Department of Pharmacology, Uniformed Services University of Health Sciences, 4301 Jones Bridge Rd, Bethesda, MD 20814, USA.
Spine J. 2010 Jan;10(1):16-25. doi: 10.1016/j.spinee.2009.10.003. Epub 2009 Nov 14.
The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) and its indications for spinal fusion continue to be expanded with recent reports citing spinal trauma application. However, there are no data establishing the effects of rhBMP-2 on the injured spinal cord.
The purpose of this study was to evaluate the extent of bone morphogenetic protein (BMP)-specific intrathecal signaling after application to the spine at various time points after a spinal cord injury (SCI).
This is an in vivo rat study using a combination of the dorsal hemisection SCI and the posterolateral arthrodesis animal models.
Sixty-five female Sprague-Dawley rats underwent either a T9-T10 dorsal hemisection SCI (n=52) or laminectomy only (n=13). Spinal cord injury animals were further subdivided into four follow-up groups (n=13/group): 30 minutes, 24 hours, 7 days, and 21 days, at which time one of two secondary surgeries were performed: Eight rats per time point received either 43 microg of rhBMP-2 per side or sterile water control over T9-T11 on absorbable collagen sponges (ACSs). Animals were perfused after 24 hours, and spinal cords were immunohistochemically analyzed. Sections of the lesion were stained with BMP-specific pSmad 1, 5, 8 antibody and costained with cell-specific markers. pSmad-positive cells were then counted around the lesion. The remaining five rats (n=5/time point) had luciferase (blood spinal cord barrier [BSCB] permeability marker) injected through the jugular vein. Subsequently, spinal cords were collected and luciferase activity was quantified around the lesion and in the cervical samples (controls) using a luminometer.
After injury, a significant increase in the number of pSmad-positive cells was observed when rhBMP-2 was implanted at the 30-minute, 24-hour, and 7-day time points (p<.05). Costaining revealed BMP-specific signaling activation in neurons, glial cells, macrophages, and fibroblasts. Spinal cord permeability to luciferase was significantly increased at 30 minutes, 24 hours, and 7 days post lesion (p<.05). A significant linear regression was established between the extent of BSCB permeability and pSmad signaling (r(2)=0.66, p=.000).
Our results indicate that rhBMP-2 use around a spinal cord lesion elicits a robust signaling response within the spinal cord parenchyma. All CNS cell types and the invading fibroblasts are activated to the extent dependent on the integrity of the meningeal and BSCB barriers. Therefore, in the presence of a SCI and/or dural tear, rhBMP-2 diffuses intrathecally and activates a signaling cascade in all major CNS cell types, which may increase glial scarring and impact neurologic recovery.
骨形成蛋白-2（rhBMP-2）的使用及其在脊柱融合方面的适应证不断扩大，最近有报道称其可应用于脊柱创伤。然而，目前尚无数据表明 rhBMP-2 对受损脊髓的影响。
本研究旨在评估 rhBMP-2 在脊髓损伤（SCI）后不同时间点应用于脊柱时，脊髓内骨形态发生蛋白（BMP）特异性信号转导的程度。
这是一项使用大鼠背侧半切 SCI 和后路关节融合动物模型的体内研究。
65 只雌性 Sprague-Dawley 大鼠接受 T9-T10 背侧半切 SCI（n=52）或单纯椎板切除术（n=13）。SCI 动物进一步分为四个随访组（n=13/组）：30 分钟、24 小时、7 天和 21 天，此时进行两种二次手术中的一种：8 只大鼠/时间点，在吸收性胶原海绵（ACS）上接受 43μg rhBMP-2 双侧或无菌水对照。24 小时后进行灌注，对脊髓进行免疫组织化学分析。病变部位用 BMP 特异性 pSmad1、5、8 抗体染色，并与细胞特异性标志物共染色。然后计算病变周围的 pSmad 阳性细胞数。其余 5 只大鼠（n=5/时间点）通过颈静脉注射荧光素（血脊髓屏障[BSCB]通透性标志物）。随后，使用发光计收集脊髓，并在病变周围和颈段样本（对照）中定量测定荧光素的活性。
损伤后，在 rhBMP-2 植入 30 分钟、24 小时和 7 天时，pSmad 阳性细胞数显著增加（p<.05）。共染色显示 BMP 特异性信号转导在神经元、神经胶质细胞、巨噬细胞和成纤维细胞中激活。脊髓损伤后 30 分钟、24 小时和 7 天，BSCB 对荧光素的通透性显著增加（p<.05）。BSCB 通透性与 pSmad 信号之间建立了显著的线性回归（r（2）=0.66，p=.000）。
我们的结果表明，rhBMP-2 用于脊髓损伤部位会引起脊髓实质内强烈的信号反应。所有中枢神经系统细胞类型和侵入的成纤维细胞均被激活，其程度取决于脑膜和 BSCB 屏障的完整性。因此，在存在 SCI 和/或硬脑膜撕裂的情况下，rhBMP-2 可鞘内扩散，并在所有主要中枢神经系统细胞类型中激活信号级联反应，这可能会增加神经胶质瘢痕形成并影响神经功能恢复。
