Neutralization of the chemokine CXCL10 reduces apoptosis and increases axon sprouting after spinal cord injury

Spinal cord injury (SCI) is followed by a secondary degenerative process that includes cell death. We have previously demonstrated that the chemokine CXCL10 is up-regulated following SCI and plays a critical role in T-lymphocyte recruitment to sites of injury and inhibition of angiogenesis; antibody-mediated functional blockade of CXCL10 reduced inflammation while enhancing angiogenesis. We hypothesized, based on these findings, that the injury environment established by anti-CXCL10 antibody treatment would support greater survival of neurons and enhance axon sprouting compared with the untreated, injured spinal cord. Here, we document gene array and histopathological data to support our hypothesis. Gene array analysis of treated and untreated tissue from spinal cord-injured animals revealed eight apoptosis-related genes with significant expression changes at 3 days postinjury. In support of these data, quantification of TUNEL-positive cells at 3 days postinjury indicated a 75% reduction in the number of dying cells in treated animals compared with untreated animals. Gene array analysis of treated and untreated tissue also revealed six central nervous system growth-related genes with significant expression changes in the brainstem at 14 days postinjury. In support of these data, quantification of anterograde-labeled corticospinal tract fibers indicated a 60-70% increase in axon sprouting caudal to the injury site in treated animals compared with untreated animals. These findings indicate that anti-CXCL10 antibody treatment provides an environment that reduces apoptosis and increases axon sprouting following injury to the adult spinal cord.     

物理治疗促进坐骨神经损伤再生的实验研究

目的周围神经损伤是临床常见疾病,评估物理治疗对坐骨神经损伤后功能恢复的影响,为临床治疗提供一定参考。方法雌性Wistar大鼠64只,体重252～365g,随机分为A、B、C、D4组,每组16只。各组分离右侧坐骨神经后,B、C、D组钳夹坐骨神经造成损伤模型,A组不进行钳夹作为对照。术后第2天,B组未治疗,C组采用单纯电刺激治疗,D组采用电刺激、分米波和红外线综合治疗。分别于治疗后0、7、14、30d行坐骨神经功能指数(sciatic nerve function index,SFI)、神经传导速度(motor nerve conduction velocity,MNCV)检测,并取材行组织形态学观察和透射电镜观察,取治疗后30d切片行轴突图像分析。结果治疗后0、7d,B、C、D组SFI显著高于A组(P<0.05),B、C、D组间差异无统计学意义(P>0.05);治疗后14、30d,D组SFI显著降低,30d时与A组比较差异无统计学意义(P>0.05),B、C组SFI有所降低,但与A组比较差异仍有统计学意义(P<0.05)。治疗后0、7、14d,B、C、D组MNCV显著低于A组(P<0.05),C、D组与B组比较差异有统计学意义(P<0.05),14d时D组高于C组(P<0.05);治疗后30d,B、C组仍显著低于A组(P<0.05),但D组与A组比较差异无统计学意义(P>0.05)。治疗后0、7d,透射电镜观察各组仅见胶原蛋白和脂质成分;治疗后14、30d,B、C、D组可见大量雪旺细胞和再生神经纤维,D组最显著。治疗30d时轴突图像分析示,D组有髓神经纤维数、轴突直径及髓鞘厚度与A组比较差异均无统计学意义(P<0.05),有髓神经纤维数量和轴突直径显著高于B、C组(P<0.05),髓鞘厚度与C组比较差异无统计学意义(P>0.05)。结论物理治疗有促进大鼠损伤周围神经再生的作用。     

体内血管化组织工程室内进行组织工程研究的进展

目的介绍一种利用在体血管转移入组织工程室(血管化组织工程室)进行体内组织工程研究的新方法,总结目前血管化组织工程室内进行组织工程研究的进展。方法查阅国内外近年来在血管化组织工程室内进行组织工程研究的文献,并进行综合分析。结果血管化组织工程室内能构建出具有血管网络的脂肪组织、心肌组织等,常用的血管化组织工程室模型有动静脉分流环路模型和腹壁下浅动脉模型。结论体内利用血管化组织工程室进行组织工程研究的方法有望应用于临床。     

Repair of rat cranial bone defects with nHAC/PLLA and BMP‐2‐related peptide or rhBMP‐2

An ideal artificial substitute has good biocompatibility properties and is able to provide for rapid bone formation. Bone morphogenetic protein‐2 (BMP‐2) is considered as one of the most important growth factors for bone regeneration. In this study, a synthetic BMP‐2‐related peptide (designated P24) corresponding to residues of the knuckle epitope of BMP‐2 was introduced into a bioactive scaffold based on nano‐hydroxyapatite/collagen/poly(L ‐lactic acid) (nHAC/PLLA); its in vitro release kinetics was then measured. A 5 mm diameter cranial bone defect was created in the calvariae of 30 rats and randomly implanted with three groups of biomaterials: Group A (nHAC/PLLA alone); Group B (P24/nHAC/PLLA composite); and Group C (recombinant human BMP‐2 (rhBMP‐2)/nHAC/PLLA composite). The P24/nHAC/PLLA implants significantly stimulated bone growth similarly to the rhBMP‐2/nHAC/PLLA implants based on the radiographic and three‐dimensional CT evaluation and histological examination, thereby confirming the enhanced bone healing rate of these compounds compared with the stand‐alone nHAC/PLLA scaffold material. The osteoinductive ability of 3 mg P24 was similar to that of 1 µg rhBMP‐2. P24/nHAC/PLLA is a promising scaffold biomaterial for bone tissue regeneration. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1745–1752, 2011     

大鼠小体积肝移植后肝再生障碍的实验研究

目的探讨部分肝移植后肝再生状况和小体积移植物发生肝再生障碍的可能机制。方法实验分为大鼠全肝移植组（OLT）、50%部分肝移植组（50%PLT）和30%部分肝移植组（30%PLT）。分析各组术后肝功能的变化,通过免疫组化观察移植肝标本Cyclin D1和PCNA的表达,并对移植肝组织结构进行电镜观察。结果各组ALT和AST于术后24hr达到峰值,且30%PLT组上升显著。Cyclin D1和PCNA的免疫组化表达中,50%PLT组表达明显上调,而30%PLT组表达则明显抑制。电镜观察发现30%PLT组术后微观结构改变显著。结论 30%PLT组术后肝细胞增殖再生受到明显抑制,推测与肝细胞微环境的恶化和叠加的缺血再灌注损伤有关。     

锂剂促进周围神经损伤修复的初步研究

目的 探讨锂剂对周围神经损伤后神经再生的影响.方法 取48只雌性SD大鼠,制作大鼠右侧坐骨神经损伤动物模型,通过腹腔注射氯化锂,在不同时间点观察动物下肢活动情况,检测小腿三头肌神经电生理及肌湿重,并对损伤远端神经纤维的神经丝蛋白(NF200)、单核巨噬细胞抗原(ED1)、P-75和运动终板进行免疫组织化学染色观察.结果 损伤后4周,实验组动物下肢已接近正常行走步态,对照组右侧肢体仍明显跛行;损伤后2周和4周,实验组的复合肌肉动作电位(CMAP)波幅较对照组明显增大,两组间的差异有统计学意义(P＜0.05);损伤后4周、8周,实验组的小腿三头肌重量较对照组明显增大,两组间差异有统计学意义(P＜0.05);损伤后3 d,在距离损伤远端5 mm处,实验组坐骨神经纤维内NF200呈连续丝状染色,而对照组仍然是颗粒状染色;损伤后4周,在神经肌肉接头处,可观察到实验组肌肉运动终板有新生神经纤维支配,而对照组运动终板上无神经纤维支配,两组ED1及P75染色未见明显差别.结论 锂剂可有效促进周围神经损伤后的再生,但其机制仍需进一步研究.

Abstract：

Objective To evaluate the effect of lithium on nerve regeneration after peripheral nerve injury.Methods Sciatic nerve crash injury model was created on the right side of 48 female SD rats.Lithium was administered after the injury by intraperitoneal injection.Locomotion of the lower limbs, electromyography and wet muscle weight of the triceps muscles were measured at different time points after the injury.Changes of NF200, ED1, P-75 and motor end plate at the distal part of the injured nerve were detected by immunohistostaining.Results Four weeks after sciatic nerve crash injury, animals that received lithium injection restored near normal gait, whereas the control animals were limping.Two and four weeks after the injury, the lithium injection group had significantly higher CMAP amplitude than the control group.Four and eight weeks after the injury, the wet muscle weight in lithium injection group was significantly heavier than the control group.Three days after crush injury, continuous NF200 positive fibers were found 5 mm distal to the injury site in the lithium injection group, whereas in the control group, only granular NF200 positive staining was observed Four weeks after crush injury, new innervations to the motor end plate were detected in the neuromuscular junction in the lithium injection group, but not in the control group.No differences in ED1 and P75 staining were detected.Conclusion Lithium could significantly promote axon regeneration after peripheral nerve crush injury.Its mechanism is subject to further investigation.     

人羊膜上皮细胞对大鼠坐骨神经再生影响的实验研究

目的 通过实验研究了解人羊膜上皮细胞(human amniotic epithelial cells,HAECs)对大鼠坐骨神经再生的促进作用.方法 取60只SD大鼠随机分为两组,羊膜细胞组和对照组,各组再分为2周和4周组,每组15只.制作大鼠周围神经损伤再生室模型,HAECs组局部应用培养的HAECs,对照组局部使用等量的生理盐水.术后分别于2周、4周检测神经传导功能和HE染色观察神经纤维形态学变化.结果 术后2周两组的神经电生理及组织学检测比较差异无统计学意义;4周HAECs组的大鼠坐骨神经传导功能明显恢复,HE染色显示术后坐骨神经手术部位出现大量炎性肉芽组织,呈现纤维性修复,吻合口以远HAECs组神经纤维形态结构较对照组完整,神经纤维与髓鞘直径均较对照组大.结论 HAECs移植可加速大鼠坐骨神经损伤后神经传导功能恢复,有助于有髓神经纤维损伤后的轴突与髓鞘的再生修复.

Abstract：

Objective To study the effects of human amniotc epithelial cells (HAECs) on regeneration of rat sciatic nerve. Methods Sixty SD rats were randomly divided into two groups, the HAECs group and control group. Each group was further divided into two week group and four week group, with 15 rats each. The sciatic nerve was cut and the regeneration chamber was created. The HAECs were implanted into the regeneration chamber in the HAECs group and normal saline was injected into the regeneration chamber in the control group.Electro-physiological and the histological study was done at two weeks and four weeks after the surgery. The results of nerve conduction study and HE staining of regenerating nerve fibers were analyzed statistically.Results There were no statistically significant differences between the two groups at two weeks post-operatively.At four weeks, the experimental group showed significantly higher nerve conduction velocity and amplitude of evoked potentials comparing to those of the control group. Nerve fibers and inflammatory granulation tissue was seen near the lesion site by HE staining in the control group, whereas the HAECs group showed more integrated nerve fihers and mature myelination distal to the lesion site. Conclusion The application of human amniotic epithelial cells may enhance nerve regeneration in rats.     

High doses of ionizing radiation on bone repair: is there effect outside the irradiated site?

Local ionizing radiation causes damage to bone metabolism, it reduces blood supply and cellularity over time. Recent studies indicate that radiation promotes biological response outside the treatment field. The aim of this study was to investigate the effects of ionizing radiation on bone repair outside the irradiated field. Ten healthy male Wistar rats were used; and five animals were submitted to radiotherapy on the left femur. After 4 weeks, in all animals were created bone defects in the right and left femurs. Seven days after surgery, animals were euthanized. The femurs were removed and randomly divided into 3 groups (n = 5): Control (C) (right femur of the non-irradiated animals); Local ionizing radiation (IR) (left femur of the irradiated animals); Contralateral ionizing radiation (CIR) (right femur of the irradiated animals). The femurs were processed and embedded in paraffin; and bone histologic sections were evaluated to quantify the bone neoformation. Histomorphometric analysis showed that there was no significant difference between groups C (24.6 ± 7.04) and CIR (25.3 ± 4.31); and IR group not showed bone neoformation. The results suggest that ionizing radiation affects bone repair, but does not interfere in bone repair distant from the primary irradiated site.     

Osteoclast depletion with clodronate liposomes delays fracture healing in mice

Osteoclasts are abundant within the fracture callus and also localize at the chondro‐osseous junction. However, osteoclast functions during fracture healing are not well defined. Inhibition of osteoclast formation or resorptive activity impairs callus remodeling but does not prevent callus formation. Interestingly, though anti‐osteoclast therapies differentially affect resolution of callus cartilage into bone. Treatments that inhibit osteoclast formation or viability tend to impair callus cartilage resolution, while treatments that target inhibition of bone resorption generally do not affect callus cartilage resolution. Here, we tested whether depletion of osteoclasts by systemic treatment with clodronate liposomes would similarly impair callus cartilage resolution. ICR mice were treated by intraperitoneal injections of clodronate‐laden liposomes or control liposomes and subjected to closed femur fracture. Femurs were resected at multiple times after fracture and analyzed by radiography, histology, and mechanical testing to determine effects on healing. Clodronate liposome treatment did not prevent callus formation. However, radiographic scoring indicated that clodronate liposome treatment impaired healing. Clodronate liposome treatment significantly reduced callus osteoclast populations and delayed resolution of callus cartilage. Consistent with continued presence of callus cartilage, torsional mechanical testing found significant decreases in callus material properties after 28 days of healing. The results support a role for osteoclasts in the resolution of callus cartilage into bone. Whether the cartilage resolution role for osteoclasts is limited to simply resorbing cartilage at the chondro‐osseous junction or in promoting bone formation at the chondro‐osseous junction through another mechanism, perhaps similar to the reversal process in bone remodeling, will require further experimentation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1699–1706, 2017.

     

CTLA4-Ig-modified dendritic cells inhibit lymphocyte-mediated alloimmune responses and prolong the islet graft survival in mice

The induction of antigen specific tolerance is critical for prevention and treatment of allograft rejection. In this study, we transfected CTLA4-Ig gene into dendritic cells (DCs), and investigated their effect on inhibition of lymphocyte activity in vitro and induction of immune tolerance on pancreatic islet allograft in mice. An IDDM C57BL/6 murine model induced by streptozotocin is as model mouse. The model mice were transplanted of the islet cells isolated from the BALB/c mice to their kidney capsules, and injected of CTLA4-Ig modified DCs (mDCs). The results showed that mDCs could significantly inhibit T lymphocyte proliferation and induce its apoptosis; whereas, unmodified DCs (umDCs) promoted the murine lymphocyte proliferation. Compared with injection of umDCs and IgG1 modified DCs, the injection of mDCs prolonged IDDM mice's allograft survival, and normalized their plasma glucose (PG) levels within 3 days and maintained over 2 weeks. The level of IFN-gamma was lower and the level of IL-4 was higher in mDCs treated recipient mice than that in control mice, it indicated that mDCs led to Th1/Th2 deviation. After 7 days of islet transplantation, HE stain of the renal specimens showed that the islets and kidneys were intact in structure, and islet cells numbers are increased in mDCs treated mice. Our studies suggest that DCs expressing CTLA4-Ig fusion protein can induce the immune tolerance to islet graft and prolong the allograft survival through the inhibition of T cell proliferation in allogeneic mice.