Thymosinβ4及β-aescin早期应用对脊髓损伤的保护作用
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摘要
实验一、Thymosinβ4早期应用对脊髓损伤的保护作用
【研究背景】脊髓损伤是一个引起全球广泛关注的问题,尤其是近十年来。重度脊髓损伤往往导致患者终身瘫痪。来自临床研究和动物实验的大量证据均表明,脊髓损伤的预后,不仅取决于原发性损伤的严重程度,更是与其所引起的一系列细胞和分子水平的继发性损伤过程的发展程度密切相关。这些继发性病理过程包括缺血,缺氧,免疫反应,兴奋性毒性,自由基损伤,神经细胞变性死亡,轴突脱髓鞘和waller变性等。脊髓高度血管化,以满足它对氧和葡萄糖的高需求,因此缺血往往导致严重的破坏性后果。我们以往的研究结果发现在不断扩张的脊髓损伤区的边缘存在两个缺血区,临近的缺血区大量神经元正在变性或者已经消失,稍远的缺血区大多数神经元显得相对正常并且认为可以被拯救。有报道称,改善创伤后脊髓血流能显著促进神经功能恢复。
免疫反应被认为在脊髓损伤中发挥类似双刃剑的作用。浸润的免疫细胞可以释放神经营养因子,介导神经保护和促进神经再生。但是已有大量研究表明,伤后早期炎症反应以及脊髓损伤后激活的巨噬细胞可能参与损伤诱导的神经病变和运动功能损害。
神经元和少突胶质细胞均对各种继发性有害因素非常敏感而最终经历变性死亡。其中,少突胶质细胞凋亡导致原发性损伤后残存的轴突进一步脱髓鞘,使那些轴突的传导能力受损,这必然显著影响病人日后神经功能的恢复。
Tβ4是一种由43个氨基酸组成的高度保守的小分子多肽,分子量为4.9 kD,最初是从小牛胸腺组织中分离得到的。它广泛存在于几乎所有的细胞,尤其在血小板和白细胞内水平相对较高。Tβ4具有促进血管发生,抗炎,促进细胞存活和创伤修复等特性,而这些均与脊髓损伤修复密切相关。但是目前尚没有Tβ4应用于脊髓损伤的相关报道。本实验主要关注脊髓损伤后,Tβ4促进血管发生,神经元保护和抗炎三方面的作用,并观察运动功能恢复情况。
【目的】利用我们研究所自行设计的脊髓损伤模型,观察Tβ4对脊髓损伤的治疗作用。
【方法】所以大鼠分为3组:假手术组(n=5),Tβ4(n=15)和生理盐水组(n=15)。将大鼠麻醉后,使用改良的眼科显微镊制作大鼠脊髓夹伤模型。伤后30 min,腹腔注射Tβ4溶液,以后每3 d给药1次。至第7 d,处死所有大鼠,行组织化学检查, RECA1标记血管,NeuN标记神经元,ED1标记活化小胶质细胞和血源性巨噬细胞,GFAP标记胶质瘢痕。Western blotting检测活化小胶质细胞和血源性巨噬细胞的特异性标记物ED1和成熟少突胶质细胞的特异性标记物MBP的表达水平。所有大鼠存活7 d,在相应的时间点对大鼠进行BBB评分和足迹实验。生理盐水替代Tβ4作为对照组。
【结果】组织化学结果显示Tβ4组大鼠脊髓空洞面积较盐水组减少32.1%。血管长度和神经元的变化主要存在于距损伤边界吻、尾侧各1 mm范围内。血管总长度,Tβ4组吻、尾侧分别是14.72±0.17 mm/mm~2和13.35±0.13 mm/mm~2,盐水组吻、尾侧分别是12.97±0.45 mm/mm~2和11.40±0.39 mm/mm~2;神经元数量,Tβ4组吻尾侧分别是335.1±11.1/mm~2和317.7±9.3/mm~2,盐水组吻、尾侧分别是284.8±15.4/mm~2和262.1±16.6 /mm~2。Tβ4和盐水两组之间密切相关。Western blotting结果显示,Tβ4组ED1表达量较盐水组下降了35.9%;Tβ4组MBP水平仅下降了20.1%,较盐水组高44.3%。各种运动功能评估方法均显示,Tβ4组大鼠运功功能较盐水组明显改善。
【结论】在本实验,我们率先发现应用Tβ4可以显著减少SCI后损伤面积,加速运动功能恢复,很可能与其刺激血管发生,发挥神经保护和减少炎症浸润有关。Tβ4的临床安全性已经明确,因此它可以作为未来SCI治疗的选择之一。
实验二、β-aescin早期应用对脊髓损伤的保护作用
【研究背景】在SCI,除了原发性创伤造成的直接损伤外,在其周围还将发生不断扩张的继发性病理改变,包括缺血,自由基损伤,炎症反应,兴奋性毒性,神经元变性坏死及凋亡等。脂质过氧化(lipid peroxydation LPO)是脊髓损伤后最早出现的生化改变之一,是自由基损伤的延续,被认为是导致原始损伤区域向外恶化蔓延的重要因素。丙二醛(MDA)是膜结构中多不饱和脂肪酸过氧化的主要终产物,被认为是LPO的生物学标记。炎症反应在SCI中的作用类似双刃剑,炎症细胞早期广泛聚集并分泌大量毒性细胞因子和自由基而加重SCI,后期合成保护性细胞因子及生长因子介导神经保护和促进神经再生。
Aescin属三萜类化合物,是从欧洲七叶树中提取的主要活性成分。Aescin含α和β两种异构体,β-aescin作为主要的活性异构体,具有抗炎、抗水肿、清除活性氧、增强静脉张力和保护内皮细胞的作用,已广泛应用于药学研究及临床治疗。
本实验我们采用成年SD大鼠建立脊髓中度撞击伤模型。检测伤后24 h脊髓MDA的水平和髓过氧化物酶(MPO)的活性,观察伤后14 d炎症细胞的变化。观察β-aescin对它们的影响,并探讨β-aescin的SCI早期治疗作用机制。
【目的】采用成年SD大鼠建立脊髓中度撞击伤模型,观察β-aescin对大鼠脊髓损伤后继发性损害的保护作用。
【方法】所有大鼠分为3组:假手术组(n=10),Tβ4(n=18)和生理盐水组(n=18)。造模成功后30 min,开始经腹腔给予β-aescin(1.0 mg/kg)或生理盐水,3次/d,连续给药3 d。假手术组为正常对照。于各时间点采用BBB评分和足迹实验对大鼠双后肢运动功能进行评估;术后24 h,对损伤部位脊髓组织MDA含量及MPO活性进行检测;术后14 d行免疫组织化学检查,利用GFAP观察损伤星形胶质瘢痕及其包围的坏死空洞,利用ED1观察活性小胶质细胞和巨噬细胞。
【结果】β-aescin组运动功能明显改善。伤后24 h,β-aescin组大鼠脊髓组织中MDA水平降低67%,MPO活性降低50%。伤后14 d,β-aescin组空洞面积减小29.3%,ED1染色阳性面积减少32.3%。
【结论】β-aescin可以显著减轻SCI后自由基的氧化损伤作用,减少炎症细胞的活化和浸润,减小空洞面积,促进SCI后神经功能的恢复。
【Background】Spinal cord injury (SCI) is an issue that has aroused worldwide interest, particularly in recent decades. Severe SCI often leads to lifelong paralysis. Evidence from both clinical and experimental studies indicates that, the pathologic process following SCI is divided into two chronological phases: the primary mechanical injury and a wave of secondary injury triggered by the initial insult The multifaceted secondary injury that results in evolution of the pathological changes are mainly associated with ischemia, edema, immune responses, excitotoxicity, free radical injury, and apoptosis. The spinal cord is highly vascularized to meet its high demand of oxygen and glucose supply, thus ischemia may cause devastating outcomes. Our previous study found that, there were two ischemic zones at the front of the expanding secondary injury, a zone neighboring the lesion where most of the neurons are degenerating or have disappeared, and a farther zone in which most of the neurons appeared relatively normal and considered possible to be rescued. In fact, it has been reported that improving posttraumatic spinal cord blood flow can significantly facilitate functional recovery. That the immune responses considered as a double-edged sword in SCI has been well documented. Although infiltrating immune cells produce neurotrophic factors mediating neuroprotection and regeneration after SCI, it has also been shown that an early inflammatory response and activated macrophages initiated by SCI may participate in neuropathology and loss of motor function. In those secondary damaging processes after SCI, both neurons and oligodendrocytes are vulnerable to various harmful factors. Due to the loss of oligodendrocytes and subsequent axonal demyelination of surviving axons and impair their conductive capacity, it seems reasonable to expect that reducing oligodendrocyte death and improving axonal remyelination holds potential for the treatment of SCI.
Tβ4, a highly conserved 4.9 kD polypeptide, comprises of 43 amino acids, which was first isolated from bovine thymus. It is a natural polypeptide that is ubiquitously distributed in almost all cells with relatively higher levels in platelets and white blood cells. The angiogenesis, anti-inflammation, wound repair and cell survival properties of Tβ4 are highly relevant to the SCI. We have recently formulated an idea of early neurosurgery of spinal cord contusion with great success. The patients received only surgery and rehabilitation. No medication that might improve recovery was given to avoid confusion of credit of the surgery. Therefore, search for effective drugs that may fill in the big space left for improving the outcome of operation may contribute substantially to the treatment of spinal cord contusion. The present study was focused on its angiogenic, neuronal protective and anti-inflammatory effects and the results showed that these three aspects of Tβ4 worked well in reducing the secondary injury and improve locomotion.
【Objective】The present study was aimed to investigate the therapeutic value for SCI of its angiogenic, neuroprotective and anti-inflammatory properties. We used a rat SCI model of bilateral compression of the spinal cord.
【Methods】The SD rats were randomly divided into 3 groups: sham (n = 5), Tβ4 (n = 15), and normal saline (n = 15). After anesthesia, a rat SCI model of lateral compression of the spinal cord was produced using a pair of modified forceps. Tβ4 or normal saline was administrated i.p. 30 min after injury, and 2 doses on the first day of every other 3 days. Histological tests were performed to assess blood vessels, neuronal survival, and lesion size. Western blot assay was executed to detect the expression of specifical marker ED1 for activated microglia and infiltrated macrophages and specifical marker MBP for mature oligodendrocytes. All rats were allowed to surviv for 7 days. All rats underwent behavioral assessments at every time point.
【Results】Our data demonstrated that the size of lesion area delineated by GFAP was decreased by 32.1% in the Tβ4 group compared with the saline group. The changes of total length of the blood vessels and total number of neurons within the gray matter occurred in the areas 1 mm rostral and caudal to the lesion edge. The density of total length of blood vessels in the rostral 1 mm was 14.72±0.17 mm/mm~2 in the Tβ4 treated group versus 12.97±0.45 mm/mm~2 in the saline group, in the caudal 1 mm was 13.35±0.13 mm/mm~2 versus 11.40±0.39 mm/mm~2, respectively. The number of neurons in the Tβ4 treated group in the rostral and caudal 1 mm zones were 335.1±11.1/mm~2 and 317.7±9.3 /mm~2, respectively, whereas in the saline group the rostral and caudal zones hosted far less NeuN-immunoreactive cells, 284.8±15.4/mm~2 and 262.1±16.6 /mm~2, respectively. Furthermore, there was a close correlation between the effects on blood vessel length and neuronal survival. Western blotting assay showed that, the ED1 expression in the Tβ4 group was 35.9% less than that in the saline group; the MBP level was only decreased by 20.1% in the Tβ4 group but clearly decreased by 44.3% in the saline group compared with normal control. Locomotor recovery was tested using the BBB score and footprint analysis. All of the behavioral assessments were markedly improved.
【Conclusions】Here, for the first time, our present study shows that, systemic administration of Tβ4 after acute SCI in rats, remarkably reduces the lesion size and some pathological changes of the injured spinal cord, and accelerates locomotion recovery, most probably, by stimulating angiogenesis, increasing neuronal survival, reducing inflammatory infiltration and demyelination. Given its known safety in clinical uses and the beneficial effects on spinal cord injury, Tβ4 can well be a good candidate for treating spinal cord contusion.
Part II: Beneficial effects ofβ-aescin on spinal cord injury in the rat
【Background】The severity of neurological disturbance, induced by traumatic SCI, is not only associated with the degree of the mechanical insult to the tissue, but also closely correlated to progressive tissue loss beyond the primary injury site. Multiple secondary damaging factors are involved in the latter process, including ischemia, immune response, glutamate excitotoxicity, free radical generation, reactive oxygen species, lipid peroxidation (LPO), and apoptosis, to name a few. LPO is a self-perpetuating form of free radical damage that is believed to be a major factor in the spread of deterioration after SCI to regions of spared tissue outside the original zone of trauma. Malondialdehyde (MDA), the principal end product of polyunsaturated fatty acid peroxidation in cell membranes, considered as a biological marker of LPO, is a highly toxic molecule, aggravating the damage beyond membrane lipids by interacting with amino acids, proteins and DNA. The immune system likely plays a critical role in the secondary degenerative process. An intense local inflammatory response, typed by activation of resident microglia and influx of hematogenous neutrophils and macrophages, is rapidly triggered by traumatic SCI. Those cells produce free radicals including superoxide anion and nitric oxide during these processes inducing apoptosis in neurons and glia via the irreversible oxidation of proteins, lipids and nucleic acids. The LPO and inflammatory responses are therefore good targets for the exploration of pharmacological strategies to treat acute SCI. At present, the methylprednisolone having the properties of anti-peroxidation and anti-inflammation remains the only neuroprotective agent in clinic pharmacotherapy for acute SCI, but its use existing extensive controversies due to undesirable side effects. Thus we are interested in seeking for a novel therapeutic approach for acute phase SCI without significant side effects.
Aescin, the major active principle from aesculus hippocastanum, was subjected to a natural mixture of triterpene saponins. It has two isomers ofαandβ. Among which,β-aescin shown numerous pharmacological properties of anti-edema, anti-inflammation, anti-oxidation, increasing venous tone, and protecting vascular endothelial cells.β-aescin appears to be the active component of the mixture and is the molecular form present in major available pharmaceutical products. It has been widely applied in haemorrhoids, chronic venous insuf?ciency, peripheral vascular diseases, and postoperative, postburn or posttraumatic edema in clinical practices with very few adverse effects. The aim of this study was to investigate the possible effects ofβ-aescin on LPO and cellular in?ammation after traumatic SCI when the agent was administered intraperitoneally. We also determined whetherβ-aescin could improve functional deficits.
【objective】To investigate the protective effects ofβ-aescin on acute traumatic spinal cord injury (SCI) in rats
【Methods】We examined this hypothesis in a rat model of moderate spinal cord contusion. All rats were assigned into 3 groups: 1. Sham-operated control group (n = 10): only received a laminectomy with no additional treatment; 2.β-aescin group (n = 18): SCI plus 1 mlβ-aescin.i.p; 3. Saline control group (n = 18): SCI plus 1 ml saline.i.p. 30 min after injury, the spinal cord-injured rats were treated withβ-aescin (1.0 mg/kg bw i.p.) or normal saline, and received additional dose t.i.d for 3 consecutive days. Locomotor deficits induced by SCI were assessed using the BBB score and footprint analysis. Biochemical assays were executed to detect the level of malondialdehyde (MDA) and the activity of myeloperoxydase (MPO). Histological tests were performed to analyze the lesion size, and activated microglia and recruited macrophages.
【Results】Our data indicated that all of the behavioral assessments were markedly improved. In addition,β-aescin treatment significantly reduced MDA level by 67% and MPO activity by 50% at 24 hr after injury, decreased lesion cavitation by 29.3% and ED1 positive area by 32.3% at 14 days after injury.
【Conclision】The present study showed thatβ-aescin treatment strikingly reduced lipid peroxydation (LPO), suppressed microglia activation and hematogenous leukocyte infiltration, and facilitated reparation after SCI.
【研究背景】脊髓损伤是一个引起全球广泛关注的问题,尤其是近十年来。重度脊髓损伤往往导致患者终身瘫痪。来自临床研究和动物实验的大量证据均表明,脊髓损伤的预后,不仅取决于原发性损伤的严重程度,更是与其所引起的一系列细胞和分子水平的继发性损伤过程的发展程度密切相关。这些继发性病理过程包括缺血,缺氧,免疫反应,兴奋性毒性,自由基损伤,神经细胞变性死亡,轴突脱髓鞘和waller变性等。脊髓高度血管化,以满足它对氧和葡萄糖的高需求,因此缺血往往导致严重的破坏性后果。我们以往的研究结果发现在不断扩张的脊髓损伤区的边缘存在两个缺血区,临近的缺血区大量神经元正在变性或者已经消失,稍远的缺血区大多数神经元显得相对正常并且认为可以被拯救。有报道称,改善创伤后脊髓血流能显著促进神经功能恢复。
免疫反应被认为在脊髓损伤中发挥类似双刃剑的作用。浸润的免疫细胞可以释放神经营养因子,介导神经保护和促进神经再生。但是已有大量研究表明,伤后早期炎症反应以及脊髓损伤后激活的巨噬细胞可能参与损伤诱导的神经病变和运动功能损害。
神经元和少突胶质细胞均对各种继发性有害因素非常敏感而最终经历变性死亡。其中,少突胶质细胞凋亡导致原发性损伤后残存的轴突进一步脱髓鞘,使那些轴突的传导能力受损,这必然显著影响病人日后神经功能的恢复。
Tβ4是一种由43个氨基酸组成的高度保守的小分子多肽,分子量为4.9 kD,最初是从小牛胸腺组织中分离得到的。它广泛存在于几乎所有的细胞,尤其在血小板和白细胞内水平相对较高。Tβ4具有促进血管发生,抗炎,促进细胞存活和创伤修复等特性,而这些均与脊髓损伤修复密切相关。但是目前尚没有Tβ4应用于脊髓损伤的相关报道。本实验主要关注脊髓损伤后,Tβ4促进血管发生,神经元保护和抗炎三方面的作用,并观察运动功能恢复情况。
【目的】利用我们研究所自行设计的脊髓损伤模型,观察Tβ4对脊髓损伤的治疗作用。
【方法】所以大鼠分为3组:假手术组(n=5),Tβ4(n=15)和生理盐水组(n=15)。将大鼠麻醉后,使用改良的眼科显微镊制作大鼠脊髓夹伤模型。伤后30 min,腹腔注射Tβ4溶液,以后每3 d给药1次。至第7 d,处死所有大鼠,行组织化学检查, RECA1标记血管,NeuN标记神经元,ED1标记活化小胶质细胞和血源性巨噬细胞,GFAP标记胶质瘢痕。Western blotting检测活化小胶质细胞和血源性巨噬细胞的特异性标记物ED1和成熟少突胶质细胞的特异性标记物MBP的表达水平。所有大鼠存活7 d,在相应的时间点对大鼠进行BBB评分和足迹实验。生理盐水替代Tβ4作为对照组。
【结果】组织化学结果显示Tβ4组大鼠脊髓空洞面积较盐水组减少32.1%。血管长度和神经元的变化主要存在于距损伤边界吻、尾侧各1 mm范围内。血管总长度,Tβ4组吻、尾侧分别是14.72±0.17 mm/mm~2和13.35±0.13 mm/mm~2,盐水组吻、尾侧分别是12.97±0.45 mm/mm~2和11.40±0.39 mm/mm~2;神经元数量,Tβ4组吻尾侧分别是335.1±11.1/mm~2和317.7±9.3/mm~2,盐水组吻、尾侧分别是284.8±15.4/mm~2和262.1±16.6 /mm~2。Tβ4和盐水两组之间密切相关。Western blotting结果显示,Tβ4组ED1表达量较盐水组下降了35.9%;Tβ4组MBP水平仅下降了20.1%,较盐水组高44.3%。各种运动功能评估方法均显示,Tβ4组大鼠运功功能较盐水组明显改善。
【结论】在本实验,我们率先发现应用Tβ4可以显著减少SCI后损伤面积,加速运动功能恢复,很可能与其刺激血管发生,发挥神经保护和减少炎症浸润有关。Tβ4的临床安全性已经明确,因此它可以作为未来SCI治疗的选择之一。
实验二、β-aescin早期应用对脊髓损伤的保护作用
【研究背景】在SCI,除了原发性创伤造成的直接损伤外,在其周围还将发生不断扩张的继发性病理改变,包括缺血,自由基损伤,炎症反应,兴奋性毒性,神经元变性坏死及凋亡等。脂质过氧化(lipid peroxydation LPO)是脊髓损伤后最早出现的生化改变之一,是自由基损伤的延续,被认为是导致原始损伤区域向外恶化蔓延的重要因素。丙二醛(MDA)是膜结构中多不饱和脂肪酸过氧化的主要终产物,被认为是LPO的生物学标记。炎症反应在SCI中的作用类似双刃剑,炎症细胞早期广泛聚集并分泌大量毒性细胞因子和自由基而加重SCI,后期合成保护性细胞因子及生长因子介导神经保护和促进神经再生。
Aescin属三萜类化合物,是从欧洲七叶树中提取的主要活性成分。Aescin含α和β两种异构体,β-aescin作为主要的活性异构体,具有抗炎、抗水肿、清除活性氧、增强静脉张力和保护内皮细胞的作用,已广泛应用于药学研究及临床治疗。
本实验我们采用成年SD大鼠建立脊髓中度撞击伤模型。检测伤后24 h脊髓MDA的水平和髓过氧化物酶(MPO)的活性,观察伤后14 d炎症细胞的变化。观察β-aescin对它们的影响,并探讨β-aescin的SCI早期治疗作用机制。
【目的】采用成年SD大鼠建立脊髓中度撞击伤模型,观察β-aescin对大鼠脊髓损伤后继发性损害的保护作用。
【方法】所有大鼠分为3组:假手术组(n=10),Tβ4(n=18)和生理盐水组(n=18)。造模成功后30 min,开始经腹腔给予β-aescin(1.0 mg/kg)或生理盐水,3次/d,连续给药3 d。假手术组为正常对照。于各时间点采用BBB评分和足迹实验对大鼠双后肢运动功能进行评估;术后24 h,对损伤部位脊髓组织MDA含量及MPO活性进行检测;术后14 d行免疫组织化学检查,利用GFAP观察损伤星形胶质瘢痕及其包围的坏死空洞,利用ED1观察活性小胶质细胞和巨噬细胞。
【结果】β-aescin组运动功能明显改善。伤后24 h,β-aescin组大鼠脊髓组织中MDA水平降低67%,MPO活性降低50%。伤后14 d,β-aescin组空洞面积减小29.3%,ED1染色阳性面积减少32.3%。
【结论】β-aescin可以显著减轻SCI后自由基的氧化损伤作用,减少炎症细胞的活化和浸润,减小空洞面积,促进SCI后神经功能的恢复。
【Background】Spinal cord injury (SCI) is an issue that has aroused worldwide interest, particularly in recent decades. Severe SCI often leads to lifelong paralysis. Evidence from both clinical and experimental studies indicates that, the pathologic process following SCI is divided into two chronological phases: the primary mechanical injury and a wave of secondary injury triggered by the initial insult The multifaceted secondary injury that results in evolution of the pathological changes are mainly associated with ischemia, edema, immune responses, excitotoxicity, free radical injury, and apoptosis. The spinal cord is highly vascularized to meet its high demand of oxygen and glucose supply, thus ischemia may cause devastating outcomes. Our previous study found that, there were two ischemic zones at the front of the expanding secondary injury, a zone neighboring the lesion where most of the neurons are degenerating or have disappeared, and a farther zone in which most of the neurons appeared relatively normal and considered possible to be rescued. In fact, it has been reported that improving posttraumatic spinal cord blood flow can significantly facilitate functional recovery. That the immune responses considered as a double-edged sword in SCI has been well documented. Although infiltrating immune cells produce neurotrophic factors mediating neuroprotection and regeneration after SCI, it has also been shown that an early inflammatory response and activated macrophages initiated by SCI may participate in neuropathology and loss of motor function. In those secondary damaging processes after SCI, both neurons and oligodendrocytes are vulnerable to various harmful factors. Due to the loss of oligodendrocytes and subsequent axonal demyelination of surviving axons and impair their conductive capacity, it seems reasonable to expect that reducing oligodendrocyte death and improving axonal remyelination holds potential for the treatment of SCI.
Tβ4, a highly conserved 4.9 kD polypeptide, comprises of 43 amino acids, which was first isolated from bovine thymus. It is a natural polypeptide that is ubiquitously distributed in almost all cells with relatively higher levels in platelets and white blood cells. The angiogenesis, anti-inflammation, wound repair and cell survival properties of Tβ4 are highly relevant to the SCI. We have recently formulated an idea of early neurosurgery of spinal cord contusion with great success. The patients received only surgery and rehabilitation. No medication that might improve recovery was given to avoid confusion of credit of the surgery. Therefore, search for effective drugs that may fill in the big space left for improving the outcome of operation may contribute substantially to the treatment of spinal cord contusion. The present study was focused on its angiogenic, neuronal protective and anti-inflammatory effects and the results showed that these three aspects of Tβ4 worked well in reducing the secondary injury and improve locomotion.
【Objective】The present study was aimed to investigate the therapeutic value for SCI of its angiogenic, neuroprotective and anti-inflammatory properties. We used a rat SCI model of bilateral compression of the spinal cord.
【Methods】The SD rats were randomly divided into 3 groups: sham (n = 5), Tβ4 (n = 15), and normal saline (n = 15). After anesthesia, a rat SCI model of lateral compression of the spinal cord was produced using a pair of modified forceps. Tβ4 or normal saline was administrated i.p. 30 min after injury, and 2 doses on the first day of every other 3 days. Histological tests were performed to assess blood vessels, neuronal survival, and lesion size. Western blot assay was executed to detect the expression of specifical marker ED1 for activated microglia and infiltrated macrophages and specifical marker MBP for mature oligodendrocytes. All rats were allowed to surviv for 7 days. All rats underwent behavioral assessments at every time point.
【Results】Our data demonstrated that the size of lesion area delineated by GFAP was decreased by 32.1% in the Tβ4 group compared with the saline group. The changes of total length of the blood vessels and total number of neurons within the gray matter occurred in the areas 1 mm rostral and caudal to the lesion edge. The density of total length of blood vessels in the rostral 1 mm was 14.72±0.17 mm/mm~2 in the Tβ4 treated group versus 12.97±0.45 mm/mm~2 in the saline group, in the caudal 1 mm was 13.35±0.13 mm/mm~2 versus 11.40±0.39 mm/mm~2, respectively. The number of neurons in the Tβ4 treated group in the rostral and caudal 1 mm zones were 335.1±11.1/mm~2 and 317.7±9.3 /mm~2, respectively, whereas in the saline group the rostral and caudal zones hosted far less NeuN-immunoreactive cells, 284.8±15.4/mm~2 and 262.1±16.6 /mm~2, respectively. Furthermore, there was a close correlation between the effects on blood vessel length and neuronal survival. Western blotting assay showed that, the ED1 expression in the Tβ4 group was 35.9% less than that in the saline group; the MBP level was only decreased by 20.1% in the Tβ4 group but clearly decreased by 44.3% in the saline group compared with normal control. Locomotor recovery was tested using the BBB score and footprint analysis. All of the behavioral assessments were markedly improved.
【Conclusions】Here, for the first time, our present study shows that, systemic administration of Tβ4 after acute SCI in rats, remarkably reduces the lesion size and some pathological changes of the injured spinal cord, and accelerates locomotion recovery, most probably, by stimulating angiogenesis, increasing neuronal survival, reducing inflammatory infiltration and demyelination. Given its known safety in clinical uses and the beneficial effects on spinal cord injury, Tβ4 can well be a good candidate for treating spinal cord contusion.
Part II: Beneficial effects ofβ-aescin on spinal cord injury in the rat
【Background】The severity of neurological disturbance, induced by traumatic SCI, is not only associated with the degree of the mechanical insult to the tissue, but also closely correlated to progressive tissue loss beyond the primary injury site. Multiple secondary damaging factors are involved in the latter process, including ischemia, immune response, glutamate excitotoxicity, free radical generation, reactive oxygen species, lipid peroxidation (LPO), and apoptosis, to name a few. LPO is a self-perpetuating form of free radical damage that is believed to be a major factor in the spread of deterioration after SCI to regions of spared tissue outside the original zone of trauma. Malondialdehyde (MDA), the principal end product of polyunsaturated fatty acid peroxidation in cell membranes, considered as a biological marker of LPO, is a highly toxic molecule, aggravating the damage beyond membrane lipids by interacting with amino acids, proteins and DNA. The immune system likely plays a critical role in the secondary degenerative process. An intense local inflammatory response, typed by activation of resident microglia and influx of hematogenous neutrophils and macrophages, is rapidly triggered by traumatic SCI. Those cells produce free radicals including superoxide anion and nitric oxide during these processes inducing apoptosis in neurons and glia via the irreversible oxidation of proteins, lipids and nucleic acids. The LPO and inflammatory responses are therefore good targets for the exploration of pharmacological strategies to treat acute SCI. At present, the methylprednisolone having the properties of anti-peroxidation and anti-inflammation remains the only neuroprotective agent in clinic pharmacotherapy for acute SCI, but its use existing extensive controversies due to undesirable side effects. Thus we are interested in seeking for a novel therapeutic approach for acute phase SCI without significant side effects.
Aescin, the major active principle from aesculus hippocastanum, was subjected to a natural mixture of triterpene saponins. It has two isomers ofαandβ. Among which,β-aescin shown numerous pharmacological properties of anti-edema, anti-inflammation, anti-oxidation, increasing venous tone, and protecting vascular endothelial cells.β-aescin appears to be the active component of the mixture and is the molecular form present in major available pharmaceutical products. It has been widely applied in haemorrhoids, chronic venous insuf?ciency, peripheral vascular diseases, and postoperative, postburn or posttraumatic edema in clinical practices with very few adverse effects. The aim of this study was to investigate the possible effects ofβ-aescin on LPO and cellular in?ammation after traumatic SCI when the agent was administered intraperitoneally. We also determined whetherβ-aescin could improve functional deficits.
【objective】To investigate the protective effects ofβ-aescin on acute traumatic spinal cord injury (SCI) in rats
【Methods】We examined this hypothesis in a rat model of moderate spinal cord contusion. All rats were assigned into 3 groups: 1. Sham-operated control group (n = 10): only received a laminectomy with no additional treatment; 2.β-aescin group (n = 18): SCI plus 1 mlβ-aescin.i.p; 3. Saline control group (n = 18): SCI plus 1 ml saline.i.p. 30 min after injury, the spinal cord-injured rats were treated withβ-aescin (1.0 mg/kg bw i.p.) or normal saline, and received additional dose t.i.d for 3 consecutive days. Locomotor deficits induced by SCI were assessed using the BBB score and footprint analysis. Biochemical assays were executed to detect the level of malondialdehyde (MDA) and the activity of myeloperoxydase (MPO). Histological tests were performed to analyze the lesion size, and activated microglia and recruited macrophages.
【Results】Our data indicated that all of the behavioral assessments were markedly improved. In addition,β-aescin treatment significantly reduced MDA level by 67% and MPO activity by 50% at 24 hr after injury, decreased lesion cavitation by 29.3% and ED1 positive area by 32.3% at 14 days after injury.
【Conclision】The present study showed thatβ-aescin treatment strikingly reduced lipid peroxydation (LPO), suppressed microglia activation and hematogenous leukocyte infiltration, and facilitated reparation after SCI.
引文
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