Bioelectronic Medicine: a multidisciplinary roadmap from biophysics to precision therapies.

Bioelectronic Medicine stands as an emerging field that rapidly evolves and offers distinctive clinical benefits, alongside unique challenges. It consists of the modulation of the nervous system by precise delivery of electrical current for the treatment of clinical conditions, such as post-stroke movement recovery or drug-resistant disorders. The unquestionable clinical impact of Bioelectronic Medicine is underscored by the successful translation to humans in the last decades, and the long list of preclinical studies. Given the emergency of accelerating the progress in new neuromodulation treatments (i.e., drug-resistant hypertension, autoimmune and degenerative diseases), collaboration between multiple fields is imperative. This work intends to foster multidisciplinary work and bring together different fields to provide the fundamental basis underlying Bioelectronic Medicine. In this review we will go from the biophysics of the cell membrane, which we consider the inner core of neuromodulation, to patient care. We will discuss the recently discovered mechanism of neurotransmission switching and how it will impact neuromodulation design, and we will provide an update on neuronal and glial basis in health and disease. The advances in biomedical technology have facilitated the collection of large amounts of data, thereby introducing new challenges in data analysis. We will discuss the current approaches and challenges in high throughput data analysis, encompassing big data, networks, artificial intelligence, and internet of things. Emphasis will be placed on understanding the electrochemical properties of neural interfaces, along with the integration of biocompatible and reliable materials and compliance with biomedical regulations for translational applications. Preclinical validation is foundational to the translational process, and we will discuss the critical aspects of such animal studies. Finally, we will focus on the patient point-of-care and challenges in neuromodulation as the ultimate goal of bioelectronic medicine. This review is a call to scientists from different fields to work together with a common endeavor: accelerate the decoding and modulation of the nervous system in a new era of therapeutic possibilities.

Validation of vaginal microbiome proxies for in vitro experiments that biomimic Lactobacillus-dominant vaginal cultures.

The vaginal microbiome includes diverse microbiota dominated by Lactobacillus [L.] spp. that protect against infections, modulate inflammation, and regulate vaginal homeostasis. Because it is challenging to incorporate vaginal microbiota into in vitro models, including organ-on-a-chip systems, we assessed microbial metabolites as reliable proxies in addition to traditional vaginal epithelial cultures (VECs). Human immortalized VECs cultured on transwells with an air-liquid interface generated stratified cell layers colonized by transplanted healthy microbiomes (L. jensenii- or L. crispatus-dominant) or a community representing bacterial vaginosis (BV). After 48-h, a qPCR array confirmed the expected donor community profiles. Pooled apical and basal supernatants were subjected to metabolomic analysis (untargeted mass spectrometry) followed by ingenuity pathways analysis (IPA). To determine the bacterial metabolites' ability to recreate the vaginal microenvironment in vitro, pooled bacteria-free metabolites were added to traditional VEC cultures. Cell morphology, viability, and cytokine production were assessed. IPA analysis of metabolites from colonized samples contained fatty acids, nucleic acids, and sugar acids that were associated with signaling networks that contribute to secondary metabolism, anti-fungal, and anti-inflammatory functions indicative of a healthy vaginal microbiome compared to sterile VEC transwell metabolites. Pooled metabolites did not affect cell morphology or induce cell death (∼5.5%) of VEC cultures (n = 3) after 72-h. However, metabolites created an anti-inflammatory milieu by increasing IL-10 production (p = .06, T-test) and significantly suppressing pro-inflammatory IL-6 (p = .0001), IL-8 (p = .009), and TNFα (p = .0007) compared to naïve VEC cultures. BV VEC conditioned-medium did not affect cell morphology nor viability; however, it induced a pro-inflammatory environment by elevating levels of IL-6 (p = .023), IL-8 (p = .031), and TNFα (p = .021) when compared to L.-dominate microbiome-conditioned medium. VEC transwells provide a suitable ex vivo system to support the production of bacterial metabolites consistent with the vaginal milieu allowing subsequent in vitro studies with enhanced accuracy and utility.

Handoff Tool Improves Transitions from the Operating Room to the Neonatal Intensive Care Unit.

Introduction: Standardized handoffs reduce medical errors and prevent adverse events or near misses. This article describes a quality improvement initiative implementing a unique standardized handoff tool and process to transition from the operating room to the neonatal intensive care unit (NICU) at a level-four regional center with many inpatients requiring surgical intervention. Before this project, there was no standardized handoff tool or process for postsurgical transitions. The primary aim was to achieve 80% compliance with completing a structured postoperative OR to NICU handoff tool within 12 months of implementation. Methods: An interdisciplinary team developed and implemented a standardized NICU postoperative handoff tool and process that requires face-to-face communication, defines team members who should be present, and highlights communication with the family. In addition, the handoff tool compliance and process measures were monitored, evaluated, and audited. Results: Although not consistent, we achieved eighty percent compliance with the outcome measures using the handoff tool. We did not sustain 80% of appropriate providers present at handoff. In addition, insufficient data assess overall parental satisfaction with the surgical experience. Although improved, the process measure of immediate postoperative family updates did not reach the targeted goal. However, the balancing measure of staff experience and satisfaction did improve. Conclusion: Implementing a standardized handoff tool and process with an interdisciplinary and interdepartmental collaboration improves critical patient transitions from the operating room to the NICU.

Characterization of fetal microchimeric immune cells in mouse maternal hearts during physiologic and pathologic pregnancies.

Introduction: During pregnancy, fetal cells can be incorporated into maternal tissues (fetal microchimerism), where they can persist postpartum. Whether these fetal cells are beneficial or detrimental to maternal health is unknown. This study aimed to characterize fetal microchimeric immune cells in the maternal heart during pregnancy and postpartum, and to identify differences in these fetal microchimeric subpopulations between normal and pregnancies complicated by spontaneous preterm induced by ascending infection. Methods: A Cre reporter mouse model, which when mated with wild-type C57BL/6J females resulted in cells and tissues of progeny expressing red fluorescent protein tandem dimer Tomato (mT+), was used to detect fetal microchimeric cells. On embryonic day (E)15, 104 colony-forming units (CFU) E. coli was administered intravaginally to mimic ascending infection, with delivery on or before E18.5 considered as preterm delivery. A subset of pregnant mice was sacrificed at E16 and postpartum day 28 to harvest maternal hearts. Heart tissues were processed for immunofluorescence microscopy and high-dimensional mass cytometry by time-of-flight (CyTOF) using an antibody panel of immune cell markers. Changes in cardiac physiologic parameters were measured up to 60 days postpartum via two-dimensional echocardiography. Results: Intravaginal E. coli administration resulted in preterm delivery of live pups in 70% of the cases. mT + expressing cells were detected in maternal uterus and heart, implying that fetal cells can migrate to different maternal compartments. During ascending infection, more fetal antigen-presenting cells (APCs) and less fetal hematopoietic stem cells (HSCs) and fetal double-positive (DP) thymocytes were observed in maternal hearts at E16 compared to normal pregnancy. These HSCs were cleared while DP thymocytes persisted 28 days postpartum following an ascending infection. No significant changes in cardiac physiologic parameters were observed postpartum except a trend in lowering the ejection fraction rate in preterm delivered mothers. Conclusion: Both normal pregnancy and ascending infection revealed distinct compositions of fetal microchimeric immune cells within the maternal heart, which could potentially influence the maternal cardiac microenvironment via (1) modulation of cardiac reverse modeling processes by fetal stem cells, and (2) differential responses to recognition of fetal APCs by maternal T cells.

Next generation 3D-printed intravaginal ring for prevention of HIV and unintended pregnancy.

Globally, there are 20 million adolescent girls and young women living with HIV who have limited access to long-acting, effective, women-controlled preventative methods. Additionally, although there are many contraceptive methods available, globally, half of all pregnancies remain unintended. Here we report the first 3D-printed multipurpose prevention technology (MPT) intravaginal ring (IVR) for HIV prevention and contraception. We utilized continuous liquid interface production (CLIP™) to fabricate MPT IVRs in a biocompatible silicone-based resin. Etonogestrel (ENG), ethinyl estradiol (EE), and islatravir (ISL) were loaded into the silicone poly(urethane) IVR in a controlled single step drug loading process driven by absorption. ENG/EE/ISL IVR promoted sustained release of drugs for 150 days in vitro and 14 days in sheep. There were no adverse MPT IVR-related findings of cervicovaginal toxicity or changes in vaginal biopsies or microbiome community profiles evaluated in sheep. Furthermore, ISL IVR in macaques promoted sustained release for 28 days with ISL-triphosphate levels above the established pharmacokinetic benchmark of 50-100 fmol/106 PBMCs. The ISL IVR was found to be safe and well tolerated in the macaques with no observed mucosal cytokine changes or alterations in peripheral CD4 T-cell populations. Collectively, the proposed MPT IVR has potential to expand preventative choices for young women and girls.

Modulating anesthetic emergence with pathway-selective dopamine signaling.

PURPOSE OF REVIEW: To summarize the recent preclinical findings investigating dopaminergic circuits for their involvement in reversing anesthetic-induced unconsciousness. RECENT FINDINGS: The release of dopamine from the ventral tegmental area onto dopamine D1 receptor-expressing neurons in the nucleus accumbens promotes emergence following general anesthesia. Two relevant targets of dopamine D1 receptor-expressing neurons in the nucleus accumbens include the lateral hypothalamus and ventral pallidum. Activating mesocortical dopaminergic projections from the ventral tegmental area to the prelimbic cortex has also been shown to hasten emergence from general anesthesia. In contrast, the nigrostriatal dopamine pathway is not involved in regulating anesthetic emergence. The role of the tuberoinfundibular endocrine dopamine pathway remains to be tested; however, recent studies have identified an important function of neuroendocrine signaling on modulating general anesthesia. SUMMARY: Potential avenues for accelerating anesthetic emergence may be found through targeting specific arousal-promoting pathways in the brain. Accumulating evidence from rodent studies manipulating cell type- and circuit-specific signaling pathways have identified dopamine as a potent modulator of general anesthesia. Specifically, dopamine signaling along the mesolimbic and mesocortical pathways plays a fundamental role in regulating consciousness.

PEGylated Polymeric Nanoparticles Loaded with 2-Methoxyestradiol for the Treatment of Uterine Leiomyoma in a Patient-Derived Xenograft Mouse Model.

Leiomyomas, the most common benign neoplasms of the female reproductive tract, currently have limited medical treatment options. Drugs targeting estrogen/progesterone signaling are used, but side effects and limited efficacy in many cases are major limitation of their clinical use. Previous studies from our laboratory and others demonstrated that 2-methoxyestradiol (2-ME) is promising treatment for uterine fibroids. However, its poor bioavailability and rapid degradation hinder its development for clinical use. The objective of this study is to evaluate the in vivo effect of biodegradable and biocompatible 2-ME-loaded polymeric nanoparticles in a patient-derived leiomyoma xenograft mouse model. PEGylated poly(lactide-co-glycolide) (PEG-PLGA) nanoparticles loaded with 2-ME were prepared by nanoprecipitation. Female 6-week age immunodeficient NOG (NOD/Shi-scid/IL-2Rγnull) mice were used. Estrogen-progesterone pellets were implanted subcutaneously. Five days later, patient-derived human fibroid tumors were xenografted bilaterally subcutaneously. Engrafted mice were treated with 2-ME-loaded or blank (control) PEGylated nanoparticles. Nanoparticles were injected intraperitoneally and after 28 days of treatment, tumor volume was measured by caliper following hair removal, and tumors were removed and weighed. Up to 99.1% encapsulation efficiency was achieved, and the in vitro release profile showed minimal burst release, thus confirming the high encapsulation efficiency. In vivo administration of the 2-ME-loaded nanoparticles led to 51% growth inhibition of xenografted tumors compared to controls (P < 0.01). Thus, 2-ME-loaded nanoparticles may represent a novel approach for the treatment of uterine fibroids.

Oestrous cycle affects emergence from anaesthesia with dexmedetomidine, but not propofol, isoflurane, or sevoflurane, in female rats.

BACKGROUND: Although sex differences in anaesthetic sensitivity have been reported, what underlies these differences is unknown. In rodents, one source of variability in females is the oestrous cycle. Here we test the hypothesis that the oestrous cycle impacts emergence from general anaesthesia. METHODS: Time to emergence was measured after isoflurane (2 vol% for 1 h), sevoflurane (3 vol% for 20 min), dexmedetomidine (50 µg kg-1 i.v., infused over 10 min), or propofol (10 mg kg-1 i.v. bolus) during proestrus, oestrus, early dioestrus, and late dioestrus in female Sprague-Dawley rats (n=24). EEG recordings were taken during each test for power spectral analysis. Serum was analysed for 17ß-oestradiol and progesterone concentrations. The effect of oestrous cycle stage on return of righting latency was assessed using a mixed model. The association between righting latency and serum hormone concentration was tested by linear regression. Mean arterial blood pressure and arterial blood gases were assessed in a subset of rats after dexmedetomidine and compared in a mixed model. RESULTS: Oestrous cycle did not affect righting latency after isoflurane, sevoflurane, or propofol. When in the early dioestrus stage, rats emerged more rapidly from dexmedetomidine than in the proestrus (P=0.0042) or late dioestrus (P=0.0230) stage and showed reduced overall power in frontal EEG spectra 30 min after dexmedetomidine (P=0.0049). 17ß-Oestradiol and progesterone serum concentrations did not correlate with righting latency. Oestrous cycle did not affect mean arterial blood pressure or blood gases during dexmedetomidine. CONCLUSIONS: In female rats, the oestrous cycle significantly impacts emergence from dexmedetomidine-induced unconsciousness. However, 17ß-oestradiol and progesterone serum concentrations do not correlate with the observed changes.

Loss of lumbar disc height with age and its impact on pain and sensitivity associated behaviors in mice.

PURPOSE: Aging is a risk factor for several debilitating conditions including those related to chronic back pain and intervertebral disc degeneration, both of which have no cure. Mouse models are useful tools for studying disc degeneration and chronic back pain in a tightly controlled and clinically relevant aging environment. Moreover, mice offer the advantage of carrying out longitudinal studies to understand the etiology and progression of disc pathology induced by genetic or surgical strategies. Previously, age-related behavioral trends of discomfort and enhanced nociception in mice were reported; however, whether these measures are mediated by structural and pathological changes in the disc is unknown. METHODS: The goal of the present observational study was to identify behavioral correlates of age-related degenerative changes in the disc. Towards this, we collected radiographs from 150 mice (77 females) between three and 23 months of age and measured the disc height index for each level of lumbar disc. Behavioral measures were collected on several of these mice which included rearing and distance travelled in an open field test; time spent in rearing, reaching, immobile, and self-suspended in the tail suspension test; bilateral hind paw licking in response to cold allodynia using acetone; and unilateral hind paw licking in response to heat hyperalgesia using capsaicin. RESULTS: Results show that the lower lumbar discs lose height with age and these changes are independent of body composition measures including body weight, bone mineral density, fat mass, lean weight mass, percent fat mass, and percent lean mass. Disc height positively correlates with rearing and mobility in the open field test, immobility in the tail suspension test, and thermal hyperalgesia. Disc height negatively correlates with cold allodynia and rearing in the tail suspension test. Furthermore, mediation analysis shows that the lumbosacral disc significantly mediates the effect of age on rearing in the open field test, but not cold allodynia, suggesting this behavior is a useful measure of age-related axial discomfort due to disc degeneration. CONCLUSION: In summary, the findings from the current study show that disc height are associated with measures of axial discomfort and nociception in mice.

Lactic acid, citric acid, and potassium bitartrate non-hormonal prescription vaginal pH modulator (VPM) gel for the prevention of pregnancy.

INTRODUCTION: A non-hormonal prescription vaginal pH modulator (VPM) gel (Phexxi®), with active ingredients lactic acid, citric acid and potassium bitartrate, has recently been approved for prevention of pregnancy in the United States. The objective of this review is to compile the evidence available from published preclinical and clinical trials to support its use. AREAS COVERED: PubMed was searched for published literature on VPM gel. Two Phase III trials were found on clinicaltrials.gov database. The results demonstrated that VPM gel is safe, with minimal side effects, and effective (cumulative 6-7 cycle pregnancy rate of 4.1-13.65%, (Pearl Index 27.5) as a contraceptive. Microbicidal effects suggest potential for the prevention of sexually transmitted infections (STIs); currently a Phase III clinical trial is being conducted to evaluate prevention of chlamydia and gonorrhea. EXPERT OPINION: Non-hormonal reversible contraceptive options have been limited to the highly effective copper-releasing intrauterine device that requires insertion by a trained clinician, and less effective coitally associated barrier and spermicide options which are typically available over-the-counter. Spermicides, which improve efficacy of barrier devices, may increase the risk of HIV/STIs. VPM gel provides a new safe, effective non-hormonal contraceptive option, with potential for prevention of STIs.

Simvastatin-loaded liposome nanoparticles treatment for uterine leiomyoma in a patient-derived xenograft mouse model: a pilot study.

Uterine leiomyomas are complex tumours with limited medical treatment options. Simvastatin is used to treat hypercholesterolaemia and has shown promising effects as a treatment option for leiomyomas. Previously, our group demonstrated a promising effect of simvastatin treatment in a patient-derived xenograft mouse model. Here, we tested the efficacy of simvastatin liposomal nanoparticles (NPs). After bilateral leiomyoma xenograft implantation, mice (N = 12) were divided into three treatment arms: control, simvastatin and simvastatin-loaded liposome NPs (simvastatin-NPs). Treatment with simvastatin significantly reduced tumour volume and inhibited the Ki67 expression when compared to the control group. There was a trend of reduced tumour volume and Ki67 expression after treatment with simvastatin-NP; however, the results were not significant. Due to low bioavailability and short half-life of simvastatin, liposomal NPs have the potential to enhance drug delivery, however, in this study NP did not provide improvement over simvastatin, but did demonstrate their potential for the delivery of simvastatin.Impact statementWhat is already known on this subject? Simvastatin treatment in a patient-derived xenograft mouse model reduced tumour growth and decreased proliferation.What do the results of this study add? Treatment with simvastatin significantly reduced tumour volume and inhibited the Ki67 expression when compared to the control group. There was a trend of reduced tumour volume and Ki67 expression after treatment with simvastatin-NP, however, it did not improve the efficacy of simvastatin at reducing tumour growth and proliferation.What are the implications of these findings for clinical practice and/or further research? More studies are needed to optimise the formulation of NPs to further enhance the sustainable delivery of simvastatin.

Return of the Righting Reflex Does Not Portend Recovery of Cognitive Function in Anesthetized Rats.

As the number of individuals undergoing general anesthesia rises globally, it becomes increasingly important to understand how consciousness and cognition are restored after anesthesia. In rodents, levels of consciousness are traditionally captured by physiological responses such as the return of righting reflex (RORR). However, tracking the recovery of cognitive function is comparatively difficult. Here we use an operant conditioning task, the 5-choice serial reaction time task (5-CSRTT), to measure sustained attention, working memory, and inhibitory control in male and female rats as they recover from the effects of several different clinical anesthetics. In the 5-CSRTT, rats learn to attend to a five-windowed touchscreen for the presentation of a stimulus. Rats are rewarded with food pellets for selecting the correct window within the time limit. During each session we tracked both the proportion of correct (accuracy) and missed (omissions) responses over time. Cognitive recovery trajectories were assessed after isoflurane (2% for 1 h), sevoflurane (3% for 20 min), propofol (10 mg/kg I.V. bolus), ketamine (50 mg/kg I.V. infusion over 10 min), and dexmedetomidine (20 and 35 µg/kg I.V. infusions over 10 min) for up to 3 h following RORR. Rats were classified as having recovered accuracy performance when four of their last five responses were correct, and as having recovered low omission performance when they missed one or fewer of their last five trials. Following isoflurane, sevoflurane, and propofol anesthesia, the majority (63-88%) of rats recovered both accuracy and low omission performance within an hour of RORR. Following ketamine, accuracy performance recovers within 2 h in most (63%) rats, but low omission performance recovers in only a minority (32%) of rats within 3 h. Finally, following either high or low doses of dexmedetomidine, few rats (25-32%) recover accuracy performance, and even fewer (0-13%) recover low omission performance within 3 h. Regardless of the anesthetic, RORR latency is not correlated with 5-CSRTT performance, which suggests that recovery of neurocognitive function cannot be inferred from changes in levels of consciousness. These results demonstrate how operant conditioning tasks can be used to assess real-time recovery of neurocognitive function following different anesthetic regimens.

Hexavalent sperm-binding IgG antibody released from vaginal film for development of potent on-demand nonhormonal female contraception.

Nonhormonal products for on-demand contraception are a global health technology gap; this unmet need motivated us to pursue the use of sperm-binding monoclonal antibodies to enable effective on-demand contraception. Here, using the cGMP-compliant Nicotiana-expression system, we produced an ultrapotent sperm-binding IgG antibody possessing 6 Fab arms per molecule that bind a well-established contraceptive antigen target, CD52g. We term this hexavalent antibody "Fab-IgG-Fab" (FIF). The Nicotiana-produced FIF had at least 10-fold greater sperm-agglutination potency and kinetics than the parent IgG, while preserving Fc-mediated trapping of individual spermatozoa in mucus. We formulated the Nicotiana-produced FIF into a polyvinyl alcohol-based water-soluble contraceptive film and evaluated its potency in reducing progressively motile sperm in the sheep vagina. Two minutes after vaginal instillation of human semen, no progressively motile sperm were recovered from the vaginas of sheep receiving FIF Film. Our work supports the potential of multivalent contraceptive antibodies to provide safe, effective, on-demand nonhormonal contraception.

Inflammatory response elicited by Ureaplasma parvum colonization in human cervical epithelial, stromal, and immune cells.

Ureaplasma parvum is a commensal bacterium in the female reproductive tract but has been associated with pregnancy complications such as preterm prelabor rupture of membranes and preterm birth (PTB). However, the pathologic effects of U. parvum in the cervix, which prevents ascending infections during pregnancy, are still poorly understood. To determine the impact of U. parvum on the cervix, ectocervical (ecto) and endocervical (endo) epithelial and stromal cells were incubated with U. parvum. Macrophages were also tested as a proxy for cervical macrophages to determine the antigenicity of U. parvum. The effects of U. parvum, including influence on cell cycle and cell death, antimicrobial peptide (AMP) production, epithelial-to-mesenchymal transition (EMT), and inflammatory cytokine levels, were assessed. U. parvum colonized cervical epithelial and stromal cells 4 h post-infection. Like uninfected control, U. parvum neither inhibited cell cycle progression and nor caused cell death in cervical epithelial and stromal cells. U. parvum increased the production of the AMPs cathelicidin and human ß-defensin 3 and exhibited weak signs of EMT evidenced by decreased cytokeratin 18 and increased vimentin expression in cervical epithelial cells. U. parvum induced a proinflammatory environment (cytokines) and increased MMP-9 in cervical epithelial cells but promoted pro- and anti-inflammatory response in cervical stromal cells and macrophages. U. parvum may colonize the cervical epithelial layer, but induction of AMPs and anti-inflammatory response may protect the cervix and may prevent ascending infections that can cause PTB. These findings suggest that U. parvum is a weak inducer of inflammation in the cervix.

Engineering sperm-binding IgG antibodies for the development of an effective nonhormonal female contraception.

Many women risk unintended pregnancy because of medical contraindications or dissatisfaction with contraceptive methods, including real and perceived side effects associated with the use of exogenous hormones. We pursued direct vaginal delivery of sperm-binding monoclonal antibodies (mAbs) that can limit progressive sperm motility in the female reproductive tract as a strategy for effective nonhormonal contraception. Here, motivated by the greater agglutination potencies of polyvalent immunoglobulins but the bioprocessing ease and stability of immunoglobulin G (IgG), we engineered a panel of sperm-binding IgGs with 6 to 10 antigen-binding fragments (Fabs), isolated from a healthy immune-infertile woman against a unique surface antigen universally present on human sperm. These highly multivalent IgGs (HM-IgGs) were at least 10- to 16-fold more potent and faster at agglutinating sperm than the parent IgG while preserving the crystallizable fragment (Fc) of IgG that mediates trapping of individual spermatozoa in mucus. The increased potencies translated into effective (>99.9%) reduction of progressively motile sperm in the sheep vagina using as little as 33 µg of the 10-Fab HM-IgG. HM-IgGs were produced at comparable yields and had identical thermal stability to the parent IgG, with greater homogeneity. HM-IgGs represent not only promising biologics for nonhormonal contraception but also a promising platform for engineering potent multivalent mAbs for other biomedical applications.

High-Resolution Quantitative Mapping of Macaque Cervicovaginal Epithelial Thickness: Implications for Mucosal Vaccine Delivery.

Vaginal mucosal surfaces naturally offer some protection against sexually transmitted infections (STIs) including Human Immunodeficiency Virus-1, however topical preventative medications or vaccine designed to boost local immune responses can further enhance this protection. We previously developed a novel mucosal vaccine strategy using viral vectors integrated into mouse dermal epithelium to induce virus-specific humoral and cellular immune responses at the site of exposure. Since vaccine integration occurs at the site of cell replication (basal layer 100-400 micrometers below the surface), temporal epithelial thinning during vaccine application, confirmed with high resolution imaging, is desirable. In this study, strategies for vaginal mucosal thinning were evaluated noninvasively using optical coherence tomography (OCT) to map reproductive tract epithelial thickness (ET) in macaques to optimize basal layer access in preparation for future effective intravaginal mucosal vaccination studies. Twelve adolescent female rhesus macaques (5-7kg) were randomly assigned to interventions to induce vaginal mucosal thinning, including cytobrush mechanical abrasion, the chemical surfactant spermicide nonoxynol-9 (N9), the hormonal contraceptive depomedroxyprogesterone acetate (DMPA), or no intervention. Macaques were evaluated at baseline and after interventions using colposcopy, vaginal biopsies, and OCT imaging, which allowed for real-time in vivo visualization and measurement of ET of the mid-vagina, fornices, and cervix. P value ≤0.05 was considered significant. Colposcopy findings included pink, rugated tissue with variable degrees of white-tipped, thickened epithelium. Baseline ET of the fornices was thinner than the cervix and vagina (p<0.05), and mensing macaques had thinner ET at all sites (p<0.001). ET was decreased 1 month after DMPA (p<0.05) in all sites, immediately after mechanical abrasion (p<0.05) in the fornix and cervix, and after two doses of 4% N9 (1.25ml) applied over 14 hrs in the fornix only (p<0.001). Histological assessment of biopsied samples confirmed OCT findings. In summary, OCT imaging allowed for real time assessment of macaque vaginal ET. While varying degrees of thinning were observed after the interventions, limitations with each were noted. ET decreased naturally during menses, which may provide an ideal opportunity for accessing the targeted vaginal mucosal basal layers to achieve the optimum epithelial thickness for intravaginal mucosal vaccination.

Sex, drugs, and anaesthesia research.

In this issue of the British Journal of Anaesthesia, Joksimovic and colleagues report significant sex differences in sensitivity to the behavioural and neurophysiological effects of 3ß-OH, a novel neurosteroid anesthetic. Female rats were more sensitive to the effects of 3ß-OH than male rats, although the mechanims remain unclear. Sex differences have been understudied in anaesthesia research, and this article by Joksimovic and colleagues emphasizes the need to devote more effort to understanding these differences.

D-Amphetamine Rapidly Reverses Dexmedetomidine-Induced Unconsciousness in Rats.

D-amphetamine induces emergence from sevoflurane and propofol anesthesia in rats. Dexmedetomidine is an α2-adrenoreceptor agonist that is commonly used for procedural sedation, whereas ketamine is an anesthetic that acts primarily by inhibiting NMDA-type glutamate receptors. These drugs have different molecular mechanisms of action from propofol and volatile anesthetics that enhance inhibitory neurotransmission mediated by GABAA receptors. In this study, we tested the hypothesis that d-amphetamine accelerates recovery of consciousness after dexmedetomidine and ketamine. Sixteen rats (Eight males, eight females) were used in a randomized, blinded, crossover experimental design and all drugs were administered intravenously. Six additional rats with pre-implanted electrodes in the prefrontal cortex (PFC) were used to analyze changes in neurophysiology. After dexmedetomidine, d-amphetamine dramatically decreased mean time to emergence compared to saline (saline:112.8 ± 37.2 min; d-amphetamine:1.8 ± 0.6 min, p < 0.0001). This arousal effect was abolished by pre-administration of the D1/D5 dopamine receptor antagonist, SCH-23390. After ketamine, d-amphetamine did not significantly accelerate time to emergence compared to saline (saline:19.7 ± 18.0 min; d-amphetamine:20.3 ± 16.5 min, p = 1.00). Prefrontal cortex local field potential recordings revealed that d-amphetamine broadly decreased spectral power at frequencies <25 Hz and restored an awake-like pattern after dexmedetomidine. However, d-amphetamine did not produce significant spectral changes after ketamine. The duration of unconsciousness was significantly longer in females for both dexmedetomidine and ketamine. In conclusion, d-amphetamine rapidly restores consciousness following dexmedetomidine, but not ketamine. Dexmedetomidine reversal by d-amphetamine is inhibited by SCH-23390, suggesting that the arousal effect is mediated by D1 and/or D5 receptors. These findings suggest that d-amphetamine may be clinically useful as a reversal agent for dexmedetomidine.

The Neural Circuits Underlying General Anesthesia and Sleep.

General anesthesia is characterized by loss of consciousness, amnesia, analgesia, and immobility. Important molecular targets of general anesthetics have been identified, but the neural circuits underlying the discrete end points of general anesthesia remain incompletely understood. General anesthesia and natural sleep share the common feature of reversible unconsciousness, and recent developments in neuroscience have enabled elegant studies that investigate the brain nuclei and neural circuits underlying this important end point. A common approach to measure cortical activity across the brain is electroencephalogram (EEG), which can reflect local neuronal activity as well as connectivity among brain regions. The EEG oscillations observed during general anesthesia depend greatly on the anesthetic agent as well as dosing, and only some resemble those observed during sleep. For example, the EEG oscillations during dexmedetomidine sedation are similar to those of stage 2 nonrapid eye movement (NREM) sleep, but high doses of propofol and ether anesthetics produce burst suppression, a pattern that is never observed during natural sleep. Sleep is primarily driven by withdrawal of subcortical excitation to the cortex, but anesthetics can directly act at both subcortical and cortical targets. While some anesthetics appear to activate specific sleep-active regions to induce unconsciousness, not all sleep-active regions play a significant role in anesthesia. Anesthetics also inhibit cortical neurons, and it is likely that each class of anesthetic drugs produces a distinct combination of subcortical and cortical effects that lead to unconsciousness. Conversely, arousal circuits that promote wakefulness are involved in anesthetic emergence and activating them can induce emergence and accelerate recovery of consciousness. Modern neuroscience techniques that enable the manipulation of specific neural circuits have led to new insights into the neural circuitry underlying general anesthesia and sleep. In the coming years, we will continue to better understand the mechanisms that generate these distinct states of reversible unconsciousness.