An n-3 fatty acid deficient diet affects mouse spatial learning in the Barnes circular maze.

Deficiency in n-3 fatty acids has been accomplished through the use of an artificial rearing method in which ICR mouse pups were hand fed a deficient diet starting from the 2nd day of life. There was a 51% loss of total brain DHA in mice with an n-3 fatty acid-deficient diet relative to those with a diet sufficient in n-3 fatty acids. n-3 fatty acid adequate and deficient mice did not differ in terms of locomotor activity in the open field test or in anxiety-related behavior in the elevated plus maze. The n-3 fatty acid-deficient mice demonstrated impaired learning in the reference-memory version of the Barnes circular maze as they spent more time and made more errors in search of an escape tunnel. No difference in performance between all dietary groups in the cued and working memory version of the Barnes maze was observed. This indicated that motivational, motor and sensory factors did not contribute to the reference memory impairment.

Method for hand-feeding mouse pups with nursing bottles.

The purpose of this study was to establish a hand-feeding method for the mouse by using nursing bottles. Five different-sized nipples were tested. The largest nipple used for pups from 12 to 15 days of age was 5.5 mm in diameter and 10 mm long, with a tip of 1.5 mm in diameter. The size of the nipple was much larger than that of the mothers, and bloating due to air accumulation was observed in the artificially-reared pups. To prevent bloat, the nipple size was increased, and a piece of sponge was stuffed in the nipple. Among the 30 pups fed from 1 day of age, 26 were weaned and 4 died. Three pups died due to aspiration of milk into the lung, and the remaining one died due to bloating. The development of nursing bottles with nipples may be potentially useful for the studies of nutrition, behavior, immunology, psychology, and toxicology.

Glutamine depletion induces murine neonatal melena with increased apoptosis of the intestinal epithelium.

AIM: To investigate the possible biological outcome and effect of glutamine depletion in neonatal mice and rodent intestinal epithelial cells. METHODS: We developed three kinds of artificial milk with different amounts of glutamine; Complete amino acid milk (CAM), which is based on maternal mouse milk, glutamine-depleted milk (GDM), and glutamine-rich milk (GRM). GRM contains three-fold more glutamine than CAM. Eighty-seven newborn mice were divided into three groups and were fed with either of CAM, GDM, or GRM via a recently improved nipple-bottle system for seven days. After the feeding period, the mice were subjected to macroscopic and microscopic observations by immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) and Ki-67 as markers of cell proliferation, and for cleaved-caspase-3 as a marker of apoptosis. Moreover, IEC6 rat intestinal epithelial cells were cultured in different concentrations of glutamine and were subject to a 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate cell proliferation assay, flow cytometry, and western blotting to examine the biological effect of glutamine on cell growth and apoptosis. RESULTS: During the feeding period, we found colonic hemorrhage in six of 28 GDM-fed mice (21.4%), but not in the GRM-fed mice, with no differences in body weight gain between each group. Microscopic examination showed destruction of microvilli and the disappearance of glycocalyx of the intestinal wall in the colon epithelial tissues taken from GDM-fed mice. Intake of GDM reduced BrdU incorporation (the average percentage of BrdU-positive staining; GRM: 13.8%, CAM: 10.7%, GDM: 1.14%, GRM vs GDM: P < 0.001, CAM vs GDM: P < 0.001) and Ki-67 labeling index (the average percentage of Ki-67-positive staining; GRM: 24.5%, CAM: 22.4% GDM: 19.4%, GRM vs GDM: P = 0.001, CAM vs GDM: P = 0.049), suggesting that glutamine depletion inhibited cell proliferation of intestinal epithelial cells. Glutamine deprivation further caused the deformation of the nuclear membrane and the plasma membrane, accompanied by chromatin degeneration and an absence of fat droplets from the colonic epithelia, indicating that the cells underwent apoptosis. Moreover, immunohistochemical analysis revealed the appearance of cleaved caspase-3 in colonic epithelial cells of GDM-fed mice. Finally, when IEC6 rat intestinal epithelial cells were cultured without glutamine, cell proliferation was significantly suppressed after 24 h (relative cell growth; 4 mmol/L: 100.0% ± 36.1%, 0 mmol/L: 25.3% ± 25.0%, P < 0.05), with severe cellular damage. The cells underwent apoptosis, accompanied by increased cell population in sub-G0 phase (4 mmol/L: 1.68%, 0.4 mmol/L: 1.35%, 0 mmol/L: 5.21%), where dying cells are supposed to accumulate. CONCLUSION: Glutamine is an important alimentary component for the maintenance of intestinal mucosa. Glutamine deprivation can cause instability of the intestinal epithelial alignment by increased apoptosis.

Artificial rearing of infant mice leads to n-3 fatty acid deficiency in cardiac, neural and peripheral tissues.

The ability to control the fatty acid content of the diet during early development is a crucial requirement for a one-generation model of docosahexaenoic acid (DHA; 22:6n3) deficiency. A hand feeding method using artificial rearing (AR) together with sterile, artificial milk was employed for feeding mice from postnatal day 2-15. The pups were fed an n-3 fatty acid adequate (3% alpha-linolenic acid (LNA; 18:3n3) + 1% 22:6n3) or a deficient diet (0.06% 18:3n3) with linoleic acid (LA; 18:2n6) as the only dietary source of essential fatty acids by AR along with a dam-reared control group (3.1% 18:3n3). The results indicate that restriction of n-3 fatty acid intake during postnatal development leads to markedly lower levels of brain, retinal, liver, plasma and heart 22:6n3 at 20 weeks of age with replacement by docosapentaenoic acid (DPAn6; 22:5n6), arachidonic acid (ARA; 20:4n6) and docosatetraenoic acid (DTA; 22:4n6). A detailed analysis of phospholipid classes of heart tissue indicated that phosphatidylethanolamine, phosphatidylcholine and cardiolipin were the major repositories of 22:6n3, reaching 40, 29 and 15%, respectively. A novel heart cardiolipin species containing four 22:6n3 moieties is described. This is the first report of the application of artificially rearing to mouse pup nutrition; this technique will facilitate dietary studies of knockout animals as well as the study of essential fatty acid (EFA) functions in the cardiovascular, neural and other organ systems.

Decreased development of necrotizing enterocolitis in IL-18-deficient mice.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease predominantly of prematurely born infants, characterized in its severest from by extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. Proinflammatory cytokines have been implicated in the development of NEC, and we have previously shown that IL-18 is significantly elevated in the well-established neonatal rat model of NEC. To determine whether IL-18 contributes to intestinal pathology in NEC, we subjected IL-18 knockout mice to the protocol used to develop experimental NEC in newborn rats. Newborn B6.129P2-Il18(tm1Aki)/J (NEC IL-18(-/-)) and wild-type (NEC WT) mice were hand fed every 3 h with cow's milk-based formula and exposed to asphyxia and cold stress twice daily. After 72 h, animals were killed and distal ileum and liver were removed. Disease development was determined via histological changes in the ileum as scored by a blinded evaluator. The number of TNF-alpha-, IL-12-, and IL-1beta-positive cells and macrophages were determined in both ileum and liver via immunohistology. IkappaB-alpha and IkappaB-beta were determined from protein extracts from both ileum and liver using Western blot analysis. The incidence and severity of NEC was significantly reduced in NEC IL-18(-/-) mice compared with NEC WT. Furthermore, mean ileal macrophages and hepatic IL-1beta were significantly reduced in IL-18(-/-) mice subjected to the NEC protocol. There were no statistically significant changes in Kupffer cells, hepatic TNF-alpha, ileal IL-1beta, or IL-12. IkappaB-alpha and IkappaB-beta were significantly increased in NEC IL-18(-/-) mice ileum and liver, respectively. These results confirm that IL-18 plays a crucial role in experimental NEC pathogenesis.

Reduced thymic size and numbers of splenic CD4+ and CD8+ cells in artificially reared mouse pups.

The effect of early nutrition on the development of the immune tissue and T cells of mouse pups was examined. Newborn mice were divided into three experimental groups: mother-reared (MR) pups, pups that were fed on a milk substitute from the first day (AR-0), and the third day (AR-2), using a hand-feeding system. The average thymic size of the AR-2 pups was respectively significantly larger and smaller than that of the AR-0 and MR pups. In contrast, the splenic sizes of the AR-0 and AR-2 pups were greater than that of the MR pups. The numbers of CD4+CD8- and CD4-CD8+ cells in the spleen of the MR pups were significantly higher than those in the AR-0 pups. These results indicate that early nutrition affected the sizes of the thymus and spleen and the composition of CD4+CD8- or CD4-CD8+ T cells in the spleen.

An extraordinary degree of structural specificity is required in neural phospholipids for optimal brain function: n-6 docosapentaenoic acid substitution for docosahexaenoic acid leads to a loss in spatial task performance.

This study was conducted to determine whether provision of preformed dietary docosapentaenoic acid (DPAn-6) can replace docosahexaenoic acid (DHA) for brain function as assessed by spatial task performance. A newly modified artificial rearing method was employed to generate n-3 fatty acid-deficient rats. Newborn pups were separated from their mothers at 2 days of age and given artificial rat milk containing linoleic acid (LA), or LA supplemented with 1% DHA (DHA), 1% DPAn-6 (DPA) or 1% DHA plus 0.4% DPAn-6 (DHA/DPA). The animals were then weaned onto similar pelleted diets. At adulthood, behavioural tasks were administered and then the brains were collected for fatty acid analysis. The LA and DPA groups showed a lower (63-65%) brain DHA than the dam-reared, DHA and DHA/DPA groups and this loss was largely compensated for by an increase in brain DPAn-6. The brain fatty acid composition in the DPA group was the same as that in the LA group at adulthood. In the Morris water maze, the LA and DPA groups exhibited a longer escape latency than the dam-reared and DHA groups and had a defect in spatial retention. In conclusion, DPAn-6 could not replace DHA for brain function, indicating a highly specific structural requirement for DHA.

N-3 fatty acid deficiency induced by a modified artificial rearing method leads to poorer performance in spatial learning tasks.

Docosahexaenoic acid (DHA) is a major structural component of the nervous system, and depletion may lead to losses in neural function. Our objective was to demonstrate a deficit in spatial task performance in rats with low brain DHA due to a low n-3 fatty acid intake using a first-generational artificial rearing technique. Newborn rat pups were separated on d 2 and assigned to two artificial rearing groups or a dam-reared control group. Pups were hand fed artificial milk via custom-designed nursing bottles containing either 0.02% (n-3 Def) or 3.1% (n-3 Adq) of total fatty acids as LNA. At d 21, rats were weaned to either n-3 Def or n-3 Adq pelleted diets and several behavioral tasks were evaluated at 9 wk of age. Brain DHA was lower (58% and 61%, p < 0.001) in n-3 Def in comparison to n-3 Adq and dam-reared rats, respectively. At adulthood, the n-3 fatty acid-deficient rats had a significantly greater moving time than the dam-reared group (p < 0.05), but there were no differences among the three groups in the elevated plus maze test. The n-3 fatty acid deficient rats exhibited a longer escape latency (p < 0.05) and poorer memory retention in the Morris water maze compared with n-3 fatty acid adequate and dam-reared rats. We concluded that artificial rearing can be used to produce n-3 fatty acid deficiency in the first generation. This deficiency was associated with significantly reduced spatial learning. Adequate brain DHA levels are required for optimal spatial learning.

Effects of an n-3-deficient diet on brain, retina, and liver fatty acyl composition in artificially reared rats.

Rat pups born to dams fed a diet with 3.1% of total fatty acids as alpha-linolenic acid (LNA) were fed, using an artificial rearing system, either an n-3-deficient (n-3-Def) or an n-3-adequate (n-3-Adq) diet. Both diets contained 17.1% linoleic acid, but the n-3-Adq diet also contained 3.1% LNA. The percentage of brain docosahexaenoic acid (DHA) continuously decreased (71%) with time over the 29 days of the experiment, with concomitant increases in docosapentaenoic acid (DPAn-6). In the retina, the percentage of DHA rose in the n-3-Adq group, with an apparent increased rate around the time of eye opening. However, there was a flat curve for the percentage of DHA in the n-3-Def group and a rising DPAn-6 with time. Liver DHA was highest at the time of birth in the n-3-Adq group but fell off somewhat over the course of 29 days. This decrease was more pronounced in the n-3-Def group, and the DPAn-6 rose considerably during the second half of the experiment. This method presents a first-generation model for n-3 deficiency that is more similar to the case of human nutrition than is the commonly employed two-generation model.