An adult myometrial pluripotential precursor that promotes healing of damaged muscular tissues.

The use of adult stem cells for tissue and organ regeneration constitutes a promising alternative therapy in many human diseases that are currently not treatable. We have isolated a new cell type from mouse adult uterine biopsies (murine adult myometrial precursors or mAMPs) by means of using a simple and non-invasive approach. These cells have been characterized by surface markers, being positive for CD31, CD34, CD44, CD117, Stro-1 and Sca-1. A similar cell population (hAMPs) was isolated from human biopsies. AMPs can differentiate in vitro into a number of mesodermal (smooth and skeletal muscle, osteoblasts and adipocytes) as well as epidermal lineages (all neural lineages). AMPs are unusual adult stem cells as they still express some embryonic antigens and remain undifferentiated through a high number of passages before entering senescence. Importantly, when injected into animal models of muscular disease, AMPs can regenerate new muscle fibers, and promote functional muscular recovery. Moreover, these cells can regenerate the uterine lining after wound healing, reconstructing the uterine muscular architecture. In addition, these cells can form new vessels both in vitro and in vivo. We believe that these cells have superior features to other known adult stem cells and, consequently, their use holds great promise for regenerative medicine, drug development and basic research.

Uterine myometrium as a cell patch as an alternative graft for transplantation to infarcted cardiac myocardium: a preliminary study.

PURPOSE: Currently, only a small fraction of patients are able to receive reperfusion therapy for myocardial infarctions. We hypothesize that myometrial cell patch transplantation could be an alternative approach for the treatment of myocardial infarction. DESIGN: We performed a preliminary study to determine the feasibility of this novel therapeutic approach in a rabbit model. PROCEDURES: Six adult female New Zealand rabbits were used. Myocardial infarction was induced by left anterior descending artery ligation. A segment of uterus was removed via a laparotomy incision, and this uterine segment was transplanted as an autologous graft over the infarcted myocardium, which was then reinforced by greater omentum. Statistical methods and outcome measures: Hemodynamic measurements and histological studies. MAIN FINDINGS: All uterine myometrial patches survived in the test animals. Fluoroscopic hemodynamic measurements were made for ejection fractions at 8 weeks after the application of the uterine patch. Histological study demonstrated well-healed myometrial-myocardium junctions with minimum scar tissue. Angiogenesis occurred in the transplanted myometrium. Connexin 43 expression was demonstrated in the transplanted patches. CONCLUSION: Our noncontrolled preliminary rabbit experiments indicate that patches of uterine myometrium reinforced by greater omentum can be used as autologous transplant therapy for infracted myocardium. This is an innovative technique that could lead to future treatment for individuals who may suffer from an infarcted myocardium and may not be eligible for traditional reperfusion therapy.

A quantitative study of rat uterine sympathetic innervation during pregnancy and post partum.

In mammals, pregnancy induces a transient and extensive degeneration of uterine sympathetic innervation. We used the models of unilateral oviduct ligation and in oculo myometrium transplant in pregnant rats to address the role of stretching forces and/or hormone milieu in the loss of sympathetic innervation. The sympathetic fibres of the uterine horn and in oculo myometrial transplants were quantified on tissue sections processed by the glyoxylic acid technique. In normal pregnant rats, the density of uterine horn innervation was significantly reduced at late pregnancy and recovery took place during post partum. The empty horn of pregnant rats showed no significant changes in density of myometrial innervation during pregnancy or post partum. In oculo myometrial transplants were organotypically reinnervated in virgin animals. When the transplants were exposed to gestational hormonal milieu, few or no fibres were observed to the end of pregnancy; however, a significant increase at post partum was observed. Results showed that both the effects of stretching and the hormone milieu derived from the fetus-placenta complex play a role as inductors of changes on sympathetic myometrial innervation during pregnancy and support the idea that immature muscular uterine fibres are more susceptible to the effects of pregnancy than those originating from adult animals.

The oestrogenized rat myometrium inhibits organotypic sympathetic reinnervation.

Chronic administration of oestrogen to rats during the infantile/prepubertal period provokes, at 28 days of age, complete loss of noradrenaline-labelled intrauterine sympathetic nerves. It is not known whether oestrogen inhibits the growth or causes the degeneration of developing uterine sympathetic nerves, or whether the uterus recovers its innervation following cessation of infantile/prepubertal oestrogen treatment. In the present study, we analysed the time-course of the effects of oestrogen on the development of uterine sympathetic nerves in the rat, using histochemical methods. In addition, the pattern of sympathetic reinnervation of the uterus of intact and ovariectomised females was assessed 3 and 6 months after cessation of chronic oestrogen treatment. The ability of sympathetic nerves to reinnervate the oestrogenized uterine tissue was assessed in intraocular transplants of uterine myometrium into ovariectomised host rats. Early exposure to oestrogen did not inhibit the approach of sympathetic nerves to the uterus, but prevented the normal growth and maturation of intrauterine sympathetic fibres and abolished the innervation that reached the organ before initiation of treatment. Three or six months following cessation of oestrogen treatment, most of the sympathetic nerves were restricted to the mesometrium and mesometrial entrance, whereas intrauterine innervation remained persistently depressed as a consequence of a sustained oestrous-like state provoked by ovarian dysfunction (polycystic ovary). An organotypic regrowth of uterine sympathetic nerves was observed in ovariectomised infantile/prepubertal oestrogen-treated animals. After 5 weeks in oculo, the innervation of oestrogenized myometrial transplants was reduced by 50%, and substantial changes in the pattern of reinnervation were observed. In control transplants, 86% of the nerves were terminal varicose myometrial and perivascular nerve fibres, whereas 14% were preterminal nerve bundles. In oestrogenized myometrial transplants, 83% of the noradrenaline-labelled intercepting nerves were enlarged preterminal bundles and only 17% were terminal fibres. These results indicate that the oestrogenized myometrium is unattractive for sympathetic nerves and inhibits organotypic sympathetic reinnervation.

Induction of endometriotic nodules in an experimental baboon model mimicking human deep nodular lesions.

OBJECTIVE: To establish an experimental model for the study of deep nodular endometriosis. DESIGN: Induction of nodular endometriosis in baboons by grafting different uterine specimens to the peritoneal cavity. SETTING: Research and university facilities. ANIMAL(S): Ten baboons, to develop a model of induced deep nodular endometriosis. INTERVENTION(S): Biopsies of endometrium, and endometrium plus the junctional zone (JZ), full uterine thickness, and myometrium grafted to the peritoneum. MAIN OUTCOME MEASURE(S): Macroscopic descriptions recorded for observed induced lesions; staining with hematoxylin and eosin for histological evaluation and specific antibodies (CK22, CD10) for immunohistochemical studies; and analysis of surface area and volume of lesions, glandular density, and invasion of surrounding organs. RESULT(S): The incidence of induced nodular endometriosis was 100%, but the extent depended on the tissue grafted. Lesions induced after grafting specimens containing the JZ were statistically significantly larger than those not containing the JZ. Surrounding organ invasion was reported in more than 40% of lesions after grafting specimens containing the JZ. CONCLUSION(S): The first experimental model of nodular endometriosis allows investigation of deeper nodular lesions as well as invasion phenomena associated with nodular lesions.

Local mechanical effects and humoral factors evoke degeneration of guinea pig uterine innervation.

Small porcelain pellets were inserted into one of the uterine horns of virgin guinea pigs. Mating caused pregnancies only in contralateral, non-pellet-containing uterine horns. The number and morphology of uterine nerve structures was studied immunohistochemically. In pellet-surrounding tissues of virgin animals the number of nerves was reduced and remaining nerve structures displayed signs of degeneration. This nerve reduction was further advanced in early to mid-pregnancy when almost no nerve structures could be detected. Myometrial strips were transplanted to the anterior eye chamber and became reinnervated by adrenergic nerves. The pattern and structure of the adrenergic reinnervation did not differ between strips taken from virgin animals, untreated or following chemical sympathectomy, or taken from primiparous animals following pregnancy induced sympathectomy. The adrenergic reinnervation of transplants from virgin animals was not affected by pregnancy of the recipient animal. It is suggested that the uterine innervation can be influenced by local, mechanically-induced effects, which during pregnancy co-operate with conceptus-related humoral factors to cause an almost total uterine nerve degeneration. The ability of uterine adrenergic reinnervation is probably not significantly influenced by local environmental tissue factor(s), but is related rather to extent of nerve damage.

Auto-transplantation of rabbit myometrium into ear-chamber and recording of its activity.

A transparent ear-chamber and its installation in the rabbit is described as well as the subsequent autografting of myometrial tissue into the connective tissue formed in the chamber. Of the rabbits initially provided with ear-chambers 65% were found to be suitable for grafting while finally only 40% of these presented activity either spontaneously or induced by stretch or oxytocin. Qualitative evaluation of the mechanical activity was obtained by photo-electric recording of the transmission of light through the graft. An evaluation of the electrical activity was made possible by recording volume conducted potentials from wire electrodes in contact with the grafted tissue. A close time interrelationship between electrical potentials and change in transmission of light of the graft was found. Oxytocin produced electrical activity and blanching of the graft. It is concluded that recording of the electrical activity provides the most reliable way of expressing the activity of grafted myometrium.

Function of myometrial graft at time of parturition in the rabbit.

Thirteen rabbits equipped with ear chambers containing myometrial grafts were studied during late pregnancy, spontaneous and induced parturition and early puerperium. Spontaneous electrical activity of the graft was recorded by means of F.-M. telemetry transmission in the unrestrained animals. Furthermore the reaction to intravenously injected oxytocin (threshold determination) was recorded daily. During the last 5 days before spontaneous delivery increasing spontaneous activity of the tissue was observed. During the same period the response to oxytocin increased. Concomitantly with the delivery of the litter a period of rhythmic activity was observed. This period had a sudden onset and lasted 12-55 min, while the delivery itself only lasted 6-16 min. Similar activity patterns could be evoked in the early post-partum period by 100 mU oxytocin given iv. In 3 rabbits, doses from 80-300 mU oxytocin induced complete delivery. During the 48 hours' period after delivery spontaneous activity (single spikes and series of bursts) was observed. The response to oxytocin was further increased. It is concluded that as the graft is not influenced by nervous regulation, mechanical influence or local humoral factors the spontaneous parturition and the change in excitability must therefore be due to one (or more) systemic, humoral factor(s).

Evidence for an intrinsic control of myometrial contractile periodicity in sheep during pregnancy.

Reduction in concentration of prostaglandins in plasma by administration of sodium meclofenamate to pregnant sheep failed to alter the frequency or duration of electromyographic activity bursts or the response to oxytocin of myometrial tissue transplanted to the omentum. However, a significant (P < 0.05) delay (8.6 +/- 3.8 versus 1.3 +/- 0.3 min) in the myometrial response to oxytocin was observed when the hormone was administered 1 min after a spontaneous burst of electromyographic activity compared with 15 min after a burst, indicating a period of refractoriness. Similarly, the myometrial threshold for electrical stimulation was higher at 10-25% of the interval between contractions than close to the expected time of the next contraction. Stimulation of the myometrium at intervals of 30 s revealed a cycling of the electrical stimulation threshold: significantly higher voltages were required to elicit responses between spontaneous bursts of electromyographic activity (18.0 +/- 2.2 V) than during bursts (11.3 +/- 1.6 V). In contrast, there was no voltage differential in animals close to labour (< 24 h). These data provide no evidence to support a role for prostaglandins in the generation of contractions during pregnancy, but suggest that periodicity of contractions is associated with inherent changes in myometrial responsiveness to stimulation, which could occur as a result of a cycling of the resting membrane potential.

Vasoactive intestinal polypeptide (VIP) inhibits prostaglandin-F2 alpha-induced activity of the rabbit myometrium.

Seven female rabbits ahd myometrial autografts transplanted into ear-chambers. The electrical activity of the graft was recorded and mechanical activity observed. Vasoactive intestinal polypeptide (VIP) dose-dependently inhibited myometrial activity induced by prostaglandin-F2 alpha. This evidence and the occurrence of "VIPergic" nerve fibres, which seem to innervate the myometrial cells, suggest that VIP may play a physiological role in the local control of the myometrial activity.

In oculo transplants of myometrium from postpartum guinea pigs fail to support sympathetic reinnervation.

Sympathetic nerves to the enlarged fetus-containing region of the uterus undergo degenerative changes during late pregnancy and show slow regrowth after parturition. It is not known whether this unusual response of sympathetic nerves to smooth muscle hypertrophy is due to the sensitivity of short adrenergic neurons to hormonal changes, or whether the nerves respond to changes in the neurotrophic capacity of the target. We have investigated this question using in oculo transplantation. Small pieces of myometrium from the uterine horn of virgin guinea pigs, or from the region previously occupied by the placenta and fetus in postpartum guinea pigs, were transplanted into the anterior eye chamber. After 3 wk in oculo, the pattern of reinnervation of the transplants was assessed on whole mount stretch preparations stained for tyrosine hydroxylase. The histology of the transplants was examined in toluidine blue-stained semithin sections. Myometrial transplants from virgin donors and uterine artery transplants from both virgin and postpartum donors became organotypically reinnervated by sympathetic fibres from the host iris. In contrast, sympathetic nerves did not reinnervate myometrial transplants from postpartum donors, although they approached the transplants and became distributed in the surrounding connective tissue. All transplanted tissues showed a normal histological appearance. Both the myometrium and uterine artery from postpartum donors retained a hypertrophic appearance after 3 wk in oculo. We interpret these results to indicate that the degeneration of sympathetic nerves in late pregnancy, as well as their slow regrowth to the uterus after delivery, may be due to changes in uterine smooth muscle rather than a particular sensitivity of short adrenergic neurons to hormonal changes.

Effects of chronic oestrogen treatment are not selective for uterine noradrenaline-containing sympathetic nerves: a transplantation study.

Previous studies have shown that chronic administration of oestrogen during postnatal rat development dramatically reduces the total content of noradrenaline in the uterine horn, abolishes myometrial noradrenergic innervation and reduces noradrenaline-fluorescence intensity of intrauterine perivascular nerve fibres. In the present study we analysed if this response is due to a direct and selective effect of oestrogen on the uterine noradrenaline-containing sympathetic nerves, using the in oculo transplantation method. Small pieces of myometrium from prepubertal rats were transplanted into the anterior eye chamber of adult ovariectomised host rats. The effect of systemic chronic oestrogen treatment on the reinnervation of the transplants by noradrenaline-containing sympathetic fibres from the superior cervical ganglion was analysed on cryostat tissue sections processed by the glyoxylic acid technique. In addition, the innervation of the host iris was assessed histochemically and biochemically. The histology of the transplants and irises was examined in toluidine blue-stained semithin sections. These studies showed that after 5 wk in oculo, the overall size of the oestrogen-treated transplants was substantially larger than controls, and histology showed that this change was related to an increase in the size and number of smooth muscle cells within the transplant. Chronic oestrogen treatment did not provoke trophic changes in the irideal muscle. Histochemistry showed that control transplants had a rich noradrenergic innervation, associated with both myometrium and blood vessels. Conversely, in oestrogen-treated transplants only occasional fibres were recognised, showing a reduced NA fluorescence intensity. No changes in the pattern and density of innervation or in the total content of noradrenaline of the host irises were detected after chronic exposure to oestrogen. We interpreted these results to indicate that the effects of oestrogen on uterine noradrenaline-containing sympathetic nerves are neither selective or direct, but result from an interaction between sympathetic nerve fibres with the oestradiol-primed uterine tissue. A potential effect of oestrogen on the neurotrophic capacity of the uterus is discussed.