Cell mediated calcification in collagen gel cultures of fetal rat calvaria cells. Loss of gap junctions precedes calcification

Summary To analyze the mechanism of initiation of cell-mediated calcification in hard tussue and its relationship to the frequency of gap junctions, enzymatically isolated cells from fetal rat calvaria cultured in collagen gels were observed ultrastructurally over a time course. Calcification was observed at 2–3 weeks after the initiation of culture when the seeding cellularity and the concentration of β-glycerophosphate were sufficiently high. In the collagen gels, round cells (R), spindle or stellate cells (S), and fat cells (F) were characterised morphologically. The ultrastructural features of initial calcification could be classified into 4 subtypes: 1) a large mass greater than 10 μm in diameter (Type I), 2) deposition associated with dead R cells or matrix vesicles (Type II), 3) intracellular deposition (Type III), and 4) other than Types I–III (Type IV). Type II was the most frequent (44.5%) and Type III was the least (6.8%). Gap junction was observed frequently between 1) R cells, 2) S cells, 3) between R cells and S cells. The frequency of gap junctions in collagen gels decreased statistically (X²-test; p<0.001), when calcification was initiated. This cell culture system can be regarded as a useful model to analyze the initiation of cell mediated calcification in hard tissue. Gap junctions might function in cell communication and a decrease in their numbers could lead to cell death and, subsequently to calcification.     

Crystal dissolution of biological and ceramic apatites

Summary High resolution transmission electron microscopy (Hr TEM) studies on biological and synthetic calcium phosphate have provided information on the dissolution process at the crystal level. The purpose of this study was to investigate the dissolution of ceramic hydroxyapatite (HA) after implantation using Hr TEM. Recovered HA ceramic implanted in bony and nonbony sites in animals and in periodontal pockets in humans were used for the study. For comparison, sections of human fluorotic enamel with caries and sections of shark enameloid previously exposed to 0.1 HCl were similarly investigated. Hr TEM studies demonstrated that in both the biological and ceramic apatites, the lattice and atomic defects were the starting points in the dissolution process. However, significant differences in the process of dissolution were observed: (1) biological apatite crystals showed preferential core dissolution whereas ceramic apatite crystals showed nonspecific dissolution at the cores and at the surfaces; (2) the dissolution of biological apatites appeared to consistently extend along the crystal's c-axis whereas dissolution of the ceramic HA did not appear to be correlated with the crystal's c-axis. The observed differences in crystal dissolution between biological and ceramic apatites may be attributed to the following: (1) the unique crystal/protein interaction present with biological apatites but absent in ceramic HA; (2) differences in defect distribution between biological and ceramic apatites which are due to the differences in the original of these defects; and (3) the longer morphological c-axis of biological apatites compared with that of ceramic apatites. This study provided for the first time, information on the dissolution process of implanted ceramic HA crystals and suggests that the crystal defects resulting from the sintering processes during the preparation of ceramic HA affect itsin vivo degradation and performance.     

针刺对戊四唑致痫大鼠海马神经元超微结构的影响

目的观察针刺对戊四唑诱发癫痫大鼠海马神经元损伤的保护作用。方法将SD大鼠72只随机分为模型组、针刺组、正常组。正常组腹腔注射0.9%氯化钠注射液(2 mL)、其余各组腹腔注射戊四唑(50 mg/kg),于注射后4 h与24 h两个时间段取脑组织。分别于光镜下和电镜下观察海马结构改变。结果癫痫发作后4 h光镜、电镜均可见大鼠海马神经元损伤,24 h后损伤进一步加重;针刺组大鼠海马神经元损伤明显减轻。结论针刺具有明显保护癫痫继发脑神经元损伤作用。     

Increase in types IV and VI collagen in cherry haemangiomas

Summary The capillaries in cherry haemangiomas show perivascular hyalinized sheaths. In order to clarify the nature of this sheath material, the extracellular matrix of cherry haemangiomas from 20 normal volunteers (age range 30–64 years) was investigated using immunohistochemical and electronmicroscopical methods. Antibodies against collagen types III, IV and VI and laminin were used. Hyaluronic acid was visualized using the hyaluronic acid binding region of the cartilage proteoglycan as ligand. Electronmicroscopically, the sheaths contained multilaminated basement membrane-like material, collagen fibres 20–25 nm thick with a periodicity of 67 nm and broad-banded aggregates with a periodicity of 100 nm (zebra bodies or fibrous long-spacing fibres). Immunohistochemically, type IV collagen was stained throughout the whole sheath material. Staining for laminin was more confined to the endothelial side of the sheath. Intense staining for type III collagen and hyaluronic acid was found in the connective tissue of the subpapillary layer and between the cherry haemangioma capillaries. Much weaker staining for type III collagen and no staining for hyaluronic acid were found invariably in an area 4–10 m thick directly around the capillaries. Both sheath material and intercapillary connective tissue of the haemangiomas showed pronounced staining for collagen type VI. Immunogold staining revealed that type VI collagen was localized to microfibrils 5–6 nm thick and to the broad-banded aggregates with 100 nm periodicity. These findings further underline the assumption that the broad-banded aggregates consist of type VI collagen.This paper was presented in part at the XVIIIth Annual Meeting of the Arbeitsgemeinschaft Dermatologische Forschung (ADF), 9–11 November 1990, Mannheim, FRG     

Electron microscopy and morphometry enhances differentiation of epidermolysis bullosa subtypes. With normal values for 24 parameters in skin

Electron microscopy combined with morphometry was used to establish values for 24 parameters in normal skin. These results were compared with those similarly obtained from samples of epidermolysis bullosa with a view to facilitating classification of the disease. Six of the eight subtypes of epidermolysis bullosa investigated could be differentiated. Four subtypes showed values for structural components in intact skin which were outside the normal range: (1) epidermolysis bullosa simplex generalisata gravis (hemidesmosomes); (2) epidermolysis bullosa dystrophica Pasini and (3) Cockayne-Touraine (anchoring fibrils); and (4) epidermolysis bullosa acquisita (anchoring fibrils, hemidesmosomes, and lamina lucida and lamina densa aspects of the dermoepidermal junction). Two subtypes revealed specific features which could be assessed qualitatively: distinctive, circumscribed, clumped tonofilament bodies were present in basal keratinocytes from epidermolysis bullosa herpetiformis Dowling-Meara and thick (30 nm diameter) cross-striated anchoring fibrils were absent in epidermolysis bullosa dystrophica generalisata gravis. Epidermolysis bullosa simplex Köbner and Weber-Cockayne forms could not be distinguished.     

电针“百会”“神庭”对血管性痴呆大鼠学习记忆能力和海马突触结构与相关蛋白表达水平的影响

目的 观察电针“百会”“神庭”对血管性痴呆（vascular dementia,VD）大鼠学习和记忆功能的影响,并从突触结构及突触相关蛋白表达水平的角度揭示其作用机制。方法 将35只雄性SD大鼠随机分为假手术组、模型组、电针穴位组、电针非穴位组和奥拉西坦组,每组7只。采用改良双侧颈动脉结扎模型,电针穴位组大鼠选择“百会”“神庭”两穴治疗,电针非穴位组大鼠选择固定非穴位刺激,每次电针30 min,每日1次,连续干预14 d;奥拉西坦组大鼠选择腹腔注射奥拉西坦,50 mg/kg,每日1次,连续14 d。采用Morris水迷宫检测各组大鼠学习和空间记忆能力;透射电子显微镜观察各组大鼠海马CA1区突触结构;Western blot检测各组大鼠海马突触后致密蛋白95（postsynaptic density protein 95, PSD95）、GluA1、GluN2B和磷酸化GluN2B蛋白表达水平。结果 与假手术组比较,模型组大鼠学习期逃避潜伏时间延长,测试期跨越平台次数减少,目标象限停留时间显著缩短,大脑质量显著增加,海马CA1区突触结构数明显减少,海马PSD95、GluA1、GluN2B和磷酸化GluN2B蛋白表达水平均显著降低,差异均有统计学意义（P＜0.05）;与模型组比较,电针穴位组大鼠的学习期逃避潜伏时间缩短,测试期跨越平台次数增加,目标象限停留时间延长,大脑质量降低,CA1区突触结构数增多,海马PSD95、GluA1、GluN2B和磷酸化GluN2B蛋白表达水平增加,差异均有统计学意义（P＜0.05）。结论 电针“百会”“神庭”能改善VD大鼠的学习记忆功能,改变海马突触结构,分子机制可能和增加突触蛋白PSD95、GluA1和GluN2B的蛋白表达水平相关。     

Dynamic ultrastructural changes of the connective tissue sheath of human hair follicles during hair cycle

Summary Ultrastructural changes of the connective tissue sheath (CTS), including the hyaline membrane, of human hair follicles during the hair cycle, were studied in normal scalp skin specimens. In early anagen, the CTS was composed of a thin basal lamina and surrounding collagen tissue. The collagen tissue gradually thickened during the development of the hair and hair follicle. In mature anagen hair follicles, the collagen tissue was separated into three layers. The inner collagen layer, just outside the basal lamina, was thin and composed of collagen fibres running longitudinally parallel to the hair axis. The middle collagen layer was very thick with its collagen fibres running transversely against the hair axis and surrounding the inner hair tissue. Many fibroblasts were present among the collagen fibres in the middle layer, whereas the inner layer contained almost none. In the outer collagen layer, collagen fibres ran in various directions parallel to the outer surface of the outer root sheath cells. In late anagen, the basal lamina became very thick. In catagen, the basal lamine and the inner collagen layer became corrugated and showed oedematous change and degeneration. Surrounding fibroblasts showed active production of new collagen fibres, which seemed to fill the spaces left by the retraction of the hair follicle and hyaline membrane. These ultrastructural changes of the CTS show that there may be dynamic metabolic changes of the connective tissue around human hair follicles during the hair cycle.     

缬沙坦和卡托普利干预高血压大鼠左室心肌纤维化及超微结构改变的比较研究

目的：比较缬沙坦和卡托普利对心肌纤维化及超微结构改变的干预作用。方法：14周龄自发性高血压大鼠(SHR)25只随机分为5组：缬沙坦组(SHRv)、卡托普利组(SHRc)、卡托普利组 缬沙坦组(SHRc v)、假治对照组(SHR20)和高血压基础对照组(SHR14)。同周龄SD大鼠5只设为正常对照组(SD20)。疗程为6周。处死后测算左室重量／体重比(LVM／BW)、光镜观察心肌胶原分布，测算胶原容积分数(CVF)、血管周围胶原面积／管腔面积比(PVCA／LA)和组织匀浆胶原浓度，电镜观察心肌超微结构改变。结果：三个治疗组反映心肌纤维化的多项指标均较SHR20和SHR14组显著改善(P＜0．0l＆0．05)。SHRv与SHRc v组的改善相似，均优于SHRc组(P＜0．05)，但仍差于SD20组(P＜0．05)。心肌超微结构改变均较SHR20组显著好转。结论：缬沙坦和卡托普利均能抑制高血压心肌纤维化及超微结构改变，但短期治疗不能恢复正常。缬沙坦的作用优于卡托普利，两者联用优于单用卡托普利，但并不显著优于单用缬沙坦。     

R56865, a Na- and Ca-Overload Inhibitor, Reduces Myocardial Ischemia-Reperfusion Injury in Blood-Perfused Rabbit Hearts

The cardioprotective effects of R56865 were studied in isolated rabbit hearts, blood-perfused with a support rabbit system. The effect on ischemic injury was evaluated by comparing myocardial contracture and contents of ATP catabolites and of lactate during 60 min of normothermic ischemia in untreated hearts (group I) and in hearts treated with 0.63 mg/kg of R56865 starting 20 min before ischemia (group II; n = 5 in each group). R56865 delayed the onset, and decreased the extent of ischemic contracture, but had no effect on the myocardial content of ATP, of its catabolites or of lactate. The effect on reperfusion injury was studied by monitoring left ventricular function during 80-min reperfusion after the 60-min ischemia in three groups (n = 6 in each): an untreated group (group I) and two groups treated with R56865 given either before (group II) or after ischemia (group III). Ultrastructural changes and cellular calcium distribution after reperfusion were also studied. R56865 improved the recovery of function and prevented contracture during reperfusion. Left ventricular end-diastolic pressure was 13.2 ± 2.8 mmHg in group II and 31.3 ± 8.1 mmHg in group III vs 45.0 ± 2.6 mmHg in group I (P < 0.0001 for II vs I; P > 0.05 for III vs I). Left ventricular developed pressure, maximum dP/dt and minimum dP/dt recovered to 71.0 ± 5.4%, 98.9 ± 6.1%, 85.3 ± 4.8% of baseline values, respectively, in group II, to 64.5 ± 3.0% (P > 0.05), 76.8 ± 3.0%, 70.2 ± 4.0% in group III, vs 52.0 ± 6.5%, 58.9 ± 6.9% and 53.6 ± 5.8% in untreated hearts (P < 0.05 for II or III vs I). Coronary flow was 24.5 ± 2.2 ml/min and 19.8 ± 1.8 ml/min in groups II and III vs 14.8 ± 0.7 ml/min (P < 0.05) in the untreated group. On histology the myocardium in hearts treated either before after ischemia was well protected and calcium distribution was almost normal after reperfusion, while in untreated hearts, most of the myocardium displayed irreversible damage accompanied by massive intracellular calcium accumulation. We conclude that R56865 could attenuate Ca²⁺-overload, thereby reducing myocardial ischemia-reperfusion injury after an extended period of ischemia.