Development and characterization of human fetal female reproductive tract organoids to understand Müllerian duct anomalies.

Müllerian ducts are paired tubular structures that give rise to most of the female reproductive organs. Any abnormalities in the development and differentiation of these ducts lead to anatomical defects in the female reproductive tract organs categorized as Müllerian duct anomalies. Due to the limited access to fetal tissues, little is understood of human reproductive tract development and the associated anomalies. Although organoids represent a powerful model to decipher human development and disease, such organoids from fetal reproductive organs are not available. Here, we developed organoids from human fetal fallopian tubes and uteri and compared them with their adult counterparts. Our results demonstrate that human fetal reproductive tract epithelia do not express some of the typical markers of adult reproductive tract epithelia. Furthermore, fetal organoids are grossly, histologically, and proteomically different from adult organoids. While external supplementation of WNT ligands or activators in culture medium is an absolute requirement for the adult reproductive tract organoids, fetal organoids are able to grow in WNT-deficient conditions. We also developed decellularized tissue scaffolds from adult human fallopian tubes and uteri. Transplantation of fetal organoids onto these scaffolds led to the regeneration of the adult fallopian tube and uterine epithelia. Importantly, suppression of Wnt signaling, which is altered in patients with Müllerian duct anomalies, inhibits the regenerative ability of human fetal organoids and causes severe anatomical defects in the mouse reproductive tract. Thus, our fetal organoids represent an important platform to study the underlying basis of human female reproductive tract development and diseases.

Neonatal Chlamydia muridarum respiratory infection causes neuroinflammation within the brainstem during the early postnatal period.

Respiratory infections are one of the most common causes of illness and morbidity in neonates worldwide. In the acute phase infections are known to cause wide-spread peripheral inflammation. However, the inflammatory consequences to the critical neural control centres for respiration have not been explored. Utilising a well characterised model of neonatal respiratory infection, we investigated acute responses within the medulla oblongata which contains key respiratory regions. Neonatal mice were intranasally inoculated within 24 h of birth, with either Chlamydia muridarum or sham-infected, and tissue collected on postnatal day 15, the peak of peripheral inflammation. A key finding of this study is that, while the periphery appeared to show no sex-specific effects of a neonatal respiratory infection, sex had a significant impact on the inflammatory response of the medulla oblongata. There was a distinct sex-specific response in the medulla coincident with peak of peripheral inflammation, with females demonstrating an upregulation of anti-inflammatory cytokines and males showing very few changes. Microglia also demonstrated sex-specificity with the morphology of females and males differing based upon the nuclei. Astrocytes showed limited changes during the acute response to neonatal infection. These data highlight the strong sex-specific impact of a respiratory infection can have on the medulla in the acute inflammatory phase.

Properties of sodium currents in neonatal and young adult mouse superficial dorsal horn neurons.

BACKGROUND: Superficial dorsal horn (SDH) neurons process nociceptive information and their excitability is partly determined by the properties of voltage-gated sodium channels. Recently, we showed the excitability and action potential properties of mouse SDH neurons change markedly during early postnatal development. Here we compare sodium currents generated in neonate (P0-5) and young adult (≥P21) SDH neurons. RESULTS: Whole cell recordings were obtained from lumbar SDH neurons in transverse spinal cord slices (CsF internal, 32°C). Fast activating and inactivating TTX-sensitive inward currents were evoked by depolarization from a holding potential of -100 mV. Poorly clamped currents, based on a deflection in the IV relationship at potentials between -60 and -50 mV, were not accepted for analysis. Current density and decay time increased significantly between the first and third weeks of postnatal development, whereas time to peak was similar at both ages. This was accompanied by more subtle changes in activation range and steady state inactivation. Recovery from inactivation was slower and TTX-sensitivity was reduced in young adult neurons. CONCLUSIONS: Our study suggests sodium channel expression changes markedly during early postnatal development in mouse SDH neurons. The methods employed in this study can now be applied to future investigations of spinal cord sodium channel plasticity in murine pain models.

Contrasting alterations to synaptic and intrinsic properties in upper-cervical superficial dorsal horn neurons following acute neck muscle inflammation.

BACKGROUND: Acute and chronic pain in axial structures, like the back and neck, are difficult to treat, and have incidence as high as 15%. Surprisingly, most preclinical work on pain mechanisms focuses on cutaneous structures in the limbs and animal models of axial pain are not widely available. Accordingly, we developed a mouse model of acute cervical muscle inflammation and assessed the functional properties of superficial dorsal horn (SDH) neurons. RESULTS: Male C57/Bl6 mice (P24-P40) were deeply anaesthetised (urethane 2.2 g/kg i.p) and the rectus capitis major muscle (RCM) injected with 40 µl of 2% carrageenan. Sham animals received vehicle injection and controls remained anaesthetised for 2 hrs. Mice in each group were sacrificed at 2 hrs for analysis. c-Fos staining was used to determine the location of activated neurons. c-Fos labelling in carrageenan-injected mice was concentrated within ipsilateral (87% and 63% of labelled neurons in C1 and C2 segments, respectively) and contralateral laminae I - II with some expression in lateral lamina V. c-Fos expression remained below detectable levels in control and sham animals. In additional experiments, whole cell recordings were obtained from visualised SDH neurons in transverse slices in the ipsilateral C1 and C2 spinal segments. Resting membrane potential and input resistance were not altered. Mean spontaneous EPSC amplitude was reduced by ~20% in neurons from carrageenan-injected mice versus control and sham animals (20.63 ± 1.05 vs. 24.64 ± 0.91 and 25.87 ± 1.32 pA, respectively). The amplitude (238 ± 33 vs. 494 ± 96 and 593 ± 167 pA) and inactivation time constant (12.9 ± 1.5 vs. 22.1 ± 3.6 and 15.3 ± 1.4 ms) of the rapid A type potassium current (IAr), the dominant subthreshold current in SDH neurons, were reduced in carrageenan-injected mice. CONCLUSIONS: Excitatory synaptic drive onto, and important intrinsic properties (i.e., IAr) within SDH neurons are reduced two hours after acute muscle inflammation. We propose this time point represents an important transition period between peripheral and central sensitisation with reduced excitatory drive providing an initial neuroprotective mechanism during the early stages of the progression towards central sensitisation.

The medulla oblongata shows a sex-specific inflammatory response to systemic neonatal lipopolysaccharide.

Early life inflammation has been linked to long-term modulation of behavioural outcomes due to the central nervous system, but it is now becoming apparent it is also linked to dysfunction of visceral physiology. The medulla oblongata contains a number of nuclei critical for homeostasis, therefore we utilised the well-established model of neonatal lipopolysaccharide (LPS) exposure to examine the immediate and long-term impacts of systemic inflammation on the medulla oblongata. Wistar rats were injected with LPS or saline on postnatal days 3 and 5, with tissues collected on postnatal days 7 or 90 in order to assess expression of inflammatory mediators and microglial morphology in autonomic regions of the medulla oblongata. We observed a distinct sex-specific response of all measured inflammatory mediators at both ages, as well as significant neonatal sex differences in inflammatory mediators within saline groups. At both ages, microglial morphology had significant changes in branch length and soma size in a sex-specific manner in response to LPS exposure. This data not only highlights the strong sex-specific response of neonates to LPS administration, but also the significant life-long impact on the medulla oblongata and the potential altered control of visceral organs.

Alterations in brainstem respiratory centers following peripheral inflammation: A systematic review.

Immune mediators upregulated in peripheral to central immune communication can modulate respiratory function by direct action on brainstem respiratory circuits. In this systematic review we consolidated findings from independent studies examining the relationship between peripheral and neuro- inflammation within brainstem respiratory centres. Microglia and astrocytes modulate brainstem neuroinflammation in response to peripheral immune mediators which then regulates neuronal activity and ultimately respiratory behaviours. Overall, respiratory brainstem nuclei showed increases in several key immune factors, and glia showed an increased response following peripheral inflammation. However, the functional impact of this neuroinflammation remains unclear.

Differentiation of Sensory Neuron Lineage During the Late First and Early Second Trimesters of Human Foetal Development.

Sensory neurons within DRGs are broadly divided into three types that transmit nociceptive, mechanical, and proprioceptive signals. These subtypes are established during in utero development when sensory neurons differentiate into distinct categories according to a complex developmental plan. Most of what we know about this developmental plan comes from studies in rodents and little is known about this process in humans. The present study documents the expression of key genes involved in human sensory neuron development during the late first and early second trimesters (9-16WG). We observed a decrease in the expression of SOX10 and BRN3A, factors associated with migration and proliferation of sensory neurons, towards the end of the first trimester. Small and large sensory neuron populations also emerged at the end of the first trimester, as well as the transcription factors responsible for defining distinct sensory neuron types. NTRK1, which is expressed in nociceptive neurons, emerged first at ~11 WG followed by NTRK2 in mechanoreceptors at ~12 WG, with NTRK3 for proprioceptors peaking at ~14 WG. These peaks were followed by increased expression of their respective neurotrophic factors. Our results show significant differences in the expression of key signalling molecules for human DRG development versus that of rodents, most notably the expression of neurotrophins that promote the survival of sensory neuron types. This highlights the importance of examining molecular changes in humans to better inform the application of data collected in pre-clinical models.

Expression and Physiology of Voltage-Gated Sodium Channels in Developing Human Inner Ear.

Sodium channel expression in inner ear afferents is essential for the transmission of vestibular and auditory information to the central nervous system. During development, however, there is also a transient expression of Na+ channels in vestibular and auditory hair cells. Using qPCR analysis, we describe the expression of four Na+ channel genes, SCN5A (Nav1.5), SCN8A (Nav1.6), SCN9A (Nav1.7), and SCN10A (Nav1.8) in the human fetal cristae ampullares, utricle, and base, middle, and apex of the cochlea. Our data show distinct patterns of Na+ channel gene expression with age and between these inner ear organs. In the utricle, there was a general trend toward fold-change increases in expression of SCN8A, SCN9A, and SCN10A with age, while the crista exhibited fold-change increases in SCN5A and SCN8A and fold-change decreases in SCN9A and SCN10A. Fold-change differences of each gene in the cochlea were more complex and likely related to distinct patterns of expression based on tonotopy. Generally, the relative expression of SCN genes in the cochlea was greater than that in utricle and cristae ampullares. We also recorded Na+ currents from developing human vestibular hair cells aged 10-11 weeks gestation (WG), 12-13 WG, and 14+ WG and found there is a decrease in the number of vestibular hair cells that exhibit Na+ currents with increasing gestational age. Na+ current properties and responses to the application of tetrodotoxin (TTX; 1 µM) in human fetal vestibular hair cells are consistent with those recorded in other species during embryonic and postnatal development. Both TTX-sensitive and TTX-resistant currents are present in human fetal vestibular hair cells. These results provide a timeline of sodium channel gene expression in inner ear neuroepithelium and the physiological characterization of Na+ currents in human fetal vestibular neuroepithelium. Understanding the normal developmental timeline of ion channel gene expression and when cells express functional ion channels is essential information for regenerative technologies.

Generation of Vestibular Tissue-Like Organoids From Human Pluripotent Stem Cells Using the Rotary Cell Culture System.

Hair cells are specialized mechanosensitive cells responsible for mediating balance and hearing within the inner ear. In mammals, hair cells are limited in number and do not regenerate. Human pluripotent stem cells (hPSCs) provide a valuable source for deriving human hair cells to study their development and design therapies to treat and/or prevent their degeneration. In this study we used a dynamic 3D Rotary Cell Culture System (RCCS) for deriving inner ear organoids from hPSCs. We show RCCS-derived organoids recapitulate stages of inner ear development and give rise to an enriched population of hair cells displaying vestibular-like morphological and physiological phenotypes, which resemble developing human fetal inner ear hair cells as well as the presence of accessory otoconia-like structures. These results show that hPSC-derived organoids can generate complex inner ear structural features and be a resource to study inner ear development.

Excitability of Rat Superficial Dorsal Horn Neurons Following a Neonatal Immune Challenge.

Previous studies have shown that neonatal exposure to a mild inflammatory challenge, such as lipopolysaccharide (LPS, Salmonella enteriditis) results in altered pain behaviors later in life. To further characterize the impact of a neonatal immune challenge on pain processing, we examined the excitability of superficial dorsal horn (SDH) neurons following neonatal LPS exposure and subsequent responses to noxious stimulation at three time-points during early postnatal development. Wistar rats were injected with LPS (0.05 mg/kg i.p.) or saline on postnatal days (PNDs) 3 and 5, and later subjected to the formalin test at PNDs 7, 13, and 22. One hour after formalin injection into the plantar hindpaw, animals were euthanized (Ketamine, 100 mg/kg i.p.) and transverse slices from the lumbosacral spinal cord were prepared. Whole-cell patch-clamp recordings were made from SDH neurons (KCH3SO4-based internal, 22-24°C) on the ipsi- and contralateral sides of the spinal cord. Depolarising current steps were injected into SDH neurons to categorize action potential (AP) discharge. In both saline- and LPS-treated rats we observed age-related increases the percentage of neurons exhibiting tonic-firing, with concurrent decreases in single-spiking, between PND 7 and 22. In contrast, neonatal exposure to LPS failed to alter the proportions of AP discharge patterns at any age examined. We also assessed the subthreshold currents that determine AP discharge in SDH neurons. The rapid outward potassium current, IAr decreased in prevalence with age, but was susceptible to neonatal LPS exposure. Peak IAr current amplitude was greater in ipsilateral vs. contralateral SDH neurons from LPS-treated rats. Spontaneous excitatory synaptic currents (sEPSCs) were recorded to assess network excitability. Age-related increases were observed in sEPSC frequency and time course, but not peak amplitude, in both saline- and LPS-treated rats. Furthermore, sEPSC frequency was higher in ipsilateral vs. contralateral SDH neurons in LPS-treated animals. Taken together, these data suggest a neonatal immune challenge does not markedly affect the intrinsic properties of SDH neurons, however, it can increase the excitability of local spinal cord networks via altering the properties of rapid A-type currents and excitatory synaptic connections. These changes, made in neurons within spinal cord pain circuits, have the capacity to alter nociceptive signaling in the ascending pain pathway.

Altered nociceptive, endocrine, and dorsal horn neuron responses in rats following a neonatal immune challenge.

The neonatal period is characterized by significant plasticity where the immune, endocrine, and nociceptive systems undergo fine-tuning and maturation. Painful experiences during this period can result in long-term alterations in the neurocircuitry underlying nociception, including increased sensitivity to mechanical or thermal stimuli. Less is known about the impact of neonatal exposure to mild inflammatory stimuli, such as lipopolysaccharide (LPS), on subsequent inflammatory pain responses. Here we examine the impact of neonatal LPS exposure on inflammatory pain sensitivity and HPA axis activity during the first three postnatal weeks. Wistar rats were injected with LPS (0.05mg/kg IP, Salmonella enteritidis) or saline on postnatal days (PNDs) 3 and 5 and later subjected to the formalin test at PNDs 7, 13, and 22. One hour after formalin injection, blood was collected to assess corticosterone responses. Transverse spinal cord slices were also prepared for whole-cell patch clamp recording from lumbar superficial dorsal horn neurons (SDH). Brains were obtained at PND 22 and the hypothalamus was isolated to measure glucocorticoid (GR) and mineralocorticoid receptor (MR) transcript expression using qRT-PCR. Behavioural analyses indicate that at PND 7, no significant differences were observed between saline- or LPS-challenged rats. At PND 13, LPS-challenged rats exhibited enhanced licking (p<.01), and at PND 22, increased flinching in response to formalin injection (p<.05). LPS-challenged rats also displayed increased plasma corticosterone at PND 7 and PND 22 (p<.001) but not at PND 13 following formalin administration. Furthermore, at PND 22 neonatal LPS exposure induced decreased levels of GR mRNA and increased levels of MR mRNA in the hypothalamus. The intrinsic properties of SDH neurons were similar at PND 7 and PND 13. However, at PND 22, ipsilateral SDH neurons in LPS-challenged rats had a lower input resistance compared to their saline-challenged counterparts (p<.05). These data suggest neonatal LPS exposure produces developmentally regulated changes in formalin-induced behavioural responses, corticosterone levels, and dorsal horn neuron properties following noxious stimulation later in life. These findings highlight the importance of immune activation during the neonatal period in shaping pain sensitivity later in life. This programming involves both spinal cord neurons and the HPA axis.

Preliminary characterization of voltage-activated whole-cell currents in developing human vestibular hair cells and calyx afferent terminals.

We present preliminary functional data from human vestibular hair cells and primary afferent calyx terminals during fetal development. Whole-cell recordings were obtained from hair cells or calyx terminals in semi-intact cristae prepared from human fetuses aged between 11 and 18 weeks gestation (WG). During early fetal development (11-14 WG), hair cells expressed whole-cell conductances that were qualitatively similar but quantitatively smaller than those observed previously in mature rodent type II hair cells. As development progressed (15-18 WG), peak outward conductances increased in putative type II hair cells but did not reach amplitudes observed in adult human hair cells. Type I hair cells express a specific low-voltage activating conductance, G K,L. A similar current was first observed at 15 WG but remained relatively small, even at 18 WG. The presence of a "collapsing" tail current indicates a maturing type I hair cell phenotype and suggests the presence of a surrounding calyx afferent terminal. We were also able to record from calyx afferent terminals in 15-18 WG cristae. In voltage clamp, these terminals exhibited fast inactivating inward as well as slower outward conductances, and in current clamp, discharged a single action potential during depolarizing steps. Together, these data suggest the major functional characteristics of type I and type II hair cells and calyx terminals are present by 18 WG. Our study also describes a new preparation for the functional investigation of key events that occur during maturation of human vestibular organs.

Low formalin concentrations induce fine-tuned responses that are sex and age-dependent: a developmental study.

The formalin test is increasingly applied as a model of inflammatory pain using high formalin concentrations (5-15%). However, little is known about the effects of low formalin concentrations on related behavioural responses. To examine this, rat pups were subjected to various concentrations of formalin at four developmental stages: 7, 13, 22, and 82 days of age. At postnatal day (PND) 7, sex differences in flinching but not licking responses were observed with 0.5% formalin evoking higher flinching in males than in females. A dose response was evident in that 0.5% formalin also produced higher licking responses compared to 0.3% or 0.4% formalin. At PND 13, a concentration of 0.8% formalin evoked a biphasic response. At PND 22, a concentration of 1.1% evoked higher flinching and licking responses during the late phase (10-30 min) in both males and females. During the early phase (0-5 min), 1.1% evoked higher licking responses compared to 0.9% or 1% formalin. 1.1% formalin produced a biphasic response that was not evident with 0.9 or 1%. At PND 82, rats displayed a biphasic pattern in response to three formalin concentrations (1.25%, 1.75% and 2.25%) with the presence of an interphase for both 1.75% and 2.25% but not for 1.25%. These data suggest that low formalin concentrations induce fine-tuned responses that are not apparent with the high formalin concentration commonly used in the formalin test. These data also show that the developing nociceptive system is very sensitive to subtle changes in formalin concentrations.