Cell adhesion molecule contactin-associated protein 3 is expressed in the mouse basal ganglia during early postnatal stages.

Cell adhesion molecules play important roles in the development of the nervous system. Among the contactin-associated protein (Caspr; also known as Cntnap) family, which belongs to the neurexin superfamily of proteins, Caspr and Caspr2 are indispensable for the formation and maintenance of myelinated nerves. In contrast, a physiological role for Caspr3 remains to be elucidated. This study examines the expression and localization of Caspr3 in the mouse brain using newly generated Caspr3 antibodies. Caspr3 was expressed abundantly between the first and the second postnatal weeks. During this period, Caspr3 was localized especially to the basal ganglia, including the striatum, external segment of the globus pallidus, and substantia nigra, and no gross abnormalities were apparent in the basal ganglia of Caspr3 knockout mice. In the striatum, Caspr3 was expressed by a subpopulation of medium spiny neurons that constitute the direct and indirect pathways. Caspr3 immunostaining was observed as punctate around the cell bodies as well as in the soma. These Caspr3 signals did not, however, overlap with those of synaptic markers. Our findings suggest that Caspr3 may play an important role in basal ganglia development during early postnatal stages.

Evaluation for Peritoneal Injury at an Early Stage Using Dual Macromolecular Markers.

Long-term peritoneal dialysis (PD) frequently produces morphological and functional changes of the peritoneum, making continuation of PD difficult. Therefore, it is necessary to evaluate peritoneal injury at an early stage and develop appropriate therapies. The aims of the present study were to evaluate peritoneal injury at an early stage and assess a drug for prevention of peritoneal injury using our previously developed novel evaluation method. Peritoneal injury was induced in model animals by intraperitoneal injection of methylglyoxal (MGO) for 1 to 5 consecutive days or chlorhexidine digluconate (CG) for 1 to 14 consecutive days. Tetramethylrhodamine-dextran (RD)-10 and fluorescein isothiocyanate-dextran (FD)-2000 were then injected into the peritoneal cavity and recovered after 120 min to evaluate peritoneal injury. The ratio of the concentration of RD-10 to FD-2000 (RD-10/FD-2000 ratio) significantly decreased in animals that had been treated with MGO or CG for 1 d. Moreover, the RD-10/FD-2000 ratio significantly increased in CG- and thalidomide-treated animals. The RD-10/FD-2000 ratio can be used to evaluate peritoneal injury at an early stage and assess the drug efficacy of thalidomide for prevention of peritoneal injury. This study will contribute to the development of therapeutic treatments for peritoneal injury.

Evaluation of hypothermia on the in vitro metabolism and binding and in vivo disposition of midazolam in rats.

The effect of hypothermia on the in vivo pharmacokinetics of midazolam was evaluated, with a focus on altered metabolism in the liver and binding to serum proteins. Rat primary hepatocytes were incubated with midazolam (which is metabolized mainly by CYP3A2) at 37, 32 or 28 °C. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of midazolam were estimated using the Michaelis-Menten equation. The Km of CYP3A2 midazolam remained unchanged, but the Vmax decreased at 28 °C. In rats, whose temperature was maintained at 37, 32 or 28 °C by a heat lamp or ice pack, the plasma concentrations of midazolam were higher, whereas those in the brain and liver were unchanged at 28 °C. The tissue/plasma concentration ratios were, however, increased significantly. The unbound fraction of midazolam in serum at 28 °C was half that at 37 °C. These pharmacokinetic changes associated with hypothermic conditions were due to reductions in CYP3A2 activity and protein binding.

Novel diagnostic method of peritoneal injury using dual macromolecular markers.

Long-term peritoneal dialysis (PD) frequently produces morphological and functional changes of the peritoneum, which makes continuation of PD difficult. Moreover, the progression of peritoneal injury causes complications and poor prognosis. Since therapeutic treatments for peritoneal injury during PD have yet to be established, it is important to diagnose peritoneal injury as early as possible. The aim of this study was to develop a method of monitoring peritoneal function to diagnose peritoneal injury. Model rats of peritoneal injury were prepared by intraperitoneal injection of methylglyoxal (MGO) for five consecutive days. Then, marker substances of various molecular weights (phenolsulfonphthalein, fluorescein isothiocyanate-dextran (FD)-10, FD-40, FD-70, FD-2000 or tetramethylrhodamine-dextran (RD)-10) were injected into the peritoneal cavity. At 120 min after injection, the remaining amounts of all marker substances were significantly decreased in the MGO-treated rats compared with those in the vehicle-treated rats. Molecular weight dependence of the peritoneal permeability was observed. A substance with a molecular weight of approximately 10000 was found to be suitable to diagnose peritoneal injury. Moreover, coadministration of RD-10 with FD-2000 enabled us to monitor enhanced peritoneal permeability and the transfer of water simultaneously, without the recovery of whole PD fluid, even in the case of different ultrafiltration volumes. We demonstrated the usefulness of administering substances to evaluate peritoneal permeability and the transfer of water simultaneously to diagnose peritoneal injury. This study should be valuable for safe and effective PD.

Human myo-inositol monophosphatase 2 rescues the nematode thermotaxis mutant ttx-7 more efficiently than IMPA1: functional and evolutionary considerations of the two mammalian myo-inositol monophosphatase genes.

Mammals express two myo-inositol monophosphatase (IMPase) genes, IMPA1/Impa1 and IMPA2/Impa2. In this study, we compared the spatial expression patterns of the two IMPase gene transcripts and proteins in mouse tissues. Results indicated discrete expression of the two IMPase genes and their protein products in various organs, including the brain. In Caenorhabditis elegans, loss of the IMPase gene, ttx-7, disrupts cellular polarity in RIA neurons, eliciting abnormal thermotaxis behavior. We performed a rescue experiment in mutant nematodes using mammalian IMPases. Human IMPA2 rescued the abnormal behavioral phenotype in the ttx-7 mutants more efficiently than IMPA1. These results raise a question about the phylogenetic origin of IMPases and the biological roles of mammalian IMPase 2 in mammals. Impa2 knockout mice generated in our laboratory, exhibited neither behavioral abnormalities nor a significant reduction in myo-inositol content in the brain and other examined tissues. Given the ability of human IMPA2 to rescue the ttx-7 mutant, and its genetic association with multiple neuropsychiatric disorders, close scrutiny of IMPA2 function and the evolutionary origin of IMPase genes is warranted.

Caspr3-Deficient Mice Exhibit Low Motor Learning during the Early Phase of the Accelerated Rotarod Task.

Caspr3 (Contactin-associated protein-like 3, Cntnap3) is a neural cell adhesion molecule belonging to the Caspr family. We have recently shown that Caspr3 is expressed abundantly between the first and second postnatal weeks in the mouse basal ganglia, including the striatum, external segment of the globus pallidus, subthalamic nucleus, and substantia nigra. However, its physiological role remains largely unknown. In this study, we conducted a series of behavioral analyses on Capsr3-knockout (KO) mice and equivalent wild-type (WT) mice to investigate the role of Caspr3 in brain function. No significant differences were observed in most behavioral traits between Caspr3-KO and WT mice, but we found that Caspr3-KO mice performed poorly during the early phase of the accelerated rotarod task in which latency to falling off a rod rotating with increasing velocity was examined. In the late phase, the performance of the Caspr3-KO mice caught up to the level of WT mice, suggesting that the deletion of Caspr3 caused a delay in motor learning. We then examined changes in neural activity after training on the accelerated rotarod by conducting immunohistochemistry using antibody to c-Fos, an indirect marker for neuronal activity. Experience of the accelerated rotarod task caused increases in the number of c-Fos-positive cells in the dorsal striatum, cerebellum, and motor cortex in both Caspr3-KO and WT mice, but the number of c-Fos-positive cells was significantly lower in the dorsal striatum of Caspr3-KO mice than in that of WT mice. The expression of c-Fos in the ventral striatum of Caspr3-KO and WT mice was not altered by the training. Our findings suggest that reduced activation of neural cells in the dorsal striatum in Caspr3-KO mice leads to a decline in motor learning in the accelerated rotarod task.

Evaluation for effect of hypothermia on the disposition of 4-nitrophenol in rats by in-vitro metabolism study and rat liver perfusion system.

OBJECTIVES: The aim of this study was to evaluate the effect of hypothermia on the in-vivo pharmacokinetics of 4-nitrophenol (4NP) using rat liver homogenate and rat liver perfusion system. METHODS: Rat liver homogenate was incubated with 4NP, which is mainly metabolized by cytochrome P450 2E1, at 37, 34, 32 or 28°C. The Michaelis constant (Km ) and maximum elimination velocity (Vmax ) of 4NP were calculated by a Hanes-Woolf plot. The hepatic extraction ratio (Eh ) of 4NP was evaluated in a rat liver perfusion study at 37, 34, 32 or 28°C. Moreover, the plasma concentration profiles of 4NP after its intravenous (i.v.) administration to rats were analysed by the moment theory and were compared with in-vitro parameters. KEY FINDINGS: While the Km of 4NP was not changed, the Vmax and Eh were reduced at low temperatures. The plasma concentrations of 4NP after its i.v. administration to rats were significantly increased at 28°C. CONCLUSION: Changes in the pharmacokinetics of 4NP under hypothermic conditions were caused by alterations in Vmax and Eh . We may be able to predict the disposition of a drug by in-vitro studies.

Evaluation of changes in hepatic disposition of phenolsulfonphthalein, indocyanine green and fluorescein isothiocyanate-dextran at low temperatures using a rat liver perfusion system.

OBJECTIVES: The aim of this study was to determine the factor changing the hepatic disposition of a drug during hypothermia using a rat liver perfusion system. METHODS: The livers of male Wistar rats were perfused at 37, 32 or 28°C in the single-pass mode. Venous outflow dilution patterns and biliary excretion rate patterns of phenolsulfonphthalein (PSP), indocyanine green (ICG) and fluorescein isothiocyanate (FITC)-dextran (FD-4, MW 4400) after the injection of a bolus into the perfused rat liver were analysed based on statistical moment theory. KEY FINDINGS: The first-pass extraction ratio (E(h) ) of PSP was significantly decreased at 32 and 28°C compared with 37°C. The biliary recovery of PSP and its conjugate was decreased and the biliary excretion was kept at a high concentration and was prolonged by low perfusion temperatures. ICG was almost extracted by a single-pass through the liver even at 32 and 28°C. The biliary recovery of ICG was significantly decreased at low temperature. Although the distribution volume of FD-4 as a vascular reference was not changed by perfusion temperature, the E(h) of FD-4 was decreased at 28°C although not markedly. CONCLUSION: The change in hepatic disposition of a drug at low perfusion temperatures differed according to disposition processes under hypothermia.

Effect of viscous additives on the absorption and hepatic disposition of 5-fluorouracil (5-FU) after application to liver surface in rats.

Objectives The aim was to study the effect of viscous additives on the absorption and hepatic disposition of 5-fluorouracil (5-FU) after application to the liver surface in rats. Methods 5-FU solution with or without viscous additives was applied to the rat liver surface with a cylindrical diffusion cell. Then, blood and the remaining solution in the diffusion cell were collected at selected times, followed by excision of the liver. The excised liver was divided into three sites and assayed for 5-FU content. Key findings The absorption rate of 5-FU from the liver surface was decreased in the presence of carboxymethylcellulose sodium (CMC-Na) and polyvinyl alcohol (PVA) as compared with the control. The k(a) values of PVA 15% and CMC-Na 1% were reduced to about 80 and 67% of the control. The maximum plasma concentration of 5-FU was decreased by incorporation of viscous additives. The 5-FU concentration at the diffusion cell attachment site of the liver (site 1) plateaued at 180 min in the absence of viscous additives. On the other hand, the concentration of 5-FU at site 1 increased in a time-dependent manner until 360 min in the presence of viscous additives. Conclusion Viscous additives might be useful for retaining drugs at their application site and controlling the rate of absorption from the liver surface.