腰椎间盘损伤后MRI表现及组织学改变的实验研究

目的：探讨山羊腰椎间盘损伤后不同时间点MRI表现和组织学改变。方法：15只山羊．人为刺伤L5／6椎间盘前纤维环全层和L6／7后部纤维环内层，在伤后3周、3个月、6个月进行腰椎MRI检查观察髓核和纤维环的信号改变，光镜下观察实验椎间盘后部纤维环组织学改变。结果：L6H椎间盘损伤后部纤维环内层后．在实验期间未发生愈合，通过MRI定性、定量分析发现，椎间盘出现退变，并随时间进行性加重（3周，3、6个月）．且退变过程较L5／6加速。他像上未发现一例出现HIZ。结论：经山羊椎间盘前方损伤后部纤维环内层后．纤维环内层很难愈合，早期出现椎间盘退变。     

腰椎间盘退变动物模型的建立

目的　建立腰椎间盘退变的动物模型并行组织学和MRI观察。　方法　健康成年恒河猴 8只 ,手术组 6只 ,在其腰4 ,5、腰5,6、腰6,7椎间盘分别作一 5mm× 3mm切口 ,分别于术前及术后 1、4、8、13、2 0周对手术椎间盘行MRI观察 ;8只恒河猴分别于术前 (对照组 )及术后 4、8、2 0周随机取 2只处死后行椎间盘组织学观察。　结果　正常成年恒河猴腰椎间盘组织学结构特点与人类相似 ;手术椎间盘高信号区域面积的百分比于术后 8、13、2 0周显著减少 (P <0 0 5 ) ;术后 4周椎间盘切口外被纤维组织覆盖 ,术后 8周 ,切口外层纤维样愈合 ,其内无愈合迹象 ,术后 2 0周 ,切口向椎间盘内延伸 ,部分髓核出现纤维化。　结论　恒河猴是一种生理特性与人类相接近的动物 ,以其为对象应用纤维环切开法可以获得可靠的腰椎间盘退变动物模型 ;通过测量MRI轴位T2加权像椎间盘高信号区域面积百分比的方法可于早期无损伤地发现椎间盘退变。     

可控轴向压力致兔腰椎间盘退变模型的建立及评价

[目的]建立可控轴向压力诱发的椎间盘退变动物模型并考察其特性.[方法]取4个月龄日本大白兔40只,将其随机分为5组(每组8只).采用基于横穿椎体的克氏针的自制加压装置,分别对2～4组动物腰椎间盘施加10 kg压力1、2、4周及8周,第1组作为假手术对照组.Thompson分级法及兔腰椎间盘磁共振(MRI)评价退变程度,HE染色及Ⅰ、Ⅱ型胶原免疫组化染色考察其组织学改变.另外构建纤维环损伤致兔腰椎间盘退变模型,并对标本行HE染色以供比较.[结果]①Thompson分级:第2组7只均为Ⅱ级,第3组6只Ⅲ级,第4组5只为Ⅳ级,第5组4只动物退变程度达到V级.②MRI评分显示,第2组椎间盘以轻度退变为主;第3组椎间盘主要为中度退变.第4组、第5组则以重度退变为主.③HE染色显示,该模型椎间盘各区域的退变情况较纤维环损伤模型更为均匀.④免疫组化染色显示,退变过程中椎间盘基质成分退化的趋势与人体相符.[结论]可控轴向压力致兔腰椎间盘退变模型可以高效诱发椎间盘退变,尤其在轻、中度椎间盘退变方面具有优势.该模型可供深入研究,并用于椎间盘退变的治疗研究.     

纤维环穿刺诱导椎间盘退变动物模型的实验研究

目的：探讨纤维环穿刺诱导椎间盘退变建立动物模型的可行性。方法：新西兰大白兔24只，用持针器夹持18G皮肤穿刺针从左前外侧刺人L3/4、L4/5、L5/6椎间盘的纤维环，深度控制在5mm。术前及术后3、6、10周对造模后的椎间盘及对照的椎间盘（L2／3）行MRI检查，并行免疫组化及组织学观察。结果：术后第3周到第10周，造模后的椎间盘MRI T2WI信号呈现持续减弱趋势，免疫组化及组织学观察发现髓核细胞的数量及Ⅱ型胶原含量较对照间盘进行性减少（P〈0．01）。结论：纤维环穿刺法可以诱导兔椎间盘的缓慢退变，为研究椎间盘的退行性变提供有效的动物模型。     

纤维环穿刺抽吸法和纤维环切开法建立兔椎间盘退变模型的比较

[目的]通过纤维环穿刺抽吸法和纤维环切开法建立兔椎间盘退变模型,分析此两种方法的可行性及特点.[方法]取10个月龄健康日本大耳白兔24只,随机分为2组,其中A组为纤维环穿刺抽吸组,利用18G穿刺针分别在兔L3、4、L4、5、L5、6椎间盘刺入并抽吸髓核8～ 12 mg;B组为纤维环切开组,利用手术刀分别在兔L3、4、L4、5、L5、6椎间盘纤维环上做水平位切口;造模完毕,两组兔分别于术后4、8、12、16周进行MRI检查,病理组织学观察和免疫组化染色观察.[结果]A、B两组退变椎间盘MRI T2加权像信号随时间延长呈现持续减弱趋势,术后4、8周纤维环穿刺组T2加权像信号强度评分较纤维环切开组低(P＜0.05),组织学观察发现造模组髓核细胞逐渐减少;免疫组化染色观察Ⅱ型胶原和蛋白聚糖表达含量随时间延长逐渐降低,且B组含量明显低于A组.[结论]两种方法均能建立兔椎间盘退变模型,但纤维环切开法椎间盘的退变程度较纤维环穿刺法出现较早且较为剧烈.     

经皮穿刺纤维环建立兔腰椎间盘退变模型

目的探讨使用自制穿刺针经皮穿刺纤维环制备兔腰椎间盘退变模型的可行性。方法将18只新西兰大白兔分为实验组、假手术组及空白对照组。实验组及假手术组使用自制穿刺针穿刺L_(3~4)、L_(4~5)和L_(5~6)椎间盘位置,实验组穿刺椎间盘深度为5 mm,假手术组钝性穿刺但不损伤椎间盘,空白对照组不作处理。术后3、6、9周每组取2只兔麻醉后行腰椎MRI检查,处死行大体观察并取椎间盘行HE染色及髓核蛋白多糖含量测定。结果术后3周开始实验组MRI信号强度、髓核蛋白多糖含量与其他两组相比明显下降(P0.05),其后呈逐渐下降趋势,大体观察及HE染色显示实验组髓核及纤维环呈逐渐退变趋势。结论自制穿刺针经皮穿刺纤维环法能成功建立兔腰椎间盘退变模型,具有操作简单、损伤小、动物存活率高等优点。     

椎体前路钢板加压诱导羊椎间盘退变的实验研究

目的 建立新型的山羊腰椎间盘退变动物模型.方法 8只6月龄山羊,取腰椎右侧腹膜后入路,L3～4、L4～5椎间盘为目标椎间盘,于L3、L5椎体前外侧给予重建钢板加压固定.术后6个月对正常及目标椎间盘行MRI检查和组织学检查,观察MRI T2加权像上的椎间盘信号区的变化和HE染色下椎间盘组织形态学变化.结果 7只山羊存活,1只山羊因麻醉原因死亡.6个月时MRI检查显示目标椎间盘T2加权像信号明显降低,组织学观察发现目标椎间盘出现髓核纤维化和纤维环退化破裂的退变表现.结论 山羊椎间盘组织学结构特点与人类相似,通过椎体前路钢板加压的方法可以建立羊的椎间盘退变模型.     

腰椎间盘退变动物模型的建立及影像学改变

目的建立腰椎间盘退变的动物模型。方法取新西兰大白兔6只,采用腹膜后入路进行腰椎手术,L3/L4椎间盘作为对照椎间盘;L4/L5椎间盘作为假手术椎间盘,只进行暴露;L5/L6椎间盘作为处理椎间盘,手术暴露后用24 G针头从椎间盘的前外侧针刺3次。4周后通过X线片观察针刺损伤对椎间盘高度的影响,通过MRI观察椎间盘在针刺损伤后退变的程度。结果腹膜后入路可以很好的显露新西兰大白兔腰椎间盘,所有动物均成活至术后4周,统计学分析说明针刺、手术暴露以及对照对术后4周椎间盘高度、MRI的T2加权信号强度的影响不同。用Newman-Keuls法对3样本均数两两比较结果显示：L3/L4与L4/L5之间的均数比较P〉0.05;L3/L4和L5/L6之间的均数比较P〈0.01;L4/L5和L5/L6之间的均数比较P〈0.01。结论术后4周针刺损伤实验动物椎间盘可以造成椎间盘高度、MRI的T2加权信号强度的降低,具有统计学意义。椎间盘损伤是造成椎间盘退变的原因之一。     

应用微创技术建立恒河猴腰椎间盘早期退变模型

目的 应用CT定位,经皮穿刺纤维环诱导恒河猴腰椎间盘退变,建立灵长类动物腰椎间盘早期退变模型.方法 恒河猴13只,随机分为三组:(1)造模组:在CT定位下,用20G穿刺针从左侧后方入路经皮穿刺L1,2:(n=12),L2,3、L3,4、L4,5、L5,6(n=13)椎间盘的纤维环全层至椎间盘髓核正中,共64个椎间盘.(2)穿刺对照组:15G穿刺针穿刺1只猴的L1,2椎间盘.(3)正常对照组:L6,7,L7-S1,共26个椎间盘.造模前及造模后4、8、12周对各组椎间盘行MRI检查,并行HE、Masson、番红O、免疫组织化学染色组织学观察.结果 (1)MRI:20G穿刺针穿刺的造模组椎间盘造模前及造模后4、8、12周,椎间盘信号强度按Pfirmann分级均为Ⅰ级.15G穿刺针穿刺椎间盘4周时信号降低(Pfirrmann Ⅲ级),8周时为黑色椎间盘(Pfirmann Ⅳ级).正常对照组椎间盘为Pfirmann Ⅰ级.(2)组织学:造模组椎间盘造模后4周未见改变,8周时HE染色示髓核内细胞数减少,12周时较为明显.Masson染色4周未见改变,8周时各层纤维间出现裂隙,12周时裂隙增宽.番红O染色见8、12周髓核内蛋白聚糖进行性减少.免疫组织化学结果显示4周和8周时同正常椎间盘比较差异无统计学意义(P＞0.05),12周时,Ⅱ型胶原合成减少(P＜0.05).15G穿刺对照组在8周时HE染色见髓核内细胞减少明显,Masson染色见纤维环各层间裂隙明显,呈波浪状.番红O染色示髓核内蛋白聚糖数量明显减少.免疫组织化学染色示Ⅱ型胶原合成减少.正常对照组在各时间点未见到形态学改变.结论 20G穿刺针可以诱发椎间盘缓慢进展的轻度退变.MRI平均信号强度观察椎间盘轻度退变时,不是敏感的指标,需要依靠组织学证实.     

纤维环穿刺法建立兔椎间盘退变模型

[目的]通过纤维环穿刺法建立兔椎间盘退变模型,分析其特点.[方法]将16只新西兰大白兔随机分为2组.经腹膜外入路暴露X2、3、L3、4椎间隙.A组为纤维环穿刺组,采用16号针头穿刺,B组为假手术对照组.术后2,4,6,8周通过MRI和组织病理学检查观察髓核变性及组织病理情况.[结果)纤维环穿刺组髓核信号强度在术后呈现逐渐降低趋势,髓核面积逐渐缩小,椎间隙高度也逐步下降,从术后4周开始两组差异有统计学意义(p＜0.O5 ).随着时间的进展,纤维环穿刺组髓核内细胞含量逐渐减少.[结论]纤维环穿刺法可成功建立椎间盘退变模型,比较真实地模拟了人类椎间盘损伤后的退变过程.     

FasL在家兔退变髓核组织中的表达及其与白细胞介素-6和肿瘤坏死因子-α的相关性

目的 观察生理解剖学屏障与FasL的分子生物学效应之间的关系,探讨椎间盘退变的发病机制.方法 采用18规格的皮肤穿刺针穿刺家兔纤维环,在术后的3、6、10周收集正常及穿刺后的椎间盘组织,免疫组织化学染色观察FasL及白细胞介素(IL)-6和肿瘤坏死因子(TNF)-α等炎性因子的表达.结果 正常髓核组织中可见少许髓核细胞质中FasL呈弱阳性染色(8.7%),实验组髓核细胞质则呈强阳性染色(41.6%、48.8%、46.4%).FasL阳性细胞百分比在正常组与各实验组之间的两两比较,差异有统计学意义(P<0.01).实验组髓核细胞FasL阳性表达百分比与IL-6及TNF-α阳性表达百分比之间显著相关(r=0.424,P<0.05及r=0.527,P<0.01).结论 FasL与理解剖学屏障的共同作用,可能是使髓核组织产生免免效应的重要因素.当生理解剖学屏障受到损伤后(如穿刺纤维环),FasL、IL-6及TNF-α的表达程度增强,FasL所介导的免疫炎性反应是引起椎间盘退变的重要机制之一.     

Effect of anular repair on the healing strength of the intervertebral disc: a sheep model

STUDY DESIGN: The intervertebral disc, in a sheep model, was used to assess the effect of directly repairing three different anular incisions on the subsequent healing strength of the intervertebral disc. OBJECTIVES: To assess whether directly repairing an anular defect, made at the time of lumbar discectomy, could influence the healing rate and strength of the anulus fibrosus. METHODS: Twenty-four sheep underwent a retroperitoneal approach to five lumbar disc levels. An anular incision, followed by partial discectomy was done at each exposed level. Anular incisions used in this study consisted of 1) a straight transverse slit, 2) a cruciate incision, and 3) a window or box excision. Healing strength was measured at three time intervals: 2 weeks, 4 weeks, and 6 weeks. Each anular incision type was performed on 30 lumbar discs, 10 discs in each time interval. Five discs in each time interval underwent direct repair, and five discs were left unrepaired to heal as controls. The sheep were killed at 2, 4, and 6 weeks after surgery. The lumbar spines were removed en bloc, and the intervertebral discs were subjected to pressure-volume testing to assess the anular strength of repaired versus unrepaired disc injuries at each time interval. RESULTS: Statistical analysis was performed to evaluate the effects of healing time, incision technique, and repair on the pressure-volume characteristics of the involved discs. Pressure-volume testing showed trends of stronger healing for repaired discs, but at no time interval was any significant difference found between repaired and nonrepaired anular strength. Of the nonrepaired discs, the box incision was only 40 to 50% as strong as the slit or cruciate incised discs during early healing. CONCLUSION: Direct repair of anular incisions in the lumbar spine does not significantly alter the healing strength of the intervertebral disc after lumbar discectomy.     

Needle puncture injury of the rat intervertebral disc affects torsional and compressive biomechanics differently

Needle puncture is a common method of inducing intervertebral disc (IVD) degeneration in small animal models and may have some similarities to IVD injury conditions such as herniation. Yet, the influence of puncture injuries on IVD biomechanics is not well understood. This study quantified the acute effects of anular injury on the biomechanics of rat caudal IVDs in compression and torsion following puncture with 30, 25 and 21 G needles. In compression, puncture injury reduced elastic stiffness by 20% for all needle sizes, but differences between control and punctured discs did not remain after compressive overload. In contrast, torsional parameters associated with anular fiber tension were affected proportionally with needle size. We conclude that IVD injuries that penetrate through the thickness of the annulus affect IVD biomechanics through different mechanisms for compression and torsion. Anular injuries affect torsional properties in a manner directly related to the amount of fiber disruption and compressive properties in a manner that affects pressurization.     

两种骨水泥渗漏对椎间盘影响的实验研究

目的 探讨椎体成形术时骨水泥渗漏是否会引起椎间盘退变，以及椎间盘退变程度与骨水泥类型是否相关。方法 选用8只成年家犬，以每只犬L2-3、L3-4、L4-5椎间盘为实验对象，随机分为对照组、聚甲基丙烯酸甲酯（polymethylmethacrylate，PMMA）与磷酸钙骨水泥（calcium phosphate cement，CPC）3组。对照组仅行椎间盘穿刺，不注入任何物质，PMMA组及CPC组均各向椎间盘注入0．1ml骨水泥。术前及术后24周摄正、侧位X线片，计算椎间盘高度指数百分数（disc height index percentage，DHIP）。术后24周行MR检查，计算MRI指数。组织学检查参照Masuda标准对椎间盘退变程度评分并分析。结果 术后24周X线片显示对照组椎间隙无狭窄，病理学检查未见椎间盘退变。PMMA、CPC组椎间盘MRI显示：椎间隙有狭窄，R加权像髓核信号不同程度降低且不均一，其相对高信号区面积减小，髓核形态不规则，纤维环与髓核界限不清。组织学检查显示髓核细胞数量不同程度减少，空泡变小。髓核的细胞外基质不同程度压缩，纤维环断裂或扭转。3组DHIP、MRI指数、组织学评分的差异均有统计学意义（P〈0.01）。结论 PMMA、CPC注入椎间盘会导致椎间盘退变，PMMA所致椎间盘退变较CPC更为严重.     

Part 2: quantitative proton t2 and sodium magnetic resonance imaging to assess intervertebral disc degeneration in a rabbit model

STUDY DESIGN.: Comparison of sodium concentration ([Na]) and proton T2 relaxation time between normal and degenerated discs in a rabbit model. OBJECTIVE.: The purpose of this article was to evaluate quantitative [Na] and T2 characteristics of discs associated with degenerative changes. SUMMARY OF BACKGROUND DATA.: Intervertebral disc degeneration is a common chronic condition that may lead to back pain, limited activity, and disability. Noninvasive imaging method to detect early intervertebral disc degeneration is vital to follow disease progression and guide clinical treatment and management. METHODS.: Dual-tuned magnetic resonance imaging of rabbit discs was performed using 3T. Thirteen rabbits were included in the study; 6 control rabbits (24 normal discs) and 7 rabbits with annular puncture-induced disc degeneration (9 degenerated discs, 19 intact internal-control discs). Dual-tuned magnetic resonance imaging of discs was performed at baseline and 12-week poststab. [Na] and T2 were measured and compared among 3 groups of discs. RESULTS.: The mean [Na] were 274.8 ± 40.2 mM for the normal discs, 247.2 ± 27.7 mM for the internal-control discs, and 190.6 ± 19.1 mM for the degenerated discs. The corresponding T2 for 3 groups were 97.1 ± 12.1 ms, 93.7 ± 11.9 ms, and 79.0 ± 9.1 ms, respectively. The [Na] is highly correlated with the T2 in the degenerated discs (r = 0.90, P < 0.01). The mean percent decreases from the normal to degenerated discs were in 30.6% in [Na] and 18.6% in T2, whereas those from the internal-control to degenerated discs were 22.9% in [Na] and 15.6% in T2. CONCLUSION.: Although both [Na] and T2 changes in discs were associated with the disc-punctured rabbits, greater change in [Na] is observed at 12-week poststab compared with T2 change. Because T2 and [Na] reflect different disc properties, performing both imaging under same condition will be helpful in the evaluation of disc degeneration.     

The effect of needle size inducing degeneration in the rat caudal disc: evaluation using radiograph,magnetic resonance imaging,histology, and immunohistochemistry

Background context

The rat caudal disc has been increasingly used in studying of disc degeneration because of its simplicity, low cost, and efficiency. However, the reproducibility and standardization are essential to facilitate the investigations of biologic therapeutics at different stages of degeneration.

Purpose

To identify the effect of different needle gauges to the degenerative response in rat caudal discs and to examine its pathogenesis by looking at the cellular and matrix changes.

Study design

In vivo study of injury-induced rat caudal disc degeneration using needle puncture.

Patient sample

Thirty-six Lewis rats aged 12–14 weeks.

Outcome measures

The induced degenerative discs were analyzed by plain radiograph, magnetic resonance imaging (MRI) and histological examination. Proteoglycan content was assessed by alcian blue stain. Immunohistochemistry using aggrecan, collagen II, and Sox-9 was also evaluated to investigate cell differentiation and matrix changes.

Methods

All rats were divided into three groups according to different needle gauges (18G, 20G, and 22G). Caudal discs were punctured percutaneously under image guidance. Radiographs and MRI were obtained at 2 weeks interval until 8 weeks. At each time point, three rats from each group were sacrificed for histological analysis and immunohistochemistry.

Results

Larger needle gauges, especially 18G, produced more deterioration of the disc when compared with smaller sizes, particularly with time. Significant differences were identified in almost all parameters compared between 18G and 22G at the 8-week time point. For the effect of time in the same needle size, the differences occurred between 2- or 4-week and 8-week time point in the 18G and 20G groups. The proteoglycan and aggrecan stain gradually decreased over time. Chondrogenic differentiation was identified within the degenerative disc by detecting Sox-9 positive cells and collagen II accumulation increased as degeneration progressed.

Conclusions

The puncture-induced degenerative changes in rat caudal discs can imitate the human degenerative cascade as observed in plain radiograph, MRI, histology, and immunohistochemistry. We suggest that needle size affects the occurrence of progression of degeneration; thus, the large needle size was required to accelerate the deterioration. The size of needle and time point after injury should be considered when investigating the effect of therapeutic materials to retard degeneration or regenerate the intervertebral disc.     

Biomechanical factors influencing nuclear disruption of the intervertebral disc

STUDY DESIGN: A disc model with full anular division was used to investigate how different biomechanical parameters influence the severity of nuclear disruption during compressive loading. OBJECTIVE: To quantify the manner in which flexion, hydration, and loading rate contribute to the breakdown in the intrinsic cohesive structure of the nucleus pulposus. SUMMARY OF BACKGROUND DATA: The risk of disc herniation is known to increase when the disc is loaded in flexed positions. However, there is a lack of experimental data showing how a combination of flexion with different loading rates and hydration levels affects the extent of nuclear disruption. METHODS: A reproducible state of full hydration was established for isolated bovine caudal discs. A period of static preloading at an applied stress of 1 MPa was used to obtain a consistent state of partial hydration. Then 96 discs were subjected to a full-thickness division of the anulus fibrosus and compressed while hydration level, degree of flexion, and rate of loading were varied systematically. RESULTS: A full spectrum of nuclear damage was observed in the tests, ranging from no detectable disruption to sudden sequestration of the entire nucleus. These results were quantified, and a general correlation was established between the severity of disruption and the different loading parameters. CONCLUSIONS: The degree of flexion and the level of hydration were shown to play an important role in influencing the tendency of the nucleus to break loose and extrude through a preexisting anular division. Interestingly, the rate of loading appeared to have only a minor effect on the severity of damage induced in discs that incorporated a full depth anular division.