Etsumori Harada

Possible regulatory mechanism of DHA-induced anti-stress reaction in rats

To determine whether docosahexaenoic acid (DHA) affects stress responses in rats, we investigated its influence on several behavioral tests. Female rats were fed a diet deficient in (n-3) fatty acid from mating through pregnancy and lactation. Male pups fed the same diet as their dams were used for experiments. The effects of dietary (n-3) fatty acid deficiency and supplementation with DHA on psychological stress and conditioned-fear stress were investigated. The effect of DHA on psychological stress was examined by an elevated plus-maze test. The (n-3) deficient rats spent significantly (P<0.05) less time in the open arms; after 1 week of supplementation with DHA, they showed a significant (P<0.01) improvement. We then examined the paired effects of DHA and CRH on stress manifestations by an intracerebroventricular (i.c.v.) cannulation and behavior testing. An i.c.v. infusion of CRH (500 pmol) under resting conditions was shown to have stress-inducing effects on behavior such as decreases of rearing, smelling and feeding, and increases of face washing; the supplementation of DHA significantly improved these distress behaviors. Finally, conditioned fear was induced by 40 min forced exposure to a cage in which the rat had experienced footshocks (30 x 1 mA x 1 s) 1 day before. Freezing behavior was dramatically suppressed by the supplementation of DHA, even 48 h after the conditioning treatment. Furthermore, the effect of DHA on the conditioned fear stress response is maintained over a long-term period. The i.c.v. pre-treatment of rats with bicuculline, a GABA(A) receptor antagonist, enhanced the conditioned-fear-induced freezing time in a dose-dependent fashion in the (n-3) fatty acid deficient animals. Significantly, the DHA supplemented group was not affected by the pre-treatment with bicuculline. From these findings, it is concluded that the involvement of DHA in stress responses may act via a GABA(A) receptor-mediated mechanism.

Influence of a dietary n-3 fatty acid deficiency on the cerebral catecholamine contents, EEG and learning ability in rat.

Female rats were fed on a diet deficient in (n-3) fatty acid or enriched in docosahexaenoic acid (DHA) diet from mating and throughout pregnancy and lactation. Pups fed on the same diet as their dams were used for experiments. The effects of dietary (n-3) fatty acid deficiency on cerebral catecholamine contents and electroencephalogram (EEG) in rat pups during the postnatal development were investigated. The (n-3) deficient rat pups showed significantly lower levels of noradrenaline (NA) in cerebral cortex, hippocampus and striatum, compared with those in the DHA adequate rats. Dopamine (DA) contents were significantly lower in the (n-3) deficient rats until the 7th day of age. These results were consistent with observations in the EEG analysis, relative powers of fast activities in the EEG recorded from the (n-3) deficient rats were significantly lower than those in the DHA adequate rats. The effect of supplementation with DHA in (n-3) deficient rats on learning ability was also studied in a model of learning, active avoidance test and three-panel run way test, after weaning. Although the percentages of avoidance in the (n-3) deficient rats (saline group) were constantly 20% or less until the 3rd session, the percentage of avoidance in the DHA supplemented rats rapidly increased to 53% following the first administration. While in the three-panel runway test, there were no significant differences between two groups. These results suggest that chronic consumption of a (n-3) fatty acid deficient diet could modify the biosynthesis of catecholamine in the brain, and might induce the behavioral disturbances. Furthermore, the decreased learning ability induced by (n-3) deficiency in the active avoidance test is a reversible following a supplementing DHA after the weaning.

Characteristic transport of lactoferrin from the intestinal lumen into the bile via the blood in piglets

Lactoferrin is a major iron-binding protein in milk from several species, such as humans, monkeys, mice and sows. Using neonatal and weaner piglets, the characteristic transfer of lactoferrin from intestinal lumen into bile via the circulation was investigated. Bovine lactoferrin (1 or 3 g/kg body weight) was infused into the stomach through a polyethylene tube or into the duodenum through a duodenal catheter over 5 min. Peripheral blood and bile samples were collected after the infusion. Lactoferrin absorbed into plasma and bile were assayed quantitatively by double-antibody enzyme-linked immunosorbent assay, and homogeneity of bovine lactoferrin in plasma and bile was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting methods. Morphological investigation was carried out according to the peroxidase anti-peroxidase method. Following oral administration in neonatal pigs, bovine lactoferrin appeared in the blood circulation and reached a peak level after 2 h. It was confirmed immunohistochemically that lactoferrin was transported by endocytosis via the epithelial cells. Lactoferrin absorbed into the blood was also detected in the bile and reached a peak value 12 h after oral administration. Transportation of lactoferrin from the intestinal lumen into the bile via the bloodstream was also observed in weaner piglets. Lactoferrin transported into plasma and bile was confirmed to be the same substance as administrated lactoferrin by electrophoresis and immunoblotting methods. Lactoferrin transported into bile was re-absorbed into the blood in neonatal pigs. These results demonstrate that lactoferrin contained in milk is transported into the circulation from the intestinal lumen and excreted into the bile, suggesting the possibility of entero-hepatic circulation of lactoferrin in neonatal pigs.

Evidence of lactoferrin transportation into blood circulation from intestine via lymphatic pathway in adult rats

Using adult rats, the characteristic transporting system for lactoferrin (LF) from intestinal lumen into the blood circulation was investigated. The rats were randomly divided into two groups, a non‐collected thoracic lymph (NC) group and a collected thoracic lymph (LC) group. Peripheral blood and thoracic lymph were collected from a jugular vein and a thoracic lymph duct, respectively, under anaesthesia. Bovine LF (bLF) was infused into the duodenal lumen by needle over a 1‐min period at a dose of 1 g kg−1. The transported bLF in the plasma and lymph was assayed quantitatively by double‐antibody enzyme‐linked immunosorbent assay (ELISA). Morphological investigation was also carried out in the intestine, lymph node, and liver. Following intraduodenal administration of bLF, the transported bLF in the NC group was detected in the plasma, and reached a peak value at 2 h. Furthermore, the bLF concentration in the thoracic duct lymph fluid in the LC group increased significantly, and peaked 2 h after the administration. In addition, bLF was not detected in the plasma of the LC group. Immunohistochemical analysis clearly showed anti‐bLF positive particles in the epithelial cells of the apical villi. The striated border and baso‐lateral membrane were also bLF positive. These results suggest that intraduodenally infused bLF is transported into the blood circulation via the lymphatic pathway, not via portal circulation in adult rats.

Novel function of bovine milk-derived lactoferrin on antinociception mediated by μ-opioid receptor in the rat spinal cord

Lactoferrin (LF) is a multifunctional protein that is found in milk, neutrophils, and other biological fluids. Although LF and the LF receptor have been identified in the central nervous system (CNS), the physiological role of LF remains unknown. We found that bovine milk-derived LF (BLF) reduces nociception in various pain models, as shown by the formalin test, hot plate test, and acetic acid writhing test in rats. Intraperitoneal (i.p.) administration of BLF significantly inhibited nociception in these pain models. These antinociceptive effects were also confirmed in BLF-fed rats. The antinociceptive effects of BLF were blocked by naloxone treatment, even though prostaglandin E(2) (PGE(2)) production in the ascites fluid that accumulated during the writhing test was not affected by BLF. Intrathecal (i.t.) application of BLF caused marked antinociceptive effects that were reversed by co-administration of a specific mu-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-NH(2) (CTOP), or by naloxone during the formalin test. We conclude that LF possesses mu-opioid receptor-mediated antinociceptive activity in the spinal cord.

Simplified preparation of a refined milk formula comparable to rat's milk : Influence of the formula on development of the gut and brain in artificially reared rat pups

BACKGROUND Milk formulas for artificially reared (AR) rat pups are mostly based on complex cows milk products, prepared by laborious and time-consuming processes. The aim of this study was to develop a simplified procedure for preparing a refined formula and to examine its influences on gut and brain development. METHODS The formula comprised a combination of purified cows casein and whey proteins, five kinds of edible oil, minerals, and vitamins. Detailed analyses showed that the composition of macro- and micro-nutrients, osmolarity, and pH of the new formula closely resembled those of rats milk. Rat pups, each with an intragastric cannula implanted at age 5 days, were artificially reared for the following 10-15 days. RESULTS The body weight gain of AR pups matched that of mother-reared (MR) pups. Histoplanimetrical analyses showed that the small intestine in AR pups was more developed in relation to area of a transverse section, number and length of villi, and thickness of tunica muscularis than that of MR pups. Fat components in the formula influenced the fatty acid composition and the cholesterol-to-phospholipid ratio in the small intestinal microvillus membrane (MVM) of AR pups, but not the MVM fluidity. Brain weight was not significantly different between the two groups at age 15-20 days. CONCLUSION This formula is useful for artificial rearing of rats and for identifying dietary components contributing to metabolic adaptation during the suckling period.

Characteristic Transfer of Colostral Components into Cerebrospinal Fluid via Serum in Neonatal Pigs

In order to evaluate the possibility of modification of brain function by colostral suckling, the characteristic transfer of colostral components into serum and cerebrospinal fluid (CSF) has been studied by SDS electrophoresis, immunoblot and ELISA methods in nonsuckling pigs. Total protein concentrations in the serum increased immediately after oral administration of bovine colostrum, reaching a peak value (7.0 ± 0.7 g/dl) at 24 h after administration, corresponding to a 3-fold increase compared to preinfusion levels. IgG and other macromolecular components (MW 19,000–58,000) were recognized in serum by electrophoretic and ELISA analysis. Total protein concentrations in the CSF collected from the cisterna magna also increased steeply after colostral administration, reaching a maximal value (54.1 ± 5.0 mg/dl) at 4 h, corresponding to a 4-fold increase compared to preinfusion levels. Two colostral components (MW 19,000 and 31,000) in serum were confirmed to be present in the CSF by electrophoresis. The component of MW 19,000 was identified by immunoblot as β-lactoglobulin. IgG in serum transferred from colostrum could not be detected in the CSF by ELISA. Lactoferrin administered into the intestine was also detected in the CSF via serum. These results indicate that some components of colostrum can be transported into the CSF via the serum, suggesting the possibility of modification of immature brain functions by colostral suckling in neonatal pigs.

Bovine lactoferrin reduces plasma triacylglycerol and NEFA accompanied by decreased hepatic cholesterol and triacylglycerol contents in rodents

In the present study we examined whether oral administration of bovine lactoferrin (bLF) reduces plasma or hepatic triacylglycerol and cholesterol in mice. When bLF mixed with a standard commercial diet (10 g/kg) was given to mice for 4 weeks, plasma triacylglycerol and NEFA decreased, while plasma HDL-cholesterol levels increased (P<0.01). These changes in plasma lipid profiles were accompanied by significant decreases in hepatic cholesterol and triacylglycerol contents. When mice were fed a high-fat diet containing 300.0 g lard, 10.0 g cholesterol and 2.5 g bovine bile powder/kg for 4 weeks, bovine LF did not have any significant effects on plasma or hepatic cholesterol and triacylglycerol concentrations. Furthermore, bLF had no significant effects on faecal excretion of total bile acids in mice. Interestingly, bLF showed a suppressive effect on the lymphatic triacylglycerol absorption in chronically treated rats. We conclude that bLF has a beneficial effect on plasma cholesterol levels and retards hepatic lipid accumulation in mice fed a standard diet.

Opioid mediated suppressive effect of milk-derived lactoferrin on distress induced by maternal separation in rat pups

The present study assessed the effects of bovine milk-derived lactoferrin (bLf) on distress activities induced by maternal separation in 5- to 18-day-old rat pups. The rat pups were injected with BSA (100 mg/kg, i.p.; control) or bLf (100 mg/kg, i.p.) 30 min before the behavioral test. Distress activity was estimated by means of recording body movements or ultrasonic vocalizations (USVs). After 5 min of maternal separation, bLf significantly (P<0.01) suppressed body movements, particularly in the 10-day-old pups. This suppressive effect of bLf was reversed by pretreatment with naloxone, CTOP, and norBNI at doses of 0.1-1 mg/kg. Additionally, USVs were also suppressed by bLf, which was reversed by pretreatment with naloxone. Inhibition of nitric oxide synthase (NOS) with nitro-L-arginine methyl ester (L-NAME) dose dependently (3-10 mg/kg) suppressed separation-induced USV production in 10-day-old pups. Interestingly, the suppressive effect of bLf was completely reversed by pretreatment with a low dose (1 mg/kg) of L-NAME, which did not affect the USVs with single application. These findings demonstrate that milk-derived bLf suppresses distress induced by maternal separation via an opioid-mediated mechanism. Furthermore, bLf possibly activates NOS, and an elevated nitric oxide may cause some modification of the opioid system.

Suppressive effects of milk-derived lactoferrin on psychological stress in adult rats

Lactoferrin (LF) is known as an iron-binding glycoprotein. It has been shown that bovine LF (bLF) is transported into cerebrospinal fluid via blood although its physiological effects in the central nervous system (CNS) are still unclear. In this study, a suppressive effect of bLF on psychological distress was investigated in adult rats. Intraperitoneal injection of bLF (100 mg/kg) reduced stressful behaviors in a conditioned fear-induced freezing test and an elevated plus-maze test. Interestingly, the suppressive effect of bLF was enhanced by pretreatment with electric foot-shock (FS). This suppressive effect of bLF in the elevated plus-maze test was reversed by pretreatment with naloxone, an opioid receptor antagonist, at a dose of 1 mg/kg (ip). N(omega)-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase (NOS) inhibitor, also blocked the suppressive effect of bLF and foot-shock. In addition, combined application of a low dose of bLF (30 mg/kg, ip) and l-arginine (30 and 100 mg/kg, ip) showed significant potentiated effects on psychological stress. These results suggest that bLF has suppressive effects on psychological distress, especially under the condition of moderate stress. Furthermore, it is suggested that bLF possibly activates an endogenous opioidergic system via nitric oxide synthase activation.