| BackgroundTrauma spinal cord injury (SCI) is a common disease in the orthopaedic. In recent years, with the development of economic society and transportation network expands, the incidence of accidents increased year by year, and the rate of spinal cord injury also has a tendency to rise year by year. According to the previous report, it was about 10000 SCI new patients every year in the United States, because of China’s population base number are the number of times of the United States, so SCI new patients every year occurred in our country should be far more than 10000 patients. For SCI patients, currently, it often cannot obtain satisfactory results due to the lack of obvious effective treatment, and it can increase social and economic burden of the families. For young people with serious SCI, which means that their life cannot be independent life by themselves, and SCI also causes an enormous effect on their body and mind. The treatment of SCI began in the Second World War in England, subsequently, a number of SCI treatment centers occurred in the United States. Even after decades of development and the study of etiology and treatment methods for SCI patients, however, currently, how to reduce the disability caused by SCI, and how to improve the cure rate after SCI, it is still a worldwide problem. At present, the commonly used drug therapy methods include:GM-1 ganglioside, brain-derived neurotrophic factor (BDGF), methylprednisolone (MP), etc.GM-1 ganglioside is an important part of the normal cell membrane and very rich in the mammalian central nervous system, especially, its content is much higher in the synapse. Previous studies have suggested that the changes of GM-1 in concentrations on the cell membrane play an important role in adjusting neurons and cells inside and outside information. The animal studies have shown that GM-1 could improve locomotor function for SCI animals. It is possible that the mechanisms of GM-1 include:GM-1 could protect neurons after SCI by up-regulation MAP-2 (microtubule-associated protein 2) expression, this is helpful to improve locomotor function, and it also could reduce apoptosis by increasing the BCL-2 the expression and reduce the degree of tissue edema through stabling the balance of intracellular Ca2+, and reduce the lipid peroxidation and the toxicity of excitatory amino acids of EAA, promote the nerve regeneration, etc. A randomized, placebo-controlled trial with GM-1 ganglioside on the treatment of SCI patients was conducted by Geisler and co-workers,37 patients were included in the study, but finally only 34 patients completed the whole experiment:23 patients with cervical SCI and 11 patients with thoracic SCI, in addition, each SCI patient received GM-1 treatment within 72 h after injury, subsequently, all patients were followed up for 1 year and Frankle rating scale and the neurological recovery of each patient was assessed with the Frankel scale and the American Spinal Injury Association (ASIA) motor scale, the study found that GM-1 ganglioside could improve motor function in patients with SCI, however, this study sample size is less, it is not able to provide strong clinical evidence that GM-1 ganglioside has obvious therapeutic effect on SCI patients, in addition, The price of GM-1 ganglioside is very expensive, so not all families can undertake long-term treatment costs, its application is limit to some extent. Find a drug which price is moderate and obvious effectivity for SCI patients become very important in the clinical practice. In addition, the effects of GM-1 ganglioside treatment on SCI patients still need further clinical research.BDNF could protect the damage of spinal cord, promote the nerve regeneration and repair, improve the motor function, and spinal cord also produces a certain amount of nerve growth factor after spinal cord injury, but the quantity is a little. BDGF is not well used for the clinical practice, because of BDGF in the blood is extremely easy to be decomposed before it through the blood brain barrier, it can not meet the concentration effect of drug treatment, because the vast majority of BDGF were inactivated. although Liang and co-workers reported:SCI rats of spinal cord were buried pipes under the section of the spinal cord through the hose regular injections of BDGF treatment and achieved good results, but it is lack of large-scale clinical trial results to prove that it is effective for SCI patients, in addition, even if the injury medication buried pipes under the section of the spinal cord have a certain effect, but it is very difficulty achieved for SCI patients in the clinical practice. For the spinal cord under the buried pipe buried, on the one hand, it needs high piercing technology in order to avoid aggravating SCI, on the other hand, it easily caused infection, requires great care, once the infection happened, it not only can aggravate the condition and even death in SCI patients. Currently, BDGF is very difficult to application for the clinical practice, unless researchers can find the good shipping carrier, which make BDGF won’t decompose in the blood.MP is a preferred method of clinical treatment for patients with acute SCI, and it must be given to patients within 8 hours after injury, otherwise treatment effect will be greatly reduced. But the application of high-dose of MP can cause many serious complications, such as gastrointestinal bleeding, intestinal perforation, aseptic necrosis of femoral head, increasing the chances of infection and so on. Moreover, a lot of clinical treatment centers are no longer as a treatment of SCI at present, and even some treatment centers completely give up this kind of treatment method. Currently, the previous study has shown that the application of recombinant human erythropoietin (rhEPO) treatment of acute spinal cord injury in rats could better improve the motor function when compared with high-dose of MP therapy.Erythropoietin (EPO) which is a glycoprotein and cytokine produced mainly by the fetal liver and the adult kidney in the regulation of red blood cell production has recently received more attention from researchers, and the hypoxia is a vital factor to significantly induce the expression of EPO. RhEPO has similar effect to EPO biology function. Currently, EPO and its analogues have been widely used to treat some diseases in the clinical practice, such as chronic kidney disease, anemia, cancer, hemodialysis patients, surgery treatment, etc. Many different types of animal models of SCI had confirmed rhEPO protective effect of acute thoracic SCI, but the experimental animals got rhEPO treatment within 1 hour after injury. However, two previous studies didn’t find that the effects of rhEPO administration on the rat spinal cords were effective, and Mann and co-workers delayed treatment of SCI with rhEPO didn’t exhibit neuroprotective efficacy. So further systemic study is necessary to identify whether rhEPO therapy is effective, especially, SCI animals receive rhEPO treatment more than 1 h after injury. The aim of this study was to comprehensively and systemically evaluate the effectiveness of different does of rhEPO in the experimental acute thoracic SCI in a rat model of compression injury through locomotion recovery assessment, histopathological evaluation, apoptotic index, inflammatory index, electron microscopy and volume of areas of demyelination, when SCI animals received rhEPO treatment at 2 h after injury. Transmission electron microscopy was used to observe the ultrastructure of the SCI after the administration of rhEPO on days 4 after injury.PurposeTo comprehensively and systemically evaluate the effects of different dosage of rhEPO on rats acute spinal cord injury.MethodThe whole experiment was divided into two parts:the first stage and second stage. In the first stage, thirty-two SD rats were randomly divided into four groups: control group (group A; N=8), rhEPO-3,000U group (group B; N=8), rhEPO-4,000U group (group C; N=8) and rhEPO-5,000U group (group D; N=8). All rats with acute SCI were observed for 4 days after injury in order to locomotion recovery assessment, histopathological evaluation, apoptotic index, inflammatory index and electron microscopy. In the second stage, twenty-four SD rats were also randomly divided into four groups:control group (group Aa; n=6), rhEPO-3,000U group (group Bb; n=6), rhEPO-4,000U group (group Cc; n=6) and rhEPO-5,000U group (group Dd; n=6). The twenty-four rats with acute SCI were observed for 28 days after injury in order to locomotion recovery assessment and Luxol Fast Blue (LFB). Control group (group A and Aa):the spinal cord was clamped for one minute duration, and single dose 0.9% saline solution with infusion [5 ml/kg of intraperitoneal (i.p.)] was given at 2 h after injury. The same dosage 0.9% saline solution with infusion was given to rats on days 1 and 3 after injury by i.p. injection. In addition, the rats of group Aa also got the same dosage 0.9% saline solution with infusion (i.p.) on days 8,15 and 22. For preventing infection of the surgical incision, penicillin [200,000 U/kg of intramuscular (i.m.)] was given on day 1 after injury;rhEPO-3,000U group (group B and Bb):The spinal cord was clamped for one minute duration, and rhEPO (3,000 U/kg, i.p.) was given at 2 h after injury. The same dosage rhEPO (i.p.) was given on days 1 and 3 after injury. In addition, the rats of group Bb also got the same dosage rhEPO (i.p.) on days 8,15 and 22. For preventing infection of the surgical incision, penicillin (200,000 U/kg, i.m.) was given on day 1 after injury; rhEPO-4,000U group (group C and Cc):The spinal cord was clamped for one minute duration, and rhEPO (4,000 U/kg) with i.p. infusion was given at h 2, on days 1 and 3 after injury. In addition, the rats of group Cc also got the same dosage rhEPO (i.p.) on days 8,15 and 22. Penicillin (200,000 U/kg, i.m.) was given on day 1 after injury in order to prevent infection of the surgical incision; rhEPO-5,000U group (group D and Dd):The spinal cord was clamped for one minute duration, and rhEPO (5,000 U/kg) with i.p. infusion was given to rats at h 2, on days 1 and 3 after injury. In addition, the rats of group Bb also got the same dosage rhEPO (i.p.) on days 8,15 and 22. Penicillin (200,000 U/kg, i.m.) was given on day 1 after injury in order to prevent infection of the surgical incision.Building model of SCIPreoperatively, penicillin (200,000U/kg) was given to animals by i.m. injection in order to prevent infection of the surgical incision. All animals were fasted for 12 hours before surgery and humanely restrained. Animals were anesthetized with pentobarbital (70 mg/kg) by i.p. injection and then they were fixed in the prone position. Using scissors removed their back hair in the surgical area.3% Iodophor was used to disinfect surgical area for prophylaxis of infection. An midline incision about 2 centimeter was made from T8 to T12 regions, the overlying musculature separated laterally and a complete level (T10) laminectomy was performed using an operating microscope to expose the spinal cord. The extradural compression with a temporary aneurysm clip was performed on the spinal cord for 1 minute to induce crush injury. The surgical site was closed using nondegradable sutures after removing aneurysm clip, and then the closed skin incision was disinfected again by 3% iodophor. During procedure, body temperatures were maintained by exposing the animal to a heat lamp for recovery. After surgery, all rats received 2ml of 10% glucose solution and 5ml of 0.9% sodium chloride injection immediately and they were returned to their cages (2 rats/cage) when they completely recovered from anesthesia. In addition, the rats were underwent manual bladder evacuation, two or three times a day.Curative Effect Evaluation IndexBBB locomotion recovery assessment、histopathological evaluation、apoptotic index、inflammatory index, electron microscopy and volume of areas of demyelination.Statistical AnalysisSPSS version 19.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The data were expressed as means±standard deviation (SD) except for histopathological neuronal ischemic grade. Statistically significant differences between data were evaluated by one-way analysis of variance (ANOVA) followed by post-hoc least significant difference (LSD) test. For comparison between BBB scores of groups at different time points, the data were analyzed by two-way ANOVA with repeated measures and post-hoc LSD test. For nonparametric data, the Kruskal-Wallis test was used for comparing differences between groups. When ANOVA showed significant differences, the post-hoc multiple comparison was performed using the Nemenyi test. P<0.05 was accepted as statistically significant.ResultsIn the first stage of the whole experiment,32 SD rats were observed for 4 days, the different groups of BBB movement function score on days 4 after acute SCI were recorded:Group A (0.375±0.518), Group B (2.625±0.744), Group C (3.125±0.641) and Group D (4.500±0.756), respectively, in addition, The most significant locomotory functional improvements are observed after the administration of rhEPO (5,000 U/kg). Importantly, the mean BBB score of the experiment groups were better than group A, it were statistically significant between the experiment groups and control group (P<0.01). In the second stage of the whole experiment, a total of 24 SD rats was observed for 4 weeks, the rhEPO-treated groups and control group of BBB movement function score on days 28 after acute SCI were recorded:Group Aa (7.833±1.472), Group Bb(12.333±0.816), Group Cc(12.833±0.753) and Group Dd (14.500±0.548), respectively, moreover, the most significant locomotory functional improvements are observed in group Dd. In addition, the mean BBB score of the experiment groups were better than control group, it were statistically significant between group Bb, Cc, Dd and Aa (P<0.001).Importantly, according to the first and second of the experimental results, our study suggested that there was no significant between 3000U and 4000U group (P>0.05), although mean BBB score of 4000U group was slightly higher than 3000U group. It meant that 3000U and 4000U group had the similar results to improve BBB score. In the terms of histopathological evaluation, the most significant histopathological improvements was observed after the administration of rhEPO (5,000U/kg), in addition, there was no significant between group B and C (P>0.05), whereas the experimental groups of results were better than control group (P<0.05). The experimental groups of apoptosis and inflammation index were better than control group (P<0.05), but there was no significant between these experimental groups (P> 0.05). Moreover, The experimental groups of the ultrastructure score were better than control group (P< 0.001), but there was no significant between these experimental groups (P>0.05). In terms of volume of areas of demyelination,5000U got the best results and rhEPO-treated groupos markedly alleviate volume of areas of demyelination as compared with control group.Conclusion(1)、Our study suggests that delayed treatment of thoracic spinal cord injury with rhEPO exerts neuroprotection;(2)、Our data further confirm that the administration of rhEPO could reduce apoptosis, regulate inflammation, improve histopathology and motor fuctions and promote the regeneration of the spinal cord;(3)、The most significant locomotory functional and histopathological improvements and the best myelin protection were observed after administration of 5,000 U/kg rhEPO;(4)、3,000 U/kg,4,000 U/kg and 5,000 U/kg of rhEPO show the similar ultrastructural neuroprotection at 4 days post-injury;(5)、3,000 U/kg,4,000 U/kg and 5,000 U/kg of rhEPO show the similar results to inhibit apoptosis and regulate inflammation;... |
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