The effect of altered fractionation schedule on the spinal cord response: radiobiological considerations

Increased fractionation spares late reacting normal tissues more than acute reacting normal tissues. A linear quadratic model is valid from large dose per fraction down to dose per fraction of 2 Gy. Experimental studies on animals and clinical studies on the spinal cord tolerance have shown incidences of myelopathy at doses lower than 50 Gy. The α/β value of the linear quadratic model have been lower for low doses per fraction, indicating a sparing effect of altered fractionation for spinal cord myelitis. Animal data, clinical and radiobiological explanations suggest limitation of the radiobiological models. Further data suggest that one must not assume the spinal cord to have a greater tolerance at doses per fraction below the conventional dose per fraction of 2 Gy.     

Tolerance and cross tolerance to morphine after chronic spinal D-Ala2-D-Leu5-enkephalin infusion

The development of tolerance and cross tolerance to morphine at spinal cord levels on the tall flick inhibition was studied in rats tolerant to D-Ala2-D-Leu5-enkephalin (DADL). The long term intrathecal infusion of DADL was accomplished by means of osmotic minipumps. Chronic intrathecal infusion of DADL for 5 days caused a shift of dose response curves of both DADL and morphine sulfate injected intrathecally to the right indicating that tolerance and cross tolerance to morphine had developed after long term intrathecal infusion of DADL. The shift of the dose response curve of DADL was parallel, whereas that of morphine was non-parallel and flattening. Concomitant intrathecal infusion of naloxone which was more sensitive in blocking mu-opioid receptor than delta-opioid receptor blocked the development of cross tolerance to morphine while the development of tolerance to DADL was left unaffected. The studies present the evidence that two types of opioid receptors, delta- and mu-opioid in the spinal cord of rats are involved in the development of tolerance by chronic DADL exposure.     

Early radiation-induced endothelial cell loss and blood-spinal cord barrier breakdown in the rat spinal cord

Using a rat spinal cord model, this study was designed to characterize radiation-induced vascular endothelial cell loss and its relationship to early blood-brain barrier disruption in the central nervous system. Adult rats were given a single dose of 0, 2, 8, 19.5, 22, 30 or 50 Gy to the cervical spinal cord. At various times up to 2 weeks after irradiation, the spinal cord was processed for histological and immunohistochemical analysis. Radiation-induced apoptosis was assessed by morphology and TdT-mediated dUTP nick end labeling combined with immunohistochemical markers for endothelial and glial cells. Image analysis was performed to determine endothelial cell and microvessel density using immunohistochemistry with endothelial markers, namely endothelial barrier antigen, glucose transporter isoform 1, laminin and zonula occludens 1. Blood-spinal cord barrier permeability was assessed using immunohistochemistry for albumin and (99m)Tc-diethylenetriamine pentaacetic acid as a vascular tracer. Endothelial cell proliferation was assessed using in vivo BrdU labeling. During the first 24 h after irradiation, apoptotic endothelial cells were observed in the rat spinal cord. The decrease in endothelial cell density at 24 h after irradiation was associated with an increase in albumin immunostaining around microvessels. The decrease in the number of endothelial cells persisted for 7 days and recovery of endothelial density was apparent by day 14. A similar pattern of blood-spinal cord barrier disruption and recovery of permeability was observed over the 2 weeks, and an increase in BrdU-labeled endothelial cells was seen at day 3. These results are consistent with an association between endothelial cell death and acute blood-spinal cord barrier disruption in the rat spinal cord after irradiation.     

Time-dependent neurosphere-forming ability of adult rat spinal cord after irradiation

To determine whether there was evidence for long-term time-dependent changes in neurosphere-forming ability of rat spinal cord after irradiation, a 15-mm length of spinal cord (C2-T2) of 10-week-old female rats was irradiated with a single dose of 2, 5, 10 or 19 Gy. Cells were isolated from the central 10-mm segment of the irradiated spinal cord immediately or at 0.5, 1, 2 or 5 months to form neurospheres. The number and sizes of neurospheres were determined at day 10, 12, 14 and 16 in vitro. The multipotential properties of neurosphere cells were assessed by immunocytochemistry using lineage-specific markers for neurons and glia. In nonirradiated controls, the number and size of the neurospheres decreased with increasing age of the animals. Regardless of the time after irradiation, there was a dose-dependent decrease in the number and size of neurospheres obtained from the irradiated cord compared to age-matched controls. Using three-way ANOVA, the number of neurospheres was dependent on radiation dose (P < 0.0001), time after irradiation (P < 0.0001), and day of counting in vitro (P < 0.0001). Compared to cells cultured immediately after irradiation, there was an increase in the relative plating efficiency of neurospheres cultured 1 month after irradiation. However, no further increase was apparent up to 5 months after irradiation. The multipotential properties of neurosphere cells in vitro remained unchanged with increasing time after irradiation. These results may suggest a time-dependent recovery of radiation damage using neurosphere-forming ability as the end point and agree with data that show time-dependent recovery of radiation damage in spinal cord using histological or functional end points.     

三七总皂苷对脊髓损伤后BDNF 的表达及运动功能的影响

目的:探讨三七总皂苷(total panax notoginseng saponins,tPNS)对脊髓半横断损伤后对脑源性神经营养因子(Brain-derivedneurotrophic factor,BDNF)表达以及运动功能恢复的作用的影响。方法:大鼠随机分为正常组和实验组,实验组大鼠脊髓T10右侧半横断模型,损伤后15min,腹腔注射三七总皂苷,剂量为20mg.kg-1,以后每天给药一次,溶媒对照组注射等量生理盐水。术后进行BBB评分和斜板实验检测;动物分别存活1d、3d、7d、14d、28d后,采用免疫荧光化学方法检测脊髓损伤远侧端BDNF表达的变化。结果:BBB评分及斜板实验结果显示,三七总皂苷能明显促进脊髓损伤后运动功能的恢复,尤其是损伤后7d和14d,三七总皂苷组评分明显高于溶媒对照组。免疫组化结果显示:脊髓半横断损伤后,损伤远侧端损伤侧BDNF的表达强于对侧,损伤侧BDNF的表达呈现出1d,3d逐渐增强,7d达高峰的趋势,14dBDNF的表达逐渐下降,至28d仍略高于正常组。三七总皂苷组和溶媒对照组相比,BDNF表达的时间趋势相同,但相同时间点BDNF的表达强于对照组,尤其是3d、7d。结论:三七总皂苷能增强脊髓半横断损伤后BDNF的表达,这可能是其改善脊髓再生的微环境,促进脊髓损伤后运动功能恢复的机制之一。     

Proteomic analysis of spinal protein expression in rats exposed to repeated intrathecal morphine injection

Repeated administration of morphine for treating severe chronic pain may lead to neuroadaptive changes in the spinal cord that are thought to underlie molecular mechanisms of the development of morphine tolerance and physical dependence. Here, we employed a 2-D gel-based proteomic technique to detect the global changes of the spinal cord protein expression in rats that had developed morphine tolerance. Morphine tolerance at the spinal cord level was induced by repeated intrathecal injections of morphine (20 microg/10 microL) twice daily for 5 days and evaluated by measurements of paw withdrawal latencies and maximal possible analgesic effect at day 5. After behavioral tests, the lumbar enlargement segments of spinal cord were harvested and proteins resolved by 2-DE. We found that eight proteins were significantly up-regulated or down-regulated in spinal cord after morphine tolerance development, including proteins involved in targeting and trafficking of the glutamate receptors and opioid receptors, proteins involved in oxidative stress, and cytoskeletal proteins, some of which were confirmed by Western blot analysis. Morphine-induced expressional changes of these proteins in the spinal cord might be involved in the central mechanisms that underlie the development of morphine tolerance. It is very likely that these identified proteins may serve as potential molecular targets for prevention of the development of morphine tolerance and physical dependence.     

A Surgery Protocol for Adult Zebrafish Spinal Cord Injury

Adult zebrafish has a remarkable capability to recover from spinal cord injury,providing an excellent model for studying neuro-regeneration. Here we list equipment and reagents,and give a detailed protocol for complete transection of the adult zebrafish spinal cord. In this protocol,potential problems and their solutions are described so that the zebrafish spinal cord injury model can be more easily and reproducibly performed.In addition,two assessments are introduced to monitor the success of the surgery and functional recovery:one test to assess free swimming capability and the other test to assess extent of neuroregeneration by in vivo anterograde axonal tracing.In the swimming behavior test,successful complete spinal cord transection is monitored by the inability of zebrafish to swim freely for 1 week after spinal cord injury,followed by the gradual reacquisition of full locomotor ability within 6 weeks after injury.As a morphometric correlate,anterograde axonal tracing allows the investigator to monitor the ability of regenerated axons to cross the lesion site and increasingly extend into the gray and white matter with time after injury,confirming functional recovery.This zebrafish model provides a paradigm for recovery from spinal cord injury,enabling the identification of pathways and components of neuroregeneration.     

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.     

bFGF通过抑制Nogo-A信号减少脊髓损伤后胶质瘢痕形成

脊髓损伤后胶质瘢痕的形成是阻碍神经恢复的关键原因之一。碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）具有良好的神经保护及促进脊髓损伤的修复作用,然而其对于胶质瘢痕的影响及其机制仍不清楚。本研究通过采用血管动脉夹（30 g）夹闭雌性SD大鼠脊髓2 min造成急性脊髓损伤模型并予以每天皮下注射bFGF（80 μg/kg）,探讨bFGF促进脊髓损伤的恢复作用是否涉及到胶质瘢痕调控和Nogo-A/NgR信号的相关机制。通过检测损伤后28 d,各组BBB评分和斜板试验,发现bFGF显著促进脊髓损伤后大鼠运动功能的恢复。HE及尼氏染色显示,bFGF处理组相对于生理盐水处理组,其神经元明显增多,空洞面积减少。同时,星形胶质细胞标记物GFAP免疫荧光结果表明,bFGF减少胶质瘢痕形成,抑制星形胶质细胞过度激活。同样,通过Western 印迹检测发现,bFGF处理后,胶质瘢痕相关蛋白（如GFAP, neurocan）以及神经突生长抑制蛋白（Nogo-A）信号通路相关蛋白质表达量下降。上述结果表明,bFGF可能通过抑制Nogo-A信号蛋白的表达,从而抑制胶质瘢痕的形成,促进脊髓损伤的恢复。此机制研究为脊髓损伤的治疗和恢复提供全新的思路和药物靶点。     

DNA Changes in Spinal Cords of Rats with Experimental Allergic Encephalomyelitis

DNA levels were measured in the spinal cords of Lewis rats during the development of and recovery from experimental allergic encephalomyelitis (EAE). Spinal cord DNA was first increased 11 days after immunizing the rats with guinea pig myelin and rose to levels four times that of the Freund's adjuvant controls at day 14, then subsided after day 22. Spinal cord DNA was still 150% of control levels 60 days after immunization. These DNA changes were compared with fluctuations in spinal cord acid proteinase in the same animals. Acid proteinase activity in EAE spinal cord increased later than the rise in DNA and attained a level of 170% of control at days 15-17, then subsided. Spinal cord DNA was higher in rats immunized with whole myelin than in those administered equivalent amounts of purified myelin basic protein. Furthermore DNA was higher in spinal cords of rats immunized with a larger dose of myelin (1.0 mg) than with a lower amount (0.5 mg). Various protease inhibitors including pepstatin, nitrophenyl p-guanidino benzoate, polylysine, and dipropionyl rhein, previously shown to protect Lewis rats against EAE, suppressed the increase of DNA in the spinal cord. Measurement of DNA increases in the spinal cord of EAE animals provides a convenient reproducible measurement of the severity of inflammation in the CNS and provides an objective criterion for assessment of the efficacy of various agents screened as possible therapeutic treatment for multiple sclerosis.     

Arylsulfatase B Improves Locomotor Function after Mouse Spinal Cord Injury

Bacterial chondroitinase ABC (ChaseABC) has been used to remove the inhibitory chondroitin sulfate chains from chondroitin sulfate proteoglycans to improve regeneration after rodent spinal cord injury. We hypothesized that the mammalian enzyme arylsulfatase B (ARSB) would also enhance recovery after mouse spinal cord injury. Application of the mammalian enzyme would be an attractive alternative to ChaseABC because of its more robust chemical stability and reduced immunogenicity. A one-time injection of human ARSB into injured mouse spinal cord eliminated immunoreactivity for chondroitin sulfates within five days, and up to 9 weeks after injury. After a moderate spinal cord injury, we observed improvements of locomotor recovery assessed by the Basso Mouse Scale (BMS) in ARSB treated mice, compared to the buffer-treated control group, at 6 weeks after injection. After a severe spinal cord injury, mice injected with equivalent units of ARSB or ChaseABC improved similarly and both groups achieved significantly more locomotor recovery than the buffer-treated control mice. Serotonin and tyrosine hydroxylase immunoreactive axons were more extensively present in mouse spinal cords treated with ARSB and ChaseABC, and the immunoreactive axons penetrated further beyond the injury site in ARSB or ChaseABC treated mice than in control mice. These results indicate that mammalian ARSB improves functional recovery after CNS injury. The structural/molecular mechanisms underlying the observed functional improvement remain to be elucidated.     

Molecular targets for therapeutic intervention after spinal cord injury

In an effort to develop therapies for promoting neurological recovery after spinal cord injury, much work has been done to identify the cellular and molecular factors that control axonal regeneration within the injured central nervous system. This review summarizes the current understanding of a number of the elements within the spinal cord environment that inhibit axonal growth and outlines the factors that influence the neuron's ability to regenerate its axon after injury. Recent insights in these areas have identified important molecular pathways that are potential targets for therapeutic intervention, raising hope for victims of spinal cord injury.     

A Threshold Dose of Heavy Ion Radiation that Decreases the Oxidative Enzyme Activity of Spinal Motoneurons in Rats

The effect of heavy ion radiation exposure of the spinal cord on the properties of the motoneurons innervating the slow soleus and fast plantaris muscles was investigated. A 15-, 20-, 40-, 50-, or 70-Gy dose of carbon ions (5 Gy/min) was applied to the 2nd to the 6th lumbar segments of the spinal cord in rats. After a 1-month recovery period, the number and cell body size of the irradiated motoneurons innervating the soleus and plantaris muscles did not differ from that of the non-irradiated controls, irrespective of the dose received. However, the oxidative enzyme activity of these motoneurons was decreased by heavy ion radiation at doses of 40, 50, and 70 Gy compared to that of the non-irradiated controls. This decrease in oxidative enzyme activity levels in the motoneurons returned to that of the non-irradiated controls after a 6-month recovery period. We conclude that heavy ion radiation at doses of 40–70 Gy reversibly decreases the oxidative enzyme activity of motoneurons in the spinal cord of rats.     

5-Hydroxytryptophan-induced respiratory recovery after cervical spinal cord hemisection in rats.

The present study investigates the role of serotonin in respiratory recovery after spinal cord injury. Experiments were conducted on C(2) spinal cord hemisected, anesthetized, vagotomized, paralyzed, and artificially ventilated rats in which end-tidal CO(2) was monitored and maintained. Before drug administration, the phrenic nerve ipsilateral to hemisection showed no respiratory-related activity due to the disruption of the descending bulbospinal respiratory pathways by spinal cord hemisection. 5-Hydroxytryptophan (5-HTP), a serotonin precursor, was administrated intravenously. 5-HTP induced time- and dose-dependent increases in respiratory recovery in the phrenic nerve ipsilateral to hemisection. Although the 5-HTP-induced recovery was initially accompanied by an increase in activity in the contralateral phrenic nerve, suggesting an increase in descending respiratory drive, the recovery persisted well after activity in the contralateral nerve returned to predrug levels. 5-HTP-induced effects were reversed by a serotonin receptor antagonist, methysergide. Because experiments were conducted on animals subjected to C(2) spinal cord hemisection, the recovery was most likely mediated by the activation of a latent respiratory pathway spared by the spinal cord injury. The results suggest that serotonin is an important neuromodulator in the unmasking of the latent respiratory pathway after spinal cord injury. In addition, the results also suggest that the maintenance of 5-HTP-induced respiratory recovery may not require a continuous enhancement of central respiratory drive.     

吗啡耐受增加福尔马林致痛大鼠脊髓Fos和NADPH-d阳性神经元的表达

运用Ｆｏｓ免疫组织化学、ＮＡＤＰＨ－ｄ组织化学及Ｆｏｓ／ＮＡＤＰＨ－ｄ双标技术,研究了吗啡耐受对福尔马林致痛大鼠脊髓Ｆｏｓ、ＮＡＤＰＨ－ｄ阳性及Ｆｏｓ／ＮＡＤＰＨ－ｄ双标神经元表达的影响。结果观察到：在非吗啡耐受大鼠,福尔马林诱发的Ｆｏｓ－ｌｉｋｅ ｉｍｍｕｎｏｒｅａｃｔｉｖｉｔｙ（Ｆｏｓ－ＬＩ）主要分布在同侧脊髓背角浅层和颈部,急性静注吗啡可减少Ｆｏｓ－ＬＩ表达;长时间应用吗啡导致福尔马林诱发的     

The crossed phrenic phenomenon: a model for plasticity in the respiratory pathways following spinal cord injury.

Hemisection of the cervical spinal cord rostral to the level of the phrenic nucleus interrupts descending bulbospinal respiratory pathways, which results in a paralysis of the ipsilateral hemidiaphragm. In several mammalian species, functional recovery of the paretic hemidiaphragm can be achieved by transecting the contralateral phrenic nerve. The recovery of the paralyzed hemidiaphragm has been termed the "crossed phrenic phenomenon." The physiological basis for the crossed phrenic phenomenon is as follows: asphyxia induced by spinal hemisection and contralateral phrenicotomy increases central respiratory drive, which activates a latent crossed respiratory pathway. The uninjured, initially latent pathway mediates the hemidiaphragm recovery by descending into the spinal cord contralateral to the hemisection and then crossing the midline of the spinal cord before terminating on phrenic motoneurons ipsilateral and caudal to the hemisection. The purpose of this study is to review work conducted on the crossed phrenic phenomenon and to review closely related studies focusing particularly on the plasticity associated with the response. Because the review deals with recovery of respiratory muscles paralyzed by spinal cord injury, the clinical relevance of the reviewed studies is highlighted.     

Up-regulation of Μ-opioid receptors in the spinal cord of morphine-tolerant rats

Though morphine remains the most powerful drug for treating pain, its effectiveness is limited by the development of tolerance and dependence. The mechanism underlying development of tolerance to morphine is still poorly understood. One of the factors could be an alteration in the number of Μ-receptors within specific parts of the nervous system. However, reports on changes in the Μ-opioid receptor density in the spinal cord after chronic morphine administration are conflicting. Most of the studies have used subcutaneously implanted morphine pellets to produce tolerance. However, it does not simulate clinical conditions, where it is more common to administer morphine at intervals, either by injections or orally. In the present study, rats were made tolerant to morphine by injecting increasing doses of morphine (10-50 mg/kg, subcutaneously) for five days.In vitro tissue autoradiography for localization of Μ-receptor in the spinal cord was done using [³H]-DAMGO. As compared to the spinal cord of control rats, the spinal cord of tolerant rats showed an 18.8% increase or up-regulation in the density of Μ-receptors in the superficial layers of the dorsal horn. This up-regulation of Μ-receptors after morphine tolerance suggests that a fraction of the receptors have been rendered desensitized, which in turn could lead to tolerance     

Spinal cord injury reveals multilineage differentiation of ependymal cells

Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury.     

Effects of spinal cord stimulation in angina pectoris induced by pacing and possible mechanisms of action.

OBJECTIVE--To investigate the effects of spinal cord stimulation on myocardial ischaemia, coronary blood flow, and myocardial oxygen consumption in angina pectoris induced by atrial pacing. DESIGN--The heart was paced to angina during a control phase and treatment with spinal cord stimulation. Blood samples were drawn from a peripheral artery and the coronary sinus. SETTING--Multidisciplinary pain centre, department of medicine, Ostra Hospital, and Wallenberg Research Laboratory, Sahlgrenska Hospital, Gothenburg, Sweden. SUBJECTS--Twenty patients with intractable angina pectoris, all with a spinal cord stimulator implanted before the study. RESULTS--Spinal cord stimulation increased patients'' tolerance to pacing (p < 0.001). At the pacing rate comparable to that producing angina during the control recording, myocardial lactate production during control session turned into extraction (p = 0.003) and, on the electrocardiogram, ST segment depression decreased, time to ST depression increased, and time to recovery from ST depression decreased (p = 0.01; p < 0.05, and p < 0.05, respectively). Spinal cord stimulation also reduced coronary sinus blood flow (p = 0.01) and myocardial oxygen consumption (p = 0.02). At the maximum pacing rate during treatment, all patients experienced anginal pain. Myocardial lactate extraction reverted to production (p < 0.01) and the magnitude and duration of ST segment depression increased to the same values as during control pacing, indicating that myocardial ischaemia during treatment with spinal cord stimulation gives rise to anginal pain. CONCLUSIONS--Spinal cord stimulation has an anti-anginal and anti-ischaemic effect in severe coronary artery disease. These effects seem to be secondary to a decrease in myocardial oxygen consumption. Furthermore, myocardial ischemia during treatment gives rise to anginal pain. Thus, spinal cord stimulation does not deprive the patient of a warning signal.     

Cervical spinal cord deformation during simulated head-first impact injuries

The relationship between bony spinal column and spinal cord injury during an injury event is not well understood. While several studies have measured spinal canal occlusion during axial impact, there has been limited work done to quantify the spinal cord compression or deformation during simulated injury. Because the cord is a viscoelastic solid it may provide resistance to bone fragments, ligaments or other elements that move into the canal and impinge it during column injury. This would differentiate the measurement of cord compression from the measurement of occlusion of an empty canal. In the present study, a novel method of visualizing and quantifying spinal cord deformation during dynamic head-first impact of ex vivo human cervical spine specimens (N=6) was developed. A radiodense, biofidelic surrogate spinal cord was imaged in the spinal canal using high speed cineradiography at 1000 frames per second. The dorsal-ventral diameter of the cord was measured at 1.5mm increments along its length for each frame of the radiographic footage. The resulting cord deformations were used to determine the theoretical neurological outcome of the impact based on published in vivo ferret studies. The corresponding probability of recovery for the spinal cord deformations in these tests ranged between 8% for atlantoaxial dislocation injury and 95% for mid-cervical spine hyperextension injury (based on the ferret data). Clinically relevant spinal column fracture patterns were produced in this study.