Combined estrogen and ghrelin administration decreases expression of p27<Superscript>kip1</Superscript> and proportion of isomyosin type I in the striated urethral and anal sphincters and levator ani of old ovariectomized rats

We compared estrogen and/or ghrelin effects on pelvic floor muscles in old versus young adult ovariectomized rats. Ovariectomized Fisher 344 rats (18 and 3 months old, n = 24 x 2) received 42 daily intraperitoneal 17-beta estradiol (10 mug kg(-1)), ghrelin (2 mug kg(-1)), both, or vehicle (n = 6 x 4/group). Cytoplasmic p27(kip1) expression and isomyosin I proportion in striated urethral and anal sphincters and levator ani were measured, respectively, by Western blot analysis and gel electrophoresis with immunohistochemistry of muscle ghrelin receptors and radioimmunoassay of circulating growth hormone. In young adult rats, estrogen significantly decreased cytoplasmic p27(kip1) and isomyosin I signal intensities. In old rats, ghrelin and estrogen/ghrelin significantly decreased both intensities with greater estrogen/ghrelin effect. Ghrelin receptors were not immunostained in any muscle. Estrogen and/or ghrelin significantly increased or decreased, respectively, circulating growth hormone in old and young adult rats. Estrogen/ghrelin administration reversed pelvic floor muscle ageing changes in old ovariectomized rats through growth hormone production.     

The effect of ovariectomy on biomarkers of urogenital ageing in old versus young adult rats

The aim of this study was to compare the effects of ageing and ovariectomy on biomarkers of urogenital ageing in old and young-adult rats. Fisher 344 rats (18- and 3-months-old, n = 6 x 2) underwent ovariectomy. Age-matched sham animals received no intervention (n = 6 x 2). One month later, biomarkers of urogenital ageing were evaluated (light microscopic count of urethral and anal canal submucosal blood vessels, Western blot analysis of urethral, and anal canal submucosal collagen I and III and cytoplasmic p27(kip1) expression in the striated urethral and anal sphincters and levator ani and gel electrophoresis of isomyosin I proportion in these muscles) and compared in all groups (n = 24). All biomarkers of urogenital ageing studied were significantly increased in old compared to young-adult sham rats. Ovariectomy significantly increased these changes further in old versus young-adult rats with either smaller or larger differential effect than ageing compared to young-adult sham animals. Ovariectomy significantly exacerbates normative urogenital ageing changes in rats.     

Estrogen and ghrelin decrease cytoplasmic expression of p27<Superscript>kip1</Superscript>, a cellular marker of ageing,in the striated anal sphincter and levator muscle of ovariectomized rats

A study was carried out to investigate the effect of estrogen and/or ghrelin on the cellular marker of ageing, p27^kip1, in pelvic floor muscles of ovariectomized rats. Virgin Wistar rats (13 months old) underwent ovariectomy followed (1 month) by 42 daily intraperitoneal 17-β estradiol (10 μg/kg), ghrelin (2 μg/kg), both hormones, or placebo vehicle (n=6×4 groups). Six more age-matched animals underwent sham surgery without ovariectomy. Cytoplasmic expression of p27^kip1 in the striated urethral and anal sphincters and levator muscle was measured by Western blot analysis in all animals (n=30). p27^kip1 signal intensity significantly increased postovariectomy in all muscles compared to sham animals. In the anal sphincter and levator, signal intensity decreased to sham levels with ghrelin or estrogen and decreased further after estrogen or ghrelin and estrogen/ghrelin administration. Urethral sphincter signal intensity decreased without reaching sham levels after drug administration. Estrogen and/or ghrelin replacement reverses the ovariectomy-induced exacerbation of biochemical cellular ageing in the anal sphincter and levator muscle of middle-aged rats.     

雌激素预防性给药对去卵巢大鼠骨和脏器的影响

目的采用双侧卵巢切除术建立绝经后骨质疏松(postmenopausal osteoporosis,PMOP)大鼠模型,探讨雌激素预防性给药对绝经后大鼠骨和脏器的影响。方法将SD大鼠分为假手术(sham-operated,Sham)组、去卵巢(ovariectomized,OVX)组、雌二醇组(β-estradiol-treated OVX,OVX/E2)组。术后第10 d开始皮下注射给药并称量大鼠体重,术后61 d处死大鼠,取脏器和骨,称量脏器重量,计算脏器指数。制备组织切片,进行HE染色。结果组织形态学观察表明,OVX组大鼠的股骨和胫骨均出现骨小梁断裂、间距变大、结构紊乱等骨质疏松症状,而OVX/E2组并未出现明显的发病症状。相较于Sham组,OVX组大鼠子宫内膜固有层中的子宫腺数目增多,腺腔增大,子宫黏膜上皮明显增厚,而OVX/E2组的大鼠子宫形态结构并未发生明显病变。大鼠体重和脏器指数分析表明,摘除卵巢不仅会引起大鼠术后早期的体重增加,还会导致大鼠肝、肺、肾和脾的脏器指数增加,而雌激素预防性给药能一定程度上缓解去卵巢手术引发的脏器指数的异常变化。结论适时进行一定剂量的雌激素给药能够较好地预防绝经后骨质疏松症的发生,为绝经后骨质疏松症的预防和治疗提供参考。     

Effects of ovariectomy and hormone replacement on collagen and blood vessels of the urethral submucosa of rats

Collagen and blood vessels of the urethral submucosa of ovariectomized rats were studied following 28 daily subcutaneous injections of 17-beta estradiol (n=6, group 1), medroxy-progesterone acetate (n=6, group 2), both drugs (n=6, group 3) or vehicle (n= 6, control) and after sham surgery without castration or injection (n=6). Investigations included the immunohistochemistry of estrogen and progesterone receptors and collagen fibres, Western blot analysis of collagen types I and III and counting periurethral vessels by light microscopy. Our results showed positive immunostaining with estrogen, progesterone and collagen types I and III in all samples. Collagen type I and III levels were lower in the controls than in the sham group. The other groups showed increases (2>3>1) over the controls with a relatively higher increase in type III. The type I/III collagen ratio progressively decreased (control>1>2>3) below sham levels. The mean vessel count was significantly lower in control than in sham animals (P<0.00001). However, only estrogen treatment significantly increased the vessel number compared to controls (P=0.04). Our results indicate that estrogen and progesterone, alone or in combination, have an effect on collagen types I and III, and that estrogen has an effect on blood vessels of the urethral submucosa in female rats.     

Effects of ovariectomy and hormone replacement on submucosal collagen and blood vessels of the anal canal of rats

OBJECTIVE: To study the effects of oestrogen and progesterone on submucosal collagen fibres and vascular plexus of the anal canal. MATERIALS AND METHODS: Experiments were performed on sections of the anal canal of ovariectomized rats following 28 daily subcutaneous injections of 17-beta oestradiol (n = 6, OVX + E, Group 1), medroxyprogesterone acetate (n = 6, OVX + P, Group 2), both drugs (n = 6, OVX + E + P, Group 3) or vehicle (n = 6, OVX) and after sham surgery without castration or injection (n = 6). Investigations included immunohistochemistry of oestrogen and progesterone receptors and collagen fibres, Western blot analysis of collagen types I and III and counting of perianal vessels by light microscopy. RESULTS: There was positive immunostaining for oestrogen and progesterone receptors in the mucosa and for collagen types I and III in the submucosa in all samples. Type I collagen levels increased significantly with ovariectomy but were normalized with treatment with oestrogen and progesterone. Type III collagen levels decreased after ovariectomy. Administration of oestrogen and progesterone appeared to restore level to near sham values. Semi-quantitative measurement of Type I/III collagen ratios by signal intensity demonstrated a very high ratio after ovariectomy. This appeared to be restored by both oestrogen and progesterone administration either individually or in combination. Mean vessel count was significantly lower in sham animals compared to values in OVX animals (P = 0.006). However, while only oestrogen treatment increased significantly the number of vessels compared to sham animals (P = 0.04), replacement with progesterone did not affect and in combination with oestrogen reduced submucosal vessel number. CONCLUSION: Oestrogen and progesterone have synergistic effects on collagen types I and III and probably antagonistic effects on the vascular plexus of the anal canal submucosa in adult female rats.     

Improved collagen type I/III ratio at the interface of gentamicin-supplemented polyvinylidenfluoride mesh materials

Background Formation of recurrent inguinal and incisional hernia shows an underlying defect in the wound-healing process with an insufficient quality of scar formation. Even after mesh repair an altered collagen formation and insufficient mesh integration has been found as main reason for recurrences. Therefore, the development of bioactive mesh materials to achieve a local modification of the scar formation to improve patients outcome is advisable. Materials and methods A polyvinylidenfluoride mesh material (PVDF) was constructed and surface modified by plasma-induced graft polymerization of acrylic acid (PVDF + PAAc). Surface supplementation was sought by binding of gentamicin to the provided active sites of the grafted mesh surfaces (PVDF+PAAc+Gentamicin). In vivo modulation of collagen formation was evaluated in a standardized animal model where an abdominal wall replacement was performed in 45 Sprague–Dawley rats. Seven, 21, and 90 days after mesh implantation, collagen/protein ratio and the collagen type I/III ratio as well as the expression of type I alpha 1 collagen mRNA (SYBR Green real-time RT-PCR) were analyzed at the perifilamentary region. Additionally, expression of matrix metalloproteinases (MMP-8/-13) has been investigated immunohistochemically. Results Implantation of the PVDF + PAAc + Gentamicin mesh induced a significantly decreased expression of MMP-8 and MMP-13 at the interface 21 and 90 days after implantation compared to the other groups. Whereas no significant effect was observed comparing the overall collagen/protein ratio, the quality of collagen formation expressed by the collagen type I/III ratio showed significantly higher ratios around the PVDF + PAAc + Gentamicin mesh 21 and 90 days after implantation. Correspondingly, an up to 5.3-fold expression of type I alpha 1 collagen mRNA was found. Conclusion The present data confirm that a surface modification of PVDF mesh samples using plasma-induced graft polymerization of acrylic acid and supplementation of gentamicin is able to improve scar quality and mesh integration.     

Evidence for an extensive collagen type III/VI proximal domain in the rat femur. I. Diminution with ovariectomy

Collagenous proteins other than Type I have received little attention in hypogonadal bone loss. Using femora from 25 young (2.5 months) and older (11 months) control and ovariectomized adult rats killed 1–4 months postoperation, cancellous atrophy was histologically confirmed, and the immunolocalization of collagen Type III was examined. This occurred as numerous immunofluorescent Sharpey-like fibers, 5–25 μm thick, regularly associated with collagen Type VI, which ramified the femoral cortex. Sequential transverse cryosections enabled the mapping of the fibers in three-dimensions, demonstrating that they constituted an extensive subperiosteal domain which may be a lasting legacy of early skeletal development. Fiber density was greatest in the trochanters and femoral neck. The domain tapered distally and was apparently anchored into the mid-shaft by intracortical cartilaginous islands, staining for collagen Type VI (as well as Type II and fibronectin). Ovariectomy caused disconnection of the fibers and reduced the proximal domain of both young and older animals, previously positive areas of the cortex becoming negative. It is concluded that collagen Type III/VI occupies a substantial, discrete domain in the rat proximal femur as a complex extension of the periosteum. Diminution of this cortical domain with trabecular atrophy suggests that it has a proactive or reactive role in determining bone mass and strength by facilitating musculoskeletal exchange in a form that is disengaged by ovariectomy.     

温肾固疏方对去势大鼠骨组织中Ⅰ型胶原及组织蛋白酶 K 表达的影响

目的 研究温肾固疏方对绝经后骨质疏松症的防治作用。方法 通过切除大鼠双侧卵巢建立绝经后骨质疏松大鼠模型。造模后,SD大鼠随机分为模型组、雌激素组、温肾固疏方高、中、低剂量组,每组8只。假手术组仅切除卵巢旁脂肪组织。干预2个月后,通过HE染色观察骨组织、肾组织形态;ELISA法测定血清PICP、ICTP、CTX、NTX的含量;免疫组化法测定Col I、Cath K表达率的变化。结果 与假手术组比较,模型组大鼠血清ICTP、NTX、CTX含量均明显升高（P＜0.01）,骨组织形态改变与骨质疏松症一致,肾组织出现炎性改变,骨组织中Cath K含量明显增高（P＜0.01）,Col I蛋白含量显著降低（P＜0.01）;与模型组相比,血清ICTP、NTX、CTX含量显著降低（P＜0.05）,且温肾固疏方高剂量组比中、低剂量组表达明显。温肾固疏方中、高剂量组血清PICP含量显著增高（P＜0.01）。各治疗组骨组织形态改善,肾组织炎性改变减轻。各治疗组骨组织Cath K蛋白的表达均降低（P＜0.01）,Col I蛋白表达显著升高（P＜0.01）。结论 温肾固疏方能够通过下调破骨细胞Cath K的表达来抑制骨基质中Col I的分解,起到治疗绝经后骨质疏松症的作用。     

Immunohistochemical analysis of collagen types I,III, IV and α-actin in the urethra of sexually intact and ovariectomized beagles

Urinary incontinence is a widespread problem in both postmenopausal women and ovariectomized dogs. The objective of this study was to investigate the influence of ovariectomy on the immunoreactivity and the distribution pattern of collagens I, III, IV and α-actin in the canine urethra. The immunohistochemical results were evaluated in five sexually intact and five ovariectomized beagles. The immunostaining of both collagens I and III delineated urethral connective tissue fibres and co-localized within in the fibres of both groups. The basement membranes of smooth muscle cells and sinusoids showed marked type IV collagen expression, whereas only faint immunoreactivity was present at the urothelial–stromal interface. No differences could be detected in the expression or distribution of the assessed collagen types and actin between ovariectomized and control animals. In conclusion, ovariectomy does not appear to have an effect on urethral collagens I, III, IV and smooth muscle actin in the dog, as ascertained by immunohistochemistry.     

烧伤创面成纤维细胞周期,羟腈氨酸和胶原比例动态变化

目的 探讨深Ⅱ度烧伤创面愈合规律。方法 通过４８只大鼠深Ⅱ度烧伤模型,动态观察烧伤后不同时间点创面成纤维细胞增殖周期,羟脯氨酸（ＯＨＰ）含量和Ⅰ／Ⅲ型胶原比值变化及病理组织学改变。结果 伤后１０ｄ真皮细胞ｓ期细胞百分比达高峰,伤后２１ｄ仍显著高于正常对照组。ＯＨＰ含量伤后逐渐增加,人务后１４ｄ达高峰,伤后２１ｄ仍显著高于正常对照组。伤后Ⅰ／Ⅲ型胶原幽会逐渐降低,并显著低于正常对照组,病理切片观察伤     

【原创论文】前列腺组织弹性成像与间质中I、III型胶原纤维的关系

本文探讨前列腺弹性成像显示的组织硬度情况与I、III型胶原纤维的含量及二者相互走行方式的关系。对62例患者进行经直肠组织弹性成像检查（TRTE）,常规12针系统穿刺活检后,对前列腺进行TRTE引导下靶向穿刺,所取组织进行天狼腥红染色,观察I、III型胶原纤维的的分布情况,并分别计算二者的容积指数（CVF）。结果得出I型胶原纤维的CVF在质硬、软组分别为：0．05±0．02和0．02±0．01;两组之间的差异显著（P=0．002）;III型胶原纤维的CVF在质硬、软组分别为：0．05±0．04与0．07±0．03;两组之间的差异不显著（P=0．13）。圆分布的统计结果表明胶原纤维在质软组和质硬组均走行紊乱,I型胶原和III型胶原之间主要呈相互交叉走行,部分呈平行走行,二者的相互走行关系在质软、质硬组之间的差异显著（P=0．03）。前列腺弹性成像显示的组织硬度主要取决于组织中I型胶原纤维的含量,另外,I型、III型胶原纤维的相互走行方式也可影响组织的弹性。     

Evaluating adhesion reduction efficacy of type I/III collagen membrane and collagen-GAG resorbable matrix in primary flexor tendon repair in a chicken model

Background

Reduction of peritendinous adhesions after injury and repair has been the subject of extensive prior investigation. The application of a circumferential barrier at the repair site may limit the quantity of peritendinous adhesions while preserving the tendon’s innate ability to heal. The authors compare the effectiveness of a type I/III collagen membrane and a collagen-glycosaminoglycan (GAG) resorbable matrix in reducing tendon adhesions in an experimental chicken model of a “zone II” tendon laceration and repair.

Methods

In Leghorn chickens, flexor tendons were sharply divided using a scalpel and underwent repair in a standard fashion (54 total repairs). The sites were treated with a type I/III collagen membrane, collagen-GAG resorbable matrix, or saline in a randomized fashion. After 3 weeks, qualitative and semiquantitative histological analysis was performed to evaluate the “extent of peritendinous adhesions” and “nature of tendon healing.” The data was evaluated with chi-square analysis and unpaired Student’s t test.

Results

For both collagen materials, there was a statistically significant improvement in the degree of both extent of peritendinous adhesions and nature of tendon healing relative to the control group. There was no significant difference seen between the two materials. There was one tendon rupture observed in each treatment group. Surgical handling characteristics were subjectively favored for type I/III collagen membrane over the collagen-GAG resorbable matrix.

Conclusion

The ideal method of reducing clinically significant tendon adhesions after injury remains elusive. Both materials in this study demonstrate promise in reducing tendon adhesions after flexor tendon repair without impeding tendon healing in this model.     

Expression of type I,type II,and type X collagen genes during altered endochondral ossification in the femoral epiphysis of osteosclerotic (oc/oc) mice

The osteosclerotic (oc/oc) mouse, a genetically distinct murine mutation that has a functional defect in its osteoclasts, also has rickets and shows an altered endochondral ossification in the epiphyseal growth plate. The disorder is morphologically characterized by an abnormal extension of hypertrophic cartilage at 10 days after birth, which is later (21 days after birth) incorporated into the metaphyseal woven bone without breakdown of the cartilage matrix following vascular invasion of chondrocyte lacunae. In situ hybridization revealed that the extending hypertrophic chondrocytes expressed type I and type II collagen mRNA, as well as that of type X collagen and that the osteoblasts in the metaphysis expressed type II and type X collagen mRNA, in addition to type I collagen mRNA. The topographic distribution of the signals suggests a possible co-expression of each collagen gene in the individual cells. Immunohistochemically, an overlapping deposition of type I, type II, and type X collagen was observed in both the extending cartilage and metaphyseal bony trabeculae. Such aberrant gene expression and synthesis of collagen indicate that pathologic ossification takes place in the epiphyseal/metaphyseal junction of oc/oc mouse femur in different way than in normal endochondral ossification. This abnormality is probably not due to a developmental disorder in the epiphyseal plate but to the failure in conversion of cartilage into bone, since the epiphyseal plate otherwise appeared normal, showing orderly stratified zones with a proper expression of cartilage-specific genes.