成人心脏传导系统中连接蛋白43的表达

目的 探讨成人心脏及传导系统窦房结,房室结,浦肯野纤维中连接蛋白43(CX43)的表达情况。方法 免疫组织化学SABC法。结果 CX43在窦房结及房室结周围细胞膜呈散在、少量表达,在浦肯野细胞膜可见线性、细长阳性颗粒,在心房及心室肌阳性颗粒主要位于端端相连部位的闰盘处。结论 CX43在传导系统的表达与心脏传导系统的电生理传导特性相符。     

心房纤颤患者心房肌Cx43的蛋白表达

目的：研究房颤患者心房肌细胞连接蛋白43(Cx43)的蛋白表达。方法：应用SP免疫组化方法,比较正常和房颤患者心房肌Cx43的表达。结果：(1)正常心房肌细胞Cx43呈斑点状或带状,大量分布于细胞侧连接处。部分位于闰盘处。(2)房颤患者心房肌细胞Cx43颗粒发生重排,杂乱地分布于心肌的各个部位。房颤时间长者,部分Cx43移入细胞质中,表现为某些区域极度增加或减少。(3)房颤患者心房肌细胞Cx43的蛋白表达量未发生显著变化。结论：Cx43在房颤患者心房肌细胞发生重排,呈无序分布,可能是心房纤颤发病的原因之一;并提示房颤仅对Cx43的表达形式产生影响。     

大鼠脑出血移植施万细胞的实验研究

目的 探讨施万细胞（SCs）移植于大鼠脑出血部位对神经再生的影响。方法体外培养SCs，弘溴脱氧尿嘧啶（BrdU）标记后移植于脑出血模型大鼠的脑出血部位，不同时间处死动物，免疫组织化学双染检测BrdU／髓鞘碱性蛋白（MBP）和BrdU／/生长相关蛋白-43（GAP-43）的表达。结果SCs移植1周后至13周，脑内可见BrdU／MBP双染阳性细胞；移植后第12周可见BrdU和GAP-43阳性细胞，海马部位GAP-43阳性细胞尤其多见。结论 移植的SCs参与髓鞘形成和神经再生。     

ATP对人白血病细胞U937凋亡的影响

目的　探讨ATP诱导人白血病细胞U937细胞凋亡的机理。　方法　应用ATP(0 2 3g L)作用于培养的U937细胞 ,应用免疫荧光细胞化学方法检测连接蛋白Cx4 3、F 肌动蛋白 (F actin)、纽蛋白 (vinculin)的表达。　结果　ATP诱导U937细胞凋亡小体形成的同时Cx4 3表达增强、F 肌动蛋白重组 ,纽蛋白组装改善。　结论　ATP诱导人白血病细胞凋亡时 ,凋亡小体的形成与间隙连接蛋白Cx4 3表达、F 肌动蛋白重组 ,纽蛋白组装有着平行关系 ,表明它们在U937凋亡小体形成过程所起的重要作用     

Characterization of a prolonged regenerative attempt by diffusely injured axons following traumatic brain injury in adult cat: a light and electron microscopic immunocytochemical study

Traumatic brain injury in animals and humans is well known to cause axonal damage diffusely scattered throughout the brain without evidence of other brain parenchymal change. This observation has prompted some to posit that such damaged axons are well positioned to mount a regenerative attempt. The present study uses an immunocytochemical marker specific for regenerating neurites to explore this issue. Further, in an attempt to expedite and enhance any potential regenerative effort, this study evaluates the efficacy of intrathecally applied nerve growth factor. Three sets of experiments were performed in adult cats. One group of animals was subjected to moderate fluid percussion brain injury and followed for 7 or 14 days post injury, with the continuous intraventricular infusion of nerve growth factor delivered by implanted osmotic pumps. These animals were compared to a second group of time-matched, sham-operated animals receiving artificial cerebrospinal fluid infusion. To assess axonal damage immunohistochemical staining for the low molecular weight neurofilament subunit (NF-L) was carried out using an NR4 monoclonal antibody. To localize axons exhibiting a regenerative response immunohistochemical staining for the growth associated protein GAP43 was employed. In sham controls, at the light microscopic level NF-L-immunoreactive axonal swellings were numerous at 7 days, but by 14 days post injury their frequency declined markedly. In contrast, GAP43-immunoreactive, disconnected reactive axonal swellings were rarely observed at 7 days but were numerous at 14 days. Ultrastructural analysis at 14 days post injury of carefully matched sections revealed reactive axons demonstrating sprouting consistent with a regenerative effort. Analysis of tissue from animals of 14 days of survival indicated that supplementation with nerve growth factor did not appear to enhance the capacity of damaged brain axons to mount a regenerative attempt. Rather, it appears that regenerative efforts seen reflect a spontaneous response. A third group of adult cats, subjected to the same injury but not subjected to osmotic pump implantation, was allowed to survive for 22–28 days. Animals in this group also demonstrated GAP43 immunoreactivity in reactive axonal swellings in the brain stem. This study demonstrates that diffusely injured axons can mount a sustained regenerative attempt that is associated with a reorganization of their cytoskeleton and accompanied by an up-regulation of growth-associated proteins. Received: 3 March 1997 / Revised, accepted: 1 April 1997     

Phorbol ester-induced neuritic alterations in the rat neocortex

In order to explore the effect of aberrant sprouting in the CNS, phorbol 12-myristate 13-acetate (PMA) was administered into the neocortex of adult rats. PMA is a growth-promoting agent that activates and eventually downregulates protein kinase C (PKC), and induces in the rat the expression of several genes, including amyloid precursor protein (APP). We found that multiple injections of 100 nM PMA into the rat neocortex promote, in the first week postinjection, a widespread vacuolization of the neuropil with a subsequent disruption of the synapses in the injection site, followed, at d 15, by the formation of abnormally distended clusters of neurites that resembled aberrant, sprouting axons. At d 30, fewer aberrant sprouts were observed, and many degenerating neurites were found. At the ultrastructural level, the PMA-induced abnormal neurites at d 7–15 resembled growth cones, whereas the dystrophic neurites at d 30 contained abundant dense and laminated bodies. Immunohistochemical analysis indicated that the abnormal neurites in the areas of denervation and PMA administration were positive with antisynaptophysin and antigrowth-associated protein 43 (GAP-43), with an increased APP immunoreactivity surrounding them. APP immunoreactivity around the injection site was mostly associated with pyramidal neurons and glial cells. Control experiments, where saline alone or 4α-phorbol 12, 13-didecanoate (PDD, an inactive phorbol derivative) was injected, failed to show aberrant sprouting neurites. Further immunohistochemical analysis showed that the PMA-treated animals presented increased amyloidΒ immunoreactivity in the pyramidal cells at the site of injection, when compared with control injections. These findings suggest that aberrant sprouting induced by overstimulation could be followed by neurodegeneration. Alternatively, PKC downregulation could directly induce the neurodegeneration, with a secondary sprouting response.     

Exocytosis and Selective Neurite Calcium Responses in Rat Cerebellar Granule Cells During Field Stimulation

The free calcium concentration, [Ca²⁺]_c, in fura-2-loaded rat cerebellar granule cells was investigated by digital imaging during trains of uniform field stimuli in order to compare the ability of calcium channels in somata and neurites to respond to brief, physiologically relevant depolarizations. Very few somata responded to 20 Hz trains of 1 ms pulses, while virtually all neurites showed an extensive increase which was rapidly reversed when stimulation was terminated. In contrast, both somata and neurites responded when cells were depolarized with 50 mM KCl. The field stimuli evoked a tetrodotoxin-sensitive increase in Na⁺ concentration in both somata and neurites. When 4-aminopyridine, which inhibits delayed K⁺ currents in these cells, was present during the field stimulus both somata and neurites increased their [Ca²⁺]_c, suggesting that prolongation of the duration of depolarization is required for somatic Ca²⁺ channel activation. The neurite response did not depend on the orientation of the neurite relative to the applied field. The neurite response was insensitive to nifedipine (1 μM) and ω-agatoxin-IVA (30 nM) but was uniformly inhibited by ω-conotoxin-GVIA (30% inhibition at 1 μM) and ω-conotoxin-MVIIC (44% inhibition at 5 μM). The two inhibitors were not additive. The neurite [Ca²⁺]_c response was insensitive to the combination of ionotropic glutamate receptor antagonists. Field stimulation caused the exocytosis of the fluorescent probe FM1-43 previously loaded during KCl depolarization, suggesting that presynaptic Ca²⁺ channels contribute to the field-evoked neurite response.     

兔早期心肌缺血不同时段Connexin43的表达

目的 探讨急性心肌缺血不同时间心肌缝隙连接蛋白43的分布特征。方法 结扎兔左冠状动脉造成急性心肌缺血模型,于结扎后不同时间取左心室前壁心肌。用HBFP染色定位心肌缺血区域,用SP法观察心肌缺血区、交界区及非缺血区Cx43的分布,用图像分析系统分析。结果 急性心肌缺血30 min,Cx43阳性面积减少约40％,此后Cx43逐渐降低,大约480 min,Cx43几乎消失。结论 兔急性心肌缺血时,Cx43的减少有时间特点。     

X-irradiation of adult spinal cord increases its capacity to support neurite regeneration in vitro

Previous in vitro studies have shown that X-irradiation during early postnatal life can change the environment of CNS tissue in later adult life such that it becomes more supportive of neurite regeneration from adult dorsal root ganglion (DRG) neurons than non-irradiated tissue. The question arises whether or not x-irradiation during adult life can alter the CNS environment such that it also becomes more supportive of neurite regeneration. This was investigated by exposing portions of the spinal cord of adult rats to 10, 20 or 40 Gray of X-irradiation and later using this tissue to prepare cryosections suitable for use as a substrate in a cryoculture assay. Fixed cryocultures were immunolabelled using anti-glial fibrillary acidic protein (GFAP) to visualise the tissue sections and anti-growth associated protein (GAP-43) to visualise the regenerating neurites. Tissue sections from sham-irradiated animals and from those irradiated with 10 Gray did not support the regeneration of neurites. However, sections of spinal cords from rats treated with either 20 or 40 Gray of X-irradiation 4 or 32 days prior to sampling were found to support a certain degree of neurite regeneration. It is concluded that X-irradiation of adult CNS tissue can alter its environment such that it becomes more supportive of neurite regeneration and it is speculated that this change may be the result of alterations in the glial cell populations in the post-irradiated tissues.     

Relationship of a dystrophin-associated glycoprotein to junctional acetylcholine receptor clusters in rat skeletal muscle

The relationship of a member of the transmembrane dystrophin-associated glycoprotein (DAG) complex to acetylcholine receptors (AChRs) was investigated using immunofluorescence techniques at rat neuromuscular junctions (NMJs) viewed en face. These results were compared with those from a similar previous study of dystrophin and an autosomal homologue (utrophin or dystrophin-related protein, DRP) (Bewick et al. NeuroReport 1992; 3; 857–860). The region of highest 43 K DAG (43DAG) labelling projected beyond the AChRs by 0.3 μm, as does that for dystrophin. By contrast DRP labelling precisely co-localizes with the AChRs. These results suggest that at the NMJ, the region of high 43DAG concentration encompasses the area of highest intensity labelling for both DRP and dystrophin.