Effects of Passive Stretching on the Biochemical and Biomechanical Properties of Calcaneal Tendon of Rats

The role of physical activity in affecting the composition of extracellular matrix and mechanical properties of tendons has been well studied, but little is known about the role of passive stretching. The purpose of this study was to test the hypothesis that stimulation by passive stretching may change the composition and mechanical properties of tendons. Three-month-old Wistar rats were divided into three groups: the control, animals were not submitted to stretching procedures; groups that had their calcaneal tendons manually stretched three or five times a week, for 21 days. Afterward, the calcaneal tendons were removed and assayed for hydroxyproline content and biomechanical test. The hydroxyproline content in the stretched groups was higher, suggesting that more collagen was present in the tendons of these groups. These tendons also showed higher values of maximum stress and modulus of elasticity or Young's modulus. These results indicate that stretching leads to alterations in the synthesis of the extracellular matrix components and in the mechanical properties of tendons.     

Biomechanical and Biochemical Properties of Chicken Calcaneal Tendon Under Effect of Age and Nonforced Active Exercise

This study investigated if nonforced active exercise alters the biomechanical and biochemical properties of calcaneal tendon during maturation. Chickens at 1, 5, and 8 months old were divided into two groups: caged and penned. Intact tendons were used for biomechanical analysis, but they were divided into tensile and compressive regions for quantification of hydroxyproline and glycosaminoglycans. The exercise increased tendon strength after the fifth month, energy absorption in the eighth month, and ultimate tensile stress in the first month. Age increased tendon strength and energy storage and reduced stiffness but did not alter stress. There was an increase in collagen content in the fifth month. Glycosaminoglycans showed a progressive decline in the tensile region. Thus, some biomechanical and biochemical changes depend on the maturation process itself and also are influenced by spontaneous exercise, showing that mechanical stimulation of low intensity may help to improve the quality of the tendon.     

Cyclic Mechanical Stretching of Human Tendon Fibroblasts Increases the Production of Prostaglandin E 2 and Levels of Cyclooxygenase Expression: A Novel In Vitro Model Study

Prostaglandin E 2 (PGE 2 ) is a known inflammatory mediator of tendinitis, for which mechanical loading on tendons is believed to be one of the most prominent causation factors. Previous in vitro studies have shown that cyclic mechanical stretching of cells can cause changes in cell morphology and alteration of both DNA and protein syntheses. In our study, a novel system was used whereby tendon fibroblasts are cultured on microgrooved silicone surfaces and are subjected to cyclic uniaxial stretching along their long axes to mimic in vivo conditions. Using this unique model system, the cell shape and alignment can be controlled. Further, this study was designed to test the hypotheses that PGE 2 production increases in a stretching magnitude-dependent manner and that cyclooxygenase (COX) is responsible for the increased PGE 2 production in tendon fibroblasts. Human patellar tendon fibroblasts were cultured on the microgrooved silicone membranes and cyclically stretched at 4%, 8%, or 12% of nominal dish length for 24 hr. PGE 2 production was found to be increased 1.7-fold at 8% cyclic stretching and 2.2-fold at 12% cyclic stretching compared with nonstretched controls. In addition, human tendon fibroblasts had increased expression of both COX-1 and COX-2 for all three applied stretching magnitudes, with the exception of COX-1 at 4% cyclic stretching. Also, cellular PGE 2 production, after 8% cyclic stretching, was significantly decreased with the addition of indomethacin (25 &#119 M), a COX competitive inhibitor, compared with stretched cells without indomethacin treatment. These findings suggest that the increase in PGE 2 production by the human tendon fibroblasts is stretching magnitude-dependent, and that the increase in COX expression contributes to the increased production of PGE 2 after cyclic stretching. As PGE 2 is a known inflammatory mediator of tendinitis, the contribution of COX-1 and COX-2 to PGE 2 production and their roles in tendon inflammation are clearly indicated.     

Relaxin Receptor RXFP1 and RXFP2 Expression in Ligament,Tendon, and Shoulder Joint Capsule of Rats

Numerous musculoskeletal disorders are caused by thickened ligament, tendon stiffness, or fibrosis of joint capsule. Relaxin, a peptide hormone, can exert collagenolytic effect on ligamentous and fibrotic tissues. We hypothesized that local injection of relaxin could be used to treat entrapment neuropathy and adhesive capsulitis. Because hormonal effect depends on the receptor of the hormone on the target cell, it is important to confirm the presence of such hormonal receptor at the target tissue before the hormone therapy is initiated. The aim of this study was to determine whether there were relaxin receptors in the ligament, tendon, and joint capsular tissues of rats and to identify the distribution of relaxin receptors in these tissues. Transverse carpal ligaments (TCLs), inguinal ligaments, anterior cruciate ligaments (ACLs), Achilles tendons, and shoulder joint capsules were obtained from male Wistar rats. Western blot analysis was used to identify relaxin receptor isoforms RXFP1 and RXFP2. The distribution of relaxin receptors was determined by immunohistochemical staining. The RXFP1 isoform was found in all tissues examined. The RXFP2 isoform was present in all tissues but the TCLs. Its expression in ACLs tissues was relatively weak compared to that in other tissues. Our results revealed that RXFP1 and RXFP2 were distributed in distinctly different patterns according to the type of tissue (vascular endothelial cells, fibroblast-like cells) they were identified.     

Biomechanical Effects of Immobilization and Rehabilitation on the Skeletal Muscle of Trained and Sedentary Rats

Because of the scarcity of information about the comparison of training to sedentarism beforehand immobilization and rehabilitation through muscle mechanical properties, the present work investigates this theme. Seventy rats were divided into 7 groups: 1-control (C); 2-trained (T); 3-sedentary (S); 4-trained and immobilized (TI); 5-sedentary and immobilized (SI); 6-trained, immobilized and rehabilitated (TIR); 7-sedentary, immobilized and rehabilitated (SIR). Interventions: Swimming training; Sedentarism (reduced size cages); Cast immobilization (pelvic limb) and water rehabilitation. Load at the limit of proportionality (LLP), maximum limit load (MLL) and stiffness (St) were the mechanical properties determined after a mechanical test of traction of the gastrocnemius. The training improved all mechanical properties when compared to sedentarism. After immobilization, LLP and MLL were reduced in TI and SI. However, there was no difference in St between C and TI. Additionally, TI showed improved MLL when compared to SI. The comparison of TI and TIR showed significant melioration in all properties after remobilization. SIR showed an improvement only in MLL when compared to SI. Significant melioration in LLP and St was observed in TIR compared to SIR. We demonstrated that the training before immobilization and rehabilitation had a positive effect on the muscle mechanical behavior compared to sedentarism. This analysis is of fundamental importance because it helps characterize the muscle tissue under different functional demands.     

Tendon and myo-tendinous junction in an overloaded skeletal muscle of the rat

Summary Overloading of rat plantaris muscles was produced by aseptic ablation of the synergists. The morphological changes occurring after 1 or 2 weeks were investigated at the light and electron microscopical level in the distal tendon of the plantaris and at the myotendinous junction. Sham-operated rats were prepared as controls. In the tendon, quiescent fibrocytes were replaced by activated fibroblasts displaying a vesicular nucleus with prominent nucleoli and an outstanding increase in cytomembranes, particularly the rough endoplasmic reticulum and the Golgi complex. The plasmalemma of the fibroblasts was modified by the presence of caveolae and the surbsurface cytoplasm contained many membrane-bound vacuoles. In the tendon, the collagen bundles were disrupted, resulting in the formation of empty longitudinally oriented spaces; in these spaces, as in the pericapillary areas, no inflammatory cells were observed. At the myotendinous junction, fibroblast activation was consistently observed in both control and overloaded specimens. At this level, the sarcolemma of the finger-like projections of muscle fibres presented many caveolae close to clusters of large subsurface vacuoles. These observations indicate that, at the beginning of the compensatory hypertrophy, the adaptative changes to overloading include a non-inflammatory reaction of the tendon characterized by enhanced collagen synthesis and intensive membrane renewal and recycling. From the mechanical point of view this reaction can impair the tendon resistance to stretch. At the myotendinous junction the increased membrane turnover of the sarcolemma and the fibroblast activation can be considered permanent phenomena consequent to the increased stress exerted upon the interface connecting the contractile apparatus to the stroma.     

凝血酶对大鼠脑微血管内皮细胞的影响

目的 :探讨凝血酶 (Thrombin ,TM )对脑微血管内皮的影响。方法 :将大鼠脑微血管内皮细胞进行培养 ,培养液中加入 10U的TM或10U的TM + 0 .4mU的组织蛋白酶G(CaspethsinG ,CATG ) ,相差显微镜动态观察内皮细胞形态的变化 ,免疫组织化学技术检测基质金属蛋白酶 2 (MatrixMetalloproteinase 2 ,MMP 2 )表达的改变。 结果 :TM使内皮细胞发生收缩 ,细胞收缩程度具有时间依赖性 ,使内皮细胞MMP 2表达水平明显增加。TM +CATG加入培养液后 ,细胞形态、MMP 2表达与对照组比较均无明显统计学差异 (P >0 .0 5 )。结论 :TM通过激活蛋白酶激活受体 1(proteaseactivatedreceptor 1,PAR 1) ,使内皮细胞发生收缩 ,促进MMP 2表达 ,是TM增加血脑屏障 (BloodBrainBarrier,BBB)通透性的可能机制。     

Bouts of Passive Stretching after Immobilization of the Rat Soleus Muscle Increase Collagen Macromolecular Organization and Muscle Fiber Area

This study evaluated the effect of short bouts of stretching on the soleus muscle after immobilization, by measuring the birefringence of the intramuscular connective tissue (ICT) and the muscle fiber area. Thirty rats were divided into five groups: the left soleus was immobilized in the shortened position; after immobilization the animals remained free; after immobilization, the soleus was stretched daily (10 stretches of 60 sec followed by 30 sec rest); after immobilization, the soleus was stretched 3 times a week; control. Immobilization caused a loss of birefringence of the ICT and of muscle fiber area and only daily stretching increased both compared with the control (p < 0.01). In conclusion, short daily bouts of stretching after immobilization induced molecular reorganization of the collagen bundles and muscle fiber hypertrophy in the rat soleus.     

大鼠性别及血清保存条件对生化检测结果的影响研究

目的 研究大鼠不同性别、血清样本放置条件及时间对生化检验结果的影响.方法 80只大鼠编号后一次性取静脉血,放置30min后离心,每只鼠血清分成8份.首次(1h)检测后,所有血清至于-80℃冰箱保存,一份于1、2、4、7、11、23、40d时间点重复解冻、检测后复存于-80℃冰箱;其余七份分别于以上时间点单次解冻、检测后丢弃;检测项目均为谷草转氨酶(AST)、谷丙转氨酶(ALT)、尿素(UREA)、肌酐(CREA)、胆固醇(CHO)、甘油三酯(TG)、血糖(GLU)、总蛋白(TP)、白蛋白(ALB).结果 雄性、雌性大鼠血清样本-80℃冰箱保存至40d 9个生化指标均与1h差异无统计学意义;雌性大鼠血清样本保存-80℃在1d、2d、4d、7d4个时间点重复解冻,部分生化指标逐步显示差异有统计学意义,且随解冻次数及保存时间的增加,差异具有统计学意义的生化指标项数增加,第4次(7d)解冻后ALT、TP指标结果与1h测定结果比较差异具有统计学意义(均P＜0.05);第5次(11d)解冻ALT、TG、TP指标结果与1h测定结果比较差异具有统计学意义(均P＜0.05);第6次(23d)解冻ALT、CREA、TG、TP指标结果与1h测定结果比较差异具有统计学意义(均P＜0.05);第7次(40d)解冻ALT、AST、UREA、CREA、TG、GLU、TP、ALB指标结果与1h测定结果比较差异具有统计学意义(均P＜0.05).其余指标与1h测定结果比较无显著差异(P＞0.05).雄性大鼠血清样本保存-80℃重复解冻1～5次9个生化指标均与1h测定结果比较差异无统计学意义,第6次(23d)解冻TP指标测定结果比较差异具有统计学意义(P＜0.05);第7次(40d)解冻TG、TP指标测定结果比较差异具有统计学意义(均P＜0.05),其余指标与1h测定结果比较差异无统计学意义(P＞0.05).结论 不同性别大鼠血清-80℃条件下保存40d以内单次解冻后测定以及在大鼠血清解冻3次以内对生化指标测定值无影响;但重复解冻4次以上部分生化指标有显著差异,且不同性别大鼠血清指标差异情况不同,血清样本放置条件及时间及解冻次数会对大鼠生化检测指标造成影响.     

Temporal Effects of Cyclic Stretching on Distribution and Gene Expression of Integrin and Cytoskeleton by Ligament Fibroblasts In Vitro

Cyclic stretching is pivotal to maintenance of the ligaments. However, it is still not clear when ligament fibroblasts switch on expression of genes related to the mechanotransduction pathway in response to cyclic stretching. This in vitro study investigated, using ligament fibroblasts, the time-dependent changes in distribution and gene expression of β1 integrin, the cytoskeleton, and collagens after the application of 6% cyclic stretching at a frequency of 0.1 Hz for 3 hr on silicon membranes. We carried out confocal laser scanning microscopy to demonstrate changes in distribution of these components as well as quantitative real-time RT-PCR to quantify levels of these gene expression both during application of cyclic stretching and at 0, 2, 6, 12, and 18 hr after the termination of stretching. Control (unstretched) cells were used at each time point. Within 1 hr of the application of stretching, the fibroblasts and their actin stress fibers became aligned in a direction perpendicular to the major axis of stretch, whereas control (unstretched) cells were randomly distributed. In response to cyclic stretching, upregulation of actin at the mRNA level was first observed within 1 hr after the onset of stretching, while upregulation of β1 integrin and type I and type III collagens was observed between 2 and 12 hr after the termination of stretching. These results indicate that the fibroblasts quickly modify their morphology in response to cyclic stretching, and subsequently they upregulate the expression of genes related to the mechanotransduction pathway mainly during the resting period after the termination of stretching.     

In Vivo Effects of Ovarian Steroid Hormones on the Expressions of Estrogen Receptors and the Composition of Extracellular Matrix in the Anterior Cruciate Ligament in Rats

Female athletes have a significantly higher rate of anterior cruciate ligament (ACL) injury than their male counterparts. Sex steroid hormones are considered to have an influence as risk factors for female ACL injuries. We hypothesized that estrogen and progesterone have specific and synergistic influences on the composition of extracellular matrix in ACL. By ovariectomy (OVX) followed by subcutaneous estradiol (E2) and/or progesterone (P4) replacement, 40 female rats were divided into 5 groups: E2, P4, combined E2 and P4 (EP), OVX control, and sham group. After 30 days, using undecalcified sections of knee joints in conjunction with immunofluorescence staining of estrogen receptor α and β (ERα and ERβ), collagen types 1 and 3, and cartilage oligomeric matrix protein (COMP), the immunoreactivities of these proteins in two distinct parts of ACL, proximal and middle portions, were compared semiquantitatively among experimental groups. By E2 replacement, the expressions of ERα in ACL fibroblasts were elevated compared to the OVX group. At the proximal portion, the immunoreactivities of type 1 collagen by E2 replacement, type 3 collagen by P4 replacement, and COMP by E2 or P4 replacement were significantly reduced. At the middle portion, the immunoreactivity of type 3 collagen was significantly elevated by E2 replacement. However, no differences were observed between the sham and OVX groups. These findings suggest that ACL is ER-dependent and that ovarian hormones alter ligament tissue composition, especially at the proximal portion. Female hormonal influences are partly involved in the biological properties of ACL.     

Effect of Selective Estrogen Receptor Modulator/Raloxifene Analogue on Proliferation and Collagen Metabolism of Tendon Fibroblast

The selective estrogen receptor modulator raloxifene is therapeutically beneficial for postmenopausal connective tissue degradation, such as osteoporosis, vascular sclerosis, and dermal degradation; however, the effects of raloxifene on postmenopausal tendon metabolism have not been clarified. In this study, we investigated the effects of raloxifene analogue (LY117018) on cell proliferation and collagen metabolism using cultured rat Achilles tendon fibroblasts. 17β-Estradiol (E₂; 10^?11–10^?9 M) and LY117018 (10^?9–10^?7 M) had no significant effects on tendon fibroblast proliferation, based on a BrdU (5-bromo-2′-deoxyuridine) incorporation assay (24 hr) and a WST-8 colorimetric assay (2 or 6 days). Neither E₂ nor LY117018 significantly altered the expression of type I collagen, which is a main component of the tendon extracellular matrix (ECM), whereas both E₂ and LY117018 significantly increased the expression of matrix metalloproteinase (MMP)-13, which is responsible for tendon collagen degradation in rat. Also, both E₂ and LY117018 increased the expression of type III collagen and elastin, which are minor components of tendon ECM, but are considered to govern the elastic properties of tendons. These changes in collagen and MMP induced by either E₂ or LY117018 were attenuated by the estrogen receptor alpha blocker ICI 182,780. The results of this study suggest that postmenopausal estrogen deficiency might downregulate tendon collagen turnover and decrease tendon elasticity. Further, raloxifene treatment might restore these changes to premenopausal levels.     

Effect of Amplitude and Frequency of Cyclic Tensile Strain on the Inhibition of MMP-1 mRNA Expression in Tendon Cells: An In Vitro Study

To determine the effect of cyclic strain amplitude and frequency on MMP-1 (interstitial collagenase) expression in tendon cells, rat tail tendons (RTT) were immobilized or cyclically displaced to various amplitudes (1, 3, or 6% strain at 0.017 Hz) or frequencies (1% strain at 0.017, 0.17, or 1.0 Hz) for 24 hr. Stress-deprivation for 24 hr resulted in a marked upregulation in MMP-1 expression. Cyclic tensile loading at 0.017 Hz was found to significantly inhibit, but not completely eliminate, MMP-1 expression at 1% strain. MMP-1 expression was completely eliminated at 3 and 6% strain. Increasing the frequency of application of the 1% strain to 0.17 or 1.0 Hz completely eliminated MMP-1 expression. Disruption of the actin cytoskeleton with cytochalasin D abolished all inhibitory effects of cyclic strain on MMP-1 expression. The results of our study demonstrate that MMP-1 expression in tendon cells can be modulated by varying amplitudes and frequencies of cyclic tensile strain, presumably through a cytoskeletally based mechanotransduction pathway.     

头痛安胶囊对实验性大鼠软脑膜微循环障碍的影响

大鼠预防性给药,观察头痛安胶囊对高分子右旋糖酐造成的大鼠软脑膜微循环障碍的影响.结果表明,头痛安组大鼠的血流速度开始减慢的时间明显比生理盐水对照组延长,且血流形态分级数也明显优于对照组.对高分子右旋糖酐造成的血液流态障碍,头痛安组具有显著的减轻作用.     

脑康复冲剂对大鼠软脑膜微循环作用的实验研究

目的：脑康复冲剂临床用于治疗脑血栓血瘀证患者，本实验观察其对在大鼠实验性软脑膜微循环障碍的影响。方法：大鼠预防性给药，用高分子右旋糖酐水溶液静脉注射形成大鼠软脑膜的微循环障碍，观察脑康复冲剂对大鼠微循环的影响。结果：用高分子右旋糖酐造模后，大鼠软脑膜的微循环发生障碍，表现为血液流速减慢、血细胞聚集等，脑康复冲剂组血流速度开始减慢的时间明显比生理盐水对照组延长，且分级数也明显优于生理盐水组，对高分子右旋糖酐造成的血液流态障碍，脑康复组明显好于生理盐水组（P＜0．05），毛细血管网交叉点数开始减少的时间也比生理盐水对照组延长，差异显著（P＜0．01）。结论：脑康复冲剂使大鼠血流速度和血液流态得到改善，具有预防和改善动物脑微循环障碍的作用。     

Quiescence and hyporeactivity evoked by activation of cell bodies in the ventrolateral midbrain periaqueductal gray of the rat

Much evidence suggests that the midbrain periaqueductal gray region (PAG) plays a pivotal role in mediating an animal's responses to threatening, stressful, or painful stimuli. Active defensive reactions, hypertension, tachycardia and tachypnea are coordinated by a longitudinally oriented column of cells, found lateral to the midbrain aqueduct, in the caudal two-thirds of the PAG. In contrast, microinjections of excitatory amino acid (EAA) made in the ventrolateral region of the PAG in anesthetized or isolated animals evoke hypotension, bradycardia, and behavioral arrest. The aim of the present study was to examine further the effects of activation of neurons in the ventrolateral PAG. By injecting into this region low doses (40 pmol) of kainic acid (KA), a long-acting EAA, it was possible to observe a freely moving rat's behavior in a social situation (i.e., paired with a weight-matched, untreated partner). Such injected rats become quiescent, i.e., there was a cessation of all ongoing spontaneous activity. These rats were also hyporeactive: the investigative approaches of the partner failed to evoke orientation, startle reactions, or vocalization. Electroencephalographic measurements indicated that the effects of injections of KA in the ventrolateral PAG were not secondary to seizure activity. In addition to the quiescence and hyporeactivity reported here, and the hypotension and bradycardia reported previously, the ventrolateral PAG is a part of the brain from which analgesia has been readily evoked by electrical stimulation, or microinjections of either EAA or morphine. As a reaction to deep or inescapable pain, chronic injury, or defeat, animals often reduce their somatomotor activity, become more solitary, and are generally much less responsive to their environment. These data, and those from other recent studies, suggest that neurons in the ventrolateral PAG may play an important role in integrating such a passive behavioral response of which quiescence and hyporeactivity are the major components.     

单晶硅材料微型圆柱体直径及其排列间距对神经干细胞分化的影响

BACKGROUND: Different structures of matrix models, such as grating, holes and pillars make different effects on the differentiation of neural stem cells. OBJECTIVE: To explore the effects of the diameter and spacing, known as physical signals of micro pillars on neural stem cell differentiation. METHODS: Micro pillars with different diameters and spacing, both of which had four dimensions of 2.5, 5, 10 and 20 μm, were fabricated on silicon substrates by photolithographic method. Purified primary neural stem cells were incubated on the each micro pillar for 7 days in vitro. Then the differentiation of neural stem cells into neuron-like cells was observed using immunofluorescence staining and quantitative real-time PCR. RESULTS AND CONCLUSION: When the diameters of the micro pillars were constant and the spacing of micro pillars varied in the range of 2.5-10 μm, the differentiation rate of neural stem cells increased with the spacing increase. When the spacing was invariable and the diameters changed in the range of 2.5-20 μm, the differentiation rate of neural stem cells declined with the diameter increase. Especially, the micro pillars with 2.5 μm diameter and 10 μm spacing significantly promoted the differentiation of neural stem cells into neuron-like cells. These results show that specific micro pillars with small diameters and large spacing facilitate the differentiation of neural stem cells, thus providing guidance for developing tissue-engineered scaffolds.      

丹参对应激大鼠血液流变性的影响

目的：探讨丹参对应激大鼠血液流变性的影响。方法：采用血液粘度计检测血液流变学指标，荧光法检测血浆去甲肾上腺素的含量。结果：束缚应激大鼠血浆去甲状腺素的含量增高，全血比粘度、血浆比粘度、红细胞变形指数、聚集指数、压积、血浆纤维蛋白原含量均增高。经相关分析，去甲肾上腺素含量与血液流变学各指标呈中高度相关。胃灌丹参后，上述各指标均显著下降。结论：丹参能明显改善应激大鼠的血液流变性，可能是通过减少去甲肾上腺素的含量。