Structural changes of anterior horn neurons and their synaptic input caudal to a low thoracic spinal cord hemisection in the adult rat: a light and electron microscopic study

Structural changes in lumbosacral ventral horn neurons and their synaptic input were studied at 3, 10, 21, 42, and 90 days following low thoracic cord hemisection in adult rats by light microscopic examination of synaptophysin immunoreactivity (SYN-IR) and by electron microscopy. There was an ipsilateral transient decrease in SYN-IR at the somal and proximal dendritic surfaces of anterior horn neurons which extended caudally from the site of injury over a postoperative (p.o.) period of 42 days. Concomitantly, at 21 days p.o., perineuronal SYN-IR started to recover in upper lumbar segments. By 90 days p.o., a normal staining pattern of SYN was noted in upper and mid lumbar segments, but the perineuronal SYN-IR was still slightly below normal levels in low lumbar and sacral segments. Electron microscopy revealed ultrastructural changes coincident with the alterations in SYN-IR. At 3 days p.o., phagocytosis of degenerating axon terminals by activated microglial cells was observed at the somal and proximal dendritic surfaces of ventral horn neurons. These changes were most prominent up to two segments caudal to the lesion. At 10 days p.o., advanced stages of bouton phagocytosis were still detectable in all lumbosacral motor nuclei. Additionally, abnormal axon terminals, with a few dispersed synaptic vesicles and accumulations of large mitochondria, appeared at the scalloped somal surfaces of anterior horn neurons. At 21 days p.o., several large lumbosacral motoneurons had developed chromatolysis-like ultrastructural alterations and motoneuronal cell bodies had become partially covered by astrocytic lamellae. At 42 days p.o., there was a transient appearance of polyribosomes in some M-type boutons. In addition, at 42 and 90 days p.o., a few degenerating motoneurons were detected in all lumbosacral segments, but most displayed normal neuronal cell bodies contacted by numerous intact synapses as well as by astrocytic processes. In contrast to these striking alterations of synaptic input at somal and proximal dendritic surfaces of motoneurons, relatively few degenerating boutons were detected in the neuropil of motor nuclei at all the p.o. times studied. We suggest that the preferential disturbance of the predominantly inhibitory axosomatic synapses on ventral horn neurons may be involved in the mechanisms which influence the well-established increase in motoneuronal excitability after spinal cord injury.     

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.     

Astrocytes secrete basal lamina after hemisection of rat spinal cord

Basal lamina is reconstructed over the lesioned surface of the spinal cord. The following experiment (90 rats) studies the ultrastructure of the formation of this membrane and the immunohistochemistry of laminin production (a major secreted component of basal lamina). After hemisection of the spinal cord at T6 animals were prepared for electron microscopy or antilaminin-biotin-avidin-peroxidase incubation. Three-5 days posthemisection, antilaminin reaction product was observed in astrocytes and their processes which faced the lesion, endothelia of blood vessels or pia. Ultrastructurally (3 days), basal lamina was polymerizing as small projections on the surface of astrocytic membranes facing the lesion, endothelia or pia. By 5 days the basal lamina was a single membrane, folded multiple sheets or in swirls. At 6-10 days the antilaminin reaction and the basal lamina (except for duplications) did not differ from normal. Reactive astrocytes secrete laminin for at least 3-5 days after hemisection and form basal lamina on the lesioned surface of the spinal cord after spinal cord hemisection.     

大鼠脊髓半横断损伤后神经营养因子bFGF、GDNF的表达变化

目的探讨脊髓半横断损伤后早期不同时间碱性纤维母细胞生长因子(bFGF)、胶质细胞源性神经营养因子(GDNF)的表达变化. 方法在成年SD大鼠脊髓T9～T10间半横断,取损伤位点尾侧段T10节段制作冰冻切片,运用bFGF、GDNF兔抗血清以免疫组化亲合素-生物素-过氧化物酶复合物法(ABC法)染色.观察并计数腹角bFGF、GDNF的阳性神经元数.结果 bFGF、GDNF主要分布于正常大鼠脊髓腹角神经元细胞浆,损伤后腹角bFGF、GDNF阳性神经元数在3 d组(n=6)、7 d组(n=6)、21 d组(n=6)均较假手术组(n=6)明显增加(P<0.01),bFGF阳性神经元数在术后3 d时达高峰,随伤后时间的延长进行性减少(P<0.01).而GDNF阳性神经元数在术后7 d时达高峰,3 d组与21 d组比较没有显著差异.结论脊髓半横断损伤(hSCI)后bFGF、GDNF表达明显增加,提示它们可能在hSCI早期修复中发挥作用.     

Noradrenergic innervation of the rat spinal cord caudal to a complete spinal cord transection: Effects of olfactory ensheathing glia

Transplantation of olfactory bulb-derived olfactory ensheathing glia (OEG) combined with step training improves hindlimb locomotion in adult rats with a complete spinal cord transection. Spinal cord injury studies use the presence of noradrenergic (NA) axons caudal to the injury site as evidence of axonal regeneration and we previously found more NA axons just caudal to the transection in OEG- than media-injected spinal rats. We therefore hypothesized that OEG transplantation promotes descending coeruleospinal regeneration that contributes to the recovery of hindlimb locomotion. Now we report that NA axons are present throughout the caudal stump of both media- and OEG-injected spinal rats and they enter the spinal cord from the periphery via dorsal and ventral roots and along large penetrating blood vessels. These results indicate that the presence of NA fibers in the caudal spinal cord is not a reliable indicator of coeruleospinal regeneration. We then asked if NA axons appose cholinergic neurons associated with motor functions, i.e., central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more NA varicosities adjacent to central canal cluster cells, partition cells, and SMNs in the lumbar enlargement of OEG- than media-injected rats. As non-synaptic release of NA is common in the spinal cord, more associations between NA varicosities and motor-associated cholinergic neurons in the lumbar spinal cord may contribute to the improved treadmill stepping observed in OEG-injected spinal rats. This effect could be mediated through direct association with SMNs and/or indirectly via cholinergic interneurons.     

Resistance to anoxic injury in the dorsal columns of adult rat spinal cord following demyelination

In order to examine the relationship between myelination and sensitivity to anoxia in adult white matter, we studied action potential conduction in the spinal cord dorsal column of adult rats in which focal demyelinating lesions had been produced using ethidium bromide/X-irradiation. Acutely isolated spinal cords from control rats and following demyelination were maintained in vitro at 36°C and compound action potentials were studied following supramaximal stimulation. The compound action potential was totally abolished within 12 min of the onset of anoxia in normal dorsal columns, but was not abolished until 50 min following the onset of anoxia in demyelinated dorsal columns. Compound action potentials showed significantly greater recovery (to 58.1±12.2% of control amplitude) in demyelinated dorsal columns compared to controls (30.8±5.3%) following 120 min of reoxygenation. These results show that focal demyelination is associated with reduced sensitivity to anoxia within white matter of the adult spinal cord.     

Splanchnic input to thoracic spinal neurons and its supraspinal modulation in the rat

The urinary responses of 62 T8-T11 spinal neurons were recorded extracellularly following electrical stimulation of the greater splanchnic nerve (GSN) in chloralose-anesthetized rats. Recorded neurons were found in both the dorsal and ventral horns. Fifty-seven neurons increased their firing rate in response to GSN stimulation; 8 of these exhibited biphasic responses consisting of excitations followed by inhibitions. Excitatory responses to GSN stimulation consisted of either one or two bursts with latencies consistent with activation by either A delta or C fibers. GSN stimulation inhibited 5 neurons. The effects of reversible spinalization on spontaneous activity and on both synchronous and non-synchronous (afterdischarge) GSN-evoked responses were investigated using a cooling probe on the spinal cord between C1 and C2. Of 19 neurons tested in this way, 9 exhibited opposite directional changes in their spontaneous activities and their GSN-evoked responses upon spinalization. Differential effects of cold-block on first and second bursts, or on A delta- and C-fiber mediated responses, were not usually observed. However, differential effects of cold-block on synchronous and non-synchronous portions of the overall GSN-evoked response were often observed in that their magnitudes often changed independently of one another. Supraspinal pathways contributed to GSN-evoked responses of several neurons because their responses were diminished during cooling while spontaneous activity was increased or unchanged. These decreases in the magnitude of the GSN-evoked response were not always accounted for by decreases in the synchronous portions of the responses. However, most neurons did exhibit decreases in the number of non-synchronous responses, or afterdischarges, during spinal cooling, exhibiting in some cases biphasic responses. This study provides evidence for strong supraspinal regulation of splanchnic afferent input to the spinal cord of the rat. Further, this regulation exhibits some specificity toward different portions of splanchnic-evoked responses in spinal neurons.     

Membrane properties of deep dorsal horn neurons from neonatal rat spinal cord in vitro

Whole-cell patch-clamp recordings were undertaken to characterize and compare the membrane properties of deep dorsal horn neurons in transverse slices of rat lumbar spinal cord in two age groups, postnatal days (P) 3–6 and 9–16. In both age groups, significant correlations were observed between membrane time constant and cell resistance and between action potential height and its duration at half-maximal amplitude. Cell resistance and action potential half-width values were lower in the P9–16 age group. Neurons were divided into four categories based on their firing properties in response to intracellular current injection: single spike, phasic firing, repetitive firing, and delayed firing. The distribution of neurons within these categories was similar in both age groups which suggests that the firing properties of deep dorsal horn neurons are functionally differentiated at an early postnatal age.     

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.     

Paired pulse facilitation of GABAergic IPSCs in ventral horn neurons in neonatal rat spinal cord

Whole-cell patch-clamp recording of GABAergic inhibitory postsynaptic currents (IPSCs) were made in ventral horn neurons of neonatal rat lumbar spinal cord in slice. In contrast to the hippocampus where paired pulse depression is reported to be observed for GABAergic IPSCs, double pulse stimulation of GABAergic inputs resulted in enhancement in the amplitude of the second IPSC in the spinal ventral horn. The facilitation ratio was decreased during enhanced synaptic transmission by increasing Ca²⁺ concentration in the external recording solution. Baclofen and adenosine, which are reported to depress synaptic transmission by presynaptic mechanisms, depressed IPSCs and increased the facilitation ratio. A postsynaptic manipulation such as application of bicuculline or changing the driving force did not affect the facilitation ratio. These results suggest that paired pulse facilitation of GABAergic IPSCs observed in neonatal rat spinal ventral horn appears to be based upon a mechanism similar to that underlying frequency-dependent facilitation of excitatory synaptic transmission, and is sensitive to presynaptic changes in synaptic strength.     

Differential effects of chronic spinal hemisection on somatic and visceral inputs to caudal brainstem

The medullary reticular formation (MRF) receives convergent inputs from multiple somatic and pelvic/visceral territories. The effects of chronic 30-day lateral hemisections at T8 on the responses of single MRF neurons to noxious mechanical stimulation of both hindpaws was examined in urethane-anesthetized male rats. Neuronal responses on both sides of the MRF to pinching of the hindpaw on the side opposite the lesion (intact-side) were found either to be completely absent or if present, weak (i.e. hindpaw was hyposensitive). The presence or absence of intact-side responses appeared to be dependent on the lesion extent. In contrast, bilateral MRF responses to pinching the lesion-side hindpaw were present; however, responses were greater in magnitude (lower thresholds) relative to surgical sham controls suggesting hypersensitivity. Responses to lesion-side hindpaw stimulation on both sides of the MRF indicated that whereas the ascending projections are primarily crossed below the level of lesion, they are both crossed and uncrossed above. These findings are in contrast with our previous data on ascending projections from the bilaterally organized male urogenital tract. The results presented for the hindpaws correlate with clinical observations of patients with similar incomplete spinal cord injuries (Brown-Séquard syndrome).     

生物素葡聚糖胺皮质脊髓束顺行示踪技术在大鼠脊髓损伤模型中的应用

目的探讨生物素葡聚糖胺(BDA)神经示踪技术及脊髓半横断损伤模型在大鼠脊髓损伤修复的实验研究中应用。方法采用成年Sprague-Dawley大鼠,分为脊髓致伤组(n=10)和致伤对照组(n=10)。致伤组动物在相当于T7椎板水平横行剪断脊髓的后2/3;对照组动物术中仅切除椎板,不切断脊髓。术后第15d,右侧开颅,用10?A示踪剂注入右侧的感觉运动区皮质内。2周后取出大脑和脊髓组织,采用自由漂乳法行BDA染色显影。术后实验动物功能测评采用BBB运动功能评分,所得数据采用Student'st-test进行统计学原理。结果(1)脊髓损伤组动物双后肢瘫痪,BBB运动功能评分明显低于损伤对照组,统计学比较差异十分显著(P<0.01);(2)BDA顺行示踪显示大脑皮层BDA注射区内见大脑皮层的锥体细胞及其发出的轴突呈阳性染色,BDA阳性染色的皮质脊髓束神经纤维在同侧中脑、桥脑及延髓的腹侧面行走,在锥体交叉后皮质脊髓束主要在对侧脊髓白质的后索中行走。在致伤组动物中,位于脊髓白质后索中的皮质脊髓束纤维在脊髓损伤处终止;对照组皮质脊髓束BDA染色可一直延伸至L1水平。结论大鼠半脊髓切断结合应用BDA顺行示踪技术可以对脊髓损伤后的神经修复状况进行可靠的形态学评判,是研究脊髓损伤后中枢神经纤维再生修复较为理想的动物模型     

Adenosine exerts multiple effects in dorsal horn neurones of the adult rat spinal cord

In the present study, we have examined the effects of adenosine and its analogues on the electrophysiological properties of dorsal horn neurones in the rat adult spinal cord. Adenosine and the A₁ receptor agonist R-phenylisopropyl adenosine (RPIA) reversibly hyperpolarised these neurones via the generation of an outward current at −60 mV that was inhibited by pre-application of barium or Rp-adenosine 3′, 5′-cyclic monophosphothioate triethylamine. In contrast, the A_2a receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680) had no effect on the resting membrane properties of these neurones. Stimulation of the dorsal root evoked non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) at −60 mV that were completely abolished by 2,3-dihydroxy-6-nitro-7-sulophamoyl-benzo(F)quinoxalone (NBQX). Bath application of adenosine or RPIA reversibly inhibited these EPSCs in a concentration-dependent manner via a presynaptic action. In contrast, CGS21680 increased the amplitude of the EPSC in 20% of neurones tested and decreased the EPSC amplitude in 30% of neurones tested. It is concluded that adenosine exerts multiple effects upon the electrophysiological properties of dorsal horn neurones in the adult spinal cord via interaction with multiple receptors. These findings have important implications in the understanding of adenosine action in preclinical models of pain.     

Islet-1 expression in thoracic spinal motor neurons in prenatal mouse

The LIM homeodomain protein Islet-1, an embryonic marker for motoneurons in the spinal cord, has been reported to be heterogeneously expressed among motoneuron groups in mouse. In the present study, we examined Islet-1 expression in the thoracic and rostral lumbar spinal cord in prenatal mice. In the thoracic spinal cord at embryonic day 12.5 (E12.5) and E13.5, strong Islet-1 immunoreactivity was observed in the lateral group of the ventral horn, whereas weaker immunoreactivity was observed in the ventral group. Strong Islet-1 immunoreactivity was also observed in the intermediolateral area and more medial part of the intermediate zone. In the rostral lumbar spinal cord at E12.5 and E13.5, strong Islet-1 immunoreactivity was observed in the lateral group of the ventral horn, and in the intermediolateral nucleus, whereas weaker immunoreactivity was observed in the ventral, and dorsolateral groups. At E14.5, the number of Islet-1 immunoreactive neurons was reduced in the spinal cord, but the distribution pattern was similar to that at E12.5 and E13.5. At E15.5, Islet-1 immunoreactivity was almost completely confined to the intermediolateral area. Some weakly immunoreactive neurons were observed in the ventral horn. The findings of the present study indicated that Islet-1 expression at embryonic stages differs among the motoneuron groups in the thoracic and rostral lumbar spinal cord.     

实验性恒河猴脊髓半横切损伤模型的建立

目的通过脊髓半侧横切的方法建立灵长类恒河猴脊髓半横切损伤动物模型,用于神经再生的研究。方法 5只成年恒河猴称重麻醉后,颈部后正中切口显露C3-5脊髓,于C4用虹膜刀切开脊髓左半侧0．5cm,通过体感诱发电位(somatosensory evoked potential SSEP)监测,确保波形改变并观察恒河猴术后24h、7d肢体运动功能和SSEP变化。结果术后24h、7d同侧肢体瘫痪,肢体运动评分由术前的5分降到1分,SSEP显示术前潜伏期(15．88±2．53)毫秒,术后24h(22．26±4．12)毫秒;7d(23．12±3．92)毫秒延长明显(P<0．01),波幅术前(11．92±5．48)μV,术后24h(5．34±3．04) μV,7d(5．78±2．98)μV,降低明显(P<0．01)。结论恒河猴麻醉后用虹膜刀脊髓半横切能损伤皮质脊髓束,引起肢体瘫痪,结果稳定可靠,操作较为简便,可建立脊髓半侧横切损伤动物模型,重复性较好,为脊髓损伤的神经修复实验研究提供了模型基础。     

Motor neuron destruction in guinea pigs immunized with bovine spinal cord ventral horn homogenate: experimental autoimmune gray matter disease

Guinea pigs immunized with bovine spinal cord ventral horn homogenate develop muscle weakness with electromyographic and morphologic evidence of denervation. Pathological examination demonstrates a loss of motoneurons and scattered inflammatory foci primarily localized to the spinal cord. Immunohistochemical techniques document the presence of immunoglobulin G at the motor end plate and around the external membrane and within the cytoplasm of motoneurons. This syndrome of experimental autoimmune gray matter disease (EAGMD) differs from experimental autoimmune motor neuron disease induced by inoculation with purified motoneurons and also differs from experimental autoimmune encephalomyelitis. The existence of two different forms of immune-mediated motoneuron destruction suggests that a number of cytoplasmic and membrane antigens may give rise to an immunologically based attack on the motor system.     

促甲状腺释放激素类似物YM-14673对大鼠脊髓损伤后水肿的影响

目的研究促甲状腺释放激素（ＴＲＨ）类似物,ＹＭ－１４６７３大鼠脊髓损伤后水肿的影响。方法用改良Ａｌｌｅｎ氏法建立大鼠脊髓损伤模型,分设正常组、对照组和治疗组,治疗组在损伤后１５分钟注射ＹＭ－１４６７３,用称重法测量脊髓的水含量,公式：（湿重－干重）÷湿重×１００％。结果对照组示伤后２４小时脊髓水肿,治疗组显示在２４小时脊髓水肿减轻。结论早期应用ＴＲＨ类似物,ＹＭ-１４６７３可减轻脊髓损伤后的脊随水肿。     

Syngeneic grafting of adult rat DRG-derived schwann cells to the injured spinal cord

A subdural inflatable microballoon was used to induce closed traumatic contusion to adult rat spinal cord. This spinal cord injury model was associated with reproducible and graded neurological deficits and histopathological alterations. At various delays after injury, transplantations of syngeneic adult cultured dorsal root ganglion-derived Schwann cells were performed into the spinal cord lesion. The transplants were well integrated and reduced the microcystic posttraumatic cavitation as well as the gliosis. Schwann cells transplants were invaded by numerous regenerating neurites most of which, based upon their neurotransmitter contents, seem to originate from the dorsal root ganglion.     

胚胎脊髓移植在恢复损伤脊髓传导功能中的作用

目的：探讨胚胎脊髓移植在恢复损伤脊髓传导功能中的作用。方法：将Ｅ１４胚胎脊髓植入成鼠损伤脊髓后３０、４５、６０天时，用单位放电记录技术观察了正常脊髓神经元和移植物神经元的自发放电活动，及其对刺激坐骨神经、红核和同时刺激的反应。结果：正常脊髓神经元的自发单位放电多是一个低频的单发脉冲活动。无论选择那种刺激方式，都可见兴奋、抑制和无反应三种反应。术后３０天时，胚胎神经元的自发单位放电以高频电脉冲活动为主，簇状放电所占比例较大，对刺激有反应的放电单位数也较少；随着动物存活时间的延长，这些单位放电的情况逐渐向着低频、单脉冲以及高反应率的方向发展。至术后６０天时，胚胎神经元单位放电的频率、形式以及对刺激的反应情况都变得和正常神经元的相似。结论：胚胎神经元移植后经历了一个逐渐发育分化过程，在这个过程中它们有可能逐渐和宿主神经元形成了功能性突触连接。     

Assessment of axonal dysfunction in an in vitro model of acute compressive injury to adult rat spinal cord axons

An in vitro model of spinal cord injury was developed to study the pathophysiology of posttraumatic axonal dysfunction. A 25 mm length of thoracic spinal cord was removed from the adult male rat (n = 27). A dorsal column segment was isolated and pinned in a recording chamber and superfused with oxygenated (95%O₂/5% CO₂) Ringer. The cord was stimulated with a bipolar electrode, while two point responses were recorded extracellularly. Injury was accomplished by compression with a modified aneurysm clip which applied a 2 g force for 15 s. With injury the compound action potential (CAP) amplitude decreased to 53.7 ± 5.4% (P < 0.001), while the latency increased to 115.6 ± 3.1% (P < 0.0025) of control values. The absolute refractory period increased with injury from 1.7 ± 0.1 ms to 2.1 ± 0.1 ms (P < 0.001). With train stimulation (200 and 400 Hz), injured axons showed evidence of high frequency conduction failure (P < 0.05). The infusion of 5 mM 4-aminopyridine (4-AP), a blocker of voltage-sensitive ‘fast’ K channels confined to internodal regions, resulted in broadening of the CAP of injured axons to 114.9 ± 3.1% of control (P < 0.05). Ultrastructural analysis of the injured dorsal column segments revealed marked axonal and myelin pathology, including considerable myelin disruption.In conclusion, we have developed and characterized an in vitro model of mammalian spinal cord injury which simulates many of the features of in vivo trauma. Injured axons display characteristic changes in physiological function including a shift in refractory period and high frequency conduction failure. The ultrastructural data and response of injured axons to 4-AP suggest that myelin disruption with exposure of ‘fast’ K⁺ channels contributes to posttraumatic axonal dysfunction.