Reduced Gene Expression of KCC2 Accelerates Axonal Regeneration and Reduces Motor Dysfunctions after Tibial Nerve Severance and Suturing.

Gamma-aminobutyric acid and glycine (GABA/Gly) are predominantly inhibitory neurotransmitters in the mature central nervous system; however, they mediate membrane potential depolarization during development. These differences in actions depend on intracellular Cl- concentrations ([Cl-]i), which are primarily regulated by potassium chloride cotransporter 2 (KCC2). After nerve injury, KCC2 expression markedly decreases and GABA/Gly mediate depolarization. Following nerve regeneration, KCC2 expression recovers and GABA/Gly become inhibitory, suggesting that KCC2 reduction and GABA/Gly excitation may be crucial for axonal regeneration. To directly clarify their involvement in regeneration, we analyzed recovery processes after tibial nerve severance and suturing between heterozygous KCC2 knockout mice (HT), whose KCC2 levels are halved, and their wild-type littermates (WT). Compared with WT mice, the sciatic functional index-indicating lower limb motor function-was significantly higher until 28 days after operation (D28) in HT mice. Furthermore, at D7, many neurofilament-positive fibers were elongated into the distal part of the sutured nerve in HT mice only, and myelinated axonal density was significantly higher at D21 and D28 in HT animals. Electron microscopy and galanin immunohistochemistry indicated a shorter nerve degeneration period in HT mice. Moreover, a less severe decrease in choline acetyltransferase was observed in HT mice. These results suggest that nerve degeneration and regeneration proceed more rapidly in HT mice, resulting in milder motor dysfunction. Via similar microglial activation, nerve surgery may reduce KCC2 levels more rapidly in HT mice, followed by earlier increased [Cl-]i and longer-lasting GABA/Gly excitation. Taken together, reduced KCC2 may accelerate nerve regeneration via GABA/Gly excitation.

Implication of serum growth differentiation factor-15 level in patients with renal diseases.

BACKGROUND: The synthesis of growth differentiation factor-15 (GDF-15) is induced by inflammation, hypoxia, and oxidative stress and is receiving great interest as a predictive biomarker for cardiovascular disease. However, its detailed impact on patients with renal disease remains uncertain. METHODS: Patients who underwent renal biopsy for evaluation of renal disease between 2012 and 2017 in our institute were prospectively included. Serum GDF-15 levels were measured and its association with baseline characteristics and its impact on the 3-year composites of renal prognosis (composites of > 1.5 folds of serum creatinine and renal replacement therapy) were investigated. RESULTS: A total of 110 patients (64 [42, 73] years old, 61 men) were included. The median serum GDF-15 level at baseline was 1885 (998, 3496) pg/mL. A higher serum GDF-15 level was associated with comorbidities including diabetes mellitus, anemia, renal impairment, and pathologic features including crescent formation, hyaline degeneration, and interstitial fibrosis (p < 0.05 for all). Serum GDF-15 level was a significant predictor of 3-year composite renal outcomes with an odds ratio per 100 pg/mL of 1.072 (95% confidence interval 1.001-1.103, p = 0.036) after adjustment for potential confounders. CONCLUSIONS: Serum GDF-15 levels were associated with several renal pathological features and renal prognosis in patients with renal diseases.

Reversible Transition between Discrete and 1D Infinite Architectures: A Temperature-Responsive Cu(I) Complex with a Flexible Disilane-Bridged Bis(pyridine) Ligand.

A thermoresponsive structural change based on a disilane-bridged bis(pyridine) ligand and CuI is reported. Single-crystal X-ray analysis revealed that there are two polymorphs in the Cu(I) complex: octanuclear copper(I) complex at 20 °C and 1D staircase copper(I) polymer complex at -173 °C. The formation of these polymorphs is due to the flexibility of the ligand. Cu-I bond formation is observed upon cooling the sample from -10 °C to -170 °C. The temperature-induced phase transition progression was clarified by DSC, VT-PXRD, and VT-photoluminescence measurements and indicated a reversible temperature-controlled crystal-to-crystal phase transition. Observation on a VT-stage using a high-speed camera showed crystal cracking during single-crystal to single-crystal transitions between these polymorphic forms.

Blood Taurine Dynamics in Captive Lions: Relationship with Feed and Bile Acid Composition.

Taurine is an essential nutrient for felines including lions. Various severe symptoms induced by taurine deficiency have been reported for domestic and captive felines. Particularly for captive lions in zoos, little information related to taurine requirements is available. Therefore, this study evaluated the relationship between blood taurine concentration and taurine content in prey feed given at zoos, as well as the composition, types, and conjugation properties of bile acids (BAs) in blood collected repeatedly from four lions housed at three zoos. Blood taurine concentrations in four lions were within the normal range, although individual differences and variations were found. Taurine was abundant in feed supplied at the zoos. A positive correlation between blood taurine concentration and feed amount was observed in lions housed at the same zoo. Approximately 70-80% of the total BA pool was cholic acid, with 50-70% being taurine-conjugated. Individual differences and variations were found. No correlation was found between blood taurine concentration and the compositions of BAs in the blood. Results showed that supply of taurine was sufficient for all lions fed the prey feed. Future studies must be conducted to clarify influences on individual differences, as well as individual variations in blood taurine concentration and blood BA composition.

Synthesis and characterization of tetraphenylammonium salts.

The phenyl (Ph) group is a representative substituent in the field of organic chemistry as benzene (the parent molecule) is of fundamental importance. Simple Ph-substituted compounds of common chemical elements are well known. However, extensive structural characterization of tetraphenylammonium (Ph4N+) salts has not been reported. Herein, the synthesis of Ph4N+ salts and their characterization data including the 1H and 13C nuclear magnetic resonance (NMR) spectra and the single-crystal X-ray structure have been presented. An intermolecular radical coupling reaction between an aryl radical and a triarylammoniumyl radical cation was conducted to synthesize the target moieties. The Ph4N+ salts described herein are the simplest tetraarylammonium (Ar4N+) salts known. The results reported herein can potentially help access the otherwise inaccessible non-bridged Ar4N+ salts, a new class of rigid and sterically hindered organic cations.

Developmental Formation of the GABAergic and Glycinergic Networks in the Mouse Spinal Cord.

Gamma-aminobutyric acid (GABA) and glycine act as inhibitory neurotransmitters. Three types of inhibitory neurons and terminals, GABAergic, GABA/glycine coreleasing, and glycinergic, are orchestrated in the spinal cord neural circuits and play critical roles in regulating pain, locomotive movement, and respiratory rhythms. In this study, we first describe GABAergic and glycinergic transmission and inhibitory networks, consisting of three types of terminals in the mature mouse spinal cord. Second, we describe the developmental formation of GABAergic and glycinergic networks, with a specific focus on the differentiation of neurons, formation of synapses, maturation of removal systems, and changes in their action. GABAergic and glycinergic neurons are derived from the same domains of the ventricular zone. Initially, GABAergic neurons are differentiated, and their axons form synapses. Some of these neurons remain GABAergic in lamina I and II. Many GABAergic neurons convert to a coreleasing state. The coreleasing neurons and terminals remain in the dorsal horn, whereas many ultimately become glycinergic in the ventral horn. During the development of terminals and the transformation from radial glia to astrocytes, GABA and glycine receptor subunit compositions markedly change, removal systems mature, and GABAergic and glycinergic action shifts from excitatory to inhibitory.

Slow progression of sciatic nerve degeneration and regeneration after loose ligation through microglial activation and decreased KCC2 levels in the mouse spinal cord ventral horn.

Peripheral nerve injury affects motor functions. To reveal the mechanisms underlying motor dysfunction and recovery after nerve compression, which have not been precisely examined, we investigated the temporal relationship among changes in motor function, nerve histopathology, and marker molecule expression in the spinal cord after loose ligation of the mouse sciatic nerve. After ligation, sciatic motor function suddenly declined, and axons gradually degenerated. During degeneration, galanin was localized in motor neuron cell bodies. Then, in the ventral horn, microglia were activated, and expression of choline acetyltransferase (ChAT), a synthetic enzyme of acetylcholine, and potassium chloride co-transporter 2 (KCC2), which shifts the action of γ-amino butyric acid (GABA) and glycine to inhibitory, decreased. Motor function recovery was insufficient although axonal regeneration was complete. ChAT levels gradually recovered during axonal regeneration. When regeneration was nearly complete, microglial activation declined, and KCC2 expression started to increase. The KCC2 level sufficiently recovered when axonal regeneration was complete, suggesting that the excitatory action of GABA/glycine may participate in axonal regeneration. Furthermore, these changes proceeded slower than those after severance, suggesting that loose ligation, compression, may mediate slower progression of degeneration and regeneration than severance, and these changes may cause the motor dysfunction and its recovery.

Cryofibrinogen-associated glomerulonephritis accompanied by advanced gastric cancer.

We had a 72-year-old man with advanced gastric cancer, poorly differentiated adenocarcinoma, receiving chemotherapy with S-1 (tegafur, gimeracil, and oteracil potassium) plus oxaliplatin. Ascites developed despite remission of gastric cancer and metastasis. Given no malignant cells in ascites, leg edema, renal impairment, hypoalbuminemia, and massive proteinuria, we diagnosed as nephrotic syndrome with microscopic hematuria. Renal biopsy showed membranoproliferative glomerulonephritis with no deposition of immunoglobulins and complements. Of note, electronic microscopy found organized deposits with microtubular structures in the glomerular capillary lumens and subendothelial spaces. The liquid chromatography-tandem mass spectrometry method detected fibrinogen alpha chain, beta chain, gamma chain, and fibronectin, and we eventually diagnosed cryofibrinogen-associated glomerulonephritis. Cryofibrinogen was not detected in plasma. He was expired at 5 months following renal biopsy due to the progression of refractory nephrotic syndrome. In addition to the detailed assessment of specifically organized deposits, the analysis using liquid chromatography-tandem mass spectrometry method is useful to diagnose cryofibrinogen-associated glomerulonephritis. We should consider cryofibrinogen-associated glomerulonephritis as a differential diagnosis when the patients with malignancy showed abnormal urinalysis and renal impairment, though it is a rare disease.

Renal Cell Carcinoma on the Native Kidney Following Kidney Transplantation.

A 48-year-old man with histories of IgA nephropathy for 33 years, hemodialysis for 29 years, and a kidney transplant from a deceased donor 5 years ago was admitted to our institute complaining of high fever and back pain. Although repeated follow-up of computed tomography failed to detect any de novo issues, he was eventually diagnosed as a renal cell carcinoma with multiple metastases, developing from his native-acquired cystic disease kidney with multiple cysts using a positron emission tomography. We should be cautious of de novo renal cell carcinoma in kidney transplantation recipients, and careful follow-up might be helpful to detect it.

Clinical Implications of Steroid Therapy for Crescentic Glomerulonephritis and Gemella morbillorum-associated Infective Endocarditis.

A 54-year-old man was admitted to our institute with a diagnosis of infective endocarditis (IE) with vegetation on the mitral valve and severe regurgitation due to Gemella morbillorum infection together with renal dysfunction, which was eventually diagnosed as infection-related pauci-immune necrotizing crescentic glomerulonephritis. Given the refractoriness to antibiotics, the persistent activity of nephritis, and repeated cerebral hemorrhaging, we prioritized steroid therapy over early surgical mitral valve replacement. Following steroid therapy, the glomerulonephritis completely improved. Although the administration of steroid therapy in the active phase of IE remains controversial, it might be indicated if comorbid glomerulonephritis is critical.

IgA Nephropathy with Dominant IgA2 Deposition Accompanied by Mantle Cell Lymphoma.

Malignant lymphoma is rarely complicated by secondary IgA nephropathy. We encountered a 74-year-old man with rapidly progressive glomerulonephritis due to IgA nephropathy with predominant deposition of IgA2, instead of IgA1, in the glomerulus that was eventually diagnosed as secondary IgA nephropathy due to mantle cell lymphoma. Renal impairment was improved by chemotherapy for the mantle cell lymphoma. IgA came from the colonic mucosa that was stimulated by the infiltrated lymphoma cells, instead of the tumor itself. We should consider mantle cell lymphoma as a cause of secondary IgA nephropathy, although its prevalence may not be very high.

[A case of oldest-old patient with chronic renal failure successfully treated with peritoneal dialysis].

He was a 92-year-old male patient with mild cognitive impairment while preserved activity of daily life. His renal dysfunction gradually increased due to the nephrosclerosis accompanied by systemic edema and water retention. We eventually decided to initiate peritoneal dialysis instead of standard hemodialysis for his end-stage renal dysfunction refractory to optimal medical therapy, given his preserved cognitive function and family support. He underwent an established therapeutic program for the peritoneal dialysis at home with an Information and Communication Technology (ICT).Given the increase in age of the patients with renal dysfunction, peritoneal dialysis has been receiving great attention as a home care strategy. The recent improvement in the device technology and ICT that enables us remote monitoring would reduce patients' effort in the management of peritoneal dialysis. Collaboration with home nursing and care workers would also be warranted for successful home care.

Development and persistence of neuropathic pain through microglial activation and KCC2 decreasing after mouse tibial nerve injury.

Gamma-amino butyric acid (GABA) is an inhibitory neurotransmitter in the mature brain, but is excitatory during development and after motor nerve injury. This difference in GABAergic action depends on the intracellular chloride ion concentration ([Cl-]i), primarily regulated by potassium chloride co-transporter 2 (KCC2). To reveal precise processes of the neuropathic pain through changes in GABAergic action, we prepared tibial nerve ligation and severance models using male mice, and examined temporal relationships amongst changes in (1) the mechanical withdrawal threshold in the sural nerve area, (2) localization of the molecules involved in GABAergic transmission and its upstream signaling in the dorsal horn, and (3) histology of the tibial nerve. In the ligation model, tibial nerve degeneration disappeared by day 56, but mechanical allodynia, reduced KCC2 localization, and increased microglia density remained until day 90. Microglia density was higher in the tibial zone than the sural zone before day 21, but this result was inverted after day 28. In contrast, in the severance model, all above changes were detected until day 28, but were simultaneously and significantly recovered by day 90. These results suggested that in male mice, allodynia may be caused by reduced GABAergic synaptic inhibition, resulting from elevated [Cl-]i after the reduction of KCC2 by activated microglia. Furthermore, our results suggested that factors from degenerating nerve terminals may diffuse into the sural zone, whereby they induced the development of allodynia in the sural nerve area, while other factors in the sural zone may mediate persistent allodynia through the same pathway.

Hyper-Formation of GABA and Glycine Co-Releasing Terminals in the Mouse Cerebellar Nuclei after Deprivation of GABAergic Inputs from Purkinje Cells.

GABA and glycine are inhibitory neurotransmitters. However, the mechanisms underlying the formation of GABAergic and glycinergic synapses remain unclear. The influence of GABAergic input deprivation on inhibitory terminal formation was investigated using Purkinje cell (PC)-specific vesicular GABA transporter (VGAT) knockout (L7-VGAT) mice, in which GABA release from PCs diminishes in an age-dependent manner. We compared the late development of GABAergic and glycinergic terminals in the cerebellar nucleus (CN) between control and L7-VGAT mice. In the control CN, the density of glutamate decarboxylase (GAD)-positive dots remained unchanged between postnatal 2 months (P2M) and 13 months (P13M), whereas glycine transporter 2 (GlyT2)-positive dots increased in density during this time frame. No difference in the density of GlyT2-positive dots was observed between control and L7-VGAT mice at P2M, but the density was significantly higher in the L7-VGAT fastigial nuclei (FN) than the control FN at P13M. When VGAT was absent from PC terminals, GlyT2-positive dots included GAD and VGAT and formed synapses. These results indicated that GABAergic terminals were formed by P2M, glycinergic terminals were actively formed after P2M, and more glycinergic terminals were formed in the L7-VGAT FN than in the control FN, suggesting that the increased glycinergic terminals may derive from interneurons within the FN and may also release GABA. These results suggest that the deprivation of GABAergic inputs from PCs may accelerate the formation of co-releasing terminals derived from interneurons and that the inhibitory terminal numbers and types may be regulated by the quantity of functional GABAergic inputs.

Embryonic development of GABAergic terminals in the mouse hypothalamic nuclei involved in feeding behavior.

The inhibitory neurotransmitter gamma-amino butyric acid (GABA) plays important roles in energy balance and feeding behavior in the hypothalamus. To reveal the time course of GABAergic network formation, we examined the immunohistochemical localization of glutamic acid decarboxylase (GAD), a GABAergic neuron marker, vesicular GABA transporter (VGAT), a marker of inhibitory terminals, and K+-Cl--cotransporter2 (KCC2), which shifts GABA action from excitation to inhibition, in the developing mouse hypothalamus. GABAergic terminals, seen as GAD- and VGAT-positive dots, increased in density during embryonic development. Moreover, the onset of KCC2 localization was almost concomitant with GABAergic terminal formation, and KCC2-positive profiles increased in density during development. This suggested that after the formation of GABAergic terminals, GABAergic action may change to inhibition in the hypothalamus. This maturation appears to proceed as follows: the lateral hypothalamus (LH) matures first, followed by the paraventricular nucleus (PVN) by the time of birth, while the ventromedial hypothalamus (VMH) and the arcuate nucleus (Arc) are not fully mature at the time of birth. Our findings suggest that GABAergic networks in the "feeding center" (LH) and the "exit" (PVN) may mature before birth, while those in the "satiety center" (VMH) and "higher control center" (Arc) may mature after birth.

Changes in the expression and localization of signaling molecules in mouse facial motor neurons during regeneration of facial nerves.

After injury, peripheral axons usually re-extend toward their target, and neuronal functions recover. Previous studies have reported that expression of various molecules are transiently altered in motor neurons after nerve injury, but the time course of these changes and their relationship with functional recovery have not been clearly demonstrated. We used the mouse facial nerve transection and suturing model, and examined the changes in expression of five molecules, choline acetyl transferase (ChAT), galanin, calcitonin gene-related protein (CGRP), gephyrin, and potassium chloride co-transporter 2 (KCC2) in the facial motor neurons after surgery until recovery. Number of ChAT-positive neurons was markedly decreased at days 3 and 7, and recovered to the normal level by day 60, when facial motor functions recovered. Localization of two neuropeptides, CGRP and galanin, was increased in the perikarya and axons during regeneration, and returned to the normal levels by days 60 and 28, respectively. Expression of two postsynaptic elements of γ-amino butyric acid synapses, gephyrin and KCC2, was decreased at days 3 and 7, and recovered by day 60. These results suggest that ChAT, CGRP, and KCC2 may be objective indicators of regeneration, and altering their expression may be related to the functional recovery and axonal re-extension.

Estradiol-loaded PLGA nanoparticles for improving low bone mineral density of cancellous bone caused by osteoporosis: Application of enhanced charged nanoparticles with iontophoresis.

Postmenopausal osteoporosis among older women, which occurs by an ovarian hormone deficiency, is one of the major public health problems. 17 ß-estradiol (E2) is used to prevent and treat this disease as a drug of hormone replacement therapy. In oral administration, E2 is significantly affected by first-pass hepatic metabolism, and high dose administration must be needed to obtain drug efficacy. Therefore, alternative administration route is needed, and we have focused on the transdermal drug delivery system. In this study, we have prepared E2-loaded poly(DL-lactide-co-glycolide) (PLGA) nanoparticles for osteoporosis by using a combination of an antisolvent diffusion method with preferential solvation. The average particle diameter of the nanoparticles was 110.0±41.0nm and the surface charge number density was 82 times higher than that of conventional E2-loaded PLGA nanoparticles. Therapeutic evaluation of E2-loaded PLGA nanoparticles was carried out using ovariectomized female rats. Therapeutic efficacy was evaluated to measure bone mineral density of cancellous bone using an X-ray CT system. When the E2-loaded PLGA nanoparticles were administrated once a week, bone mineral density was significantly higher than that of the non-treated group at 60days after the start of treatment. Also, in the group administered this nanoparticle twice a week, the bone mineral density increased significantly at 45days after the start of treatment. From these results, it was revealed that E2-loaded PLGA nanoparticles with iontophoresis were useful to recover bone mineral density of cancellous bone, and it was also suggested that they extend the dosing interval of E2.

Distinct development of the glycinergic terminals in the ventral and dorsal horns of the mouse cervical spinal cord.

In the spinal cord, glycine and γ-amino butyric acid (GABA) are inhibitory neurotransmitters. However, the ontogeny of the glycinergic network remains unclear. To address this point, we examined the developmental formation of glycinergic terminals by immunohistochemistry for glycine transporter 2 (GlyT2), a marker of glycinergic terminals, in developing mouse cervical spinal cord. Furthermore, the developmental localization of GlyT2 was compared with that of glutamic acid decarboxylase (GAD), a marker of GABAergic terminals, and vesicular GABA transporter (VGAT), a marker of inhibitory terminals, by single and double immunolabeling. GlyT2-positive dots (glycinergic terminals) were first detected in the marginal zone on embryonic day 14 (E14). In the ventral horn, they were detected at E16 and increased in observed density during postnatal development. Until postnatal day 7 (P7), GAD-positive dots (GABAergic terminals) were dominant and GlyT2 immunolabeling was localized at GAD-positive dots. During the second postnatal week, GABAergic terminals markedly decreased and glycinergic terminals became dominant. In the dorsal horn, glycinergic terminals were detected at P0 in lamina IV and P7 in lamina III and developmentally increased. GlyT2 was also localized at GAD-positive dots, and colocalizing dots were dominant at P21. VGAT-positive dots (inhibitory terminals) continued to increase until P21. These results suggest that GABAergic terminals first appear during embryonic development and may often change to colocalizing terminals throughout the gray matter during development. The colocalizing terminals may remain in the dorsal horn, whereas in the ventral horn, colocalizing terminals may give rise to glycinergic terminals.

Domain-based biophysical characterization of the structural and thermal stability of FliG, an essential rotor component of the Na+-driven flagellar motor.

Many bacteria move using their flagellar motor, which generates torque through the interaction between the stator and rotor. The most important component of the rotor for torque generation is FliG. FliG consists of three domains: FliGN, FliGM, and FliGC. FliGC contains a site(s) that interacts with the stator. In this study, we examined the physical properties of three FliG constructs, FliGFull, FliGMC, and FliGC, derived from sodium-driven polar flagella of marine Vibrio. Size exclusion chromatography revealed that FliG changes conformational states under two different pH conditions. Circular dichroism spectroscopy also revealed that the contents of α-helices in FliG slightly changed under these pH conditions. Furthermore, we examined the thermal stability of the FliG constructs using differential scanning calorimetry. Based on the results, we speculate that each domain of FliG denatures independently. This study provides basic information on the biophysical characteristics of FliG, a component of the flagellar motor.

Transdermal delivery of estradiol-loaded PLGA nanoparticles using iontophoresis for treatment of osteoporosis.

BACKGROUND: Estradiol is one of the therapeutic agents for osteoporosis. We have reported transdermal permeability of estradiol-loaded nanoparticles, and permeability effect of estradiol was enhanced by using nanoparticle system and iontophoresis [Colloids and Surfaces B: Biointerfaces97 (2012), 84-89]. OBJECTIVE: This study was conducted in vivo to evaluate therapeutic efficacy of the estradiol-loaded PLGA nanoparticles for osteoporosis. METHODS: Prior to the in vivo study, we have determined the surface charge density of the particles and found they have negatively charged polyelectrolyte layers on the surfaces. Ovariectomized female Sprague-Dawley rats were used as an animal model of osteoporosis. They were separated into three groups by administration route of estradiol-loaded PLGA nanoparticles, passive diffusion group, iontophoresis group and control. After treatment, we have measured bone mineral density of spine using an X-ray computed tomography system. RESULTS: Bone mineral density after iontophoresis was significantly higher than that of passive diffusion and control group. By usage of iontophoresis, the nanoparticles were permeated through follicles and migrated into capillary vessel around follicles, and the loaded drug reached effective blood concentration in plasma of rat. CONCLUSIONS: From this study, we found that the combination with charged nanoparticle system and iontophoresis is useful to osteoporosis treatment.