Fabrication and Characterization of Dissolving Microneedles for Transdermal Drug Delivery of Apomorphine Hydrochloride in Parkinson's Disease.

PURPOSE: We fabricated and characterized polyvinyl alcohol (PVA)-based dissolving microneedles (MNs) for transdermal drug delivery of apomorphine hydrochloride (APO), which is used in treating the wearing-off phenomenon observed in Parkinson's disease. METHODS: We fabricated MN arrays with 11 × 11 needles of four different lengths (300, 600, 900, and 1200 µm) by micromolding. The APO-loaded dissolving MNs were characterized in terms of their physicochemical and functional properties. We also compared the pharmacokinetic parameters after drug administration using MNs with those after subcutaneous injection by analyzing the blood concentration of APO in rats. RESULTS: PVA-based dissolving MNs longer than 600 µm could effectively puncture the stratum corneum of the rat skin with penetrability of approximately one-third of the needle length. Although APO is known to have chemical stability issues in aqueous solutions, the drug content in APO-loaded MNs was retained at 25°C for 12 weeks. The concentration of APO after the administration of APO-loaded 600-µm MNs that dissolved completely in skin within 60 min was 81%. The absorption of 200-µg APO delivered by MNs showed a Tmax of 20 min, Cmax of 76 ng/mL, and AUC0-120 min of 2,829 ng・min/mL, compared with a Tmax of 5 min, Cmax of 126 ng/mL, and AUC0-120 min of 3,224 ng・min/mL for subcutaneous injection. The bioavailability in terms of AUC0-120 min of APO delivered by MNs was 88%. CONCLUSION: APO-loaded dissolving MNs can deliver APO via skin into the systemic circulation with rapid absorption and high bioavailability.

Immunohistochemical analysis for acetylcholinesterase and choline acetyltransferase in mouse cerebral cortex after traumatic brain injury.

The regulation of glial cells, especially astrocytes and microglia, is important to prevent the exacerbation of a brain injury because over-reactive glial cells promote neuronal death. Acetylcholine (ACh), a neurotransmitter synthesized and hydrolyzed by choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), respectively, in the central nervous system, has the potential to regulate glial cells' states, i.e., non-reactive and reactive states. However, the expression levels of these ACh-related enzymes in areas containing reactive glial cells are unclear. Herein we immunohistochemically investigated the distributions of AChE and ChAT with reactive glial cells in the cryo-injured brain of mice as a traumatic brain injury model. Immunohistochemistry revealed AChE- and ChAT-immunopositive signals in injured areas at 7 days post-injury. The signals were observed in and around glial fibrillary acidic protein (GFAP)- or CD68-immunopositive cells, and the numbers of cells doubly positive for GFAP/AChE, GFAP/ChAT, CD68/AChE, and CD68/ChAT were significantly increased in injured areas compared to sham-operated areas. Enzyme histochemistry for AChE showed intensely positive signals in injured areas. These results suggest that reactive astrocytes and microglia express and secrete AChE and ChAT in brain-injury areas. These glial cells may adjust the ACh concentration around themselves through the regulation of the expression of ACh-related enzymes in order to control their reactive states.

New decolorization method produces more information from tissue sections stained with hematoxylin and eosin stain and masson-trichrome stain.

Histological analysis is a fundamental and principal method used in biological research and even for disease diagnosis. The result shows the status of cells and tissues in organs and enables us to infer the condition of the whole body. The tissue staining method known as hematoxylin and eosin staining (HE) is one of the most general methods of investigating the status of cells and tissues. Hematoxylin stains the nucleus violet and eosin stains cytosol pink. HE staining shows the unique morphologies of tissues and cells. However, after being stained with HE, tissues are very difficult to use in another histological analysis because hematoxylin is hard to remove from the sections due to its stain stability. Therefore, serial sections of the tissue are used to obtain more information through another staining, including immunohistochemistry. The adjacent tissue section is not the same as the HE-stained section, however, so the results from the adjacent sections can cause confusion or ambiguity. The present study showed that our decolorization solution can decolor the hematoxylin or iron hematoxylin stain from stained structures, including the nucleus, and the decolored section could be stained again in another staining, including immunohistochemistry. This decolorization method is very valuable, in that it can determine the accurate distribution of substances and features in cells and tissues, and thus it can improve the robustness of the resulting data.

Acetylcholine suppresses the increase of glia fibrillary acidic protein expression via acetylcholine receptors in cAMP-induced astrocytic differentiation of rat C6 glioma cells.

Astrocytes, the most common glial cells in the central nervous system, maintain neuronal functions and have roles in neurological diseases. Acetylcholine (ACh) is one of the most essential neurotransmitters, and ACh receptor (AChR) ligands were recently reported to influence astrocyte functions. However, the functions of ACh, the only endogenous agonist of AChR, in astrocytogenesis and in the expression of astrocytic marker genes have not been known. We previously demonstrated that the inhibition of acetylcholine esterase (AChE) suppressed the differentiation of rat glioma C6 cells, an astrocyte differentiation model, and we observed a suppressive effect of ACh agonists on astrocyte differentiation. Our present study revealed that in the cAMP-induced differentiation of C6 cells, an AChR antagonist alleviated the expression of glia fibrillary acidic protein (GFAP) that had been suppressed by dichlorvos (DDVP), an organophosphate and an AChE inhibitor. Our findings also demonstrated a direct effect of ACh on the GFAP expression, and that muscarinic AChR is involved in the suppressive effect of ACh on the GFAP expression in differentiation-induced C6 cells. This is the first report indicating that ACh the only endogenous agonist for AChRs functions as a mediator of astrocyte differentiation.

Embryonic cholecystitis and defective gallbladder contraction in the Sox17-haploinsufficient mouse model of biliary atresia.

The gallbladder excretes cytotoxic bile acids into the duodenum through the cystic duct and common bile duct system. Sox17 haploinsufficiency causes biliary atresia-like phenotypes and hepatitis in late organogenesis mouse embryos, but the molecular and cellular mechanisms underlying this remain unclear. In this study, transcriptomic analyses revealed the early onset of cholecystitis in Sox17+/- embryos, together with the appearance of ectopic cystic duct-like epithelia in their gallbladders. The embryonic hepatitis showed positive correlations with the severity of cholecystitis in individual Sox17+/- embryos. Embryonic hepatitis could be induced by conditional deletion of Sox17 in the primordial gallbladder epithelia but not in fetal liver hepatoblasts. The Sox17+/- gallbladder also showed a drastic reduction in sonic hedgehog expression, leading to aberrant smooth muscle formation and defective contraction of the fetal gallbladder. The defective gallbladder contraction positively correlated with the severity of embryonic hepatitis in Sox17+/- embryos, suggesting a potential contribution of embryonic cholecystitis and fetal gallbladder contraction in the early pathogenesis of congenital biliary atresia.

Potentiation of Methylmercury-Induced Death in Rat Cerebellar Granular Neurons Occurs by Further Decrease of Total Intracellular GSH with BDNF via TrkB in Vitro.

Brain-derived neurotrophic factor (BDNF) is a principal factor for neurogenesis, neurodevelopment and neural survival through a BDNF receptor, tropomyosin-related kinase (Trk) B, while BDNF can also cause a decrease in the intracellular glutathione (GSH) level. We investigated the exacerbation of methylmercury-induced death of rat cerebellar granular neurons (CGNs) by BDNF in vitro. Since methylmercury can decrease intracellular GSH levels, we hypothesized that a further decrease of the intracellular GSH level is involved in the process of the exacerbation of neuronal cell death. In the present study, we established that in CGN culture, a decrease of the intracellular GSH level was further potentiated with BDNF in the process of the methylmercury-induced neuronal death and also in GSH reducer-induced neuronal death. BDNF treatment promoted the decrease in GSH levels induced by methylmercury and also by L-buthionine sulfoximine (BSO) and diethyl maleate (DEM). The promoting effect of BDNF was observed in a TrkB-vector transformant of the rat neuroblastoma B35 cell line but not in the mock-vector transformant. These results indicate that the exacerbating effect of BDNF on methylmercury-induced neuronal death in cultures of CGNs includes a further decrease of intracellular GSH levels, for which TrkB is essential.

Anatomy of the Murine Hepatobiliary System: A Whole-Organ-Level Analysis Using a Transparency Method.

The biliary tract is a well-branched ductal structure that exhibits great variation in morphology among vertebrates. Its function is maintained by complex constructions of blood vessels, nerves, and smooth muscles, the so-called hepatobiliary system. Although the mouse (Mus musculus) has been used as a model organism for humans, the morphology of its hepatobiliary system has not been well documented at the topographical level, mostly because of its small size and complexity. To reconcile this, we conducted whole-mount anatomical descriptions of the murine extrahepatic biliary tracts with related blood vessels, nerves, and smooth muscles using a recently developed transparentizing method, CUBIC. Several major differences from humans were found in mice: (1) among the biliary arteries, the arteria gastrica sinistra accessoria was commonly found, which rarely appears in humans; (2) the sphincter muscle in the choledochoduodenal junction is unseparated from the duodenal muscle; (3) the pancreatic duct opens to the bile duct without any sphincter muscles because of its distance from the duodenum. This state is identical to a human congenital malformation, an anomalous arrangement of pancreaticobiliary ducts. However, other parts of the murine hepatobiliary system (such as the branching patterns of the biliary tract, blood vessels, and nerves) presented the same patterns as humans and other mammals topologically. Thus, the mouse is useful as an experimental model for studying the human hepatobiliary system.

Fate mapping of gallbladder progenitors in posteroventral foregut endoderm of mouse early somite-stage embryos.

In early embryogenesis, the posteroventral foregut endoderm gives rise to the budding endodermal organs including the liver, ventral pancreas and gallbladder during early somitogenesis. Despite the detailed fate maps of the liver and pancreatic progenitors in the mouse foregut endoderm, the exact location of the gallbladder progenitors remains unclear. In this study, we performed a DiI fate-mapping analysis using whole-embryo cultures of mouse early somite-stage embryos. Here, we show that the majority of gallbladder progenitors in 9-11-somite-stage embryos are located in the lateral-most domain of the foregut endoderm at the first intersomite junction level along the anteroposterior axis. This definition of their location highlights a novel entry point to understanding of the molecular mechanisms of initial specification of the gallbladder.

A novel Amh-Treck transgenic mouse line allows toxin-dependent loss of supporting cells in gonads.

Cell ablation technology is useful for studying specific cell lineages in a developing organ in vivo. Herein, we established a novel anti-Müllerian hormone (AMH)-toxin receptor-mediated cell knockout (Treck) mouse line, in which the diphtheria toxin (DT) receptor was specifically activated in Sertoli and granulosa cells in postnatal testes and ovaries respectively. In the postnatal testes of Amh-Treck transgenic (Tg) male mice, DT injection induced a specific loss of the Sertoli cells in a dose-dependent manner, as well as the specific degeneration of granulosa cells in the primary and secondary follicles caused by DT injection in Tg females. In the testes with depletion of Sertoli cell, germ cells appeared to survive for only several days after DT treatment and rapidly underwent cell degeneration, which led to the accumulation of a large amount of cell debris within the seminiferous tubules by day 10 after DT treatment. Transplantation of exogenous healthy Sertoli cells following DT treatment rescued the germ cell loss in the transplantation sites of the seminiferous epithelia, leading to a partial recovery of the spermatogenesis. These results provide not only in vivo evidence of the crucial role of Sertoli cells in the maintenance of germ cells, but also show that the Amh-Treck Tg line is a useful in vivo model of the function of the supporting cell lineage in developing mammalian gonads.

Acetylcholine esterase is a regulator of GFAP expression and a target of dichlorvos in astrocytic differentiation of rat glioma C6 cells.

The main target of neurotoxins is neurons because they comprise the main part of neural function, but glial cells may be indirect targets because they support the function of neurons. Among the glial cells, astrocytes in particular act as "nurse cells", regulating neuronal survival and functions. In the present study, to reveal whether a known neurotoxic substance, organophosphate dichlorvos (DDVP), affects the differentiation of astrocytes, we used an astrocyte differentiation model in rat glioma C6 cells. Morphological change and induction of GFAP expression in the differentiating C6 cells were suppressed by DDVP treatment. The known potential targets of DDVP are acetylcholine esterase (AChE), fatty acid amide hydrolase and methyl guanine methyl transferase. Among the specific inhibitors against these enzymes, the AChE inhibitor paraoxon successfully suppressed the cellular morphological changes and the induction of GFAP expression in differentiating C6 cells. These results indicate that DDVP inhibits differentiation in the C6 astrocyte-differentiation model, in which at least AChE inhibition is involved and that AChE is a potent regulator of the differentiation. Furthermore, considering that the main substrate of AChE is ACh, thus, ACh may act as regulators of astrocyte differentiation.

Sox17 haploinsufficiency results in perinatal biliary atresia and hepatitis in C57BL/6 background mice.

Congenital biliary atresia is an incurable disease of newborn infants, of unknown genetic causes, that results in congenital deformation of the gallbladder and biliary duct system. Here, we show that during mouse organogenesis, insufficient SOX17 expression in the gallbladder and bile duct epithelia results in congenital biliary atresia and subsequent acute 'embryonic hepatitis', leading to perinatal death in ~95% of the Sox17 heterozygote neonates in C57BL/6 (B6) background mice. During gallbladder and bile duct development, Sox17 was expressed at the distal edge of the gallbladder primordium. In the Sox17(+/-) B6 embryos, gallbladder epithelia were hypoplastic, and some were detached from the luminal wall, leading to bile duct stenosis or atresia. The shredding of the gallbladder epithelia is probably caused by cell-autonomous defects in proliferation and maintenance of the Sox17(+/-) gallbladder/bile duct epithelia. Our results suggest that Sox17 plays a dosage-dependent function in the morphogenesis and maturation of gallbladder and bile duct epithelia during the late-organogenic stages, highlighting a novel entry point to the understanding of the etiology and pathogenesis of human congenital biliary atresia.

Culture on a fragmin/protamine-coated plate suppresses the collagen type IαI and TGF-ß1 mRNA expression of rat hepatic stellate RI-T cells.

Hepatic stellate cells (HSCs) intracellularly preserve vitamin A in the normal liver. When the liver is damaged, HSCs transform into myofibroblast-like cells, and then proliferate and increase their expression of collagen. Cultured on a plastic plate, HSCs spontaneously activate. To maintain HSCs in a quiescent state with low expression of collagen, coating methods with extracellular matrixes (ECMs) such as Matrigel-coating or laminin-rich coating are commonly used for HSC cultivation. Kishimoto et al. [14] reported that Fragmin®/protamine microparticles (F/P-MPs) have the ability to absorb heparin-binding cytokines like ECMs. Therefore, we examined whether the cultivation on an F/P-MPs-coated plate maintains the quiescent state of RI-T cells (derived from rat HSCs) including the suppression of collagen expression. We found that the mRNA levels of collagen type IαI and TGF-ß1 in RI-T cells were significantly suppressed in the cultivation on F/P-MPs-coated plates compared to cultures on noncoated and Matrigel-coated plates. We conclude that the F/P-MPs coating method is useful for maintaining with low expressions of collagen IαI and TGF-ß 1 mRNA levels in HSCs.

Vitamin K has the potential to protect neurons from methylmercury-induced cell death in vitro.

Vitamin K (VK) has a protective effect on neural cells. Methylmercury is a neurotoxicant that directly induces neuronal death in vivo and in vitro. Therefore, in the present study, we hypothesized that VK inhibits the neurotoxicity of methylmercury. To prove our hypothesis in vitro, we investigated the protective effects of VKs (phylloquinone, vitamin K(1); menaquinone-4, vitamin K(2) ) on methylmercury-induced death in primary cultured neurons from the cerebella of rat pups. As expected, VKs inhibited the death of the primary cultured neurons. It has been reported that the mechanisms underlying methylmercury toxicity involve a decrement of intracellular glutathione (GSH). Actually, treatment with GSH and a GSH inducer, N-acetyl cysteine, inhibited methylmercury-induced neuronal death in the present study. Thus, we investigated whether VKs also have protective effects against GSH-depletion-induced cell death by employing two GSH reducers, L-buthionine sulfoximine (BSO) and diethyl maleate (DEM), in primary cultured neurons and human neuroblastoma IMR-32 cells. Treatment with VKs affected BSO- and DEM-induced cell death in both cultures. On the other hand, the intracellular GSH assay showed that VK(2), menaquinone-4, did not restore the reduced GSH amount induced by methylmercury or BSO treatments. These results indicate that VKs have the potential to protect neurons against the cytotoxicity of methylmercury and agents that deplete GSH, without increasing intracellular GSH levels. The protective effect of VKs may lead to the development of treatments for neural diseases involving GSH depletion.