【作者】 刘洋；

【导师】 贾堂宏；

【作者基本信息】 山东大学 ， 外科学， 2009， 博士

【摘要】 目的:由于低钠血症是颈脊髓损伤后常见、严重并发症,其发病机制不明,学术界对此长期存在争论,尚无一个确定性理论合理的解释这一临床现象。国内学者多倾向于两种学说:抗利尿激素分泌异常综合征（SIADH）和脑盐耗综合症（CSWS）。该两种学说被国外学者多用于解释颅脑疾患所致低钠血症,是否能直接或间接引用于脊髓损伤后低钠血症的解释,尚需商榷。还有学者提出心钠素学说和脊髓性盐耗学说。众多学说和相关研究均来自临床资料,无相关实验研究。在临床诊疗过程中,不可能建立一个无任何治疗干预的颈脊髓损伤并发低钠血症的自然病程进行观察和研究。因此,动物实验研究亟待开展。本研究接合自身临床经验,力图建立大鼠颈脊髓损伤并发低钠血症动物模型,希望通过动物模型为探究该疾患发病机制,开展相关治疗打下基础。方法:实验研究:1.准备阶段:对相关颈脊髓损伤并发低钠血症临床文献进行回顾性分析、总结,系统地掌握相关发病机制的推测理论和理论依据,明确热点问题,为实验研究提供理论基础,进而完成实验设计。依据低钠血症发生率与脊髓损伤程度和损伤平面高度呈正相关,确立Frankel A型颈脊髓损伤作为实验研究首选类型。低钠血症与水钠代谢密切相关,准确计量水钠摄入与排出是实验研究的关键。临床工作中,患者使用胃管鼻饲术可有效的记录和控制水钠摄入量;留置导尿术可精确计量排出水、钠量。上述两种方法均可应用于动物实验中。但长期下胃管喂养实验动物不易实现,往往采用胃造瘘术来达到上述目的。因此,采用经胃造瘘管推注营养液的方法量化水、钠摄入。2.预实验阶段:确立实验动物;对比实验动物标准饲料喂养和肠内营养乳剂（TP）喂养的异同;对比脊髓钝性打击伤、脊髓环形卡压伤和脊髓锐性横断伤三种脊髓损伤模型优缺点,选择合适脊髓损伤模型用于实验阶段研究;训练胃造瘘术和经胃造瘘管TP喂养术;训练导尿术,确立导尿对象。3.实验阶段:成年健康雌性Wistar大鼠45只,体重290±10克,随机分为无手术对照组A,手术对照组B,实验组C,每组各15只。A组大鼠未做任何手术干预,采用标准鼠粮和水喂养。术前B组和C组大鼠,均每日每只给于TP 60ml喂养。7天后,进行手术干预前,三组大鼠均鼠尾静脉取血测量血钠浓度。B组大鼠行颈后路椎旁肌剥离术和胃造瘘术,C组大鼠行颈脊髓完全横断损伤术、胃造瘘术和导尿术。术后两组大鼠均经胃造瘘管每天每只60ml TP喂养,连续每日鼠尾静脉取血测量血钠浓度。B组采用动物代谢笼的方法收集每日尿量,C组利用尿管收集大鼠每日尿量。测量每只术后大鼠每日平均尿钠浓度。实验完成后,B、C组大鼠CO₂法处死,进行尸检和组织学检查。所获数据采用SPSS13.0统计软件进行分析,数据以（?）±s表示,各项指标组间比较采用方差分析,以P＜0.05为差异有统计学意义。临床研究:自2005年2月-2008年8月住院的骨科病人中,选取32例因各种外伤致急性颈脊髓损伤或单纯颈椎骨折患者组成颈部损伤组（A组）,所有患者均于受伤后24h内入院。同时期,选出22例住院超过两周无颈脊髓疾患患者组成对照组（B组）,以上患者均排除糖尿病、高血压病、心脏病、肝肾疾病、全身肿瘤、结核等疾病。A组:男26例,女6例;年龄18-63岁,平均42.5岁,B组:男18例,女4例;年龄20-65岁,平均43.3岁。疾病种类:A组中,颈椎骨折脱位并脊髓损伤患者22例（颈4骨折2例,颈5骨折4例,颈6骨折5例,颈7骨折3例,颈5、6骨折4例,颈6、7骨折3例,颈4、5、6骨折1例）,无骨折脱位颈脊髓损伤8例（发育性颈椎管狭窄4例,后纵韧带骨化2例,颈椎间盘突出2例）,单纯颈椎锥体裂纹骨折2例（颈5、颈7各1例）。B组中,膝关节单纯外侧半月板损伤患者4例,膝关节单纯内侧副韧带Ⅰ度损伤4例,慢性腰腿疼痛8例,膝关节骨性关节炎患者6例。A组脊髓损伤分级:Frankel A级6例,B级9例,C级11例,D级4例,E级2例。对两组患者血压（BP）和脉搏,血、尿生化指标,每日出入水量,醛固酮激素（ALD）、心钠素（ANP）和抗利尿激素（ADH）进行观测。并对原发病和低钠血症开展治疗。所获数据采用SPSS13.0统计软件进行分析,数据以（?）±s表示,各项指标组间比较采用方差分析,以P＜0.05为差异有统计学意义。结果:实验结果:通过7天TP喂养大鼠,大鼠大便性状改变为粘稠糊样,每日不超过5mL,无腹泻发生。大鼠体重、行为也无明显变化。大鼠经胃造瘘管TP喂养也得到相同大便性状和排便量。C组3只大鼠于术后当日出现陈式呼吸,心脏穿刺取血后死亡,血液检测与术前血钠无明显变化后,此3只大鼠自C组排除。标准鼠粮喂养的A组大鼠血钠浓度和TP喂养的B组、C组大鼠术前血钠浓度对比,显示无明显差异（P＞0.05）,排除了TP喂养对大鼠血钠的影响。C组大鼠平均血钠浓度手术前、后减少了18.333±5.867mmol/1,B组术前、后平均血钠浓度减少了0.534±2.850mmol/1。两组大鼠手术前后血钠浓度变化值对比有明显差异（P＜0.05）,表明颈脊髓完全横断损伤干预后的C组大鼠于手术后均获得低钠血症。B组手术前、后平均血钠浓度对比无明显差异（P＞0.05）。C组术后经留置导尿准确计量尿量,自第2个24h尿量明显增高,均大于35mL,最高达50mL。C组大鼠24h平均尿钠浓度检测显示,自第3个24h平均尿钠浓度升高明显。B组大鼠按动物代谢笼的方法来收集术后尿液,尿计量不够精确,尿液易受鼠便污染。两组尿钠量仍然进行了对比,显示C组大鼠有过度尿钠流失。B组和C组大鼠所有刀口愈合良好,无红肿渗出等炎症表现。B组和C组大鼠处死后尸检均未发现胃造瘘口TP渗漏现象,造瘘管周有轻度粘连,腹腔未见炎性渗出。C组大鼠颈部肌肉缝合严密,未见脑脊液漏现象。去除颈6、7椎板后,可见机化血肿填塞于完全横断的脊髓两断端间。脊髓断端病理切片HE染色检查,脊髓正常神经结构消失,炎症细胞浸润。C组大鼠导尿管未见感染表现。临床研究结果:A组患者动脉收缩压、舒张压及心率均低于B组（P＜0.05）,A组患者坐起后收缩压与舒张压均低于平卧位（P＜0.05）,出现体位性低血压,B组患者坐起后收缩压与舒张压与平卧位相比无明显变化（P＞0.05）;A组平均血钠浓度明显低于B组（P＜0.05）,A组平均血氯浓度低于B组（p＜0.05）,但仍在正常范围内,两组间的血尿素氮、血钾浓度平均值无显著差异（P＞0.05）;A组患者每日平均尿量均明显高于B组,A组低钠血症患者呈现水代谢的负平衡（P＜0.05）,A组中24h尿氯的平均排出量高于B组（P＜0.05）,A组尿钠量可达B组两倍,观察到明显尿钠流失（P＜0.05）;A组ALD、ANP检测值均低于B组（P＜0.05）,而两组ADH检测值无显著性差异（P＞0.05）;所有低钠血症患者经治疗,血钠均获纠正,无一例死亡或出现严重脑部并发症。结论:结合临床和实验研究,颈脊髓损伤并发低钠血症的可能机制有:1.由于颈脊髓损伤使交感神经系统受到抑制,体内交感神经兴奋性下降后,通过肾交感神经抑制肾脏对肾素的合成与分泌,肾素—血管紧张素—醛固酮系统（RAS）障碍,ALD的合成、分泌也随之减少,使尿钠排出量增加,可导致低钠血症。2.颈髓损伤后,由于交感神经功能调节障碍,迷走神经支配占优势,心脏排血功能下降;瘫痪平面以下血管张力低下;躯体运动神经功能障碍,呼吸肌和下肢肌肉瘫痪,静脉血液回流减少,三者均使病人有效循环血量减少,血压降低,刺激压力感受器,ADH分泌阈值下降,ADH分泌增加,肾小管对水重吸收增加,可致稀释性低钠血症。3.RAS与ANP是作用相反、互相拮抗的两个系统,ALD分泌减少,导致对ANP的抑制减弱,使尿钠排除增多,可导致低钠血症。4.多种原因引起烦渴症状导致水的过分摄入,可致稀释性低钠。本研究认为水钠代谢调节途径是一个多方面,多机制,纷繁复杂的系统,其中任何一个方面出现问题,都会引起一系列的调节改变。颈脊髓损伤后可以产生多种导致水钠代谢紊乱的因素,这就使得颈脊髓损伤后低钠血症的发生机制必然是一个复杂的病理生理过程,决不能仅从某一角度进行分析。鉴于临床研究的局限性,我们运用大鼠作为研究对象,建立颈脊髓横断损伤模型。以符合大鼠生理需要量为基础,通过胃造瘘TP支持营养和尿管导尿,量化大鼠出入水量及钠量,观察大鼠血钠、尿钠、尿量变化。通过对照组排除干扰因素,C组大鼠均获得颈脊髓损伤后低钠血症,所用方法均是成熟实验技术,所获结果与临床观察结果相一致,首创了颈脊髓损伤后可供水钠代谢紊乱研究的动物模型,为明确发病机制打下基础。本模型具有操作技术成熟,可重复性强,经济成本低等优势。利用该模型可以进行颈脊髓损伤后长期自然病程观察;可以对调节水、电解质代谢的激素进行检测;可以建立不同脊髓损伤程度和不同脊髓损伤平面的大鼠模型,观察相应激素水平、水钠代谢的异同;可以开展神经电生理研究;还可以开展治疗干预相关研究,选择合理治疗方法,从颈脊髓损伤早期入手,预防或者减少、减轻严重并发症的出现。该动物模型应用前景广泛,不仅用于低钠血症的研究,还可针对颈脊髓损伤后其它电解质紊乱及一系列并发症进行深入研究。希望能通过它为治疗颈脊髓损伤后的严重并发症做出贡献。更多还原

【Abstract】 Objective Hyponatremia is a serious complication following cervical spinal cord injury（SCI）.The mechanism of the hyponatremia caused by cervical SCI is still in dispute now.Basically there are two explanations:One is the syndrome of inappropriate secretion of antidiuretic hormone（SIADH）.Another one is the cerebral salt wasting syndrome（CSWS）.Initially these two theories try to explain the blood sodium loss after the craniocerebral injury.Later they were also used to explain the hyponatremia after cervical SCI.The precise mechanisms underlying hyponatremia induced by cervical SCI is not clear.Somebody reported:The high plasma concentration atrial natriuretic factor（ANP） responded to excessive water and salt excretion in SCI patients.Many clinic cases were reported every year,there is no experimental detail reported to study hyponatremia after cervical SCI.So we decided to establish a novel rat model for mechanism investigation of hyponatremia following cervical spinal cord injury（SCI）,Method.Experimental study:1.Preparation phase of the experiment:Retrospective analysis of clinical reports of hyponatremia after cervical SCI.Some feature can be found.The incidence of hyponatremia was related to the degree of the spinal cord injury.So the Frankle A type spinal cord injury was used in the study.Hyponatremia is highly related with the metabolism of water and sodium.Precisely measuring the input-output amounts of water and sodium is the key of the experiment.Using gastrostomy feeding and urethral catheterization can resolve the problems.2.Test experiments:The rat has similar serum sodium（SNa） concentration with human being and has widely accepted as the experimental animal.All the experimental techniques, such as SCI surgery gastrostomy and Urethral catheterization,have successfully performed in rat formerly.Rat is omnivorous animal and very economy.Subsequently, we established the rat model for studying the hyponatremia following cervical SCI. Healthy adult Wistar Rats（16 rats,half male,half female with average body weight of 290±10g）.The rats were self-fed with enteral nutritional emulsion（TP）.The daily quantities of intake TP,the feces,as well as the changes of weight behavior and SNa concentration were recorded.The anatomy of spine and ventriculus of the rats were studied.The rat spinal cord can be experimentally transected,contused or compressed to produce a lesion.After comparison of the length of surgery,the quantity of bleeding,as well as the survival rate of various procedures to cause the cervical SCI, it was decided to use the rat SCT model was used in our study.The spinous process of T2 can reach a length of 7mm,longest of all vertebrae.According to the spinous process of T2,the 6th cervical vertebra（C6） can be exposed easily.Through the opened interval between the C6,7 spinous process,spinal cord can be intersected.The stomach capacity of the rats was measured after the gastrostomy.The TP was injected into the stomach gently and slowly through the catheter by syringe.The rat feces,as well as the changes of weight behavior and SNa concentration were recorded after gastrostomy feeding.Urethral catheterization was done with a catheter through the urethra..As male rat,its anatomy of the urethra structure is more complicated than female rat.We recommend female rat as experimental animal.3.Experiment: Forty-five female adult Wistar rats,were divided into three groups randomly,group A were fed with normal rodent food and had no surgery（control group）,group B were fed with enteral nutritional emulsion（TP） through gastrostomy after lesions of paravertebral muscles（sham group）,group C were fed with TP through gastrostomy after cervical spinal cord transaction（SCT） and urethral catheterization（SCI group）. Before done the operation,each rat both in group B and group C were fed with TP（60ml/day） for 1 week.The TP was in little battles,and the rats could catch the TP by themselves.Before operation,the blood samples were collected in group B and C, and the samples were also collected in group A at same time.After surgery,the blood samples were collected every 24 hours in group B and C.All the blood samples were collected from the tail veins and were analyzed by the electrolyte analyzer（Chiron 348,Chiron Diagnostics Corp.,MA,USA）.The urine volumes were collected every 24 hours in group C after surgery.The same thing did in group B by using metabolic cage.The dally average urinary sodium concentration was checked by the electrolyte analyzer.At the 4th day,the rats in group B and C were put to death by CO2 after blood and urine samples were collected.Autopsies were done to observe the locations with surgeries.The blood sodium concentrations of prior operation in three groups and the differences of blood sodium concentration between prior and post operation in group B and C were analyzed by one or two way ANOVA,with P＜0.05 considered significant.All data are presented as the means±S.E.M.Clinical study:54 patients admitted to the Orthopedic Department of Jinan central Hospital between 2005 and 2008 were studied.Group A:32 cases,6 were female and 26 were male,with a mean age of 42.5 years（range 18 to 63 years）;All patients suffered cervical injury and admitted in the same day of the injury,2 patients suffered only cervical fracture,30 patients of them suffered acute cervical spinal cord injury;Frankel classification: Grade-A 6 cases,Grade-B 9 cases,Grade-C 11 cases,Grade-D 4 cases,Grade-E 2 cases.Group B（control group）:22 cases,4 were female and 18 were male,with a mean age of 43.3 years（range 20 to 65 years）;4 patients suffered lateral meniscus injury,4 patients suffered medial collateral ligament injury,8 patients suffered chronically low back pain,6 patients suffered osteoarthritis of knees.All patient s had measurement including blood press（BP）,pulse,sodium,potassium,chlorine of blood and urine,blood urine nitrogen（BUN）,input-output amounts of water,ALD,ANP, ADH.All the data were analyzed by one or two way ANOVA,with P＜0.05 considered significant.All data are presented as the mean±S.E.M.Result.Experimental results:There were no changes in weights、behavior and activity after feeding with TP before surgery.At the 1st day after surgery,three rats undergone SCT were excluded from experiment because Cheyne-Stokes breathing was observed. In these three rats,the SNa concentration between prior and post of surgery had not changed much.All the other rats were in good condition during experiment. Compared to normal diet,the SNa was not changed much by TP feeding.After normal or TP feeding one week,all rats were collected blood sample before surgery.In group A,the average of SNa concentration is 140.27mmol/l;and the average of SNa concentration is 141.67mmol/1 in group B and C.There is no significant difference （P=0.185＞0.05） between these two feeding.At the fourth day after surgery,the SNa concentration loss or change was observed in group C and B.In rats undergone SCT injury,the average of SNa concentration decreased from 141.417 mmol/1 to 123.083mmol/1.In group B,the average of SNa concentration changed from141.867mmol/1 to141.333mmol/1.After surgery,the decreases of SNa concentration in group C and B was shown significant difference（P=0.000＜0.05）. The rats in group C developed hyponatremia four days after surgery.In group C,the urine was collected daily after surgery.The daily average urine volumes got obvious increase in group C since the 2nd day after operation.And the daily average urinary sodium concentrations got obvious increase since the 3rd day after operation.The urine volumes were collected every day in group B by using metabolic cage.The urine contaminated by pasty feces frequently.The differences of daily average urinal sodium between group B and C after surgery could be acquired obviously.Over natriuresis could be observed in group C compared with group B.At the 4th day,all the rats in group B and C were sacrificed by CO₂.Then autopsies were shown that there was no TP or urine leakage and infection of the gastric fistula or Urethral catheter.In the lesion area of cervical spinal cords of rats in group C,the complete ruptures of the spinal cords were observed,and hematomases were formation in the interspaces of lesion spinal cords.Clinical result:Comparison of group B that group A is correlated with a low blood pressure（BP）,high urine output,and low serum sodium, high urine sodium,low serum chlorine,high plasma ALD and ANP.There were no obvious serum potassium,urine nitrogen and plasma ADH changes in group A compared with group B.Orthostatic hypotension was observed in the patients of group A.All the cases of hyponatremia in group A was corrected during hospitalization. Conclusion:The hyponatremia induced by cervical SCI can be related to four factors. First,renal conservation of sodium may be impaired owing to the renin-angiotensin-aldosterone system（RAS） lost normal level cause by sympathetic denervation of the kidney or impaired renal blood flow.Second,The hypotension of cervical SCI is related to the disability of both the sympathetic and the somatic nervous systems in these subjects.The somatic nervous system lesion impairs breathing ability,manifested by decreasing vital capacity with progressively higher levels of paralysis.Breathing is the pulmonary pump that determines venous return and cardiac output and supports BP.The lowering of BP coincides with the loss of vital capacity as the level of paralysis becomes higher.This somatic disability also inactivates the assistance of leg muscle contraction in the return of venous blood from the lower extremities.Although the centrally disabled sympathetic system reacts by reflex to noxious stimuli with autonomic dysreflexia,it fails to respond to orthostasis with vasoconstriction to protect the circulation against gravity.ADH is secreted to exceptionally high concentrations in response to the reduced blood pressure.The stimulating effect of hypotension overcomes the inhibitory effect of hypotonicity. Thus,water is conserved despite serum hyponatremia.Third,The high plasma concentration ANP responded to excessive water and salt excretion in SCI patients. Fourth,the augmented water intake,evidenced by polyuria,provides ample substrate for the expression of ADH.In summary,the hyponatremia of SCI can be related to the effects of partially blocked sodium conservation,enhanced water conservation,and increased fluid intake.We used gastrostomy to feed the rats with TP.Urethral catheterization was used to collect the urine.The quantity of the input-output water and the sodium were precisely measured.After SCT,the changes of the urine volume, urinal sodium as well as SNa were recorded.Comparing with the data of groupB,we got the positive results,we primarily established the rat model of hyponatremia following cervical SCI.Next study also should be focused on the long term investigation and the continue change of the blood sodium concentration by this model.We will test more electrolytes and hormone changes in hyponatremia following cervical SCI by this model.Electrophysiologic and therapeutics study on hyponatremia of SCI will be done by this model.Hopefully,it will be helpful to study the mechanism and therapeutic approaches for SCI-associated hyponatremia.更多还原