Structure of the human tibialis anterior tendon

The structure and vascular pattern of the human tibialis anterior tendon was investigated using injection techniques, light and transmission electron microscopy and immunohistochemistry. From the well vascularised peritenon, blood vessels penetrate the tendon tissue and anastomose with a longitudinally oriented intratendinous network. The distribution of blood vessels within the tibialis anterior tendon was not homogenous. The posterior part of the tendon had a complete vascular network that extends from the musculotendinous junction to the insertion at the first metatarsal and medial cuneiform bones. In the anterior half, the tissue was avascular in a zone with a length of 45–67 mm. This zone was covered by a single layer (∼30 μm) of oval shaped cells. Transmission electron microscopy showed that these cells have the characteristics of chondroid cells. This region was stained by Alcian blue at pH 1 which indicates a high concentration of acid glycosaminoglycans and immunohistochemical staining for chondroitin-4-sulphate, chondroitin-6-sulphate and aggrecan was positive. However, immunostaining for the typical cartilage specific type II collagen within this zone was negative. The location of the avascular zone corresponds to the region where the tibialis anterior tendon wraps around the superior and inferior retinacula which serve as fibrous pulleys. This is the region where most spontaneous ruptures of the tibialis anterior tendon occur. The presence of fibrocartilage within gliding tendons is a functional adaptation to compressive and shearing forces. In contrast to reports from the literature about the structure of gliding tendons wrapping around a bony pulley, the gliding zone of the tibialis anterior tendon has only a narrow layer of chondroid cells and proof of type II collagen is lacking.     

蚕丝组织工程化肌腱的生物力学研究

目的探讨用蚕丝与同种异体肌腱细胞联合培养植入体内,构建组织工程化肌腱的生物力学指标。方法实验分2组,一组是植入附着了肌腱细胞的蚕丝材料组,另一组是单纯植入蚕丝材料组。分别在术后的第2,4,6,8周进行随机取材,在每次取材时每组分别取20只,对材料进行生物力学测定。所得数据均采用SPSS13．0统计软件进行处理和分析。结果在第2,4,6,8周进行取材,生物力学的测定结果显示在同时间点内,细胞组的结果明显优于非细胞组（P≮0．05）,细胞组自身在不同时间点的比较中,发现除第8周以外（P〉O．05）,时间越长,力学的结果越优秀（P〈O．05）;而在非细胞组则只有第8周的结果与前3次测定结果的差异有统计学意义（P〈0．05）。结论本实验的结果说明蚕丝材料对肌腱细胞的黏附性好,生物力学性能优越,附着肌腱细胞后可以构成组织工程化肌腱。经更深入的实验和研究,蚕丝材料可能会在肌腱缺损的治疗方面具有良好的应用前景。     

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.     

Human hamstring tenocytes survive when seeded into a decellularized porcine Achilles tendon extracellular matrix

Abstract

Tendon ruptures and defects remain major orthopaedic challenges. Tendon healing is a time-consuming process, which results in scar tissue with an altered biomechanical competence. Using a xenogeneic tendon extracellular matrix (ECM) as a natural scaffold, which can be reseeded with autologous human tenocytes, might be a promising approach to reconstruct damaged tendons. For this purpose, the porcine Achilles (AS) tendons serving as a scaffold were histologically characterized in comparison to human cell donor tendons. AS tendons were decellularized and then reseeded with primary human hamstring tenocytes using cell centrifuging, rotating culture and cell injection techniques. Vitality testing, histology and glycosaminoglycan/DNA quantifications were performed to document the success of tendon reseeding. Porcine AS tendons were characterized by a higher cell and sulfated glycosaminoglycan content than human cell donor tendons. Complete decellularization could be achieved, but led to a wash out of sulfated glycosaminoglycans. Nevertheless, porcine tendon could be recellularized with vital human tenocytes. The recellularization led to a slight increase in cell number compared to the native tendon and some glycosaminoglycan recovery. This study indicates that porcine tendon can be de- and recellularized using adult human tenocytes. Future work should optimize cell distribution within the recellularized tendon ECM and consider tendon- and donor species-dependent differences.     

Locally applied angiogenic factors – a new therapeutic tool for meniscal repair

Tears in the peripheral part of the menisci have a better healing potential than tears in the central part, because the central two-thirds of the menisci are avascular. The avascular status of the meniscus is maintained by the expression of antiangiogenic factors such as endostatin. The distribution of endostatin in the menisci correlates with the degree of vascularization. Endostatin immunostaining is strong in the avascular zone and reduced in the vascularized outer one-third. Endostatin interacts with signal transduction of the vascular endothelial growth factor (VEGF) by reducing VEGF-induced kinase (Erk1/2) phosphorylation. VEGF plays an important role in angiogenesis in fetal menisci and it is down-regulated in the adult meniscus.

We hypothesized that healing of meniscal tears in the avascular zone can be promoted by the local application of the angiogenic factor VEGF. To evaluate this hypothesis a tear was created in the avascular zone of the medial meniscus in 18 merino sheep. The tear was then repaired with an uncoated suture (group 1), a suture coated with PDLLA (group 2), and by a suture coated with PDLLA/VEGF (group 3).

After 6 weeks we observed increased factor VIII immunostaining in the VEGF-treated group. However, in this treatment group (VEGF/PDLLA) no meniscus healed. In the uncoated suture group and in the PDLLA-coated suture group partial healing was observed in three animals and complete healing in three animals, respectively. Factor VIII expression is normally restricted to vascular endothelial cells. In this study, however, single endothelial cells could be detected in the menisci of the VEGF/PDLLA group. This finding suggests that the application of VEGF might have stimulated proliferation of vascular endothelial cells but the application of VEGF was not successful in stimulating the more complex process of vasculogenesis.

Further immunohistochemical examinations of the specimen have shown that in the VEGF/PDLLA group there is strong immunostaining against matrix metalloproteinase 13 (MMP-13). In vitro studies have shown that VEGF can stimulate chondrocytes to proliferate but also to express MMP-13 via HIF1- induction. Since meniscal fibrochondrocytes express the VEGF receptor 2 (KDR) the induction of MMP expression might be another factor which inhibits healing despite increased angiogenesis.

In conclusion, the local application of VEGF via PDLLA-coated sutures does not promote meniscal healing. A single growth factor might not always be a promising tool for the promotion of tissue repair. Further studies have to find out if growth factor combinations (VEGF and angiopoitin) might be more effective in stimulating vasculogenesis during meniscal healing.     

In vivo tendon engineering with skeletal muscle derived cells in a mouse model

Engineering a functional tendon with strong mechanical property remains an aim to be achieved for its eventual application. Both skeletal muscle and tendon are closely associated during their development and both can bear strong mechanical loading dynamically. This study explored the possibility of engineering stronger tendons with mouse skeletal muscle derived cells (MDCs) and with mouse tenocytes as a control. The results demonstrated that both MDCs and tenocytes shared the gene expression of growth differentiation factor-8 (GDF-8), collagens I, III, VI, scleraxis and tenomodulin, but with MyoD gene expression only in MDCs. Quantitatively, MDCs expressed higher levels of GDF-8, collagens III and VI (p < 0.05), whereas tenocytes expressed higher levels of collagen I, scleraxis and tenomodulin (p < 0.05). Interestingly, MDCs proliferated faster with more cells in S + G2/M phases than tenocytes (p < 0.05). After been seeded on polyglycolic acid (PGA) fibers, MDCs formed better quality engineered tendons with more mature collagen structure and thicker collagen fibrils as opposed to tenocyte engineered tendons. Biochemically, more collagen VI and decorin were produced in the former than in the later. Functionally, MDC engineered tendons exhibited stronger mechanical properties than tenocyte engineered tendons, including maximal load, stiffness, tensile strength and Young's modulus (p < 0.05). Furthermore, with the increase of implantation time, MDCs gradually lost their expression of myogenic molecules of MyoD and desmin and gained the expression of tenomodulin, a marker for tenocytes. Collectively, these results indicate that MDCs may serve as a desirable alternative cell source for engineering functional tendon tissue.     

Gene Expression of Tendon Collagens and Tenocyte Markers in Long-Term Monolayer and High-Density Cultures of Rat Tenocytes

As a result of repeated movement, tendons are functionally open to traumas. According to this situation, tenocytes have already been used for tissue engineering therapies. It has been reported that long-term monolayer (ML) culture of tenocytes may lead to a phenotypic drift within passages. Depending on our previously published work, it is clearly demonstrated that high-density (HD) culture improves cell growth and differentiation of tenocytes. However, it is not yet established if HD favors the differentiated state during long-term culture. Therefore, we compared the differences in gene expression of tendon collagens and tendon markers of tenocytes from long-term ML and HD culture conditions by quantitative, real-time polymerase chain reaction (QRT-PCR) for over a period of 3 weeks. COLI, COLIII, COLV, Scx, and Tnmd were target genes as the major matrix constituents of tendons as well as being involved in matrix integrity and tenocyte phenotype. According to our results, tenocytes in HD culture synthesized less amounts of COLIII, COLV, and Tnmd, and dependent on the investigation time point, higher amounts of Scx. We consider that tenocytes produced in HD culture system may not provide sufficient efficiency during tissue engineering approaches. By the fact that most molecules showed significantly higher expression profiles in ML culture condition, it is suggested that culture and passage in ML should be taken into consideration for further tissue engineering approaches to maintain a phenotype with less amount of drift.     

Complement gene expression is regulated by pro-inflammatory cytokines and the anaphylatoxin C3a in human tenocytes

Interplay between complement factors, regulatory proteins, anaphylatoxins and cytokines could be involved in tendon healing and scar formation. The expression and regulation of complement factors by cytokines or anaphylatoxins are completely unclear in tendon.Hence, the gene expression of the anaphylatoxin receptors C3aR, C5aR and cytoprotective complement regulatory proteins (CRPs) was analysed in human tendon, cultured primary tenocytes and to directly compare the general expression level, additionally in human leukocytes. Time-dependent regulation of complement by cytokines and the anaphylatoxin C3a was assessed in cultured tenocytes.Gene expression of the anaphylatoxin receptors C3aR, C5aR and the CRPs CD46, CD55 and CD59 was detected in tendon, cultured tenocytes and leukocytes, whereas CD35 could only be found in tendon and leukocytes. Compared with cultured tenocytes, complement expression was higher in tendon and compared with leukocytes C3aR, C5aR, CD35 and CD55, but not CD46 and CD59 gene expression levels were lower in tendon. C3aR mRNA was up-regulated by both TNFα and C3a in cultured tenocytes in a time-dependent manner whereby C5aR gene expression was only induced by C3a. IL-6 or C3a impaired the CRP gene expression. C3a stimulation lead to an up-regulation of TNFα and IL-1β mRNA in tenocytes. Degenerated tendons revealed an increased C5aR and a reduced CD55 expression.The expression profile of the investigated complement components in tendon and cultured tenocytes clearly differed from that of leukocytes. Tenocytes respond to the complement split fragment C3a with CRP suppression and enhanced pro-inflammatory cytokine gene expression suggesting their sensitivity to complement activation.     

BMP12基因和间充质干细胞修复兔跟腱缺损的形态学研究

目的　观察用BMP12基因和间充质干细胞修复兔跟腱缺损的形态学变化。方法　模拟微重力条件下构建两种组织工程化肌腱。 2 4只新西兰白兔分为 4组 :①单纯人发角蛋白 (HHK对照组 )组 ;②骨髓间充质干细胞 (MSCs) /HHK组织工程化肌腱组 ;③骨形态发生蛋白 12 (BMP12 )基因诱导的腱细胞 /HHK组织工程化肌腱组 ;④pTARGET BMP12质粒 /HHK组。采用光电镜、免疫组织化学和RT PCR方法观察术后不同时期损伤肌腱的修复情况。结果　MSCs/HHK组织工程化肌腱组和基因诱导的腱细胞 /HHK组织工程化肌腱组的缺损肌腱的再生修复效果均优于单纯HHK组 ,尤以基因诱导的腱细胞 /HHK组织工程化肌腱组的修复效果最佳 ,且伴随有Ⅰ型胶原mRNA表达的增高。结论　BMP12基因和MSCs通过促进内源性愈合参与了缺损肌腱的再生修复。     

Repair of tendon defect with dermal fibroblast engineered tendon in a porcine model

Harvesting autologous tenocytes for tendon engineering may cause secondary tendon defect at the donor site. Dermal fibroblasts are an easily accessible cell source and do not cause major donor site defect. This study aims to explore the possibility of tendon engineering using dermal fibroblasts. A total of 45 hybrid pigs were randomly divided into three groups: experimental group (n = 15)--repair of tendon defect with a dermal fibroblast engineered tendon; control group 1 (n = 15)--repair of defect with a tenocyte engineered tendon; and control group 2 (n = 15)-repair of defect with a scaffold alone. Both autologous dermal fibroblasts and tenocytes were seeded on polyglycolic acid (PGA) unwoven fibers to form a cell-scaffold construct and cultured in vitro for 7 days before in vivo implantation to repair a defect of flexor digital superficial tendon. Specimens were harvested at weeks 6, 14, and 26 for gross, histological, and mechanical analyses. Microscopy revealed good attachment of both dermal fibroblasts and tenocytes on PGA fibers and matrix production. In vivo results showed that fibroblast and tenocyte engineered tendons were similar to each other in their gross view, histology, and tensile strength. At 6 weeks, parallel collagen alignment was observed at both ends, but not in the middle in histology, with more cellular components than natural tendons. At weeks 14 and 26, both engineered tendons exhibited histology similar to that of natural tendon. Collagens became parallel throughout the tendon structure, and PGA fibers were completely degraded. Interestingly, dermal fibroblast and tenocyte engineered tendons did not express type III collagen at 26 weeks, which remained observable in normal pig skin and control group 2 tissue using polarized microscopy, suggesting a possible phenotype change of implanted dermal fibroblasts. Furthermore, both fibroblast and tenocyte engineered tendons shared similar tensile strength, about 75% of natural tendon strength. At 6 weeks in control group 2, neo-tissue was formed only at the peripheral area by host cells. A cord-like tissue was formed at weeks 14 and 26. However, the formed tissue was histologically disorganized and mechanically weaker than both cell-engineered tendons (p < 0.05). These results suggest that dermal fibroblasts may have the potential as seed cells for tendon engineering.     

Engineering of extensor tendon complex by an ex vivo approach

Engineering of extensor tendon complex remains an unexplored area in tendon engineering research. In addition, less is known about the mechanism of mechanical loading in human tendon development and maturation. In the current study, an ex vivo approach was developed to investigate these issues. Human fetal extensor tenocytes were isolated, expanded and seeded on polyglycolic acid (PGA) fibers that formed a scaffold with a shape mimicking human extensor tendon complex. After in vitro culture for 6 weeks, 7 cell-scaffold constructs were further in vitro cultured with dynamic mechanical loading for another 6 weeks in a bioreactor. The other 14 constructs were in vivo implanted subcutaneously to nude mice for another 14 weeks. Seven of them were implanted without loading, whereas the other 7 were sutured to mouse fascia and animal movement provided a natural dynamic loading in vivo. The results demonstrated that human fetal cells could form an extensor tendon complex structure in vitro and become further matured in vivo by mechanical stimulation. In contrast to in vitro loaded and in vivo non-loaded tendons, in vivo loaded tendons exhibited bigger tissue volume, better aligned collagen fibers, more mature collagen fibril structure with D-band periodicity, and stronger mechanical properties. These findings indicate that an extensor tendon complex like structure is possible to generate by an ex vivo approach and in vivo mechanical loading might be an optimal niche for engineering functional extensor tendon.     

In vitro tendon engineering with avian tenocytes and polyglycolic acids: a preliminary report

Although there are many reports of in vivo tendon engineering using different animal models, only a few studies involve the short-term investigation of in vitro tendon engineering. Our previous study demonstrated that functional tendon tissue could be engineered in vivo in a hen model using tenocytes and polyglycolic acid (PGA) fibers. This current study explored the feasibility of in vitro tendon engineering using the same type of cells and scaffold material. Tenocytes were extracted from the tendons of a hen's foot with enzyme digestion and cultured in DMEM plus 10% FBS. Unwoven PGA fibers were arranged into a cord-like construct and fixed on a U-shape spring, and tenocytes were then seeded on PGA fibers to generate a cell-PGA construct. In experimental group 1, 22 cell-scaffold constructs were fixed on the spring with no tension and collected at weeks 4 (n = 7), 6 (n = 7) and 10 (n = 8); in experimental group 2, five cell-scaffold constructs were fixed on the spring with a constant strain and collected after 6 weeks of culture. In the control group, three cell-free scaffolds were fixed on the spring without tension. The collected engineered tendons were subjected to gross and histological examinations and biomechanical analysis. The results showed that tendon tissue could be generated during in vitro culture. In addition, the tissue structure and mechanical property became more mature and stronger with the increase of culture time. Furthermore, application of constant strain could enhance tissue maturation and improve mechanical property of the in vitro engineered tendon (1.302 +/- 0.404 Mpa with tension vs 0.406 +/- 0.030 Mpa without tension at 6 weeks). Nevertheless, tendon engineered with constant strain appeared much thinner in its diameter than tendon engineered without mechanical loading. Additionally, its collagen fibers were highly compacted when compared to natural tendon structure, suggesting that constant strain may not be the optimal means of mechanical load. Thus, application of dynamic mechanical load with a bioreactor to the construction of tendon tissue will be our next goal in this series of in vitro tendon engineering study.     

Effect of paratenon and repetitive motion on the gliding resistance of tendon of extrasynovial origin

We measured the gliding resistance of canine and human tendons of intrasynovial origin and tendons of extrasynovial origin with and without paratenon, and investigated the structure of paratenon using scanning electron microscopy. In the canine study, seven intrasynovial flexor digitorum profundus (FDP) tendons, seven extrasynovial fibularis (peroneus) longus (FL) tendons with paratenon, and seven FL tendons without paratenon were used. In the human study, seven intrasynovial FDP tendons and seven extrasynovial palmaris longus (PL) tendons cut in half (one half with paratenon, the other half without paratenon) were used. The gliding resistance of each tendon was measured at 1, 5, 10, 20, 50, and100 flexion/extension cycles. The canine and human FDP tendons maintained a gliding resistance significantly lower than that of the other tendons at all observation points (P < 0.05). In the canine, the gliding resistance of the FL tendon with paratenon was significantly lower than that of the FL tendon without paratenon up to 50 flexion/extension cycles (P < 0.05), but the two were not significantly different at 100 cycles. In the human, the gliding resistance of PL tendons with paratenon was significantly lower than that of the PL tendons without paratenon at all measuring points (P < 0.05). Preservation of paratenon thus appears to decrease the gliding resistance of extrasynovial tendons after repetitive motion in vitro.     

Adenovirus-mediated gene transfer of growth and differentiation factor-5 into tenocytes and the healing rat Achilles tendon

Growth and differentiation factor-5 (GDF-5) is known to induce tendon tissue and stimulate tendon healing. The hypothesis was that adenoviral GDF-5 transfer leads to transitory transgene expression and improves Achilles tendon healing. In vitro experiments were first performed with rat tenocytes. Transgene expression was evaluated by RT-PCR, Western blotting and GDF-5-ELISA. In vivo virus dosage and transgene expression were examined by a marker gene transfer (LacZ and luciferase). In the main experiment in 131 rats, adenovirus particles (3 x 10(10)) were injected into transected Achilles tendons. The time course of GDF-5 mRNA expression was assessed by real-time RT-PCR. Histology and biomechanical testing were used to evaluate tendon healing and tensile strength. In vitro GDF-5 was secreted with a maximum after 2 weeks (330 ng GDF-5/10(6) cells per 24 hr). In vivo GDF-5 transgene expression showed a maximum at 4 weeks. At 8 weeks, GDF-5 specimens were thicker (p<0.05) with a trend to higher strength (p=0,064). Histology showed greater cartilage formation in type II collagen stains than in controls. Injection of adenovirus particles successfully can deliver the GDF-5 gene in healing tendons and leads to thicker tendon regenerates after 8 weeks. This technique might become a new approach for nonsurgical treatment of tendon injuries.     

Vascular endothelial growth factor (VEGF), VEGF receptors expression and microvascular density in benign and malignant thyroid diseases

Angiogenesis is critical for the growth and metastatic spread of tumours. Vascular endothelial growth factor (VEGF) is the most potent inducer of neovasculature, and its increased expression has been related to a worse clinical outcome in many diseases. The purpose of this study was to evaluate the relation between VEGF, its receptors (VEGFR-1 and VEGFR-2) and microvessel density (MVD) in thyroid diseases. Immunostaining for VEGF and VEGF receptors was performed in 66 specimens of thyroid tissue, comprising 17 multinodular goitre (MNG), 14 Graves' disease, 10 follicular adenoma, 8 Hashimoto's thyroiditis, 7 papillary carcinoma and 10 normal thyroid specimens. Thyrocyte positivity for VEGF and VEGF receptors was scored 0-3. Immunohistochemistry for CD31, and CD34 on the same sections was performed to evaluate MVD. Immunohistochemical staining of VEGF in thyrocytes was positive in 92% of all the thyroid tissues studied. Using an immunostaining intensity cut off of 2, increased thyrocyte staining was seen in follicular adenoma specimens, MNG and normal thyroids compared with Hashimoto's thyroiditis and Graves' disease (P < 0.05). Similarly, VEGF thyrocyte expression in Graves' disease was less than other pathologies (P < 0.05). VEGFR-1 expression and the average MVD score did not differ between the different thyroid pathologies. VEGF expression was lower in autoimmune pathologies compared to autonomous growth processes. Conversely, both VEGFR-1 and VEGFR-2 were widely expressed in benign and neoplastic thyroid disease, suggesting that the up-regulation of VEGF and not its receptors occurs as tissue becomes autonomous. There was no clear relationship between MVD measurement and thyroid pathology.     

Vascular endothelial growth factor inhibits outward delayed-rectifier potassium currents in acutely isolated hippocampal neurons

In the present study, whole-cell patch-clamp recording was used to study whether vascular endothelial growth factor (VEGF) had a regulatory effect on the potassium-channel currents. The outward delayed-rectifier potassium currents (I(K)) were recorded in acutely isolated hippocampal neurons from 14-day-old rat brains. A local application of VEGF at the concentrations from 50 ng/ml to 200 ng/ml dose-dependently inhibited I(K). Administration of VEGF (100 ng/ml) to the neurons only for seconds could significantly reduce I(K) in 26 of 39 recorded cells. The currents could recover to 82.8+/-3.7% of the control level at 60 s after removing VEGF in the buffer. In the I-V curve analysis, VEGF negatively shifted the I-V curve of I(K); the inhibition was gradually enhanced as the membrane potential increased from -40 mV to 50 mV in 13 cells. Thus, the results reveal that VEGF inhibits I(K) in acute, reversible and voltage-dependent manners. Double staining combined with confocal laser scanning microscopy was used to simultaneously detect the distribution of VEGF receptors (flt-1 and flk-1) in the hippocampal section and isolated neuron. Results showed that flt-1-positive staining, but not flk-1, could be observed on the membrane of the hippocampal neuron in both preparations, suggesting the presence of neuronal membrane VEGF flt-1 receptors in the hippocampus. To investigate if the inhibition by VEGF on I(K) is related to the presence of flt-1 receptors, we further did flt-1-receptor immunostaining for the recorded neurons, which was labeled with Lucifer Yellow during the recording. Among nine recorded cells, five showing the inhibition by VEGF had detectable signals for flt-1 receptors on their membrane, whereas the other four showing no inhibition had no flt-1 receptors either. The results suggest that VEGF can acutely inhibit I(K) in the hippocampal neurons probably related to the presence of membrane flt-1 receptors in the neurons.