Understanding Immune-Driven Brain Aging by Human Brain Organoid Microphysiological Analysis Platform.

The aging of the immune system drives systemic aging and the pathogenesis of age-related diseases. However, a significant knowledge gap remains in understanding immune-driven aging, especially in brain aging, due to the limited current in vitro models of neuroimmune interaction. Here, the authors report the development of a human brain organoid microphysiological analysis platform (MAP) to discover the dynamic process of immune-driven brain aging. The organoid MAP is created by 3D printing that confines organoid growth and facilitates cell and nutrition perfusion, promoting organoid maturation and their committment to forebrain identity. Dynamic rocking flow is incorporated into the platform that allows to perfuse primary monocytes from young (20 to 30-year-old) and aged (>60-year-old) donors and culture human cortical organoids to model neuroimmune interaction. The authors find that the aged monocytes increase infiltration and promote the expression of aging-related markers (e.g., higher expression of p16) within the human cortical organoids, indicating that aged monocytes may drive brain aging. The authors believe that the organoid MAP may provide promising solutions for basic research and translational applications in aging, neural immunological diseases, autoimmune disorders, and cancer.

Intelligent acoustofluidics enabled mini-bioreactors for human brain organoids.

Acoustofluidics, by combining acoustics and microfluidics, provides a unique means to manipulate cells and liquids for broad applications in biomedical sciences and translational medicine. However, it is challenging to standardize and maintain excellent performance of current acoustofluidic devices and systems due to a multiplicity of factors including device-to-device variation, manual operation, environmental factors, sample variability, etc. Herein, to address these challenges, we propose "intelligent acoustofluidics" - an automated system that involves acoustofluidic device design, sensor fusion, and intelligent controller integration. As a proof-of-concept, we developed intelligent acoustofluidics based mini-bioreactors for human brain organoid culture. Our mini-bioreactors consist of three components: (1) rotors for contact-free rotation via an acoustic spiral phase vortex approach, (2) a camera for real-time tracking of rotational actions, and (3) a reinforcement learning-based controller for closed-loop regulation of rotational manipulation. After training the reinforcement learning-based controller in simulation and experimental environments, our mini-bioreactors can achieve the automated rotation of rotors in well-plates. Importantly, our mini-bioreactors can enable excellent control over rotational mode, direction, and speed of rotors, regardless of fluctuations of rotor weight, liquid volume, and operating temperature. Moreover, we demonstrated our mini-bioreactors can stably maintain the rotational speed of organoids during long-term culture, and enhance neural differentiation and uniformity of organoids. Comparing with current acoustofluidics, our intelligent system has a superior performance in terms of automation, robustness, and accuracy, highlighting the potential of novel intelligent systems in microfluidic experimentation.

Tubular human brain organoids to model microglia-mediated neuroinflammation.

Human brain organoids, 3D brain tissue cultures derived from human pluripotent stem cells, hold promising potential in modeling neuroinflammation for a variety of neurological diseases. However, challenges remain in generating standardized human brain organoids that can recapitulate key physiological features of a human brain. Here, we present tubular organoid-on-a-chip devices to generate better organoids and model neuroinflammation. By employing 3D printed hollow mesh scaffolds, our device can be easily incorporated into multiwell-plates for reliable, scalable, and reproducible generation of tubular organoids. By introducing rocking flows through the tubular device channel, our device can perfuse nutrients and oxygen to minimize organoid necrosis and hypoxia, and incorporate immune cells into organoids to model neuro-immune interactions. Compared with conventional protocols, our method increased neural progenitor proliferation and reduced heterogeneity of human brain organoids. As a proof-of-concept application, we applied this method to model the microglia-mediated neuroinflammation after exposure to an opioid receptor agonist. We found isogenic microglia were activated after exposure to an opioid receptor agonist (DAMGO) and transformed back to the homeostatic status with further treatment by a cannabinoid receptor 2 (CB2) agonist (LY2828360). Importantly, the activated microglia in tubular organoids had stronger cytokine responses compared to those in 2D microglial cultures. Our tubular organoid device is simple, versatile, inexpensive, easy-to-use, and compatible with multiwell-plates, so it can be widely used in common research and clinical laboratory settings. This technology can be broadly used for basic and translational applications in inflammatory diseases including substance use disorders, neural diseases, autoimmune disorders, and infectious diseases.

Beneficial effect of diosgenin as a stimulator of NGF on the brain with neuronal damage induced by Aß-42 accumulation and neurotoxicant injection.

To investigate the beneficial effects of diosgenin (DG) on the multiple types of brain damage induced by Aß-42 peptides and neurotoxicants, alterations in the specific aspects of brain functions were measured in trimethyltin (TMT)-injected transgenic 2576 (TG) mice that had been pretreated with DG for 21 days. Multiple types of damage were successfully induced by Aß-42 accumulation and TMT injection into the brains of TG mice. However, DG treatment significantly reduced the number of Aß-stained plaques and dead cells in the granule cells layer of the dentate gyrus. Significant suppression of acetylcholinesterase (AChE) activity and Bax/Bcl-2 expression was also observed in the DG treated TG mice (TG+DG group) when compared with those of the vehicle (VC) treated TG mice (TG+VC group). Additionally, the concentration of nerve growth factor (NGF) was dramatically enhanced in TG+DG group, although it was lower in the TG+VC group than the non-transgenic (nTG) group. Furthermore, the decreased phosphorylation of downstream members in the TrkA high affinity receptor signaling pathway in the TG+VC group was significantly recovered in the TG+DG group. A similar pattern was observed in p75(NTR) expression and JNK phosphorylation in the NGF low affinity receptor signaling pathway. Moreover, superoxide dismutase (SOD) activity was enhanced in the TG+DG group, while the level of malondialdehyde (MDA), a marker of lipid peroxidation, was lower in the TG+DG group than the TG+VC group. These results suggest that DG could exert a wide range of beneficial activities for multiple types of brain damage through stimulation of NGF biosynthesis.

Hepatotoxicity and nephrotoxicity of gallotannin-enriched extract isolated from Galla Rhois in ICR mice.

To evaluate the hepatotoxicity and nephrotoxicity of Galla Rhois (GR) toward the liver and kidney of ICR mice, alterations in related markers including body weight, organ weight, urine composition, liver pathology and kidney pathology were analyzed after oral administration of 250, 500 and 1,000 mg/kg body weight/day of gallotannin-enriched extract of GR (GEGR) for 14 days. GEGR contained 68.7±2.5% of gallotannin, 25.3±0.9% of gallic acid and 4.4±0.1% of methyl gallate. Also, the level of malondialdehyde (MDA), a marker of lipid peroxidation, was decreased with 19% in the serum of high dose GEGR (HGEGR)-treated mice. The body and organ weight, clinical phenotypes, urine parameters and mice mortality did not differ among GEGR-treated groups and the vehicle-treated group. Furthermore, no significant increase was observed in alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN) and the serum creatinine (Cr) in the GEGR-treated group relative to the vehicle-treated group. Moreover, the specific pathological features induced by most toxic compounds such as CCl4 were not observed upon liver and kidney histological analysis. Overall, the results of the present study suggest that GEGR does not induce any specific toxicity in liver and kidney organs of ICR at doses of 1,000 mg/kg body weight/day, indicating that this is no observed adverse effect level (NOAEL).

Toxicity of antioxidative extract collected from Styela clava tunics in ICR mice.

Some polymers and bioactive compounds derived from Styela clava tunic (SCT) have been reported as traditional medicine for the treatment of inflammation, oxidative stress and surgical wounds although there is little scientific evidence of their liver and kidney toxicity. To investigate the toxicity of ethanol extracts of SCT (EtSCT) in the liver and kidney of ICR mice, alterations in related markers including body weight, organ weight, urine composition, liver pathology and kidney pathology were analyzed following oral administration of 50 and 100 mg/kg body weight/day of EtSCT for 14 days. EtSCT showed a high level of free radical scavenging activity for DPPH (93.1%) and NO (16.2%) as well as the presence of 14.8 mg/mL of flavonoids and 36.2 mg/mL of phenolics, while EtSCT treated groups did not show any significant alterations in the body and organ weight, clinical phenotypes, urine parameters or mice mortality when compared with the vehicle treated group. In addition, constant levels of serum biochemical markers including alanine phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and serum creatinine (CRE) were maintained. Moreover, no specific histopathological features induced by most toxic compounds were observed in liver and kidney sections stained with hematoxilin and eosin. Therefore, the present results indicate that EtSCT with strong antioxidant activity cannot induce any specific toxicity in liver and kidney organs of ICR at doses of 100 mg/kg body weight/day.

Beneficial effects of ethanol extracts of Red Liriope platyphylla on vascular dysfunction in the aorta of spontaneously hypertensive rats.

Some biological effects of Red Liriope platyphylla (RLP) on various chronic diseases including Alzheimer's disease, diabetes and obesity were suggested after a report of the production from Liriope platyphylla (L. platyphylla, LP) roots using a steaming process. To examine the beneficial effects of ethanol extracts RLP (EEtRLP) on the vascular dysfunction of hypertension, alterations in key factors related to vascular regulation and antioxidant conditions were investigated in spontaneously hypertensive rats (SHR) after EEtRLP treatment for 2 weeks. High levels of 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity were detected in 500 or 1,000 mg/mL EEtRLP. Although no significant improvement of systolic blood pressure or aortic wall thickness were observed in the EEtRLP treated group, the expression level of angiotensin converting enzyme (ACE) and ACE2 increased significantly after EEtRLP treatment. Moreover, the concentration of aldosterone and K ion in serum rapidly recovered in the EEtRLP treated group relative to the vehicle treated group. Furthermore, the endothelial nitric oxide synthase (eNOS) expression and superoxide dismutase (SOD) activity were significantly increased in the EEtRLP treated group relative to the vehicle treated group, while the level of malondialdehyde (MDA) and NOx in the serum of the same group were recovered to the level of Wistar Kyoto (WKY) rats. Overall, the results presented herein provide novel evidence that EEtRLP treatment may improve vascular dysfunction in the aorta of the SHR through up regulation of the antioxidant state and down regulation of aldosterone and K ion concentration. These results also suggest that EEtRLP may be a potential candidate for treatment of various chronic diseases showing vascular dysfunction.

Characterization of allergic response induced by repeated dermal exposure of IL-4/Luc/CNS-1 transgenic mice to low dose formaldehyde.

Although formaldehyde (FA) is known to be a major allergen responsible for allergic contact dermatitis, there are conflicting reports regarding correlation between FA exposure and interleukin (IL-4) expression. To investigate whether allergic responses including IL-4 expression were induced by repeated dermal exposure to low dose FA, alterations in the luciferase signal and allergic phenotypes were measured in IL-4/Luc/CNS-1 transgenic (Tg) mice containing luciferase cDNA under control of the IL-4 promoter after exposure to 4% FA for 2 weeks. High levels of luciferase were detected in the abdominal region of the whole body and submandibular lymph node (SLN) of FA treated mice. Additionally, the ear thickness and IgE concentration were significantly upregulated in the FA treated group when compared with the acetone olive oil (AOO) treated group. FA treated mice showed enhanced auricular lymph node (ALN) weight, epidermis and dermis thickness, and infiltration of inflammatory cells. Furthermore, the expression of IL-6 among T helper 2 cytokines was higher in the FA treated group than the AOO treated group, while vascular endothelial growth factor (VEGF) levels remained constant. Overall, the results presented herein provide additional evidence that various allergic responses may be successfully induced in IL-4/Luc/CNS-1 Tg mice after exposure to low dose FA for 2 weeks. The luciferase signal under the IL-4 promoter may reflect general indicators of the allergic response induced by exposure to low dose FA.

Metabolomics approach to serum biomarker for loperamide-induced constipation in SD rats.

Loperamide has long been known as an opioid-receptor agonist useful as a drug for treatment of diarrhea resulting from gastroenteritis or inflammatory bowel disease as well as to induce constipation. To determine and characterize putative biomarkers that can predict constipation induced by loperamide treatment, alteration of endogenous metabolites was measured in the serum of Sprague Dawley (SD) rats treated with loperamide for 3 days using (1)H nuclear magnetic resonance ((1)H NMR) spectral data. The amounts and weights of stool and urine excretion were significantly lower in the loperamide-treated group than the No-treated group, while the thickness of the villus, crypt layer, and muscle layer was decreased in the transverse colon of the same group. The concentrations of aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatinine (Cr) were also slightly changed in the loperamide-treated group, although most of the serum components were maintained at a constant level. Furthermore, pattern recognition of endogenous metabolites showed completely separate clustering of the serum analysis parameters between the No-treated group and loperamide-treated group. Among 35 endogenous metabolites, four amino acids (alanine, glutamate, glutamine and glycine) and six endogenous metabolites (acetate, glucose, glycerol, lactate, succinate and taurine) were dramatically decreased in loperamide-treated SD rats. These results provide the first data pertaining to metabolic changes in SD rats with loperamide-induced constipation. Additionally, these findings correlate the changes in 10 metabolites with constipation.