The development of catecholaminergic innervation in chick spinal cord

This combined biochemical and histofluorescent study of the embryonic chick was designed to investigate the temporal and spatial development of noradrenergic pathways in the spinal cord. The sequence of catecholaminergic innervation was analyzed by measuring the specific uptake of [3H]norepinephrine, the levels of endogenous norepinephrine, and the distribution of catecholamine histofluorescence. Uptake studies showed early axons present in all levels of spinal cord by 10 days incubation with subsequent increases of uptake activity appearing in a rostrocaudal fashion. Endogenous norepinephrine values were low until day 14, at which time transmitter levels began to increase, approaching hatched values on incubation day 17. Morphologic studies demonstrated catecholaminergic terminals first in the intermediate gray matter and later concentrated in the ventral and dorsal horns. These observations were interpreted to indicate that: (1) noradrenergic axons are an early descending supraspinal pathway; (2) arborization of this system occurs in a rostral-caudal sequence; and (3) monoaminergic uptake mechanisms develop prior to and independent of neurotransmitter synthesis and storage. The development of this noradrenergic system parallels alteration in spinal cord physiology and refinements in the motility of the embryo.     

Establishment and evaluation of a rat model of complete transected spinal cord injury*☆

BACKGROUND： The establishment of a rat model of complete transected spinal cord injury lacks technological specifications. The current models lack concordance and reliability, and the death rate of the experimental animals is high. Therefore, there is a great need for a reliable model to apply clinical applications of therapy. OBJECTIVE： To construct a rat model of complete transected spinal cord injury characterized by stability, reproducibility, and a high animal survival rate. DESIGN： Completely randomized controlled study. SETTING： Department of Neurosurgery, Xiangya Hospital of Central South University. MATERIALS： Fifty-five healthy specific pathogen free grade adult female Sprague Dawley rats were provided by the Experimental Animal Department, Xiangya Medical College, Central South University. Olympus BX51 imaging collecting analytic system was provided by Olympus Company, Japan; and SEN-7203 Nihon-Kohden electrical stimulator by Nihon Kohden, Japan. METHODS： This study was performed at the Laboratory of Neurosurgery, Xiangya Hospital of Central South University from April to June 2006. Experimental grouping： 55 rats were randomly divided into model group （n = 40） and sham surgery group （n = 15）. In the model group, a self-made sliver hook was passed through the ventral side to support the spinal cord at the T12 segment and to shear it off. A complete transected spinal cord, 2 mm in length, was resected. In the sham surgery group, the spinal cord was identically exposed. The dura mater of the spinal cord was cut open, but the spinal cord was not damaged. MAIN OUTCOME MEASURES： Histopathological changes after spinal cord injury at L2 segment were observed subsequent to hematoxylin and eosin staining under optical microscopy. Olympus BX51 imaging collecting analytic system was used to count spinal cord ventral horn neurons. Motor function of rat hindlimb was evaluated with the Basso, Beattie and Bresnahan （BBB） scale. Paraplegia was evaluated as 0 point, and complete normality as     

1H-MRS in spinal cord injury: acute and chronic metabolite alterations in rat brain and lumbar spinal cord

A variety of tests of sensorimotor function are used to characterize outcome after experimental spinal cord injury (SCI). These tests typically do not provide information about chemical and metabolic processes in the injured CNS. Here, we used (1) H-magnetic resonance spectroscopy (MRS) to monitor long-term and short-term chemical changes in the CNS in vivo following SCI. The investigated areas were cortex, thalamus/striatum and the spinal cord distal to injury. In cortex, glutamate (Glu) decreased 1 day after SCI and slowly returned towards normal levels. The combined glutamine (Gln) and Glu signal was similarly decreased in cortex, but increased in the distal spinal cord, suggesting opposite changes of the Glu/Gln metabolites in cortex and distal spinal cord. In lumbar spinal cord, a marked increase of myo-inositol was found 3 days, 14 days and 4 months after SCI. Changes in metabolite concentrations in the spinal cord were also found for choline and N-acetylaspartate. No significant changes in metabolite concentrations were found in thalamus/striatum. Multivariate data analysis allowed separation between rats with SCI and controls for spectra acquired in cortex and spinal cord, but not in thalamus/striatum. Our findings suggest MRS could become a helpful tool to monitor spatial and temporal alterations of metabolic conditions in vivo in the brain and spinal cord after SCI. We provide evidence for dynamic temporal changes at both ends of the neuraxis, cortex cerebri and distal spinal cord, while deep brain areas appear less affected.     

Presence, distribution and pharmacology of conjugated catecholamines in the rat spinal cord

A method is described for the assay of conjugated catecholamines in the brain and spinal cord. Employing this method, the distribution of conjugated norepinephrine (NE) and dopamine (DA) was evaluated in the 5 main regions of the rat spinal cord and compared with those in the hypothalamus. In the spinal cord 26–35% of total catecholamines are conjugated. The percent of NE conjugated in the hypothalamus NE is about 17%, while that of DA is around 12%. Acute treatments with amphetamine, desmethylimipramine and haloperidol failed to change the concentrations of both total and conjugated NE and DA in the cervical spinal cord. Only amphetamine increased hypothalamic conjugated NE. It is concluded that evaluation of central conjugated catecholamines may provide an additional dimension to our ability to critically assess the mechanisms of action of drugs on central catecholamines.     

不同类型脊髓损伤患者和正常成人外周血白细胞糖皮质激素受体的比较及意义

目的 比较正常成人和急性脊髓损伤患者、慢性脊髓压迫症患者外周血白细胞塘皮质激素受体的结合位点数并探讨其意义。方法 采用放射配体结合法测定15例正常成人、20例急性脊髓损伤患者和21例慢性脊髓压迫症患者外周血白细胞上糖皮质激素受体结合位点数。结果 正常成人外周血白细胞精皮质激素受体结合位点数为4462±891.6个/细胞,慢性脊髓压迫症患者为4225±1271个/细胞,急性脊髓损伤患者为2517±857.8个/细胞,经统计学比较正常成人组和慢性脊髓压迫症患者组没有显著性差异,急性脊髓损伤组与其他两者相差均有显著性意义。急性脊髓损伤组中,全瘫患者为2279±921个/细胞,不全瘫患者为2806±718个/细胞,两者无统计学差异。结论 外周血白细胞上的糖皮质激素受体有高亲和力和低亲和力两种结合位点,急性脊髓损伤后外周血白细胞的精皮质激素受体结合位点数的减少主要是高亲和力位点的减少,低亲和力位点维持不变。大剂量的糖皮质激素和白细胞的低亲和力位点结合,抑制白细胞的趋向移动,减少白细胞进入损伤脊髓区,减轻损伤后的急性炎症反直,起到神经保护作用。慢性脊髓压迫症患者予以糖皮质激素治疗无疗效。     

Morphological and histochemical characterization of three types of dopamine-containing neurons in primary cultures of mouse and rat spinal cord

Although the anatomical localization and distribution of the neurotransmitter, dopamine, has been extensively studied in the vertebrate central nervous system, the cell bodies of neurons which synthesize and store this transmitter were not thought to be present in the spinal cord. Using the formaldehyde-glutaraldehyde (Faglu) method for the fluorescent visualization of catecholamines, and immunohistochemistry with antisera to the catecholamine synthetic enzymes, we have found in primary cultures of mouse and rat spinal cord three morphologically distinct types of intrinsic spinal cord neurons that contain a catecholamine and the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).     

转染绿色荧光蛋白的神经干细胞移植于半横断脊髓损伤处的体内外分化研究

目的 观察转染绿色荧光蛋白(GFP)的大鼠脊髓神经干细胞移植于半横断脊髓损伤处的体内外分化情况.方法 将表达GFP的慢病毒载体转染胎鼠脊髓神经干细胞,体外用10%胎牛血清诱导分化.转染后的神经干细胞与PLGA支架移植于大鼠半横断脊髓损伤处,术后1个月和3个月取材,行GFAP、NF和CNP免疫荧光染色.结果 转染GFP的神经干细胞球表达强烈的绿色荧光,体外分化可见GFAP/GFP、NF/GFP和CNP/GFP双阳性细胞,GFAP/GFP双阳性细胞明显多于其他两种.移植后3个月,GFP阳性细胞在脊髓内明显减少,可见少数GFAP/GFP和CNP/GFP舣阳性细胞,未见NF/GFP双阳性细胞.结论 转染GFP的神经干细胞可在体外增殖和分化,但大部分分化成胶质细胞.移植于急性期脊髓损伤处的神经干细胞不被诱导分化成神经元样细胞,可被诱导分化成神经胶质细胞.     

Neuroprotective effect of estrogen after chronic spinal cord injury in ovariectomized rats

BACKGROUND: At present, there is still lack of effective drugs for chronic spinal cord injury, whereas it is found recently that estrogen has a neuroprotective effect on brain and spinal cord injuries. OBJECTIVE: To observe the effect of estrogen on the apoptosis of nerve cells after gradual chronic spinal cord injury in ovariectomized rats. DESIGN: A randomized controlled animal trial. SETTING: Institute of Orthopaedics, the Second Hospital of Lanzhou University. MATERIALS: Sixty-five female Wistar rats of common degree, weighing 220–250 g, were provided by the experimental animal center of Lanzhou University. The rats were randomly divided into sham-operated group (n =5), estrogen-treated group (n =30) and saline control group (n =30), and the latter two groups were observed at 1, 3, 7, 14, 28 and 60 days respectively, and 5 rats for each time point. METHODS: All the rats were treated with bilateral oophorectomy 2 weeks before the experiment. T10 vertebral lamina was revolved into using plastic screw. The spinal canal impingement was not induced initially. After that, the original incision was opened to expose the screw every 7–10 days. MAIN OUTCOME MEASURES: The apoptosis and Caspase-3 positive cells in the damaged spinal cord were detected using terminal deoxynucleotidal transferase-mediated dUTP-biotin nick end labeling (TUNEL) method and Caspase-3 immunohistochemical staining at 1, 3, 7, 14, 28 and 60 days after chronic spinal cord injury respectively. RESULTS: Totally 65 rats were used, and the deleted ones during the experiment were supplemented by others. Changes of Caspase-3 expression after spinal cord injury: In the sham-operated group, only a small amount of Caspase-3 proteins were observed in the rat spinal cord, mainly located in motor neurons of spinal cord anterior horn. In the estrogen-treated group and saline control group, positive cells expressed occasionally at 1 day postoperatively, began to increase obviously at 7 days after injury, strongly expressed at 14 and 28 days, but decreased at 60 days, mainly located in the neurons of spinal cord gray matter anterior horn, and they expressed fewer in the motor neurons and white matter of ventral horn, and there were obvious differences between the estrogen-treated group and saline control group at 7, 14, 28 and 60 days (P < 0.05). CONCLUSION: Estrogen can reduce the apoptosis of nerve cells and promote the recovery of neurological function following gradual chronic spinal cord injury.     

Regional differences in catecholamine formation and metabolism in the rat spinal cord

Catecholamine metabolism was assessed from the content of norepinephrine (NE), dopamine (DA) and their metabolites in various regions of the rat spinal cord during steady-state conditions and following treatment with alpha-methyl-p-tyrosine. The content of NE was rather uniform along the cord while DA was higher in the rostral portion of the cord than in the caudal portion.For both NE and DA there was a rostrocaudal decrease of their turnover rates along the cord. In the cervical cord, DA was formed at a faster rate than NE. There was no correlation between the content of catecholamine metabolites and amine turnover rates. The non-uniformity of catecholamine turnover in the cord probably arises from the fact that different regions of brain project to different regions of cord, each having a specific physiological function. Furthermore, our study provides added support for the presence of an independent DA-containing neuronal system in the spinal cord.     

Effect of methylprednisolone on motor function and spinal cord blood flow after spinal cord compression in rats

The effect of methylprednisolone (MP) on neurologic recovery and spinal cord blood flow (SCBF) was investigated up to 4 days after a spinal cord compression injury in rats. The injury was produced at midthoracic level by applying a load of 35 g on a 2.2 x 5.0 mm compression plate for 5 min, which resulted in transient paraparesis. MP was given as a bolus dose of 30 mg/kg i.v. 60 min after injury (n = 20) and controls were given saline (n = 10). The motor performance was assessed daily as the capacity angle on the inclined plane and SCBF was measured by 14C-iodoantipyrine autoradiography on Days 1 or 4. On Day 1 the capacity angle was reduced from about 63 degrees preoperatively to 33 +/- 2 degrees (mean +/- SEM) in the control group and to 50 +/- 1 degrees in the group treated with MP (p less than 0.05). Thereafter there was a slight improvement in both groups, but the difference persisted throughout the observation period. On Day 4 both gray and white matter SCBF was better preserved in MP-treated animals than in the control group (59 +/- 4 versus 49 +/- 3 ml/min/100 g tissue for gray matter and 13.6 +/- 0.6 versus 10.7 +/- 0.8 ml/min/100 g tissue for white matter). Posttraumatic treatment with MP, thus, improved both the neurologic recovery during the first 4 days and SCBF as measured on Day 4. It is speculated that the effect of MP is at least partly exerted on the vascular bed.     

Quantitative analysis of vascularization and cytochrome oxidase following fetal transplantation in the contused rat spinal cord

In the normal adult central nervous system, a coupling between energy consumption and vascular density is well established. Likewise, the survival of fetal neural tissue grafts is highly dependent on the establishment of functional vascular integration with the host. However, to what degree graft vascularization and tissue metabolism influence the normal host response to traumatic injury has not been extensively studied. In the present report, embryonic day 14 fetal spinal cord suspension grafts were made into the lesion epicenter of subchronic (10 days) contusion-injured rats. Three months later, intraspinal transplants were analyzed using correlative cytochrome oxidase histochemistry and vascular morphometric analysis. The same approaches were applied to the host spinal cord and injured, non-transplanted animals in order to determine the ability of a graft to alter the level of post-injury vascularization and/or metabolism. In general, graft vascular density was increased over that measured in normal or injured gray matter. Vascular density in gray matter near the host/graft interface was markedly increased when compared to either gray matter of the same spinal level in injured non-grafted animals or normal control spinal gray matter. Vascular changes were not noted in gray matter 3 mm distal to the lesion epicenter (rostral or caudal) in all groups analyzed. Cytochrome oxidase was up-regulated at this time in the graft and gray matter at the host/graft interfaces when compared to either gray matter of the same spinal level in injured, non-grafted animals or that of uninjured controls. These data indicate that an intraspinal transplant placed into the contused adult rat spinal cord reaches a metabolic capacity that is likely to be associated with high levels of oxidative metabolism in the well-vascularized graft neuropil. In addition, transplantation chronically alters vascularization and metabolic patterns of adjacent spinal gray matter following contusion injury. © 1996 Wiley-Liss, Inc.     

Spread of saphenous somatotopic projection map in spinal cord and hypersensitivity of the foot after chronic sciatic denervation in adult rat

The left sciatic nerve was cut and ligated in adult rats (chronic denervation). Twenty-one days later the right sciatic nerve was cut and ligated (acute denervation). The somatotopic maps of the surviving intact saphenous nerves (left and right) were compared on day 21 by recording from single interneurons in the dorsal horn of the spinal cord. On the acute side saphenous mediated natural responses were observed only as far caudally as L3, while no natural responses were found in L4 and 5 (this silent zone in L4 and 5 had previously been sciatic territory). In contrast, after chronic sciatic denervation, L4 and L5 were not silent to natural stimulation as the saphenous natural responses had spread into the sciatic territory. Saphenous inputs always won the sciatic territory in L4 and L5 over competing thigh afferents after chronic sciatic denervation. Electrical stimulation of the saphenous nerve on the acute side produced unit responses all the way down to S1 (with no silent areas in L4 and 5). These electrically evoked unit responses in L4 to S1 of the acute side were called ‘long range’ pathways. There were no differences in ‘long range’ electrical responses in the acutely and chronically denervated cord. The caudal boundary for electrically evoked saphenous responses was S1 on both sides of the cord, and post stimulus histograms of unit responses were not statistically different on the two sides. Thus after chronic sciatic denervation natural responses mediated by saphenous spread caudally into sciatic territory, but electrically evoked responses did not change.Behaviorally, there was the expected spread of saphenous mediated responses from the medial toe and foot to more lateral regions after chronic sciatic denervation. Unexpectedly, there was an increase in sensitivity (hyperalgesia) of the medial toe and foot. We postulate that this increased sensitivity might be mediated by the spread of saphenous projections into L4 and L5 of the cord after chronic sciatic denervation. Perhaps post traumatic neuralgia in humans could be due to increased number of spinal cord cells responding to stimulation of the receptive field of the surviving nerve.     

Temporal plasticity of dorsal horn somatosensory neurons after acute and chronic spinal cord hemisection in rat

Unilateral T13 hemisection of the rat spinal cord produces a model of chronic spinal cord injury (SCI) that is characterized by bilateral hyperexcitability of lumbar dorsal horn neurons, and behavioral signs of central pain. While we have demonstrated that responsiveness of multireceptive (MR) dorsal horn neurons is dramatically increased at 28 days after injury, the effects of acute hemisection are unknown and predicted to be different than observed chronically. In the present study, the consequences of T13 hemisection are examined acutely at 45 min in MR neurons both ipsilateral and contralateral to the site of injury, and compared to the same class of cells at 28 days after injury (n=20 cells total per group: 2–3 cells/side of the cord from n=5 animals). Acutely, ipsilateral to the hemisection, both spontaneous and evoked activity of MR neurons were significantly increased, whereas contralaterally, only evoked activity was significantly increased. In animals 28 days after hemisection, spontaneous activity of MR neurons was comparable to intact levels ipsilaterally, and cells exhibited hyperexcitability to evoked stimuli bilaterally. Expansion of cutaneous receptive fields was observed only in hindpaws ipsilateral to the lesion, acutely. These results demonstrate dynamic plasticity in properties of dorsal horn somatosensory neurons after SCI.     

Ultrastructure of fetal spinal cord and cortex implants into adult rat spinal cord

The ultrastructure of cortex and spinal cord from 11-, 12-, and 15-day-old fetuses implanted into the spinal cord of adult rats was studied over 3 months. Under deep Chloropent anesthesia, a 0.5 × 1.0-mm square of fetal cortex or a 1.0-mm segment of fetal spinal cord was implanted subpially between the left dorsal column and the dorsal horn of 70 adult rats. Implants grew toward gray matter, usually interfacing with the host at the isthmus between the horns of the spinal cord. However, implants were observed that occupied the entire left dorsal and ventral horns of the left half of the host spinal cord. Implants had concentric zones: A central zone with basal lamina lined joined channels and subjacent neuroglia; a zone of differentiating implant nervous system; a zone with basal lamina lined implant with overlying pial cells on the dorsal and lateral surfaces of the implant; a zone that interfaced with the host with overlapping neuropil on the lateral and ventral surfaces of the implant. Neuron types were typical for cortical or spinal implants. Implants survived for 3 months and reached stages of neuronal and neuroglial maturation similar to controls. Both fetal spinal cord and brain were successful as implants, had delayed differentiation, and formed complex neuropils. The zone of overlapping interface of the donor and host is an anatomical indication of physiological and functional integration.     

Immunohistochemical changes of the blood-brain barrier in rat spinal cord after heavy-ion irradiation

Changes in the blood-brain barrier (BBB) of the rat spinal cord after irradiation with heavy ion were investigated ultrastructurally and immunohistochemically by using SMI 71, a monoclonal antibody against rat endothelial barrier antigen (EBA); anti-ZO-1, a polyclonal antibody against endothelial tight junctions, anti-rat serum to extravasated serum, and anti-vascular endothelial growth factor (VEGF). The lower thoracic and lumbar cord of male Wistar rats was singly irradiated with a carbon beam at a dose of 30 Gy. Rats were sacrificed before or after the onset of hind limb paralysis. Histologically, white-matter vacuolization was observed from 13 weeks after irradiation, and white-matter necrosis was first noted at 17 weeks. SMI 71 staining was decreased or lost 13 weeks after irradiation, just prior to the formation of white-matter necrosis, and was almost completely lost in the center and periphery of the white-matter necrosis. Although ZO-1 expression and tight junctions in the ultrastructure were preserved at that time, serum leakage occurred almostly completely in parallel with the changes in EBA. Therefore, carbon-ion irradiation at a dose of 30 Gy induces BBB breakdown 13 weeks after irradiation. The SMI 71-negative blood vessels were sparsely distributed throughout the entire white and gray matter, and there was no evidence of preferential localization. Immunostaining of smooth muscle actin showed that most of the SMI 71-negative blood vessels were veins or capillaries. These findings suggest that the hyper-permeability of the veins and/or capillaries that occurs after a certain latent period is one of the important factors in the pathogenesis of delayed radiation injury by carbon ions, the same as by X-rays. Radiation-induced functional disturbances of the endothelium and involvement of cytokines such as VEGF are suspected of being the cause of such vascular hyper-permeability.     

Methylprednisolone inhibits Nogo-A protein expression after acute spinal cord injury

Oligodendrocyte-produced Nogo-A has been shown to inhibit axonal regeneration. Methylprednisolone plays an effective role in treating spinal cord injury, but the effect of methylprednisolone on Nogo-A in the injured spinal cord remains unknown. The present study established a rat model of acute spinal cord injury by the weight-drop method. Results showed that after injury, the motor behavior ability of rats was reduced and necrotic injury appeared in spinal cord tissues, which was accompanied by increased Nogo-A expression in these tissues. After intravenous injection of high-dose methylprednisolone, although the pathology of spinal cord tissue remained unchanged, Nogo-A expression was reduced, but the level was still higher than normal. These findings implicate that methylprednisolone could inhibit Nogo-A expression, which could be a mechanism by which early high dose methylprednisolone infusion helps preserve spinal cord function after spinal cord injury.