Conditions of spinal cord injury model

Nine experimental animals all survived to draw materials phase points (8 hours after spinal cord injury). Three rabbits of the control group was sober and they could move bilateral hind legs freely, they could feed and drinking by themselves, and had no spinal cord injury; The rabbits in the model group and the drug group had no acupuncture reaction of bilateral hind legs after awake, their hip, knee, ankle joints and the toe had no activities with complete expression of spinal cord injury, these animals could plucked the bilateral hind legs to craw for feeding and drinking.

Testing and extracting of rabbit RNA sample in each group (Table 1 and Figure 1)

Testing results of gene microarray

Distribution of gene microarray hybrid data of the samples in the spinal cord injury group, model group and drug group is shown in Figure 2.

Distribution of differential expression genes (Figure 3)

Statistical results of differential expression genes (Table 2)

Chip detection result of Cathepsis family genes (Table 3)

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Related researches have shown that early large dosage of methylprednisolone have to be used, which can protect neuromechanism. More and more evidence explain that different kinds of proteolytic enzyme attend in histiocytic apoptosis^[1-2]. In case of physiological conditions that Cathepsis family cadherin is lysosomal proteinases, it can be released from lysosome to cytoplasm when under the pathological condition, the Cathepsis family cadherin take part in apoptosis by activating the Caspase and induce pro-apoptosis factors released by mitochondria. But it is not clear whether Cathepsis family participate in the early pathologic process of spinal cord injury and whether the high-dose methylprednisolone has protective effect on nerve cells by the lysosome apoptosis pathway.

Design
A randomized controlled animal experiment.
Time and setting
The experiment was completed in the Administration of Experimental Animal Center of Kunming General Hospital of Chengdu Military Region between March 2010 and October 2011.
Materials
Nine healthy adult Japanese white rabbits provided by the Administration of Experimental Animal Center of Kunming General Hospital of Chengdu Military Region, license No. SCXK(Dian)2010-0089, no limitation of male or female. The weight of the rabbits was (3 100±140) kg.
The main instruments and reagents are as follows:

Methods
Establishing animal model of spinal cord injury
The rabbits were divide into three groups which were control group (n=3), model group (n=3) and drug group (n=3). The control group only carried on laminectomy. Then acute spinal cord injury model^[6] was established with Allen’s falling strike method after laminectomy in the model group and drug group. L4 surface received laminectomy and beat caused acute spinal cord injury, the beating power was 250 g·cm (10 g×25 cm).
Drug therapy
The drug group received flushing-dose methylprednisolone at 2 hours after spinal cord injury with human-rabbit equivalent dose (NASCIS plan: people-rabbit equivalent dose coefficient=3.08, the weight of 30 mg/kg should be dropped off methylprednisolone in 15 minutes, after 45 minutes, methylprednisolone was dropped with 5.4 mg/(kg·h) and maintained for 23 hours). The model group was given the normal saline with the same volume. All rabbits were killed at 8 hours after modeling, and then the damaged spinal cord tissues about 8 mm in length were obtained carefully, washed the blood in tissues by pro-cooling normal saline and absorbed the excess liquid with filter, then kept in liquid nitrogen.
Total RNA extraction of spinal cord tissues
Extracted the spinal cord tissues total RNA by TRIzol reagent instructions. TRIzol reagent was put in the spinal cord
and mixed with ice-bath, then added with the chloroform and isopropyl alcohol, centrifugalization and extracted
total RNA. The proportion of total RNA 28S and 18S were tested by agarose gel electrophoresis in order to assess the integrity of total RNA; ultraviolet spectrophotometer was used to measure the absorption peak of 260 nm and 280 nm in order to calculate the purity of total RNA.
Test and analysis of gene microarray
Agilent Rabbit Oligo Microarray produced by Affymetrix Company was used, and it contained 43 603 transcripts (RNA).
Shanghai Biological Chip Co., Ltd (SBC) --National Engineering Center for Biochip at Shanghai supplied the
technology. The Lab-on system was adopted to test the nine RAN samples, and the good quality RNA samples were marked to performe the hybridization experiment. The Agilent enlarge marking method was used for marking, and proper amount of probes were used for hybridization which performed under 65 ℃ for 17 hours. The Agilent scanistor was used to scan the chips; the software of Agilent Feature Extraction was used to read the data; at last, the Feature Extration was used for Normalize analysis.
Main outcome measures
Expression and change of genes Cathepsin family in acute spinal cord injury and movement and sensation of hind limbs of the experimental animals.
Statistical analysis
The data were analyzed using 13.0 statistical software and expressed as mean±SD. A value of P < 0.05 was considered as significant.

Trauma is the most common reason for acute spinal cord injury and a series of patho-physiological course will happen after spinal cord injury. Allen has divided spinal cord injury into two parts: primary injury and secondary injury. Primary injury refers to a kind of irreversible injury firstly caused by the shift or dislocation of spinal fractures which then leads to intervertebral disc into spinal fractures and fracture fragment pricking into vertebral canal, then resulting in spinal cord compression, shock, laceration, contusion, laceration and cut injuries. While the secondary injury refers to the phenomena that after spinal cord injury, the injured area will inspire a series of neural and biochemical microcirculation disorders and inflammation reactions, which then bring about vascular spasm, blood coagulation and thrombosis. Both can determine the degree and prognosis of spinal cord injury. Mechanism of secondary injury are: (1) Vascular mechanism, namely automatic regulative vascular disorders. Due to vascular endothelial injury, basement membrane exposure, platelet adhesion and thrombosis, that lumen occlusion formation; tissue ischemia and hypoxia leads to edema and bleeding thus results in liquefaction necrosis in the spinal cord tissue. (2) Electrolyte changes, Na+ migrates into neural cells after spinal cord injury, while K+ escapes and gatheres outside the cells, which affects the neighbouring depolarization of adjacent neurons and glial cells, and resulting in nerve conduction block. Inflow of extracellular calcium and the increasing of intracellular calcium activate a variety of protease and phospholipids, which makes the cell membrane break down and lipid peroxide and then resulting in a large number of oxygen free radicals and leading to cell death^[3]. (3) Biochemical mechanisms: norepinephrine gathering, increasing of 5-hydroxytryptamine and the production of prostacyclin can damage the blood supply. The production and gathering of dynorphin and other endogenous opioids and excitatory amino acid are considered to be the factors that leading to spinal cord injury^[4]. (4) Edema: Biochemical changes of the cellular level causes cytotoxic edema, increasing of local tissue pressure and microcirculation obstruction which further reduce the usage of oxygen. (5) Energy metabolism inhibition: Adenosine triphosphate production decreases.

With regard to the whole organism and the tissue of the injured region, trauma is no doubt a powerful blow to them. Traditionally acknowledged that necrosis is the only result of trauma. However, in recent years, plenty of researches have indicated that hypoxic cells mainly end in necrosis, and at the same time, the number of apoptosis also increased. Ischemic hypoxic cells generate a great deal of oxygen free radicals and also lead to severe calcium overload, both of which will result in the peroxidation of lipid and protein and the damage of DNA. The damage of DNA will lead to mitochondrial dysfunction, disruption of cytoskeleton and activating phospholipase^[5-6]. Both of them were successively considered as the main causes to the injury even death of the ischemic hypoxic cells. However, the elimination of oxygen free radicals and calcium blocking cannot totally prevent the death of cells which already are oxygen-deficient and ischemia. Moreover, oxygen free radicals and calcium-overload can not fully explain the fact that after being oxygen-deficient and ischemia, the apoptosis of cells are increased^[7]. Recent researches indicate that oxygen free radicals and calcium-overload may be the incentive of cell death rather than the direct cause of the death of oxygen-deficient and ischemia cells. Mitochondrial damage maybe the crucial juncture which contributes to the necrosis and death of oxygen-deficient and ischemia cells^[8]. Oxygen deficient and ischemia may result two main consequences of mitochondrion: calcium overload and oxidative stress. Either the combined action or separate action of them can cause necrosis. The mechanism is related with the inducement of mitochondrial permeability transition. At present, it acknowledges that mitochondrial permeability transition is the common result of cells which has been affected by toxic material, and the essence of mitochondrial permeability transition is high-permeation status of permeability transition pore^[9-10]. Both Mitochondrial calcium overload and oxidative stress can accelerate adenine nucleotide translocase, voltage-dependent anion channel and cyclophilin D to combine into adenine nucleotide translocase-voltage-dependent anion channel-cyclophilin D trigeminal composite to stimulate the conformation alteration of adenine nucleotide translocase, voltage-dependent anion channel and then lead to the occurrence of mitochondrial permeability transition. Mitochondrial permeability transition makes permeability transition pore allows materials as high as 5 000 u molecular weight out and in mitochondrion which results in content loss of mitochondrion, decrease even disappearance of membrane potential, large-scale hydrogen entry into mitochondrion, finally leading to the swelling even disintegration of mitochondrion. With regard to cells, mitochondrial permeability transition will results in consequences as follows: (1) Disturbance will happen in the process of cellular oxidation and phosphorylation and the main energy resource of cells will disappear which make all vital movement of cells hard to continue. (2) The entry of mitochondrial cell pigment C apoptosis inducing the factors into hyalomitome can activate Caspase-9 and Caspase-3 and induce the apoptosis. (3) The accumulative Ca²⁺ obtained from mitochondrial release raise up the Ca²⁺ of hyalomitome, activate kinds of proteolytic enzyme and phospholipase intra-cellular, damage even disintegrate cell membrane and finally result in cellular necrosis. It can be concluded from the whole analysis that the occurrence of mitochondrial permeability transition is the crucial juncture which contributes to the transformation from reversible injury to irreversible injury^[11-12].

The relevant gene expression of mitochondrial apoptosis is mainly analyzed in this experience, and the result turns out that: oryctolagus cuniculus voltage-dependent anion channel 5, pseudogene mRNA and oryctolagus cuniculus solute carrier family 25 member 5 are expressed normally in the spinal cord injury group, and the expression volume is decreased after being treated by large dose of drugs, so obvious differences are displayed on the expression between drug group and spinal cord injury group. However, voltage-dependent anion channel 1, voltage-dependent anion channel 2, B-cell lymphoma 2, B-cell lymphoma 1, cytochrome b5 and RABEST086T-like cytochrome cxidase Va, Caspase 2, Caspase 3, Caspase 7, Caspase 9 and Caspase 14 appears no difference on expression between drug group and spinal cord injury group. Oryctolagus cuniculus voltage-dependent anion channel 5 pseudogene mRNA is the nuclear gene encoding which is voltage-dependent. Oryctolagus cuniculus solute carrier family 25 member 5 nuclear gene encoding mitochondrial protein mRNA is the nuclear gene encoding of mitochondrial bearer protein-adenine mononucleotide translocator. Supra citato, voltage-dependent anion channel pseudogene mRNA and solute carrier family 25 member 5 are the main components of permeability transition pore, adenine nucleotide translocase, voltage-dependent anion channel and cyclophilin D will combine into adenine nucleotide translocase-voltage-dependent anion channel-cyclophilin D trigeminal composite to stimulate adenine nucleotide translocase and voltage-dependent anion channel to change conformation and result in the occurrence of mitochondrial permeability transition which finally leads to cell death by mitochondrial mechanism. It is known to all, necrosis and apoptosis of partial histiocytes will occur after severe spinal cord injuries, and paralysis is the clinical manifestation of it. Large dose of methylprednisolone has the function of protecting neuromechanism if being applied prophase.

During this experiment, adenine nucleotide translocase and voltage-dependent anion channel increase their expressions at the prophase but decrease their expressions after being impacted by large dose of methylprednisolone. The experimental result indicates that apart from the functions of inhibiting inflammatory reaction, alleviating tissue water, repressing peroxidatic reaction of lipide and regulating the level of Ca²⁺, large dose of methylprednisolone also possibly producing a marked effect of anti-cell death by means of regulating the permeability of mitochondrial transformation hole. Obviously, this explaination just from the aspect of gene transcription level, and other gene function can be reflected on the regulation of translation or post-translation. The most important thing is that the ion permeation status of mitochondrial transformation hole essentially contributes to the occurrence of mitochondrial permeability transition. Therefore, more experiments are needed to further validate this phenomenon.

研究采用Agilent兔全基因组表达谱芯片分析技术，从基因层面探讨MPSS治疗急性SCI的分子机制。直接针对脊髓损伤组织进行研究，所得的结果更可信。评价脊髓损伤的程度和变化采用形态和功能相结合的综合评价方法，更客观、更科学。脊髓损伤动物模型的构建方法改进，更趋合理性。

甲基强的松龙是急性脊髓损伤的“有效药物”。 甲基强的松龙也是目前临床上用于治疗急性脊髓损伤的主要药物。此类药物治疗脊髓损伤的历史较长，实验和临床研究资料丰富，但争议也最多。目前仍无明确的证据表明其有效，也无法说明其无效。因此，迫切需要客观的证据明确甲基强的松龙对脊髓损伤的影响是有利还是有弊。对大剂量甲基强的松龙治疗急性脊髓损伤的机制的研究将进一步加深人类对脊髓损伤的了解和认识。脊髓损伤是人类最为严重的损伤。多年来许多学者致力于脊髓损伤的研究，取得了一定进展。然而，临床上脊髓损伤的治疗仍未取得重大突破。临床上，早期药物治疗，减压、稳定脊柱是治疗脊髓损伤的关键。其中，糖皮质激素是临床上使用最为广泛的药物。

根据美国国家脊髓损伤研究组的3次临床研究和日本相似的临床试验报告显示：甲基强的松龙是急性脊髓损伤的“有效药物”。 甲基强的松龙治疗脊髓损伤的历史较长，实验和临床研究资料最丰富，但争议也最多。目前仍无明确的证据表明其有效，也无法说明其无效，其原因在于不同的研究采用不同模型和评价的方式，更重要的是不同脊髓损伤程度之间的差异。

实验拟在成功建立脊髓损伤大白兔模型的基础上，将实验动物进行严格的分组和充分的对照，运用大剂量甲基强的松龙对脊髓完全性损伤和不完全性损伤模型进行治疗，采用病理学结合动物神经功能评定系统（BBB评分）对其疗效进行评定。在此基础上，重点探讨大剂量甲基强的松龙治疗急性脊髓损伤的分子机制：即采用Agilent兔全基因组表达谱芯片分析技术，从基因层面对大剂量甲基强的松龙治疗急性脊髓损伤的分子机制进行探讨。

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