Disruption of the anterior commissure in Olig2 deficient mice.

In the present study, we examined neural circuit formation in the forebrain of the Olig2 knockout (Olig2-KO) mouse model and found disruption of the anterior commissure at the late foetal stage. Axon bundles of the anterior commissure encountered the wall of the third ventricle and ceased axonal extension. L1-CAM immunohistochemistry showed that Olig2-KO mice lose decussation formation in the basal forebrain. DiI tracing revealed that the thin bundles of the anterior commissure axons crossed the midline but ceased further extension into the deep part of the contralateral side. Furthermore, some fractions of DiI-labelled axons were oriented dorsolaterally, which was not observed in the control mouse forebrain. The rostral part of the third ventricle was much wider in the Olig2-KO mice than in wild-type mice, which likely resulted in the delay of midline fusion and subsequent delay and malformation of the anterior commissure. We analysed gene expression alterations in the Olig2-KO mice using a public database and found multiple genes, which are related to axon guidance and epithelial-mesenchymal transition, showing subtle expression changes. These results suggest that Olig2 is essential for anterior commissure formation, likely by regulating multiple biological processes.

A Clinical Study of Histopathological and Clinical Image Changes After Sclerotherapy of Lip Venous Malformations.

Venous malformations (VMs) are histopathologically benign but can greatly impair patients' quality of life. Screlothprapy is known to be effective in improving symptoms without a scar, but surgical resection of residual lesions is sometimes necessary due to inadequate reduction. However, there is no consensus on what criteria should be used to consider switching to surgical treatment, and individualized decisions must be made for each case. To investigate the factors that contribute to the lack of efficacy of sclerotherapy in reducing lesions and how to predict this, the authors performed a retrospective clinical imaging and histopathological study of 6 cases of labial vein malformations treated with sclerotherapy and 3 cases without sclerotherapy. Clinical image investigations are based on magnetic resonance imaging before and after sclerotherapy. The authors found a significant decrease in the percentage of cystic components in the total lesion of VMs after sclerotherapy. Histopathological investigations are based on resected VMs with or without sclerotherapy. Elastica van Gieson stains suggested a significant increase in fibrotic tissue inside VMs treated with sclerotherapy compared with those without. In conclusion, magnetic resonance imaging signal changes inside the VMs after sclerotherapy was observed, and it may reflect fibrosis of the tissue. These changes in the VMs after sclerotherapy may reduce the effect of sclerotherapy on tissue reduction should be considered.

Vasodilating Effects of Antispasmodic Agents and Their Cytotoxicity in Vascular Smooth Muscle Cells and Endothelial Cells-Potential Application in Microsurgery.

This study aimed to elucidate the vasodilatory effects and cytotoxicity of various vasodilators used as antispasmodic agents during microsurgical anastomosis. Rat smooth muscle cells (RSMCs) and human coronary artery endothelial cells (HCAECs) were used to investigate the physiological concentrations and cytotoxicity of various vasodilators (lidocaine, papaverine, nitroglycerin, phentolamine, and orciprenaline). Using a wire myograph system, we determined the vasodilatory effects of each drug in rat abdominal aortic sections at the concentration resulting in maximal vasodilation as well as at the surrounding concentrations 10 min after administration. Maximal vasodilation effect 10 min after administration was achieved at the following concentrations: lidocaine, 35 mM; papaverine, 0.18 mM; nitroglycerin, 0.022 mM; phentolamine, 0.11 mM; olprinone, 0.004 mM. The IC50 for lidocaine, papaverine, and nitroglycerin was measured in rat abdominal aortic sections, as well as in RSMCs after 30 min and in HCAECs after 10 min. Phentolamine and olprinone showed no cytotoxicity towards RSMCs or HCAECs. The concentrations of the various drugs required to achieve vasodilation were lower than the reported clinical concentrations. Lidocaine, papaverine, and nitroglycerin showed cytotoxicity, even at lower concentrations than those reported clinically. Phentolamine and olprinone show antispasmodic effects without cytotoxicity, making them useful candidates for local administration as antispasmodics.

Analysis of Orbital Blowout Fracture Location and Hess Area Ratio.

ABSTRACT: This study aimed to analyze the Hess area ratio (HAR%) in cases of blowout fracture treated in our department and clarify the outline of eye movement disorders in blowout fractures. Patients who underwent surgery for orbital blowout fractures in our department were included. Fracture locations were classified into 5 types (A, outside floor; B, C, anterior and posterior floor; and D, E, anterior and posterior medial wall). The HAR% was compared before and after surgery in eligible cases. The relationship between the fracture location and preoperative HAR% was investigated using multiple regression analysis. The study involved 85 patients. Hess area ratio was higher postoperatively than preoperatively (70.75 ±â€Š18.26 versus 90.06 ±â€Š13.99, P  < 0.01). The postoperative HAR% tended to be higher when the iliac bones were compared to other materials; however, this difference was not significant (90.73 ±â€Š12.91 versus 80.30 ±â€Š17.81, P = 0.178). Fracture locations C and E significantly contributed to the prediction of HAR% as negative regression coefficients (P = 0.024 and 0.013, respectively). The posterior fracture area on both the orbital floor and medial wall contributed to the decrease in preoperative HAR%. This observation indicates that the reconstruction of the posterior region is extremely crucial.

Conserved and divergent functions of Pax6 underlie species-specific neurogenic patterns in the developing amniote brain.

The evolution of unique organ structures is associated with changes in conserved developmental programs. However, characterizing the functional conservation and variation of homologous transcription factors (TFs) that dictate species-specific cellular dynamics has remained elusive. Here, we dissect shared and divergent functions of Pax6 during amniote brain development. Comparative functional analyses revealed that the neurogenic function of Pax6 is highly conserved in the developing mouse and chick pallium, whereas stage-specific binary functions of Pax6 in neurogenesis are unique to mouse neuronal progenitors, consistent with Pax6-dependent temporal regulation of Notch signaling. Furthermore, we identified that Pax6-dependent enhancer activity of Dbx1 is extensively conserved between mammals and chick, although Dbx1 expression in the developing pallium is highly divergent in these species. Our results suggest that spatiotemporal changes in Pax6-dependent regulatory programs contributed to species-specific neurogenic patterns in mammalian and avian lineages, which underlie the morphological divergence of the amniote pallial architectures.

Pineal gland differentiation in mature teratoma: An under-recognized condition and potential pitfalls for overdiagnosis.

We herein report three cases of mature teratomas with pineal gland differentiation, which is a less recognized phenomenon. Case 1 was a 6-year-old male with a neck mass, Case 2 was a 23-year-old female with a retroperitoneal mass, and Case 3 was a 45-year-old female with a retroperitoneal mass. Each case showed the typical macroscopic and histological findings of mature teratoma, such as solid and cystic lesions mainly lined with a mature squamous epithelium. All cases also showed glial differentiation. Small foci of lobulated cell nests were detected in the center of or adjacent to mature glial tissue. Cells had a clear to pale eosinophilic cytoplasm with small round nuclei. Immunohistochemically, cells were positive for synaptophysin, neurofilament protein with a perivascular "club-shaped swelling" pattern, and cone-rod homeobox protein. To the best of our knowledge, this is the first report of pineal gland differentiation arising in mature teratoma, which may be easily overlooked or misdiagnosed as somatic-type tumors, particularly neuroendocrine tumors. To avoid overtreatment, pathologists need to be aware that pineal gland differentiation may occur in mature teratomas.

Wire Myography for Continuous Estimation of the Optimal Concentration of Topical Lidocaine as a Vasodilator in Microsurgery.

BACKGROUND: Intraoperative vasospasm during reconstructive microvascular surgery is often unpredictable and may lead to devastating flap loss. Therefore, various vasodilators are used in reconstructive microsurgery to prevent and relieve vasospasm. Lidocaine is a vasodilator commonly used in microvascular surgery. Although many reports have described its in vitro and in vivo concentration-dependent vasodilatory effects, limited studies have examined the pharmacological effects of lidocaine on blood vessels in terms of persistence and titer. METHODS: In this study, the vasodilatory effect of lidocaine was examined by using the wire myograph system. Abdominal aortas were harvested from female rats, sliced into rings of 1-mm thickness, and mounted in the wire myograph system. Next, 10, 5, 2, and 1% lidocaine solutions were applied to the artery, and the change in vasodilation force, persistence of the force, and time required to reach equilibrium were measured. RESULTS: The vasodilatory effect was confirmed in all groups following lidocaine treatment. Although strong vasodilation was observed in the 10% lidocaine group, it was accompanied by irreversible degeneration of the artery. Vasodilation in the 1% lidocaine group was weaker than that in the other groups 500 seconds after lidocaine addition (p < 0.05). Between the 5 and 2% lidocaine groups, 5% lidocaine showed a stronger vasodilatory effect 400 to 600 seconds after lidocaine addition (p < 0.01); however, there was no significant difference in these groups after 700 seconds. Additionally, there was no difference in the time required for the relaxation force to reach equilibrium among the 5, 2, and 1% lidocaine groups. CONCLUSION: Although our study confirmed the dose-dependent vasodilatory effect of lidocaine, 5% lidocaine showed the best vasodilatory effect and continuity with minimal irreversible changes in the arterial tissue.

Use of dermal fat grafts for treating anetoderma with lipoatrophy following involution of hemangiomas.

Infantile hemangioma (IH) is a benign vascular tumor that gradually involutes over several years. Rapidly involuting congenital hemangioma (RICH) is the relatively rare congenital vascular tumor that is fully grown at birth and does not undergo postnatal growth and involutes during the first year. However, after involution of both IH and RICH, some have severe sequelae, such as redundant skin or conspicuous scarring, requiring additional treatment. We present the case of a 6-year-old girl with a concave deformity due to subcutaneous atrophy, skin darkening, and altered skin texture of her left zygomatic region following involution of a hemangioma. We successfully treated this patient by transferring a dermal fat graft. This technique can be beneficial for atrophic sequelae after regression of a hemangioma and is easy to perform and cosmetically effective.

Analysis of Orbital Morphology and its Relationship With Eyelid Morphology.

Correct anatomical reconstruction of the orbital wall for function and cosmesis is important; however, this is difficult because of the structure's complexity. The authors aimed to analyze and classify orbital morphology from computed tomography (CT) images and examine the relationship between orbital morphology and eyelid morphology in the Japanese population. CT images of 60 men (right side, 29; left side, 31) and 44 women (each side, 22) were included. The lengths of the orbital medial wall and floor in the coronal plane at the anterior, middle, and posterior planes of the orbit; angle between them; simotic index; and the thickness of upper eyelid were measured. Additionally, the presence or absence of double eyelids was evaluated. Non-paired Student's t test and Pearson correlation coefficient test were used for analysis. Orbital morphology was symmetrical on both sides, and men had a larger orbit than women. Orbital morphology was classified into 2 groups according to the posterior angle, and there was a difference between the groups in the simotic index. The difference between groups may represent a genetic difference between the Jomon and Yayoi people and not only provide a new classification for the orbit of the population but also be useful in orbital reconstruction.

The evolution of basal progenitors in the developing non-mammalian brain.

The amplification of distinct neural stem/progenitor cell subtypes during embryogenesis is essential for the intricate brain structures present in various vertebrate species. For example, in both mammals and birds, proliferative neuronal progenitors transiently appear on the basal side of the ventricular zone of the telencephalon (basal progenitors), where they contribute to the enlargement of the neocortex and its homologous structures. In placental mammals, this proliferative cell population can be subdivided into several groups that include Tbr2(+) intermediate progenitors and basal radial glial cells (bRGs). Here, we report that basal progenitors in the developing avian pallium show unique morphological and molecular characteristics that resemble the characteristics of bRGs, a progenitor population that is abundant in gyrencephalic mammalian neocortex. Manipulation of LGN (Leu-Gly-Asn repeat-enriched protein) and Cdk4/cyclin D1, both essential regulators of neural progenitor dynamics, revealed that basal progenitors and Tbr2(+) cells are distinct cell lineages in the developing avian telencephalon. Furthermore, we identified a small population of subapical mitotic cells in the developing brains of a wide variety of amniotes and amphibians. Our results suggest that unique progenitor subtypes are amplified in mammalian and avian lineages by modifying common mechanisms of neural stem/progenitor regulation during amniote brain evolution.

NG2 expression in NG2 glia is regulated by binding of SoxE and bHLH transcription factors to a Cspg4 intronic enhancer.

NG2 is a type 1 integral membrane glycoprotein encoded by the Cspg4 gene. It is expressed on glial progenitor cells known as NG2 glial cells or oligodendrocyte precursor cells that exist widely throughout the developing and mature central nervous system and vascular mural cells but not on mature oligodendrocytes, astrocytes, microglia, neurons, or neural stem cells. Hence NG2 is widely used as a marker for NG2 glia in the rodent and human. The regulatory elements of the mouse Cspg4 gene and its flanking sequences have been used successfully to target reporter and Cre recombinase to NG2 glia in transgenic mice when used in a large 200 kb bacterial artificial chromosome cassette containing the 38 kb Cspg4 gene in the center. Despite the tightly regulated cell type- and stage-specific expression of NG2 in the brain and spinal cord, the mechanisms that regulate its transcription have remained unknown. Here, we describe a 1.45 kb intronic enhancer of the mouse Cspg4 gene that directed transcription of EGFP reporter to NG2 glia but not to pericytes in vitro and in transgenic mice. The 1.45 kb enhancer contained binding sites for SoxE and basic helix-loop-helix transcription factors, and its enhancer activity was augmented cooperatively by these factors, whose respective binding elements were found in close proximity to each other. Mutations in these binding elements abrogated the enhancer activity when tested in the postnatal mouse brain.

The Future Vocation of Neural Stem Cells: Lineage Commitment in Brain Development and Evolution.

Understanding the fate commitment of neural stem cells is critical to identify the regulatory mechanisms in developing brains. Genetic lineage-tracing has provided a powerful strategy to unveil the heterogeneous nature of stem cells and their descendants. However, recent studies have reported controversial data regarding the heterogeneity of neural stem cells in the developing mouse neocortex, which prevents a decisive conclusion on this issue. Here, we review the progress that has been made using lineage-tracing analyses of the developing neocortex and discuss stem cell heterogeneity from the viewpoint of comparative and evolutionary biology.

Origin of Oligodendrocytes in the Vertebrate Optic Nerve: A Review.

One of the unsolved problems in the research field of oligodendrocyte (OL) development has been the site(s) of origin of optic nerve OLs and its precursor cells (OPCs). It is generally accepted that OLs in the optic nerve are derived from the brain, and thus optic nerve OLs are immigrant cells. We previously demonstrated the brain origin of optic nerve OPCs in chick embryos. However, the site of optic nerve OPC origin has not been examined experimentally in developing rodents for the past two decades. We have recently reported that optic nerve OPCs in mice arise in the preoptic area by E12.5 and gradually migrate caudally and enter the optic nerve. These OPCs give rise to myelinating OLs in the optic nerve in the postnatal or adult stages. Surprisingly, there are species differences with respect to the origin of optic nerve OPCs between chicks and mice. Here, we summarize the site of OPC origin in the optic nerve based on our own previous and recent results, and discuss possible mechanisms underlying these species differences.

Development of the prethalamus is crucial for thalamocortical projection formation and is regulated by Olig2.

Thalamocortical axons (TCAs) pass through the prethalamus in the first step of their neural circuit formation. Although it has been supposed that the prethalamus is an intermediate target for thalamocortical projection formation, much less is known about the molecular mechanisms of this targeting. Here, we demonstrated the functional implications of the prethalamus in the formation of this neural circuit. We show that Olig2 transcription factor, which is expressed in the ventricular zone (VZ) of prosomere 3, regulates prethalamus formation, and loss of Olig2 results in reduced prethalamus size in early development, which is accompanied by expansion of the thalamic eminence (TE). Extension of TCAs is disorganized in the Olig2-KO dorsal thalamus, and initial elongation of TCAs is retarded in the Olig2-KO forebrain. Microarray analysis demonstrated upregulation of several axon guidance molecules, including Epha3 and Epha5, in the Olig2-KO basal forebrain. In situ hybridization showed that the prethalamus in the wild type excluded the expression of Epha3 and Epha5, whereas loss of Olig2 resulted in reduction of this Ephas-negative area and the corresponding expansion of the Ephas-positive TE. Dissociated cultures of thalamic progenitor cells demonstrated that substrate-bound EphA3 suppresses neurite extension from dorsal thalamic neurons. These results indicate that Olig2 is involved in correct formation of the prethalamus, which leads to exclusion of the EphA3-expressing region and is crucial for proper TCA formation. Our observation is the first report showing the molecular mechanisms underlying how the prethalamus acts on initial thalamocortical projection formation.

Avian brains: Insights from development, behaviors and evolution.

Birds are an extensively specialized animal group with unique anatomical, physiological and ecological characteristics. Sophisticated social behaviors and remarkable cognitive abilities are present in several avian lineages, driven by their enlarged brains and intricate neural networks. These unique traits could be a result of adaptive evolution under the wide range of environmental constraints; however, the intrinsic mechanisms of avian brain development and evolution remain unclear. Here, we introduce recent findings regarding developmental aspects of avian brain organization and neuronal networks for specific avian behaviors, which provide an insight into the link between the evolution of brain development and complex cognitive functions.

Three-dimensional venous visualization with phase-lag computed tomography angiography for reconstructive microsurgery.

BACKGROUND: Most free flap reconstruction complications involve vascular compromise. Evaluation of vascular anatomy provides considerable information that can potentially minimize these complications. Previous reports have shown that contrast-enhanced computed tomography is effective for understanding three-dimensional arterial anatomy. However, most vascular complications result from venous thromboses, making imaging of venous anatomy highly desirable. METHODS: The phase-lag computed tomography angiography (pl-CTA) technique involves 64-channel (virtually, 128-channel) multidetector CT and is used to acquire arterial images using conventional CTA. Venous images are three-dimensionally reconstructed using a subtraction technique involving combined venous phase and arterial phase images, using a computer workstation. RESULTS: This technique was used to examine 48 patients (12 lower leg reconstructions, 34 head and neck reconstructions, and 2 upper extremity reconstructions) without complications. The pl-CTA technique can be used for three-dimensional visualization of peripheral veins measuring approximately 1 mm in diameter. CONCLUSION: The pl-CTA information was especially helpful for secondary free flap reconstructions in the head and neck region after malignant tumor recurrence. In such cases, radical dissection of the neck was performed as part of the first operation, and many vessels, including veins, were resected and used in the first free-tissue transfer. The pl-CTA images also allowed visualization of varicose changes in the lower leg region and helped us avoid selecting those vessels for anastomosis. Thus, the pl-CTA-derived venous anatomy information was useful for exact evaluations during the planning of free-tissue transfers.

Comparative gene expression analyses reveal heterochrony for Sox9 expression in the cranial neural crest during marsupial development.

Compared to placental mammals, marsupials have short gestation period, and their neonates are relatively immature. Despite these features, marsupial neonates must travel from the birth canal to the teat, suckle and digest milk to complete development. Thus, certain organs and tissues of marsupial neonates, such as forelimbs to crawl and jaw elements to suckle, must develop early. Previous reports showed that cranial neural crest (CNC) cells, as the source of ectomesenchyme of jaw elements, are generated significantly early in gray short-tailed opossum (Monodelphis domestica) compared to other amniote models, such as mouse. In this study, we examined the expression of genes known to be important for neural crest formation, such as BMP2/BMP4 (neural crest inducer), Pax7 (neural border specifier), Snail1 and Sox9/Sox10 (neural crest specifier) in Monodelphis domestica, and compared the expression patterns with those in mouse, chicken, and gecko embryos. Among those genes, the expression of Sox9 was turned on early and broadly in the premigratory CNC cells, and persisted in the ectomesenchyme of the cranial anlagen in opossum embryos. In contrast, Sox9 expression diminished in the CNC cells of other animals at the early phase of migration. Comparison of the onset of Pax7 and Sox9 expression revealed that Sox9 expression in the prospective CNC was earlier and broader than Pax7 expression in opossum, suggesting that the sequence of border specification and neural crest specification is altered. This study provides the first clue for understanding the molecular basis for the heterochronic development of the CNC cells and jaw elements in marsupials.

Glycogenolysis-Induced Astrocytic Serping1 Expression Regulates Neuroinflammatory Effects on Hippocampal neuron.

The bacterial pathogen, lipopolysaccharide (LPS), elicits microglial response and induces cytokine secretion that subsequently activates astrocytes. Recent findings have indicated that LPS-induced activation of postnatal glial cells has led to alterations in synapse formation in hippocampal and cortical neurons, thereby resulting in a prolonged increased risk for seizure or depression. Nevertheless, its mechanisms remain to be fully elucidated. Cellular metabolism has recently gained recognition as a critical regulatory mechanism for the activation of peripheral immune cells, as it supplies the requisite energy and metabolite for their activation. In the present study, we report that LPS did not change the expression of reported astrocyte-derived synaptogenic genes in the postnatal hippocampus; however, it induced upregulation of astrocytic complement component regulator Serping1 within the postnatal hippocampus. As a regulatory mechanism, activation of glycogen degradation (glycogenolysis) governs the expression of a subset of inflammatory-responsive genes including Serping1 through reactive oxygen species (ROS)-NF-κB axis. Our study further demonstrated that glycogenolysis is implicated in neurotoxic phenotypes of astrocytes, such as impaired neuronal synaptogenesis or cellular toxicity. These findings suggested that activation of glycogenolysis in postnatal astrocytes is an essential metabolic pathway for inducing responses in inflammatory astrocytes.

Oncological Outcomes of Concurrent Chemoradiotherapy and Surgical Treatment for Patients With T3 Hypopharyngeal Cancer: A Single-Center Retrospective Analysis.

Background Since the larynx and pharynx are vital for respiration, swallowing, and speech, chemoradiotherapy (CRT) has been widely applied for T3 hypopharyngeal cancer (HPC) as an organ-preserving treatment. However, CRT can lead to severe late adverse events such as dysphagia and aspiration pneumonia, especially in patients who have difficulty swallowing and/or aspiration at the time of initial diagnosis. Patients and methods Between 2012 and 2020, 86 patients with T3 HPC treated with curative intent at Kobe University Hospital were included in this study. The average age was 69 years old, ranging from 43 to 89. Diseases were classified as Stage III in 29 patients, Stage IVA in 52 patients, and Stage IVB in five patients. Thirty-five (41%) patients were treated by CRT, and 51 (59%) patients were treated by surgery. Patients were followed up for at least two years, and the follow-up period ranged from four to 128 months (median: 45 months). Results Three-year progression-free survival (PFS) rates of patients treated by CRT and patients treated by surgery were 56.2% and 60.3%, respectively. Three-year disease-specific survival (DSS) rates of patients treated by CRT and surgically treated patients were 79.0% vs. 70.8%, respectively. Three-year overall survival (OS) rates of patients treated by CRT and surgically treated patients were 64.5% and 69.0%, respectively. Of note, a significant difference was observed between three-year DSS and three-year PFS (79.0% vs. 56.2%, p = 0.0014) in the patients treated by CRT but not in the patients treated by surgery. Conclusions No significant differences were observed between the PFS, DSS, and OS rates of patients treated by CRT and those of surgically treated patients. Locoregional recurrences after CRT were significantly successfully salvaged by surgical intervention. These results suggest that CRT can be applied as an alternative to surgery without reducing survival, especially for patients without severe clinical symptoms. Meticulous follow-up is mandatory for early detection of recurrence to salvage by surgery and for the management of late adverse events.

The Prospective Natural History Study of Patients with Intractable Venous Malformation and Klippel-Trenaunay Syndrome to Guide Designing a Proof-of-Concept Clinical Trial for Novel Therapeutic Intervention.

Background: The natural history of venous malformation (VM) and Klippel-Trenaunay Syndrome (KTS) has not been quantitatively studied. To obtain benchmarks to guide designing clinical trials to assess safety and efficacy of novel drug candidates, the clinical course of the patients was followed for 6 months. Methods and Results: This is a multicenter prospective observational study evaluating the change rate in lesion volume from baseline with magnetic resonance images, as the primary endpoint. In addition, disease severities, performance status (PS), pain visual analog scale (VAS) score, quality of life (QoL), infections, and coagulation markers were also evaluated. Thirty-four patients (VM = 17, KTS = 17, 1-53 of age; median 15.9 years) with measurable lesion volume were analyzed. There was no statistically significant difference in the lesion volume between baseline and day 180, and the mean change rate (standard deviation) was 1.06 (0.28). There were no baseline characteristics that affected the change in lesion volume over 6 months. However, there were patients who showed more than 20% volume change and it was suggested that the lesion volume was largely impacted by local infection. There were no statistically significant changes in pain VAS score, severity, PS, QoL score, D-dimer, and platelet count over 6 months within all patients analyzed. Conclusion: The results showed the representative natural course of VM and KTS for a 6-month period with objective change of lesion volume and other factors, suggesting that it is scientifically reasonable to conduct a Phase 2 proof-of-concept study without a placebo arm, using the results of this study as the control. Clinical Trial Registration: NCT04285723, NCT04589650.