Intra‐amniotic mesenchymal stem cell therapy improves the amniotic fluid microenvironment in rat spina bifida aperta fetuses

ObjectivesSpina bifida aperta (SBA) is one of the most common neural tube defects. Neural injury in SBA occurs in two stages involving failed neural tube closure and progressive degeneration through contact with the amniotic fluid. We previously suggested that intra‐amniotic bone marrow‐derived mesenchymal stem cell (BMSC) therapy for fetal rat SBA could achieve beneficial functional recovery through lesion‐specific differentiation. The aim of this study is to examine whether the amniotic fluid microenvironment can be improved by intra‐amniotic BMSC transplantation.MethodsThe intra‐amniotic BMSC injection was performed using in vivo rat fetal SBA models. The various cytokine expressions in rat amniotic fluid were screened by protein microassays. Intervention experiments were used to study the function of differentially expressed cytokines.ResultsA total of 32 cytokines showed significant upregulated expression in the BMSC‐injected amniotic fluid. We focused on Activin A, NGF, BDNF, CNTF, and CXCR4. Intervention experiments showed that the upregulated Activin A, NGF, BDNF, and CNTF could inhibit apoptosis and promote synaptic development in fetal spinal cords. Inhibiting the activity of these factors weakened the anti‐apoptotic and pro‐differentiation effects of transplanted BMSCs. Inhibition of CXCR4 activity reduced the engraftment rate of BMSCs in SBA fetuses.ConclusionBMSC transplantation can improve the amniotic fluid environment, and this is beneficial for SBA repair.

In utero intra‐amniotic BMSC or PBS microinjection in the E15 fetuses was performed in E15 rat fetuses with spina bifida aperta, and amniotic fluid was collected at E21 for protein array detection. Venn diagram shows the relationship of three biological processes (GO: 0030335, 0048699, and 0043524) and the attribution of differentially expressed proteins. Comparative analysis of five proteins with the largest fold changes in the process of generation of neurons.     

Increased levels of myosin mRNAs in spontaneously hypertensive rat heart

RNAs isolated from normal and spontaneously hypertensive (SHR) rat heart tissues were examined by direct hybridization assay using cloned DNA probes containing chicken cardiac myosin light chain and heavy chain specific cDNA sequences. In 7 weeks old SHR heart, the level of mRNA hybridizable to these probes is the same as in normal rat heart. However, at 18 weeks of age, when hypertrophy in SHR is well established as a consequence of age-related increase in blood pressure and cardiac mass, there is an increase in SHR mRNA levels consistent with the increase in the corresponding proteins. Thus, the increase in mRNAs for major myofibrillar proteins and the onset of hypertrophic state in SHR appear to occur simultaneously.     

Angiotensin-induced vasopressin release and activation of hypothalamic neuron in pre-term fetuses

Our previous studies have shown that central administration of angiotensin II (ANG II) causes vasopressin release in the near-term fetus in utero as evidence that the hypothalamic-neurohypophysial system has relatively matured before birth. However, it is still unknown whether the vasopressin controlling centers have been functionally developed in younger fetuses. This study determined fetal plasma vasopressin levels and hypothalamic vasopressin neuron activity in the chronically instrumented pre-term ovine fetuses. Introcerebroventricular (i.c.v.) administration of ANG II did not affect fetal plasma osmolality and sodium concentrations. However, fetal plasma vasopressin levels were significantly increased ( approximately 3-fold) in response to central injection of ANG II. Central ANG II also induced vasopressin-neuron activity marked with c-fos expression in the fetal hypothalamus at pre-term. In addition, the fetal organum vasculosum of the lamina terminalis and the subfornical organ were activated. The results suggest that hypothalamic-neurohypophysial system has been relatively intact and functional at 70% gestational age, and that central angiotensin is important in inducing fetal vasopressin release in utero.     

Proteomic analysis of amniotic fluid in pregnancies with Turner syndrome fetuses

Turner syndrome, occurring in 1:2500 female births, is caused by the complete or partial absence of one X chromosome. Amniotic fluid supernatant proteins from five second trimester pregnancies with Turner syndrome fetuses and five normal ones were analyzed by 2DE, MALDI-TOF-MS, and Western blot. Serotransferin, lumican, plasma retinol-binding protein, and apolipoprotein A-I were increased in Turner syndrome, while kininogen, prothrombin, and apolipoprotein A-IV were decreased. Since differentially expressed proteins are likely to cross the placenta barrier and be detected in maternal plasma, proteomic analysis may enhance research for noninvasive prenatal diagnosis of Turner syndrome.     

Increased corticosterone levels in mice subjected to the rat exposure test

In recent years, there has been a notable interest in studying prey-predator relationships to develop rodent-based models for the neurobehavioral aspects of stress and emotion. However, despite the growing use of transgenic mice and results showing important differences in the behavioral responses of rats and mice, little research has been conducted regarding the responses of mice to predators. The rat exposure test (RET), a recently developed and behaviorally validated prey-predator (mouse-rat)-based model, has proven to be a useful tool in evaluating the defensive responses of mice facing rats. To further validate the RET, we investigated the endocrine and behavioral responses of mice exposed to this apparatus. We first constructed a plasma corticosterone secretion curve in mice exposed to a rat or to an empty cage (control). Rat-exposed mice showed a pronounced rise in corticosterone levels that peaked 15 min from the beginning of the predator exposure. The corticosterone levels and behavioral responses of mice exposed to a rat or to a toy in the RET apparatus were then measured. We observed high plasma corticosterone levels along with clear avoidance behaviors represented by decreases in tunnel and surface area exploration and increases in risk assessment behaviors and freezing. This strongly suggests that the test elicits a repertoire of behavioral responses compatible with an aversion state and indicates that it is a promising model for the evaluation of prey-predator interactions. However, more physiological, neurochemical, and pharmacological studies are needed to further validate the test.     

A comparative study of plasma vasopressin levels and V1 and V2 vasopressin receptor properties in congenital hypothyroid rat under thyroxine or vasopressin therapy

The effects of propylthiouracil (PTU) treatment on the plasma vasopressin level, on the number of hepatic (V1) or renal (V2) vasopressin receptors and on the hormone-sensitive adenylate cyclase activity in the kidney of developing rats were studied in parallel. In addition, we investigated the corrective effects of thyroxine therapy on the plasma vasopressin level and parameters related to the liver, and the effects of vasopressin therapy on the parameters related to the kidney. As already reported in the case of the number of V2 receptors and adenylate cyclase activity in the kidney, the deficient plasma vasopressin level in hypothyroid rats was completely corrected by two daily physiological doses of thyroxine given from birth to the age of sacrifice (1 month). Unlike the V1 receptors, the V2 receptors are known to be highly dependent on their specific circulating ligand. Since, first of all, the deficit was similar in the numbers of V1 and V2 receptors in hypothyroid rats, and, secondly, the treatment of hypothyroid rats by two daily physiological doses of long lasting vasopressin was found ineffective to recover the deficit in the number of V2 receptors, it can be concluded that thyroid deficiency directly alters vasopressin receptor biosynthesis in both liver and kidney, instead of acting via the depressed plasma vasopressin level.     

Central cholinergic signal-mediated neuroendocrine regulation of vasopressin and oxytocin in ovine fetuses

Background  

The hypothalamic-neurohypophysial system plays a fundamental role in the maintenance of body fluid homeostasis by secreting arginine vasopressin (AVP) and oxytocin (OT) in response to a variety of signals, including osmotic and nonosmotic stimuli. It is well established that central cholinergic mechanisms are critical in the regulation of cardiovascular responses and maintenance of body fluid homeostasis in adults. Our recent study demonstrated that intracerebroventricular (i.c.v.) injection of carbachol elicited an increase of blood pressure in the near-term ovine fetuses. However, in utero development of brain cholinergic mechanisms in the regulation of the hypothalamic neuropeptides is largely unknown. This study investigated AVP and OT neural activation in the fetal hypothalamus induced by central carbachol.     

Oxytocin and vasopressin in the rat do not readily pass from the mother to the amniotic fluid in late pregnancy

In order to see whether the mother contributes to the vasopressin or oxytocin levels of amniotic fluid, these peptides were measured under conditions (1) in which the fetus lacks vasopressin (Brattleboro strain) and (2) where high maternal oxytocin and vasopressin plasma levels were induced by means of a controlled-delivery Accurel-collodion device. No vasopressin could be demonstrated in amniotic fluid of vasopressin-deficient fetuses present in a heterozygous (i.e., vasopressin-synthetizing mother). High peptide levels on the maternal side of Wistar rats generally failed to affect the amniotic fluid levels. The increase that was occasionally seen in amniotic vasopressin was probably due to fetal release concomitant with growth retardation. Amniotic vasopressin is derived from the fetus. Since amniotic fluid oxytocin is neither derived from the mother nor from the fetal brain, other fetal sources should be considered.     

ECG wave form, short term heart rate variability and plasma catecholamine concentrations in intrauterine growth-retarded guinea-pig fetuses

Electrocardiogram waveform, short term heart rate variability and catecholamine concentrations were studied with maternally-induced anesthesia in eleven growth-retarded guinea-pig fetuses and their normal-sized littermates at 63 days of gestation. Intrauterine growth retardation was induced by unilateral uterine artery ligation performed between day 32 and 35. In the growth-retarded group fetal weight was reduced by 45%. Blood gases, acid-base status and oxygen content were similar in the two groups. The growth-retarded guinea-pig fetuses were hypoglycemic and demonstrated a rise in hemoglobin concentration. The T/QRS ratio (T wave amplitude/QRS amplitude) was similar in both groups. The short-term heart rate variability was significantly reduced in the growth-retarded group. Plasma catecholamine concentrations were increased in growth-retarded fetuses but differed only significantly for noradrenaline compared to controls. We suggest that similar T/QRS ratio in both groups of fetuses indicates that aerobic myocardial metabolism is maintained among growth-retarded fetuses. The mechanism behind the reduced variability is unclear.     

Increased levels of UMP synthase protein and mRNA in pyrazofurin-resistant rat hepatoma cells

Rat hepatoma cells that have undergone stepwise selection in increasing concentrations of pyrazofurin have coordinately increased levels of both orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (EC 4.1.1.23) activity. These two activities catalyze the conversion of orotic acid to UMP in de novo pyrimidine biosynthesis. Cells selected in 50 microM pyrazofurin have over 40 times the wild type level for both activities. A single polypeptide of approximately 55,000 daltons is increased in the resistant cells in amounts corresponding to the increase in the two activities. Resistant cell lines that are grown for extended periods in the absence of pyrazofurin are unstable, losing their elevated levels of both enzyme activities and the increased specific protein. Antibody prepared against a purified protein containing both enzyme activities binds specifically to this increased protein. These results corroborate other evidence indicating the two enzyme activities are contained within a single polypeptide called UMP synthase. Poly(A+) mRNA isolated from wild type and resistant lines was analyzed by in vitro translation for production of UMP synthase protein. Immunoprecipitation of the translation products shows the resistant cells have a 17-fold increase in translatable mRNA activity coding for UMP synthase. The synthase accounts for 0.24% of the total in vitro translation products synthesized with poly(A+) mRNA from the pyrazofurin-resistant cells as opposed to 0.014% with wild type mRNA. A cloned UMP synthase cDNA sequence hybridizes strongly to a 1.8-kilobase mRNA in the resistant cells. This mRNA is only barely detectable in equivalent preparations from wild type cells. Quantitation of the mRNA by dot hybridization techniques indicates a 16-fold increase in UMP synthase mRNA in the resistant cells. Although this increase in mRNA for UMP synthase could explain most of the increased protein, it is not sufficient to totally account for the 40-fold increase in UMP synthase.     

Increased thirst and vasopressin secretion after myocardial infarction in rats

Impaired regulation of salt and water balance in left ventricular dysfunction and heart failure can lead to pulmonary and peripheral edema and hyponatremia. Previous studies of disordered water regulation in heart failure have used models of low cardiac output with normal cardiac function (e.g., inferior vena cava ligation). We investigated thirst and vasopressin (AVP) secretion in a rat myocardial infarction model of chronic left ventricular dysfunction/heart failure in response to a 24-h water deprivation period. Thirst (implied from water drunk), hematocrit, plasma renin activity, and plasma AVP concentrations increased with water deprivation vs. ad libitum water access. Thirst and plasma AVP concentrations were significantly positively correlated with infarct size after 24-h water deprivation but not under ad libitum water access conditions. The mechanism by which this occurs is unclear but could involve increased osmoreceptor sensitivity, altered stimulation of baroreceptors, the renin-angiotensin system, or altered central neural control.     

Plasma luteinizing hormone levels in normal and prenatally stressed male and female rat fetuses and their mothers

Concentrations of luteinizing hormone (LH) were measured in plasma of fetal and neonatal rats obtained from control mothers and from mothers exposed to stress from Days 14 to 21 of gestation. The regimen of stress used is known to be associated with an abnormal ontogenetic pattern of testosterone secretion from the fetal testes. The overall ontogenetic pattern of immunoreactive LH levels in plasma was similar in male and female rats, and was unaffected by stress. In all groups, LH was low from Days 16 to 20 of gestation, and then rose progressively through birth, i.e. Day 23. However, stressing the mother significantly decreased the already low levels of LH between Days 16 and 20, as indicated by a larger percentage of samples from stressed fetuses of both sexes with LH levels below the limit of sensitivity of the assay. Sex differences in both the control and stressed group became evident only after Day 20 of gestation, with plasma concentrations of females exceeding those of males from Day 21 to 23 post-conception.     

Increased plasma and tissue levels of vitamin E in the spontaneously diabetic BB rat

Increased plasma and tissue levels of vitamin E were found in spontaneously diabetic BB rats (D) as well as asymptomatic/diabetes-prone BB rats (AD) in comparison to levels in non-diabetic control rats (ND). Treatment of D rats with insulin for 30 days returned plasma and tissue values of vitamin E to control levels. The changes reported here could not be explained solely on the basis of variations in total lipid content of plasma. These data suggest the metabolism of vitamin E is altered in asymptomatic and spontaneously diabetic BB rats and this alteration returns to control values following insulin treatment. Furthermore, it might be speculated that these data indicate a relationship between vitamin E and insulin.     

Increased acridine orange uptake and lysosomal enzyme levels in rat liver after steroid administration

A steroid which stabilizes lysosomes $\text{in vitro}$ and a pyrogenic steroid which labilizes lysosomes $\text{in vitro}$ were compared with respect to their ability to modify lysosomal uptake and lysosomal enzyme levels $\text{in vivo}$. Cortisone acetate increased the uptake of acridine orange by rat liver lysosomes when the dye was administered by intrathoracic injection. The steroid increased and accelerated the uptake of acridine orange so that, in liver lysosomes from treated rats, the maximum uptake was double that of controls and was reached at 2h, whereas in controls the maximum uptake was at 4h after the injection of the dye. This large elevation of uptake is specific to the lysosomal fraction and is not seen in other subcellular fractions of rat liver. The specific activities of a lysosomal enzyme β-N-acetylglucosaminidase were increased in lysosomal fractions from cortisone acetate-treated rats. Etiocholanolone, a steroid which labilizes lysosome $\text{in vitro}$, similarly accelerated and increased acridine orange uptake by lysosomes but had little effect on lysosomal β-N-acetylglucosaminidase levels. Thus the ability of steroids to stabilize or labilize lysosomes $\text{in vitro}$ does not correlate with their effect on lysosomal uptake of injected substances $\text{in vivo}$, or with their ability to induce increased specific activities of lysosomal enzymes.