Fetoscopic transesophageal electrocardiography and stimulation in fetal sheep: a minimally invasive approach aimed at diagnosis and termination of therapy-refractory supraventricular tachycardias in human fetuses.

BACKGROUND: Therapy-refractory supraventricular tachycardia commonly results in hydrops and death in human fetuses. The purpose of this study in fetal sheep was to assess the feasibility of a minimally invasive fetoscopic approach for fetal transesophageal electrocardiography and stimulation aimed at diagnosis and termination of these tachycardias. METHODS AND RESULTS: We studied a total of 10 fetal sheep (87 to 103 days of gestation; term=145 days). We entered the amniotic cavity using a percutaneous fetoscopic approach and placed various electrophysiology catheters into the fetal esophagus. We recorded the number of animals in which fetoscopic transesophageal electrocardiography and stimulation were successful and assessed pacing success and thresholds for different catheters. In addition, we monitored for potential adverse effects from stimulation and for other complications of the operation. Recording of transesophageal electrocardiograms was successful in all fetal sheep. Capture during stimulation was successfully documented by additional fetal bipolar surface electrocardiograms in 7 fetuses. In fetuses in which fetal surface electrocardiograms were not recorded, pacing stimulus artifacts interfered with documentation of capture. Although stimulation thresholds were high, the maternal rhythm was not affected by fetal stimulation. CONCLUSIONS: Fetoscopic fetal transesophageal electrocardiography and stimulation are feasible in fetal sheep. This minimally invasive approach might have the potential to improve diagnosis and management of therapy-refractory supraventricular tachycardias in human fetuses.

Androgens transcriptionally regulate the expression of cystatin-related protein and the C3 component of prostatic binding protein in rat ventral prostate and lacrimal gland.

In this report, it is demonstrated that the C3 component of prostatic binding protein (PBP) is also expressed and androgen regulated in the exorbital lacrimal gland, as shown previously for cystatin-related protein (CRP), another abundant secretory protein from the ventral prostate. The presence of C3 messenger RNA (mRNA) could be demonstrated by both Northern blot hybridization and PCR amplification and sequencing. The mRNAs encoding the C1 and C2 components of PBP, however, were undetectable. At the protein level, the C3 component in the lacrimal gland is glycosylated and linked by disulfide bridges to a new 10-kDa component not reacting with the PBP antiserum. As shown previously for CRP, the expression of C3 in the lacrimal gland requires the simultaneous presence of androgens and a functional androgen receptor. The effects of castration and androgen treatment on CRP and C3 mRNA concentrations were studied by Northern blot and dot blot hybridization; effects on transcription rates were determined by nuclear run-on assay. Two days after castration, the relative abundance of CRP mRNA had declined significantly (P < 0.01) to 10.5 +/- 1.5% (+/-SEM) of precastration levels in the prostate and to 14.5 +/- 8.0% in the lacrimal gland; the transcription rates declined to 14.3% and 10.0%, respectively. The C3 mRNA level and transcription rate in the prostate showed a more moderate decrease (P < 0.05) to 40.6 +/- 8.5% and 41.7%, but were hardly measurable in the lacrimal gland. Androgen administration resulted in a rapid increase in the transcription rates, which reached or exceeded control levels after 6-9 h of treatment and clearly preceded the increase in mRNA levels. It is concluded that the lacrimal gland, which can be studied conveniently in female and long term androgen-depleted animals offers a suitable model for the study of androgen-regulated gene expression.

Androgenic induction of cystatin-related protein and the C3 component of prostatic binding protein in primary cultures from the rat lacrimal gland.

Cystatin-related protein (CRP) and the C3 component of prostatic binding protein (C3) are synthesized in vivo under androgen control in the lacrimal gland and ventral prostate of adult male rats [1,2]. Androgen administration to female or 7-day castrated male rats, which do not express CRP, can induce its synthesis [3]. In this study, we show androgen-dependent expression of CRP1 and C3 in primary cultures of acinar cells of the lacrimal gland of female rats. Addition of androgens to the culture medium results in the synthesis and secretion of both proteins in a time- and dose-dependent way. Estradiol or progesterone are unable to induce their expression. Dexamethasone in low concentrations and present as a basal component of the serum free defined medium, is needed to sensitize the culture system for androgens. In high concentrations, this synthetic glucocorticoid seems to play a similar role as androgens in CRP1 and C3 regulation.

Tissue-specific androgen responses in primary cultures of lacrimal epithelial cells studied by adenoviral gene transfer.

The lacrimal gland secretes most of the water and many proteins present in tear fluid. The composition of the tear fluid is affected dramatically by androgens, an observation which has been linked to the fact that more than 90% of the patients with Sjögren syndrome are female. Although the presence of androgen receptors in the lacrimal gland has been established, the molecular biology of the protective effects of androgens remains largely unknown. Here, we report the use of primary cultures of the lacrimal gland which express endogenous proteins under androgen control, as a more homologous test system for tissue-specific transcription studies. Infection with recombinant adenoviral vectors was the most efficient method to introduce foreign gene constructs in these cultures. A thus introduced mouse mammary tumor virus promoter was inducible with androgens and this effect was independent of the sexual genotype of the infected cells. By use of two recombinant adenoviral vectors containing genomic fragments of the SC gene, which is androgen responsive in the lacrimal gland, we could demonstrate the functionality of the sc promoter as well as its androgen regulation in this culture system.

Expression of cystatin-related protein and of the C3-component of prostatic-binding protein during postnatal development in the rat ventral prostate and lacrimal gland.

The expression of cystatin-related protein (CRP) and of the C3-component of prostatic-binding protein (PBP) during postnatal development of the rat was studied by Northern blotting, dot blot and in situ hybridisation, and by radioimmunoassay or immunoblotting. In intact male rats, very little or no PBP-C3 could be detected in the prostate at 10 days, but at 20 days there was already strong expression. By in situ hybridisation, the first expression of C3 mRNA was observed at 13 days in the prostate and at 22 days in the lacrimal gland. For CRP, this occurred at 16 and 22 days, respectively. Neither CRP nor C3 was expressed in prepubertal male rats castrated at day 1 or day 10 or in female rats. Androgen treatment of intact male animals did not advance the expression of both mRNAs in the prostate, but did so in the lacrimal gland with first expression of C3 at 19 instead of 22 days and of CRP at 13 instead of 22 days. Identical values were obtained in female rats. Androgen treatment of castrated adult male rats resulted in a more rapid and homogeneous secondary induction. Positive immunostaining for the androgen receptor (AR) was observed in the lacrimal gland at 7 days, but its concentration, estimated by immunoblotting, was still low at 10 days. Maximal levels, reached at 30 days, were markedly higher in male than in female rats. In conclusion, CRP and C3 are induced by androgens in prepubertal rats. The time point of induction, however, is probably determined by other tissue and differentiation-dependent factors in addition to androgens and the AR.

Primary rat lacrimal cells undergo acinar-like morphogenesis on reconstituted basement membrane and express secretory component under androgen stimulation.

Single cells or small cell clusters, isolated from the rat lacrimal gland, were incubated on reconstituted basement membrane (matrigel) in a well-defined serum-free medium. During the first days of culture, cells reassociated and reorganized in structures resembling acini. These multicellular structures, maintained in culture for 2 weeks, consisted of well-polarized cuboidal cells surrounding a central lumen and exhibiting apically located microvilli. Myoepithelial cells were observed at the periphery of the acinar structures. Both in the native lacrimal and in the cultured aggregates, epithelial cells displayed strong immunoreactivity for cytokeratin 8, while myoepithelial cells were immunoreactive for vimentin and alpha-smooth muscle isoactin. These data indicate that the cultured aggregates closely mimic the in vivo architecture of lacrimal glands both by morphology and immunohistochemistry. We further demonstrated the presence of an intact androgen receptor and the ability of the cultured aggregates to respond to androgens with increased secretion of the secretory component. Comparable androgen responses were observed in lacrimal gland cultures of 5-week-old male and female rats. In conclusion, we report a morphologically and functionally differentiated culture system of primary rat lacrimal cells, in which androgen-regulated gene expression was observed. This culture model provides a unique experimental paradigm for studying the effects of hormones, cytokines, and growth factors on the morphogenesis, growth, and functional differentiation of lacrimal glands.

Simultaneous in utero assessment of AV nodal and ventricular electrophysiologic parameters in the fetal sheep heart.

BACKGROUND: Fetal tachyarrhythmias are usually of supraventricular origin. To investigate whether specific electrophysiologic properties of the fetal heart contribute to this preponderance by either favoring supraventricular tachycardias or by rendering ventricular tachycardias unlikely, we measured fetal electrophysiologic parameters in utero using transuterine fetal transesophageal electrocardiograms in fetal sheep. Since overdrive pacing may help to establish the mechanism of an arrhythmia and may be used to treat fetal tachycardias, different modes of transesophageal pacing in utero were also assessed. METHODS AND RESULTS: Decapolar electrophysiology catheters were fetoscopically inserted into the esophagus of 9 fetal sheep (pregnancy duration 94- 105 days, term = 145 days). Electrocardiograms were recorded simultaneously from all adjacent bipoles and from two pacing wires sutured onto the fetal shoulders. Pacing was attempted either via two adjacent electrodes of the intraesophageal catheter or via the most distal and most proximal electrode. Fetal cycle length, PQ, and QT intervals were close to (approx. 75 %), but fetal QRS duration was < 20 % of maternal values, thus shifting the relation between activation and repolarization towards longer excitation wave lengths. Fetal QT dispersion was small (< or = 10 ms). Atrial pacing was achieved in all fetuses using distant electrodes, and with lower thresholds when compared to closely spaced bipolar electrodes (p < 0.05). CONCLUSIONS: (I) An altered relation between ventricular activation and repolarization and a low dispersion of ventricular repolarization may protect the fetal heart against ventricular reentrant tachycardias. (II) Relatively normal fetal AV nodal conduction delay already provides one of the prerequisites for supraventricular reentrant tachycardias involving the AV node at this stage of fetal development. (III) High-rate esophageal pacing of the fetal atria is best achieved using widely spaced bipolar pacing electrodes.