Assessment of myofascial medialization following intraoperative fascial traction (IFT) in a cadaveric model.

PURPOSE: Intraoperative fascial traction (IFT) for the treatment of large ventral hernias and loss of domain (LOD) hernias is a promising tool in abdominal wall surgery. However, little is known about the extent of gain in myofascial advancement especially for the anterior rectus sheath. We, therefore, used a cadaveric model to determine the medialization during IFT. METHODS: 4 fresh frozen specimens were used. Retromuscular preparation was carried out followed by IFT with diagonal vertical traction for 30 min. Medial advancement of the anterior rectus sheath was measured after 15 and 30 min as well as traction forces. RESULTS: Total medialization for anterior rectus sheath after 30 min of IFT was 10.5 cm (mean). The mean traction force was 16.28 kg. Total medialization was significantly higher during the first 15 min of vertical fascial traction (p < 0.05). CONCLUSIONS: IFT provides significant medialization for the anterior rectus sheath in the cadaveric model. The findings align with results from a retrospective case study. Therefore, we see IFT as a beneficial tool in abdominal wall surgery.

Accuracy of Latarjet graft and screw position after using novel drill guide.

PURPOSE: For the success of Latarjet procedure a correct graft positioning is mandatory. Furthermore, the correct screw placement is important to avoid cartilage damage and soft tissue irritation. Due to a cadaveric experimental study, the accuracy of graft and screw positioning utilizing a novel drill guide for a minimal-invasive Latarjet procedure was analyzed. METHODS: Five human fresh-frozen shoulder specimens have been treated in accordance with the Congruent-Arc Latarjet technique using the glenoid bone loss set (Arthrex, Naples, FL, USA) with 3.75 mm cannulated screws throughout a 5 cm skin incision without detachment of the scapularis tendon. All procedures were performed by one single, experienced shoulder surgeon. The accuracy of graft and screw positioning was assessed due to a CT scan. RESULTS: A noticeable learning curve was noted during the study period as the first surgery took 45 min and the last 33 min. All grafts were correctly positioned with regard to the articular line of the glenoid surface. Impingement with the maximum head circumference was not encountered. The screw positions did not affect the suprascapular nerve. A damage of the graft was not noticed. CONCLUSIONS: The authors can recommend the usage of the new drill guide tested in this study. It seems to be beneficial in the Latarjet procedure and may ease correct graft positioning and prevent screw misplacement. Compared to fully arthroscopically performed Latarjet procedures it provides much steeper learning curve and seems technically easier and quicker to handle.

Retroperitoneal anatomy of the iliohypogastric, ilioinguinal, genitofemoral, and lateral femoral cutaneous nerve: consequences for prevention and treatment of chronic inguinodynia.

PURPOSE: Chronic inguinodynia is one of the most frequent complications after groin herniorrhaphy. We investigated the retroperitoneal anatomy of the iliohypogastric, ilioinguinal, genitofemoral, and lateral femoral cutaneous nerve to prevent direct nerve injury during hernia repairs and to find the most advantageous approach for posterior triple neurectomy. METHODS: We dissected the inguinal nerves in 30 human anatomic specimens bilaterally. The distances from each nerve and their entry points in the abdominal wall were measured in relation to the posterior superior iliac spine, anterior superior iliac spine, and the midpoint between the two iliac spines on the iliac crest. We evaluated our findings by creating high-resolution summation images. RESULTS: The courses of the iliohypogastric and ilioinguinal nerve are most consistent on the anterior surface of the quadratus lumborum muscle. The genitofemoral nerve always runs on the psoas muscle. The entry points of the nerves in the abdominal wall are located as follows: the iliohypogastric nerve is above the iliac crest and lateral from the anterior superior iliac spine, the ilioinguinal nerve is with great variability, either above or below the iliac crest and lateral from the anterior superior iliac spine, the genital branch is around the internal inguinal ring, the femoral branch is either cranial or caudal to the iliopubic tract, and the lateral femoral cutaneous nerve is either medial or lateral to the anterior superior iliac spine. CONCLUSION: Nerve injury during inguinal hernia repairs can be avoided by taking the topographic anatomy of the inguinal nerves into consideration. The most advantageous plane to look for the iliohypogastric and ilioinguinal nerve during posterior neurectomy is on the anterior surface of the quadratus lumborum muscle. For the surgical treatment of severe chronic inguinodynia, especially after posterior open or endoscopic mesh repair (TAPP/TEP), the retroperitoneoscopic or open retroperitoneal approach for posterior triple neurectomy can be considered.

Estrogen up-regulates estrogen receptor alpha and synaptophysin in slice cultures of rat hippocampus.

Previous studies have shown that estrogen application increases the density of synaptic input and the number of spines on CA1 pyramidal neurons. Here, we have investigated whether Schaffer collaterals to CA1 pyramidal cells are involved in this estrogen-induced synaptogenesis on CA1 pyramidal neurons. To this end, we studied estrogen-induced expression of both estrogen receptor (ER) subtypes (ERalpha and ERbeta) together with the presynaptic marker synaptophysin in the rat hippocampus. In tissue sections as well as in slice cultures mRNA expression of ERalpha, ERbeta and synaptophysin was higher in CA3 than in CA1, and mRNA expression and immunoreactivity for both ER subtypes were found in both principal cells and interneurons. By using quantitative image analysis we found stronger nuclear immunoreactivity for ERalpha in CA3 than in CA1. In slice cultures, supplementation of the medium with 10(-8) M estradiol led to an increase of nuclear immunoreactivity for ERalpha, but not for ERbeta, which was accompanied by a dramatic up-regulation of synaptophysin immunoreactivity in stratum radiatum of CA1. Together these findings indicate that estrogen effects on hippocampal neurons are more pronounced in CA3 than in CA1 and that ER activation in CA3 neurons leads to an up-regulation of a presynaptic marker protein in the axons of these cells, the Schaffer collaterals. We conclude that estradiol-induced spine formation on CA1 pyramidal cells may be mediated presynaptically, very likely by activation of ERalpha in CA3 pyramidal cells, followed by an increase in Schaffer collateral synapses.

Role of tumor necrosis factor in preovulatory follicles of swine.

The effects of tumor necrosis factor (TNF) on cultured porcine granulosa cells that were obtained from preovulatory follicles were studied with regard to following parameters: 1) TNF receptor type I expression, 2) progesterone receptor and transforming growth factor beta receptor type II (TbetaR II) as markers of luteinization, 3) proliferation, and 4) apoptosis. For comparative purposes the effects of TNF were also studied on insulin/forskolin-treated cells, as this treatment is well established to induce luteinization. Cytochemical methods followed by semiquantitative analysis were used. Our data show that TNF treatment upregulates TNF receptor type I expression in granulosa cells. TNF downregulates the expression of TbetaR II of insulin/forskolin-stimulated and of unstimulated cells. The progesterone receptor is also downregulated by the cytokine after insulin/forskolin-induced luteinization. Supplementation of the medium with TNF leads to increased proliferation and at the same time it induces apoptosis. Our results indicate that TNF exerts an inhibitory influence on luteinization and that TNF influences the balance between follicular growth (proliferation) and atresia (apoptosis).

Steroidogenic factor-1 expression in marmoset and rat hippocampus: co-localization with StAR and aromatase.

Steroidogenic factor-1 (SF-1), an orphan nuclear receptor, was studied with respect to the expression of steroidogenic enzymes in the hippocampus of rat and marmoset, since SF-1 is a regulator of steroid biosynthesis in the gonads. We used the steroidogenic acute regulatory protein (StAR) as a marker of the first step in the cascade of oestrogen synthesis and aromatase as a marker of the last. StAR transports cholesterol to the inner mitochondrial membrane where it is converted by the cytochrome P-450 enzyme complex. This is the rate-limiting step in steroid biosynthesis. Aromatase metabolizes testosterone to oestrogen. Using an anti-SF-1 antibody we show that SF-1 is highly expressed in neuronal cells of the pyramidal layer (CA1--CA3) and in the dentate gyrus of rat and marmoset hippocampi. Binding of the antibody was seen in more than 60% of all cells in the pyramidal layer and in the fascia dentata. In situ hybridization studies revealed the same expression pattern for StAR and aromatase. StAR and aromatase-positive cells were strictly correlated with SF-1 as shown by computer-assisted confocal microscopy in double labelling experiments (immunohistochemistry and in situ hybridization). This coexpression may imply SF-1 as a possible regulator of steroidogenesis in the hippocampus. However, a few interneurones express solely SF-1 and aromatase but are negative for StAR. Since the expression of StAR represents the first step in steroidogenesis its expression is suggestive for a de novo synthesis of steroids. A small population of interneurones must import precursors for oestrogen synthesis from other sources. Responsive cells, as evidenced by the presence of oestrogen receptor transcripts, were also found in the pyramidal layer and dentate gyrus. In conclusion, (1) SF-1 could play a regulatory role in steroidogenesis in the hippocampus of marmoset and rat and (2) with respect to the capacity of steroidogenesis two populations of hippocampal neurones coexist.

Spontaneous luteinization of antral marmoset follicles in vitro.

Large non-luteinized follicles of the marmoset monkey were cultured for up to 96 h in the presence of substances that are known to induce luteinization, i.e. LH, transforming growth factor (TGF)-beta and cyclic AMP. The state of the basal lamina, and the expression of connexin-43, alpha(2) integrin subunit and TGF-beta receptor type II (TbetaR-II) were chosen as parameters to judge the progress of luteinization. Antral follicles, cultured for 1 h, were not luteinized, as shown by an intact basal lamina, strong immunoreactivity of connexin-43 in granulosa cells, and no expression of TbetaR-II in the theca layer. After 12 h, most follicles showed a dissolution of the basal lamina, a faint reactivity of connexin-43, high expression of TbetaR-II in theca- and outer granulosa cells and high expression of alpha(2) integrin subunit in granulosa cells bordering at the basement membrane; all of which indicate luteinization. After 96 h of culture, luteal structures (e.g. corpora lutea accessoria) had developed. This was true for both non-stimulated and stimulated follicles. Our results strongly suggest that antral follicles luteinize spontaneously. The decisive determinant appears to be the follicular stage.

TGF-beta could be involved in paracrine actions in the epididymis of the marmoset monkey (Callithrix jacchus).

The transforming growth factor-beta1 (TGF-beta1) and the transforming growth factor-beta receptor type II (TGF-betaRII) were studied in the epididymis of sexually mature marmoset monkeys (Callithrix jacchus) by immunohistochemical localization of the protein and by polymerase chain reaction (PCR) analysis of the mRNA level. In order to specify reactive cell types, the morphology of all three segments (caput, corpus, and cauda epididymidis) was evaluated by light microscopy. Six different cell types could be distinguished: principal, basal, apical, and clear cells, as well as intraepithelial lymphocytes and macrophages. Using immunohistochemistry, specific staining for TGF-beta1 in the caput was found in 47% of the apical cells, whereas the TGF-betaRII was located in the apical portion of 91% of all principal cells. In the corpus epididymidis, 20% of the apical cells were immunopositive for TGF-beta, and binding of the receptor antibody occurred in 17% of the principal cells (all numbers based on counts of counterstained nuclei). All differences between percentages in the caput and corpus were significant as determined by chi-square test. PCR analysis revealed detectable levels of TGF-beta1 mRNA in the marmoset epididymis. Our results indicate for the first time that TGF-beta1 is synthesized in the marmoset epididymis, possibly in a different subpopulation of epididymal cells than the TGF-beta receptor type II. Thus, TGF-beta might be of functional relevance in the primate epididymis.

Possible involvement of transforming growth factor-beta 1 and transforming growth factor-beta receptor type II during luteinization in the marmoset ovary.

The expression of transforming growth factor-beta 1 (TGF-beta 1), and transforming growth factor-beta receptor type II (T beta R-II), were evaluated in periovulatory marmoset ovaries. Histochemical methods were used, in particular double-labelling techniques, in order to correlate growth factor/receptor expression with proliferation (Ki 67), apoptosis (TUNEL method) and luteinization (3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)). The latter was used as a luteinization marker. Periovulatory ovaries are especially suited for studying all aspects since they typically consist of small non-luteinized follicles, large luteinizing follicles and corpora lutea accessoria (Clas), which have developed from large luteinizing follicles. TGF-beta 1 and T beta R-II expression was found in luteinizing theca cells of large periovulatory follicles and in all luteal cells of Clas. Non-luteinized theca cells, including those of small follicles were always devoid of any immunostaining. Granulosa cells of small follicles were immunopositive for T beta R-II. Large follicles with granulosa cell immunoreactivity of both antibodies coexisted with non-reactive follicles of comparable size. The highest activity of the luteal marker enzyme 3 beta-HSD was co-localized in the same cells that expressed TGF-beta 1 and T beta R-II. The double-labelling experiments revealed that TGF-beta 1 and T beta R-II expression is not correlated with proliferation or apoptosis of follicular cells. Our results indicate that TGF-beta 1 and T beta R-II participate in differentiation processes, i.e. luteinization, rather than proliferation. In particular, the dynamics of T beta R-II expression appear highly related to the process of luteinization.

The expression of sf-1/Ad4BP is related to the process of luteinization in the marmoset (Callithrix jacchus) ovary.

Expression of the nuclear receptor, steroidogenic factor-1 (SF-1/Ad4BP), was studied in a primate (marmoset) ovary using immunohistochemical, RT-PCR, and immunoblot techniques. The periovulatory phase was compared with the luteal phase. With PCR we found a marmoset homolog of SF-1/Ad4BP to be expressed in ovarian and other steroidogenic tissues. Characteristically, the periovulatory ovaries consisted of growing (non-luteinized) small follicles together with large luteinizing follicles and many corpora lutea accessoria (Clas), which had developed from atretic large follicles. During the luteal phase, true corpora lutea (Cls) were additionally found. In general, we found that small follicles were devoid of any immunoreactivity of SF-1/Ad4BP. In large follicles, the luteinizing theca and granulosa cells express SF-1/Ad4BP. All luteal cells of Clas showed a nuclear staining in both ovary types. In Cls, only a few luteal cells were positive. Large follicles of different sizes showed no differences in expression level, as evidenced by immunoblot analysis. Our results indicate that SF-1/ Ad4BP participates in the activation of genc transcription during the onset of luteinization and that Clas are essential for ovarian luteal function.

Regulation of oxytocin expression in the bovine corpus luteum. Orphan receptors and the oxytocin promoter.

The bovine oxytocin gene is massively up-regulated during the early development of the corpus luteum. Oxytocin transcription is induced in a highly synchronous fashion in the granulosa cells of the dominant follicle at the time of ovulation. The possibility to isolate large numbers of differentiating granulosa-luteal cells from exactly defined stages of development allows the investigation of the factors controlling oxytocin expression in vivo by molecular and cell biology methods. Using primary cultures of bovine granulosa cells the synergistic activation of oxytocin transcription by the cAMP pathway and stimulation of IGF-I or insulin receptors could be established. Analysis of transcription factors isolated from the nuclei of bovine granulosa cells and corpus luteum led to the identification of the tissue-specific orphan receptor SF-1 binding to the promoter of the actively transcribed oxytocin gene. The luteinizing bovine granulosa cells provide the only easily accessible experimental system established so far in which the endogenous oxytocin gene is expressed. Although the link between increased cAMP level and receptor tyrosine kinase activation on the one hand and the induction of oxytocin transcription on the other has not been established yet, these experiments constitute one of the few direct approaches to investigate the complexity of events that regulate oxytocin expression in vivo.

The orphan receptor SF-1 binds to the COUP-like element in the promoter of the actively transcribed oxytocin gene.

The factors regulating oxytocin expression have not yet been characterized in detail. Although direct control by ligand-dependent binding of nuclear hormone receptors to the oxytocin promoter has been suggested, the presence of these receptors in the tissues expressing oxytocin has not been shown consistently. We have analyzed nuclear proteins from preovulatory bovine granulosa cells and corpus luteum, tissues actively expressing the oxytocin gene, and describe here the characterization of a tissue-specific factor binding to the conserved element in the oxytocin promoter that has been implicated in the control of this gene. This factor is the bovine homologue of SF-1, an orphan receptor expressed specifically in steroidogenic tissues. It is suggested that SF-1 binds to the oxytocin promoter in vivo and is involved in control of oxytocin gene expression possibly by interaction with other factors.

Two orphan receptors binding to a common site are involved in the regulation of the oxytocin gene in the bovine ovary.

The peptide hormone oxytocin is highly expressed in the hypothalamus within only a small number of magnocellular neurons. However, it is also expressed in a much larger number of cells in the bovine corpus luteum at high levels in an estrous cycle-dependent manner. By using nuclear extracts from this tissue for in vitro binding studies, two protein complexes have been shown to bind to a common site in the bovine oxytocin promoter. One of these proteins has been identified as the bovine homologue of the chicken ovalbumin upstream promoter transcription factor (COUP-TF). The second protein is here characterized as the bovine homologue of a tissue-specific transcription factor, steroidogenic factor 1 (SF-1). The relative expression of these two factors during luteal development correlates with the level of luteal oxytocin gene expression, with SF-1 being the factor binding to the promoter of the oxytocin gene when this promoter is activated. Cotransfection experiments using the murine testicular cell line TM4 show that SF-1 can stimulate the expression of a transfected oxytocin gene, suggesting that SF-1 may be involved in upregulation of the oxytocin gene in vivo, possibly by transducing a stimulatory signal to the RNA polymerase.

The COUP transcription factor (COUP-TF) is directly involved in the regulation of oxytocin gene expression in luteinizing bovine granulosa cells.

Competition with specific oligonucleotides in DNA-binding experiments, polyacrylamide gel electrophoresis, and recognition by specific antibodies have identified the ubiquitous transcription factor COUP as one of the nuclear proteins binding to the promoter region of the bovine oxytocin gene in endogenously expressing bovine granulosa cells. PCR cloning of partial cDNA sequences for bovine COUP-TF I and II and development of RNase protection assays demonstrated the up-regulation of COUP-TF in bovine granulosa cells and corpus luteum under conditions where the oxytocin gene is switched off. These experimental results from in vitro and in vivo studies point to the direct involvement of COUP-TF in oxytocin gene down-regulation during luteinization of bovine granulosa cells.

Effects of gonadotropins, insulin and insulin-like growth factor I on ovarian oxytocin and progesterone production.

Oxytocin is produced in the granulosa-derived cells of the ruminant corpus luteum where its gene is dramatically up-regulated within days of ovulation. Regulation of these processes is poorly understood but oxytocin release can be increased by insulin, insulin-like growth factor I (IGF-I), and gonadotropins. Here we have assessed interactions between these regulatory systems. Follicle-stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotropin (hCG) caused dose-dependent release of oxytocin from bovine granulosa cells cultured in medium containing 100 ng/ml insulin. The gonadotropins also increased oxytocin mRNA levels and their effects were mimicked by forskolin. The effects of these stimuli on oxytocin and progesterone release were synergistically increased by insulin or IGF-I. Binding studies revealed separate binding sites with characteristics of insulin and IGF-I receptors. Insulin potentiated the effects of hCG and forskolin on oxytocin mRNA levels and release of oxytocin and progesterone in cells from follicles containing greater than 50 ng/ml estradiol. In cells from follicles containing less than 5 ng/ml estradiol these stimuli had little effect on oxytocin release although progesterone release was synergistically increased by insulin and forskolin. The data suggest that gonadotropins regulate oxytocin synthesis and release and that these effects are amplified by insulin or IGF-I acting via their own receptors. Changes associated with maturation of the target cells in vitro appear prerequisite for oxytocin production in response to increased cAMP levels in the presence of insulin or IGF-I.

Mapping of the bovine oxytocin gene control region: identification of binding sites for luteal nuclear proteins in the 5' non-coding region of the gene.

Abstract In view of the small number of hormone-producing cells, the factors regulating oxytocin gene expression in the classic site of synthesis, in the magnocellular neurons of the hypothalamus, have not yet been characterized. In the early bovine corpus luteum there is a tissue-specific oxytocin expression involving many more cells. This tissue therefore was chosen as a experimental system to identify deoxyribonucleic acid elements and nuclear proteins involved in the regulation of oxytocin gene expression. 3.2 kb from the 5'non-coding region of the bovine oxytocin gene have been sequenced and subcloned fragments used as probes for gel retardation and footprinting experiments. Binding sites for luteal as well as more ubiquitous proteins were detected in the oxytocin promoter region and in an artiodactyl-specific dispersed repeated deoxyribonucleic acid element. A binding site in the promoter region with a superficial similarity to an estrogen-responsive element (-159 to -152) was shown not to bind this steroid hormone receptor but to bind two nuclear proteins alternatively. One is a luteal protein, the other a more general transcription factor belonging to the steroid hormone receptor superfamily and similar, if not identical to the COUP protein. This alternative binding of a tissue- and phase-specifically expressed protein or an ubiquitous factor to the same site in the oxytocin promoter suggests a role for these two proteins in the transient up-regulation and subsequent down-regulation of the oxytocin gene during the differentiation of the bovine corpus luteum.