir was detected in motor neurons of the lesioned side. BDNF-ir and pERK1/2-ir were also observed on the unlesioned side when a high dose of BDNF was injected. Therefore, we examined BDNF-ir and pERK1/2-ir after injecting BDNF s.c. into normal rats. Both immunoreactivities were observed in motor nuclei on both sides. Finally, we examined pERK1/2-ir after a lower dose of BDNF was injected, which prevents the decrease in choline acetyl transferase that occurs in the motor neuron upon axotomy. Spinal motor nuclei contained a few cell bodies with pERK1/2-ir. These findings represent the first direct evidence that subcutaneously injected BDNF is transported to motor neurons and that it activates a signaling pathway in the spinal cord and exhibits neurotrophic effects in vivo.     

Direct evidence for the link between monoaminergic descending pathways and motor activity. I. A study with microdialysis probes implanted in the ventral funiculus of the spinal cord

Monoaminergic projections to the spinal cord are involved in the modulation of motor, autonomic, and sensory functions. More specifically, the increase of electrical activity of serotonergic neurons in raphe obscurus has been correlated with locomotion in t treadmill-trained cats [Jacobs,B.L. and Fornal, C.,Trends Neurosci., 9 (1993) 346–352]. In order to test the direct correlation between locomotion and the release of monoamines, microdialysis probes were permanently implanted for 45 days into the ventral funiculus of the spinal cord (white matter) of adult rats. Eight days after implantation, these rats were subjected to an endurant exercise on a treadmill, and dialysis sessions were organized in such a way that microdialysate samples of 15 min duration were collected during pre-, per- and post-exercise periods. Measurements of serotonin, 5-hydroxyindoleacetic acid, dopamine and 3-methoxy-4-hydroxyphenylethylglycol concentration in the extracellular space showed significant increases during locomotion when compared with both pre- and post-exercise values. Histological analysis shows that serotonergic axons were present close to the dialysis probe. These results demonstrate that the implantation of a microdialysis probe in the ventral funiculus, close to a potential target of monoaminergic projections, is a suitable technique for the collection of neuromediators released during spontaneous running.     

Presence of binucleate neurons in the spinal cord of young and senile rats

The presence of binucleate cells constitutes a normal feature of some animal tissues but is rare in the normal brain and has not been documented in the spinal cord. We assessed different segments of the rat spinal cord in order to determine the frequency and distribution of binucleate neurons in this structure as well as the impact of aging on this neuronal population. Young (4–5 months) and senile (32 months) female Sprague–Dawley rats were used. Sections from cervical, thoracic and lumbar segments were histochemically and immunohistochemically (NeuN) stained and the frequency and distribution of binucleate neurons was determined by manual counting. The frequency of binucleate neurons in all of the analysed segments was comparable between young and senile animals. Binucleate neurons were particularly frequent in the C5 and C6 segments. The overall distribution of binucleate neurons in the different laminae assessed was, Lm-III = 19%; Lm-VI = 17%; Lm-VII = 39%; LmVIII = 8%; Lm-IX = 11%; Lm-X = 6%, and was comparable between young and senile rats. We conclude that binucleate neurons occur as a normal feature of the rat spinal cord and that their frequency and distribution does not change with aging.     

Long-term surgical outcomes and prognostic factors of adult symptomatic spinal cord cavernous malformations

Cervical and thoracic or lumbar intramedullary spinal cavernous malformations (ISCMs) may behave differently. We conducted this retrospective study by using data from adult ISCMs to compare their natural histories and explore prognostic factors for improved clinical outcomes. Neurological functions were assessed by using the Modified McCormick Scale (MMCS) and Aminoff-Logue Disability Scale. A total of 111 study-eligible adult patients were included in this study. Patients with cervical ISCMs mostly demonstrated a shorter duration of symptoms (P = 0.026), an acute onset pattern with some recovery (P = 0.026), and a larger lesion size (P = 0.033) than their thoracic or lumbar counterparts. Thoracic or lumbar lesions had a higher proportion of motor symptoms (P = 0.001) and sphincter problems (P = 0.005), and they were usually associated with an aggressive clinical course (P = 0.001, OR = 9.491, 95% CI = 2.555–35.262) in multivariate analysis. There was no difference in age, sex distribution, hemorrhage risk between the cervical and thoracic-lumbar groups. A better preoperative neurological status (P = 0.034, OR = 2.768, 95% CI = 1.081–7.177) and improvement immediately after surgery (P < 0.001, OR = 8.756, 95% CI = 4.837–72.731) were identified as indicators for long-term improvement by multivariate analysis. Cervical lesions had a high proportion for long-term improvement, but it was not a predictor for improvement in multivariate analysis. ISCMs in the thoracic or lumbar location should be considered for surgical removal more aggressively than those in the cervical location. Surgical removal of symptomatic ISCMs can avoid further neurological deterioration and usually result in satisfactory long-term outcomes.     

Interactions of cyclic adenosine monophosphate,brain-derived neurotrophic factor,and glial cell line-derived neurotrophic factor treatment on the survival and growth of postnatal mesencephalic dopamine neurons in vitro

The survival of rat postnatal mesencephalic dopamine (DA) neurons in dissociated cell cultures was studied by examining the combinatorial effects of dibutyryl cyclic adenosine monophosphate (db-cAMP), glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF), as well as selective inhibitors of protein kinase A (PKA), and mitogen-activated protein kinase (MAPK). Postnatal DA neurons were maintained for 14 days in vitro, and were identified by immunohistochemistry using tyrosine hydroxylase antibody. The survival and growth of DA neurons was significantly increased by the inclusion of either >100 microM db-cAMP or 10 microM Forskolin plus 100 microM IBMX in the culture medium. Neither 10-50 ng/ml GDNF nor 50 ng/ml BDNF alone significantly increased DA neuron survival in vitro. However, the combined use of GDNF and BDNF did increase DA neuron survival, and the addition of either db-cAMP or IBMX/Forskolin to media containing these neurotrophins markedly increased DA neuron survival and growth. The cAMP inhibitor Rp-cAMP, the cAMP-dependent protein kinase A inhibitor H89, and the MAP kinase (MAPK) pathway inhibitor PD98059 significantly reduced the survival of DA neurons when applied alone in the absence of added growth factors. Application of GDNF plus BDNF, or db-cAMP significantly protected the DA neurons from the deleterious effects on survival of either 20 microM H89 or 20 microM PD 98059. The results suggest that BDNF, GDNF, and cAMP produce convergent signals to activate PKA and MAPK pathways which are involved in the survival of postnatal mesencephalic DA neurons in vitro.     

Diagnostic significance of motor evoked potentials in space-occupying lesions of the brain stem and spinal cord

Summary Motor evoked potentials (MEP) were examined in 50 patients with space-occupying lesions of the brain stem and spinal cord. MEP findings were correlated with the motor status as established by clinical examination. The results clearly show the high sensitivity of MEP for detection of motor deficits: 17 recordings (77%) from the thenar muscle and 42 (84%) from the anterior tibial muscle correlated correctly with the clinical motor status. False-positive results were found in 5 (23%) thenar recordings and 8 (16%) and anterior tibial recordings. False-negative correlation was not observed. The high rate of false-positive results appears to indicate that MEP detect subclinical motor deficits. This electrophysiological test is therefore recommended, especially when involvement of the descending pathways is suspected and clinical examination reveals no abnormality.     

运动诱发电位联合体感诱发电位监测在脊髓髓内肿瘤手术中的应用

目的 探讨全静脉麻醉下运动诱发电位(MEP)联合体感诱发电位(SEP)术中监测应用于脊髓髓内肿瘤手术的优越性、可靠性及临床应用价值.方法 对72例脊髓髓内肿瘤患者术中行SEP和MEP联合监测,参照McCormick评分标准对术前、术后脊髓功能的改变和诱发电位变化之间的关系进行统计分析.结果 14例脊髓神经功能改善,18例术后脊髓神经功能下降者与诱发电位监测结果具有一致性(P＜0.05).结论 对脊髓髓内肿瘤手术进行SEP与MEP监测有利于避免"假阴性/假阳性"结果及术后神经功能障碍的发生.     

Temporal neurotransmitter conditioning restores the functional activity of adult spinal cord neurons in long-term culture

The ability to culture functional adult mammalian spinal cord neurons represents an important step in the understanding and treatment of a spectrum of neurological disorders including spinal cord injury. Previously, the limited functional recovery of these cells, as characterized by a diminished ability to initiate action potentials and to exhibit repetitive firing patterns, has arisen as a major impediment to their physiological relevance. In this report, we demonstrate that single temporal doses of the neurotransmitters serotonin, glutamate (N-acetyl-DL-glutamic acid) and acetylcholine-chloride lead to the full electrophysiological functional recovery of adult mammalian spinal cord neurons, when they are cultured under defined serum-free conditions. Approximately 60% of the neurons treated regained their electrophysiological signature, often firing single, double and, most importantly, multiple action potentials.     

Treatment of chronically injured spinal cord with neurotrophic factors stimulates betaII-tubulin and GAP-43 expression in rubrospinal tract neurons

Exogenous neurotrophic factors provided at a spinal cord injury site promote regeneration of chronically injured rubrospinal tract (RST) neurons into a peripheral nerve graft. The present study tested whether the response to neurotrophins is associated with changes in the expression of two regeneration-associated genes, betaII-tubulin and growth-associated protein (GAP)-43. Adult female rats were subjected to a right full hemisection lesion via aspiration of the C3 spinal cord. A second aspiration lesion was made 4 weeks later and gel foam saturated in brain-derived neurotrophic factor (BDNF), glial cell-line derived neurotrophic factor (GDNF), or phosphate-buffered saline (PBS) was applied to the lesion site for 60 min. Using in situ hybridization, RST neurons were examined for changes in mRNA levels of betaII-tubulin and GAP-43 at 1, 3, and 7 days after treatment. Based on analysis of gene expression in single cells, there was no effect of BDNF treatment on either betaII-tubulin or GAP-43 mRNA expression at any time point. betaII-Tubulin mRNA levels were enhanced significantly at 1 and 3 days in animals treated with GDNF relative to levels in animals treated with PBS. Treatment with GDNF did not affect GAP-43 mRNA levels at 1 and 3 days, but at 7 days there was a significant increase in mRNA expression. Interestingly, 7 days after GDNF treatment, the mean cell size of chronically injured RST neurons was increased significantly. Although GDNF and BDNF both promote axonal regeneration by chronically injured neurons, only GDNF treatment is associated with upregulation of betaII-tubulin or GAP-43 mRNA. It is not clear from the present study how exogenous BDNF stimulates regrowth of injured axons.     

The organization of acromiodeltoid and spinodeltoid motor nuclei in rat spinal cord

The retrograde intraaxonal transport of fluorescent dyes (10% Bisbenzimide or 2% Nuclear Yellow) was used to identify the motoneuron populations that control two prime movers of the rat shoulder joint, the acromiodeltoid (Ad) and spinodeltoid (Sd) muscles. The Ad and Sd motor nuclei were both comprised of an average of 30–40 motoneurons distributed between spinal segments C5 and C7, in lateral regions of Rexed's lamina IX. Although there was considerable overlap among them, the center of the Ad motor nucleus was consistently located more rostral and medial than that for the Sd motor nucleus.     

Bulbospinal respiratory neurons are a source of double synapses onto phrenic motoneurons following cervical spinal cord hemisection in adult rats

The purpose of this study was to determine if the medullary neurons that provide the primary excitatiry drive to phrenic motoneurons (i.e., rostral ventral respiratory group, rVRG) are a source of double synapse formation in the phrenic nucleus after spinal cord hemisection. The axons of rVRG neurons either ipsilateral or contralateral to the hemisection were labeled by injection of a mixture of HRP and WGA-HRP into the rostral ventral respiratory group. Phrenic motoneurons ipsilateral and caudal to the hemisection were labeled by the retrograde transport of HRP. The ultrastructural results indicated that after hemisection, rVRG neurons from both sides of the medulla formed labelled double synapses in the phrenic nucleus.     

Upregulation of Group I metabotropic glutamate receptors in neurons and astrocytes in the dorsal horn following spinal cord injury

Of the glutamate receptor types, the metabotropic glutamate receptors (mGluRs) are G proteins coupled and can initiate a number of intracellular pathways leading to hyperexcitability of spinal neurons. In this study, we tested the expression of mGluRs to determine which cell types might contribute to sustained neuronal hyperexcitability in the lumbar enlargement with postoperative day (POD) 7 (early), 14 (late), and 30 (chronic phase) following spinal cord injury (SCI) by unilateral hemisection at T13 in Sprague-Dawley rats. Expression was determined by confocal analyses of immunocytochemical reaction product of neurons (NeuN positive) and astrocytes (GFAP positive) in the dorsal horn on both sides of the L4 segment. Neurons were divided into two sizes: small (<20 microm) and large (>35 microm), for physiological reasons. We report a significant increase of mGluR(1) expression in large and small neurons of the dorsal horn on both sides of the cord in late and chronic phases when compared to control sham groups. Expression of mGluR(2/3) significantly increased in large neurons on the ipsilateral (hemisected) side in the late phase. Expression of mGluR(5) significantly increased in large neurons in early, late, and chronic phases. In addition, mGluR(1) and mGluR(5) expression after hemisection was significantly increased in astrocytes in early, late, and chronic phases; whereas mGluR(2/3) did not display any significant changes. In conclusion, our data demonstrate long-term changes in expression levels of Group I mGluRs (mGluR(1) and mGluR(5)) in both neurons and astrocytes in segments below a unilateral SCI. Thus, permanent alterations in dorsal horn receptor expression may play important roles in transmission of nociceptive responses in the spinal cord following SCI.     

Detecting peripheral motor nervous system involvement in chronic spinal cord injury using two novel methods: MScanFit MUNE and muscle velocity recovery cycles

ObjectiveTo examine the peripheral nervous system (PNS) in spinal cord injured (SCI) patients using two novel methods: (1) MScanFit MUNE; a motor unit number estimation method detecting motor unit loss and (2) muscle velocity recovery cycles (MVRCs) measuring muscle membrane properties which has previously shown depolarization of the muscle membrane in denervated muscles.MethodsThirty chronic SCI patients (lesion above Th10) and twenty-five gender –and age matched healthy controls (HC) were examined. MScanFit was recorded from peroneal nerve to anterior tibial muscle (TA) and tibial nerve to abductor hallucis muscle after excluding localized mononeuropathies. MVRCs were recorded from TA.ResultsNerve conduction studies showed mononeuropathy in 8 patients (27%) (sciatic (2), -or peroneal nerve (6)). SCI patients had in average reduced motor unit number compared with HC and prolonged muscle refractory period and reduced supernormality.SignificanceA high prevalence of nerve lesion and a diffuse affection of the PNS following SCI are highly relevant findings that should be accounted for when planning neurorehabilitation for persons living with SCI.     

脊髓损伤大鼠远端神经元及骨骼肌变化的基础研究

目的观察脊髓损伤大鼠远端神经元及骨骼肌变化情况。方法 20只大鼠随机分为2组,每组10只,分别为假手术组和脊髓损伤组,假手术组行椎板切除术,脊髓损伤组行胸10完全脊髓损伤,在制成模型后1、2、4、12、24周观察大鼠坐骨神经-运动终板-内侧腓肠肌形态变化情况。结果脊髓损伤组电镜下坐骨神经术后12周有髓神经纤维髓鞘崩解,其板层结构清晰,有髓神经纤维髓鞘于术后24周模糊、碎裂髓鞘变多,12周后无髓神经纤维及薄髓增多;术后12周腓肠肌光镜下局部肌细胞多数模糊,但边界清楚,结缔组织增生明显,肌细胞核相对聚集;肌细胞于术后24周融合,融合细胞间有空隙,细胞核密集,结缔组织增生明显;术后12周电镜下运动终板突触前后及皱褶膜不可分辨,肌纤维明暗带清晰,突触结构紊乱,z线不连续,高倍镜下突触前后膜不可辨,突触皱褶未见,可见类圆形细小颗粒及突触小泡,肌板结构清晰。结论大鼠脊髓损伤后在损伤平面以下周围神经、运动终板、骨骼肌在形态上会发生规律性变化,12周后显著变化,24周后则毁损性改变。     

BDNF，NT—3对体外培养的胚鼠脊髓AChE和NADPH—d阳性神经元生长发育的影响

利用AChE和NADPH d酶组织化学染色法研究了脑源性神经营养因子 (brain derivedneurotrophicfac tor ,BDNF)和神经营养因子 3(neurotrophin 3,NT 3)对离体培养的胚胎大鼠脊髓胆碱能神经元和一氧化氮能神经元生长发育的影响。结果显示 :BDNF处理组和NT 3处理组AChE阳性神经元数和NADPH d阳性神经元数均显著高于对照组 (P <0 .0 5 )。BDNF组AChE阳性神经元和NADPH d阳性神经元胞体平均直径、每细胞突起数和最长突起长度均显著高于对照组 (P <0 .0 5 )。NT 3组NADPH d阳性神经元的生长发育与对照组无明显差异 ,仅AChE阳性神经元的每细胞突起数和最长突起长度显著高于对照组 (P <0 .0 5 ) ,对胞体发育无影响。结果提示 :BDNF ,NT 3促进脊髓神经元的存活和生长发育 ,二者的作用具有选择性和特异性。     

The dependence of spinal cord development on corticospinal input and its significance in understanding and treating spastic cerebral palsy

The final phase of spinal cord development follows the arrival of descending pathways which brings about a reorganisation that allows mature motor behaviours to emerge under the control of higher brain centres. Observations made during typical human development have shown that low threshold stretch reflexes, including excitatory reflexes between agonist and antagonist muscle pairs are a feature of the newborn. However, perinatal lesions of the corticospinal tract can lead to abnormal development of spinal reflexes that includes retention and reinforcement of developmental features that do not emerge in adult stroke victims, even though they also suffer from spasticity. This review describes investigations in animal models into how corticospinal input may drive segmental maturation. It compares their findings with observations made in humans and discusses how therapeutic interventions in cerebral palsy might aim to correct imbalances between descending and segmental inputs, bearing in mind that descending activity may play the crucial role in development.