Crimp morphology in relaxed and stretched rat Achilles tendon

Fibrous extracellular matrix of tendon is considered to be an inextensible anatomical structure consisting of type I collagen fibrils arranged in parallel bundles. Under polarized light microscopy the collagen fibre bundles appear crimped with alternating dark and light transverse bands. This study describes the ultrastructure of the collagen fibrils in crimps of both relaxed and in vivo stretched rat Achilles tendon. Under polarized light microscopy crimps of relaxed Achilles tendons appear as isosceles or scalene triangles of different size. Tendon crimps observed via SEM and TEM show the single collagen fibrils that suddenly change their direction containing knots. The fibrils appear partially squeezed in the knots, bent on the same plane like bayonets, or twisted and bent. Moreover some of them lose their D-period, revealing their microfibrillar component. These particular aspects of collagen fibrils inside each tendon crimp have been termed 'fibrillar crimps' and may fulfil the same functional role. When tendon is physiologically stretched in vivo the tendon crimps decrease in number (46.7%) (P<0.01) and appear more flattened with an increase in the crimp top angle (165 degrees in stretched tendons vs. 148 degrees in relaxed tendons, P<0.005). Under SEM and TEM, the 'fibrillar crimps' are still present, never losing their structural identity in straightened collagen fibril bundles of stretched tendons even where tendon crimps are not detectable. These data suggest that the 'fibrillar crimp' may be the true structural component of the tendon crimp acting as a shock absorber during physiological stretching of Achilles tendon.     

Development and ageing of phenotypically distinct fibrocartilages associated with the rat Achilles tendon

Summary We describe by routine histology and by immunohistochemistry three phenotypically and developmentally distinct fibrocartilages associated with the Achilles tendon of the rat. All the fibrocartilages develop after birth and show significant age-related changes in the composition of their extracellular matrix. Attachment-zone fibrocartilage occurs at the insertion of the tendon on the calcaneus. It derives from the cartilage rudiment of the calcaneus and from the region where the tendon merges with the perichondrium. The extracellular matrix contain type II collagen and chondroitin sulphate. Compressive tendon fibrocartilage occurs in the deep part of the tendon where it presses against the calcaneus, and is derived by metaplasia of tendon cells. The cells label strongly for the intermediate filament vimentin, and the extracellular matrix contains chondroitin and keratan sulphates, but type II collagen only in very old animals (>2 years). Calcaneal fibrocartilage covered the posterior surface of the calcaneus where it was in contact with the Achilles tendon. It labelled intensely for type II collagen and contained chondroitin and keratan sulphates. The cells were rich in vimentin. This fibrocartilage was derived from the calcaneal perichondrium.     

Restricted expression of the gap junctional protein connexin 43 in the arterial system of the rat

Connexin 43 (Cx43) has been reported to be expressed in vascular smooth muscle cells and endothelial cells. Evidence for possible variations in Cx43 distribution within different parts of the vascular system is limited. We have therefore investigated the expression of Cx43 in the endothelia and media of 11 vessels of different size and function in the rat, using immunofluorescence and confocal laser scanning microscopy. The results showed that punctate Cx43 staining was abundant in the endothelia and media of all of the 5 elastic arteries examined. In the media, the amount of Cx43 staining decreased as the size of the elastic arteries became smaller. In the 6 muscular arteries examined, 2 different patterns of Cx43 staining were observed. In the first type, Cx43 expression was high in the endothelium but virtually absent from the media. Mesenteric resistance, hepatic and tail arteries were examples. In the second type, Cx43 staining was absent from both the media and the endothelia. The coronary, basilar, and middle cerebral arteries showed this appearance. The results suggest that expression of Cx43 is largely restricted to elastic arteries in the arterial system of the rat. The lack of immunodetectable Cx43 from the media of all muscular arteries examined, and from the endothelia of some of these arteries, raises the possibility of significant differences in the form of expression of Cx43 in these vessels or the presence of other connexins.     

鼠胚胎癌细胞的诱导分化和间隙连接形成

在原有研究的基础上,本文进一步证实鼠胚胎癌细胞系P_(19)的突变型αaS1可以被维生素A酸(RA)诱导分化,但对DMSO不敏感。在被一定浓度的RA诱导分化时,αaS1细胞同样出现了增生和分化间呈动态平衡的平稳阶段。与其他突变型相比,αaS1,细胞形成间隙连接的能力较差,这表明EC细胞突变型的某一种间隙连接型可能会出现缺陷。P_(19)和RAJH_6的混合细胞集合物在含RA的培养液中,只有前者被不断诱导分化,但经6-TG处理后,存活的RAJH_6细胞的分数基本平稳。此项结果提示,正在分化的EC细胞和未分化的EC细胞之间可形成代谢偶联。我们的工作有助于进一步了解细胞分化、先天畸形和肿瘤逆转。     

Excitability and gap junction–mediated mechanisms in nucleus accumbens regulate self-stimulation reward in rats

The nucleus accumbens (Acb) is a part of the striatum which integrates information from cortical and limbic brain structures, and mediates behaviors which reinforce reward. Previous work has suggested that neuronal synchrony mediated by gap junctions in Acb-related areas is involved in brain pleasure and reward. In order to gain insight into functional aspects of the neural information processing at the level of the striatum, we explored the possible role of Acb gap junctional communication and chemical synapses on reward self-stimulation in rats using positive reinforcement. Rats were trained to press a lever that caused an electrical current to be delivered into the hypothalamus, which is recognized to cause pleasure/reward. Intracerebral infusion into the Acb of the gap junctional blocker carbenoxolone (CBX) decreased the lever-pressing activity. Considering that the net effect of blocking gap junctions is a reduced synchronized output of the cellular activities, which at some level represents a decrease in excitability, two other inhibitors of neuronal excitability, carbamazepine (CBZ) and tetrodotoxin (TTX), were infused into the Acb and their effects on lever-pressing assessed. All manipulations that diminished excitability in the Acb resulted in reduced lever-pressing activity. CBX and TTX were also infused into motor cortex mediating forelimb lever-pressing with no effect. However, a manipulation that has the net effect of increasing excitation, the infusion of the opiate antagonist naloxone, also decreased significantly brain self-stimulation. We conclude that reward behaviors depend to a great extent on both excitability and gap junction–mediated mechanisms in Acb neuronal networks. Thus, the Acb provides a site for the study of pleasure/reward, addiction and conscious experience.     

Changes in stiffness induced by hindlimb suspension in rat Achilles tendon

The aim of this study was to measure the effects of hindlimb suspension on mechanical properties of the rat Achilles tendon. Adult male Wistar rats were randomly assigned to groups to be either suspended, or a control. After 21 days, Achilles tendons were removed for mechanical analysis. Classical tests of tensile performance were made, and mechanical parameters were derived from a stress-strain relationship. The tendons of animals that had been suspended presented values for maximal stress and tangent modulus which were 37.5% (P < 0.01) and 41% (P < 0.01), respectively, lower than the tendons of the control rats. In a similar way, the energy absorption capacity had largely decreased in animals that had been suspended. However, the maximal strain was similar in the two groups. These results showed that hindlimb suspension in rats has an important detrimental effect on mechanical properties of the Achilles tendon. Differences in tendon stiffness obtained here, along with those found by other investigators, encourage the hypothesis that homeostatic responses of soft tissues are due to changes in limb loadings. This study may be useful in providing a better understanding of the adaptation of human skeletal muscle when exposed to microgravity. Accepted: 3 September 1999     

Regional fibrocartilage variations in human anterior cruciate ligament tibial insertion: a histological three-dimensional reconstruction

Abstract

Purpose: We studied anterior cruciate ligament (ACL) tibial insertion architecture in humans and investigated regional differences that could suggest unequal force transmission from ligament to bone. Materials and methods: ACL tibial insertions were processed histologically. With Photoshop software, digital images taken from the histological slides were collaged, contour lines were drawn, and different gray values were filled based on the structure. The data were exported to Amira software for three-dimensional reconstruction. Results: The uncalcified fibrocartilage (UF) layer was divided into three regions: lateral, medial and posterior according to the architecture. The UF zone was significantly thicker laterally than medially or posteriorly (p?<?0.05). Similarly, the calcified fibrocartilage (CF) thickness was significantly greater in the lateral part of the enthesis compared to the medial and posterior parts (p?<?0.05). Conclusions: The UF quantity (more UF laterally) corresponding to the CF quantity (more CF laterally) at the ACL tibial insertion provides further evidence suggesting that the load transferred from the ACL to the tibia was greater laterally than medially and posteriorly.     

Adipose tissue at entheses: the innervation and cell composition of the retromalleolar fat pad associated with the rat Achilles tendon

This study set out to determine whether the fat pad at the attachment of the Achilles tendon has features enabling it to function as an immune organ and a mechanosensory device, and to be a source of pain in insertional tendon injuries. Sections for histology and immunohistochemistry were cut from the Achilles tendon enthesis organ of 1 day old, 1 month, 4 month and 24 month old rats. For fluorescence and peroxidase immunohistochemistry, cryosections were labelled with primary antibodies directed against PGP9.5, substance P, neurofilament 200, calcitonin gene related peptide, CD68, CD36, myeloid related protein 14, actin and vinculin. The fat pad contained not only adipocytes, but also fibrous tissue, mast cells, macrophages, fibroblasts and occasional fibrocartilage cells. It was richly innervated with nerve fibres, some of which were likely to be nociceptive, and others mechanoreceptive (myelinated fibres, immunoreactive for neurofilament 200). The fibres lay between individual fat cells and in association with blood vessels. In marked contrast, the enthesis itself and all other components of the enthesis organ were aneural at all ages. The presence of putative mechanoreceptive and nociceptive nerve endings between individual fat cells supports the hypothesis that the fat pad has a proprioceptive role monitoring changes in the insertional angle of the Achilles tendon and that it may be a source of pain in tendon injuries. The abundance of macrophages suggests that the adipose tissue could have a role in combating infection and/or removing debris from the retrocalcaneal bursa.     

Where tendons and ligaments meet bone: attachment sites ('entheses') in relation to exercise and/or mechanical load

Entheses (insertion sites, osteotendinous junctions, osteoligamentous junctions) are sites of stress concentration at the region where tendons and ligaments attach to bone. Consequently, they are commonly subject to overuse injuries (enthesopathies) that are well documented in a number of sports. In this review, we focus on the structure-function correlations of entheses on both the hard and the soft tissue sides of the junction. Particular attention is paid to mechanical factors that influence form and function and thus to exploring the relationship between entheses and exercise. The molecular parameters indicative of adaptation to mechanical stress are evaluated, and the basis on which entheses are classified is explained. The application of the 'enthesis organ' concept (a collection of tissues adjacent to the enthesis itself, which jointly serve the common function of stress dissipation) to understanding enthesopathies is considered and novel roles of adipose tissue at entheses are reviewed. A distinction is made between different locations of fat at entheses, and possible functions include space-filling and proprioception. The basic anchorage role of entheses is considered in detail and comparisons are explored between entheses and other biological 'anchorage' sites. The ability of entheses for self-repair is emphasized and a range of enthesopathies common in sport are reviewed (e.g. tennis elbow, golfer's elbow, jumper's knee, plantar fasciitis and Achilles insertional tendinopathies). Attention is drawn to the degenerative, rather than inflammatory, nature of most enthesopathies in sport. The biomechanical factors contributing to the development of enthesopathies are reviewed and the importance of considering the muscle-tendon-bone unit as a whole is recognized. Bony spur formation is assessed in relation to other changes at entheses which parallel those in osteoarthritic synovial joints.     

全镜下跟腱清理与止点增强治疗跟腱部分撕裂的Haglund综合征的疗效对比

目的 比较全关节镜下跟腱清理与跟腱止点增强重建治疗跟腱部分撕裂的Haglund综合征患者的临床疗效。方法 回顾性分析2016年1月至2021年3月在青县人民医院应用全关节镜下治疗跟腱部分撕裂的Haglund综合征的24例患者资料,依据手术方式不同分为关节镜下跟腱止点清理组（A组）和关节镜下跟腱止点清理+跟腱止点增强重建组（B组）,每组各12例。术后定期随访,采用疼痛视觉模拟（VAS）评分、美国足踝外科协会（AOFAS）足踝功能评分及维多利亚体育研究所评分（VISA-A）评估治疗结果,进行观察对比。结果 患者平均随访时间（7.5±0.8）个月,末次随访均不存在后足跟肿痛、跟腱再次断裂等并发症。所有患者症状较术前均得到缓解。A组和B组的VAS评分、AOFAS评分、VISA-A评分均较术前有所改善;然而A组和B组术前及术后6个月的VAS、AOFAS、VISA-A评分对比,差异无统计学意义（P>0.05）;B组术后1、3个月的VAS、AOFAS、VISA-A评分优于A组,差异有统计学意义（P<0.05）。结论 全关节镜下跟腱止点增强重建治疗跟腱部分撕裂的Haglund综合征,较关节...     

Temperature dependence of gap junction properties in neonatal rat heart cells

Cell pairs of neonatal rat hearts were used to study the influence of temperature on the electrical properties of gap junctions. A dual voltage-clamp method was adopted, which allowed the voltage gradient between the cells to be controlled and the intercellular current flow to be measured. Cell pairs with normal coupling revealed a positive correlation between the conductance of the junctional membranes, g _j, and temperature. Cooling from 37° C to 14° C led to a steeper decrease in g _j, cooling from 14° C to -2° C to a shallower decrease (37° C: g _j=48.3 nS; 14° C: g _j=21.4 nS;-2°C: g _j=17.5 nS), corresponding to a temperature coefficient, Q ₁₀, of 1.43 and 1.14 respectively. The existence of two Q ₁₀ values implies that g _j may be controlled by enzymatic reactions. When g _j was low, i. e. below 5 nS (conditions: low temperature; treatment with 3 mM heptanol), it showed voltage-dependent gating. This property was not visible when g _j was large, i. e. 20–70 nS (conditions: high temperature; normal saline), presumably because of series resistances (pipette resistance). Cell pairs with weak intrinsic coupling and normally coupled cell pairs treated with 3 mM heptanol revealed a positive correlation between the conductance of single gap-junction channels, _j, and temperature (37° C: 75.6 pS; -2°C: 19.6 pS), corresponding to a Q ₁₀ of 1.41.     

Progesterone,oestradiol and oxytocin and their in vitro effect on maintaining the number of gap junction plaques in human myometrium at term

Previous studies by electron microscopy and laser confocal microscopy of immuno-histochemically stained sections have shown that during pregnancy the extent of gap junction formation in human myometrium is low, but that an increase occurs in the active stages of labour despite a high concentration of progesterone in maternal blood. The present investigation focused on the effect of in vitro exposure of isolated myometrical tissue to progesterone, oestradiol and oxytocin, on the number of gap junction plaques in human myometrium at term. Myometrial biopsies were obtained at term from 13 pregnant women who had an elective caesarean section in the 37th or 40th week of pregnancy. The biopsies were immersed immediately in Hepes buffer and buffer containing 0.5, 5.0 μg mL^-1 of progesterone, and 0.1 μg mL^-1 of oestradiol. The muscle biopsies were trimmed under a stereo microscope into strips along the bundles of smooth muscle cells and mounted in tissue baths, superfused with Hepes buffer supplemented with glucose (0.01 mm ); subsequently the strips were exposed to buffer containing different concentrations of progesterone, oestradiol and oxytocin. Incubation of the muscle strips for 180 min resulted in a significant decrease of the number of gap junctions (P <0.01). Neither oestradiol or oxytocin, alone or in combination, had a significant effect on the maintenance of the number of gap junctions. The progesterone concentration of 5.0 μg mL^-1, combined with oxytocin, and with or without oestradiol had a significantly positive effect on the number of gap junction plaques in strips of human myometrium at term (P<0.05 vs. buffer alone). The high concentration of progesterone in the superfusion medium during in vitro experiments may be responsible for the maintenance of high numbers of gap junction complexes in term human myometrium. This finding is of interest in the light of findings of persisting high progesterone levels in maternal blood during labour.     

左旋多巴诱发异动症大鼠纹状体缝隙连接蛋白CX36表达增强

目的:观察左旋多巴诱发异动症(LID)大鼠模型缝隙连接蛋白36(CX36)表达,初步探讨缝隙连接在LID形成机制中的作用。方法:制备帕金森病(PD)和LID大鼠模型,将实验动物分3组:LID模型组、PD未治疗组、正常对照组。各组大鼠分2亚组(缝隙连接阻断剂处理组和生理盐水对照组),观察系统途径给予缝隙连接阻断剂甘珀酸(carbenoxolone)对各组大鼠不自主运动行为的影响。利用免疫组化法检测各组大鼠脑皮层运动区和纹状体区CX36表达并进行分析比较。结果:腹腔注射缝隙连接阻断剂甘珀酸对阿扑吗啡诱导的LID不自主运动和PD旋转行为均无明显影响(P>0.05)。免疫组化结果显示LID组皮层运动区和纹状体区CX36表达较PD组和正常组均有显著增多(P<0.05),PD组与正常对照组亦有明显差别(P<0.05)。结论:LID大鼠基底节及大脑皮层CX36表达增加,缝隙连接可能参与了LID的形成机制。     

The plantaris tendon and a potential role in mid-portion Achilles tendinopathy: an observational anatomical study

The source of pain and the background to the pain mechanisms associated with mid-portion Achilles tendinopathy have not yet been clarified. Intratendinous degenerative changes are most often addressed when present. However, it is questionable if degeneration of the tendon itself is the main cause of pain. Pain is often most prominent on the medial side, 2-7cm from the insertion onto the calcaneus. The medial location of the pain has been explained to be caused by enhanced stress on the calcaneal tendon due to hyperpronation. However, on this medial side the plantaris tendon is also located. It has been postulated that the plantaris tendon might play a role in these medially located symptoms. To our knowledge, the exact anatomy and relationship between the plantaris- and calcaneal tendon at the level of complaints have not been anatomically assessed. This was the purpose of our study. One-hundred and seven lower extremities were dissected. After opening the superficial fascia and paratendon, the plantaris tendon was bluntly released from the calcaneal tendon moving distally. The incidence of the plantaris tendon, its course, site of insertion and possible connections were documented. When with manual force the plantaris tendon could not be released, it was defined as a 'connection' with the calcaneal tendon. In all specimens a plantaris tendon was identified. Nine different sites of insertion were found, mostly medial and fan-shaped onto the calcaneus. In 11 specimens (10%) firm connections were found at the level of the calcaneal tendon mid-portion. Clinical and histological studies are needed to confirm the role of the plantaris tendon in mid-portion Achilles tendinopathy.     

Gap junction protein expression and cellularity: comparison of immature and adult equine digital tendons

Injury to the energy-storing superficial digital flexor tendon is common in equine athletes and is age-related. Tenocytes in the superficial digital flexor tendon of adult horses appear to have limited ability to respond adaptively to exercise or prevent the accumulation of strain-induced microdamage. It has been suggested that conditioning exercise should be introduced during the growth period, when tenocytes may be more responsive to increased quantities or intensities of mechanical strain. Tenocytes are linked into networks by gap junctions that allow coordination of synthetic activity and facilitate strain-induced collagen synthesis. We hypothesised that there are reductions in cellular expression of the gap junction proteins connexin (Cx) 43 and 32 during maturation and ageing of the superficial digital flexor tendon that do not occur in the non-injury-prone common digital extensor tendon. Cryosections from the superficial digital flexor tendon and common digital extensor tendon of 5 fetuses, 5 foals (1-6 months), 5 young adults (2-7 years) and 5 old horses (18-33 years) were immunofluorescently labelled and quantitative confocal laser microscopy was performed. Expression of Cx43 and Cx32 protein per tenocyte was significantly higher in the fetal group compared with all other age groups in both tendons. The density of tenocytes was found to be highest in immature tissue. Higher levels of cellularity and connexin protein expression in immature tendons are likely to relate to requirements for tissue remodelling and growth. However, if further studies demonstrate that this correlates with greater gap junctional communication efficiency and synthetic responsiveness to mechanical strain in immature compared with adult tendons, it could support the concept of early introduction of controlled exercise as a means of increasing resistance to later injury.     

缝隙连接在血管内皮和平滑肌细胞中的作用及其与高血压的关系

细胞间的直接通讯--缝隙连接在控制和协调血管功能方面起重要作用。缝隙连接是沟通相邻细胞质膜的唯一一类通道,为离子、小分子代谢物质、第二信使等细胞间信号分子的直接交换提供一个通道。血管内皮细胞和平滑肌细胞表达多种类型缝隙连接蛋白(connexin,Cx),多种Cx 在心血管系统中的共同表达并协调一致地工作,对心血管系统的发育、平滑肌和内皮细胞功能的整合、以及对血管壁细胞功能协调等起着重要作用。在血管发生疾病时Cx表达相应发生改变,以与缝隙连接调节血管紧张度和动脉血压的作用相一致。本综述讨论了在血管内皮和平滑肌细胞在生理和病理情况下(以高血压为例)缝隙连接对血管结构和功能的影响。     

Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels

The effects of 8-bromoguanosine 3:5-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (g_j) in rat Cx43-transfected cells by 24.0±3.7% (mean±SEM, n=5), whereas g_j was not affected in human Cx43-transfected cells by the same treatment. The relaxation of g_j in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (_j) of about 20, 40–45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the _j distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [³²P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [³²P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased g_j, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.