Impaired spatial memory in adult vitamin D deficient BALB/c mice is associated with reductions in spine density, nitric oxide, and neural nitric oxide synthase in the hippocampus

Vitamin D deficiency is prevalent in adults and is associated with cognitive impairment. However, the mechanism by which adult vitamin D (AVD) deficiency affects cognitive function remains unclear. We examined spatial memory impairment in AVD-deficient BALB/c mice and its underlying mechanism by measuring spine density, long term potentiation (LTP), nitric oxide (NO), neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS) in the hippocampus. Adult male BALB/c mice were fed a control or vitamin D deficient diet for 20 weeks. Spatial memory performance was measured using an active place avoidance (APA) task, where AVD-deficient mice had reduced latency entering the shock zone compared to controls. We characterised hippocampal spine morphology in the CA1 and dentate gyrus (DG) and made electrophysiological recordings in the hippocampus of behaviourally naive mice to measure LTP. We next measured NO, as well as glutathione, lipid peroxidation and oxidation of protein products and quantified hippocampal immunoreactivity for nNOS and eNOS. Spine morphology analysis revealed a significant reduction in the number of mushroom spines in the CA1 dendrites but not in the DG. There was no effect of diet on LTP. However, hippocampal NO levels were depleted whereas other oxidation markers were unaltered by AVD deficiency. We also showed a reduced nNOS, but not eNOS, immunoreactivity. Finally, vitamin D supplementation for 10 weeks to AVD-deficient mice restored nNOS immunoreactivity to that seen in in control mice. Our results suggest that lower levels of NO, reduced nNOS immunostaining contribute to hippocampal-dependent spatial learning deficits in AVD-deficient mice.

Tissue-Specific Content of Polyunsaturated Fatty Acids in (n-3) Deficiency State of Rats

The dietary intake of fatty acids (FAs) affects the composition and distribution of FAs in the body. Here, a first-generation (n-3)-deficiency study was conducted by keeping young (age 21 ± 2 days) Sprague–Dawley male rats on a peanut-oil-based diet for 33 days after weaning in order to compare the effect of mild (n-3)-deficiency on the lipid composition of different organs and feces. Soybean-oil-based diet was used as a control. The plasma FA levels corresponded to FAs levels in the organs. Lower docosahexaenoic acid (DHA) content was detected in the plasma, brain, testis, visceral fat, heart, and lungs of the (n-3)-deficient group, whereas the DHA content of the eye and feces did not differ between the experimental groups. The DHA content of the brains of the (n-3)-deficient group was 86% of the DHA content of the brains of the (n-3)-adequate group. The DHA level of the organs was affected in the order of visceral fat > liver triacylglycerols > lung > heart > liver phospholipids > testis > eye > brain, with brain being least affected. The low levels of (n-3) FAs in the liver, brain, eye, heart, and lung were offset by an increase in the (n-6) FAs, mainly arachidonic acid. These results indicate that, in rats, adequate maternal nutrition during pregnancy and weaning does not provide enough (n-3) FAs for 33 days of an (n-3)-deficient diet. Results of this study can be used also to evaluate the conditions needed to reach mild (n-3) deficiency in the first generation of rats and to evaluate the feasibility to collect data from a variety of organs or only selected ones.

Developmental iron deficiency dysregulates TET activity and DNA hydroxymethylation in the rat hippocampus and cerebellum

Iron deficiency (ID) during neurodevelopment is associated with lasting cognitive and socioemotional deficits, and increased risk for neuropsychiatric disease throughout the lifespan. These neurophenotypical changes are underlain by gene dysregulation in the brain that outlasts the period of ID; however, the mechanisms by which ID establishes and maintains gene expression changes are incompletely understood. The epigenetic modification 5-hydroxymethylcytosine (5hmC), or DNA hydroxymethylation, is one candidate mechanism because of its dependence on iron-containing TET enzymes. The aim of the present study was to determine the effect of fetal-neonatal ID on regional brain TET activity, Tet expression, and 5hmC in the developing rat hippocampus and cerebellum, and to determine whether changes are reversible with dietary iron treatment. Timed pregnant Sprague-Dawley rats were fed iron deficient diet (ID; 4 mg/kg Fe) from gestational day (G)2 to generate iron deficient anemic (IDA) offspring. Control dams were fed iron sufficient diet (IS; 200 mg/kg Fe). At postnatal day (P)7, a subset of ID-fed litters was randomized to IS diet, generating treated IDA (TIDA) offspring. At P15, hippocampus and cerebellum were isolated for subsequent analysis. TET activity was quantified by ELISA from nuclear proteins. Expression of Tet1, Tet2, and Tet3 was quantified by qPCR from total RNA. Global %5hmC was quantified by ELISA from genomic DNA. ID increased DNA hydroxymethylation (p=0.0105), with a corresponding increase in TET activity (p<0.0001) and Tet3 expression (p<0.0001) in the P15 hippocampus. In contrast, ID reduced TET activity (p=0.0016) in the P15 cerebellum, with minimal effect on DNA hydroxymethylation. Neonatal dietary iron treatment resulted in partial normalization of these changes in both brain regions. These results demonstrate that the TET/DNA hydroxymethylation system is disrupted by developmental ID in a brain region-specific manner. Differential regional disruption of this epigenetic system may contribute to the lasting neural circuit dysfunction and neurobehavioral dysfunction associated with developmental ID.

Improvement of Growth Retardation and Related Immunodeficiency by Dietary Intervention with Crackers Containing Animal Source Ingredients in Malnourished Rats

Hidden hunger is a risk factor for many health problems, including stunting, which is one of the globally prevalent signs of malnutrition. Stunting can be reduced through feeding on animal source foods. In our study, some animal source foods (egg, butter, yoghurt, and white cheese) in addition to other nutritious ingredients (wheat flour, minced fresh carrot, wheat germ, yeast, and iodine salt) were used to prepare four samples of crackers, with different taste (cumin, paprika, tomato, and cheese). The dough from all ingredients was prepared, flattened, cut then baked to prepare the crackers. These crackers were organoleptically and physically evaluated. Antioxidant activity, total phenolic content (TPC) and total flavonoid contents of the crackers’ samples were determined. The cumin-flavored crackers (the highest in TPC, flavonoids, and antioxidant activity) was evaluated for its macro-and micronutrients and studied in malnourished rats. Two groups of rats (each of 12) were fed on a balanced diet and a protein-deficient diet, respectively for 3 weeks. Six rats from each group were sacrificed and the remaining rats were fed on a balanced diet and diet supplemented with the cumin-flavored crackers, respectively for 5 weeks. The cumin-flavored crackers (100 g) contained 15.64 g protein, 231.00 mg calcium, 4.00 mg zinc, 83.75 µg iodine, arginine (16.53 mg/g protein), and lysine (19.30 mg/g protein). Malnutrition, immunodeficiency (as evidenced by a drop in cluster of differentiation 4 (CD4), cluster of differentiation 8 (CD8) and CD4/CD8 ratio), and stunting (as evidenced by a decrease in rat length, femur length, and insulin growth factor-1) were all observed in rats fed a protein-deficient diet for 3 weeks. Also, femur calcium and magnesium decreased in the malnourished rats. The dietary intervention with the cumin-flavored crackers reversed the reduction in aforementioned parameters in the malnourished rats. The cumin-flavored crackers may improve growth retardation and related immunodeficiency in previously malnourished rats.

Effects of Memantine and High Dose Vitamin D on Gait in Male APP/PS1 Alzheimer’s Disease Mice Following Vitamin D Deprivation

Background: Altered gait is a frequent feature of Alzheimer’s disease (AD), as is vitamin D deficiency. Treatment with memantine and vitamin D can protect cortical axons from exposure to amyloid-β and glutamate toxicity, suggesting this combination may mitigate altered gait in AD. Objective: Investigate the effects of vitamin D deprivation and subsequent treatment with memantine and vitamin D enrichment on gait performance in APPswe/PS1dE9 mice. Methods: Male APPswe/PS1dE9 mice were split into four groups (n = 14 each) at 2.5 months of age. A control group was fed a standard diet throughout while the other three groups started a vitamin D-deficient diet at month 6. One group remained on this deficient diet for the rest of the study. At month 9, the other two groups began treatment with either memantine alone or memantine combined with 10 IU/g of vitamin D. Gait was assessed using CatWalk at months 6, 9, 12, and 15. Results: Vitamin D deprivation led to a 13% increase in hind stride width by month 15 (p < 0.001). Examination of the treatment groups at month 15 revealed that mice treated with memantine alone still showed an increase in hind stride width compared to controls (p < 0.01), while mice treated with memantine and vitamin D did not (p = 0.21). Conclusion: Vitamin D deprivation led to impaired postural control in the APPswe/PS1dE9 model. Treatment with memantine and vitamin D, but not memantine alone, prevented this impairment. Future work should explore the potential for treatments incorporating vitamin D supplementation to improve gait in people with AD.

Lactobacillus plantarum MA2 Ameliorates Methionine and Choline-Deficient Diet Induced Non-Alcoholic Fatty Liver Disease in Rats by Improving the Intestinal Microecology and Mucosal Barrier

Non-alcoholic fatty liver disease (NAFLD) has become a highly concerned health issue in modern society. Due to the attentions of probiotics in the prevention of NAFLD, it is necessary to further clarify their roles. In this study, the methionine and choline-deficient (MCD) diet induced NAFLD rats model were constructed and treated with strain L. plantarum MA2 by intragastric administration once a day at a dose of 1 × 108 cfu/g.bw. After 56 days of the therapeutic intervention, the lipid metabolism and the liver pathological damage of the NAFLD rats were significantly improved. The content of total cholesterol (TC) and total triglyceride (TG) in serum were significantly lower than that in the NAFLD group (p < 0.05). Meanwhile, the intestinal mucosal barrier and the structure of intestinal microbiota were also improved. The villi length and the expression of claudin-1 was significantly higher than that in the NAFLD group (p < 0.05). Then, by detecting the content of LPS in the serum and the LPS-TLR4 pathway in the liver, we can conclude that Lactobacillus plantarum MA2 could reduce the LPS by regulating the gut microecology, thereby inhibit the activation of LPS-TLR4 and it downstream inflammatory signaling pathways. Therefore, our studies on rats showed that L. plantarum MA2 has the potential application in the alleviation of NAFLD. Moreover, based on the application of the strain in food industry, this study is of great significance to the development of new therapeutic strategy for NAFLD.

Environmental and Genetic Factors Affecting Bone Diseases and Phenotypes in Mouse Models

Bone diseases and phenotypes are affected in multiple ways. We focused on studying the effects of genetic and environmental factors, especially their impact on bone properties. Firstly, we investigated the effects of β-caryophyllene (BCP), a naturally occurring dietary cannabinoid, on protecting bone from vitamin D deficiency in mice fed on a diet lacking or supplemented with vitamin D (VD). We found that the VD-deficient diet enhanced the length of femur and tibia bones (P<0.05), and increased bone volume (BV; P<0.01) and the trabecular bone volume fraction (BV/TV; P <0.01) compared to the D+ diet. When given BCP-containing diet, mice exhibited higher BV and bone mineral density (BMD; P<0.05) than the control group. The trabecular and cortical bone were also affected by VD and BCP. In addition, the inclusion of dietary BCP improved the serum concentrations of klotho (P < 0.05). In summary, these data indicate that BCP enhances the level of klotho in the serum, leading to improved bone properties and mineralization in an experimental mouse model. Under conditions lacking UV light, the D-deficient diet could affect multiple properties of bone, including trabecular and cortical bone, in mice. The D-deficient diet can also result in weight loss in mice. My second project is to evaluate the bone properties in a mouse model with Il-1rn mutation. When knockout for IL-1rn, mice of Balb/c genomic background exhibited susceptibility to spontaneous arthritis disease (SAD), while those of a DBA/1 background were resistant to developing SAD. Our progress on the study of SAD suggested that some of the bone phenotypes, BMD, BV, tibia length, and cortical thickness, were different between wildtype and IL-1rn knockout mice both in Balb/c and DBA/1 strains. The two congenic mouse strains were also evaluated for bone properties. The results revealed that IL-1rn affected BMD differently between Balb/c and DBA/1 mouse strains. The absence of IL-1rn decreased BMD in Balb/c mice and increased BMD in DBA/1 -/- mice compared with wildtype animals. QTL in DBA.B -/- which affect arthritis in congenic strains also regulated BMD, with interferon activated gene 202b (Ifi202b) being the most favored candidate gene for BMD. Our data suggest some of the bone phenotypes are affected by the regulation of gene expression in the context of IL-1ra loss. To our knowledge, this is the first study to investigate the relationship of gene interaction in bone phenotypes with the loss of IL-1ra in an animal model. In my third study, we studied the different effects between endogenously produced and diet-supplied vitamin C on spontaneous arthritis disease susceptibility. In this study, we sought to investigate whether the source of vitamin C (endogenously produced or exogenously supplied) influences the development of inflammatory arthritis using a mouse model of SAD. SAD-susceptible Balb/c IL-1rn-/- mice were bred with vitamin C-deficient Sfx mice to produce a double mutant (SAD-susceptible, vitamin C-deficient) mouse strain. The three strains were raised, with the double mutant and Sfx mice supplied with vitamin C in drinking water, and mice’s arthritis severity scores were measured biweekly. Incidence and average severity for each strain were calculated. At four months of age, the mice were sacrificed, and body measurements and leg samples were collected. X-ray microcomputed tomography was used to scan the legs to characterize the bone profile. Femur length, tibia length, and bone volume were found to be significantly lower in double mutant mice than in Balb/c IL-1rn-/- mice. There was no significant difference in bone mineral density and femur thickness between the two arthritis-susceptible strains. The double mutant mice had an earlier onset of arthritis as well as a more severe disease than that of the Balb/c knockout (KO) strain. Our findings suggest that the source of vitamin C could affect both the susceptibility of mice to SAD and the severity of disease. In addition, we examined the sex differences in several mouse models of inflammatory arthritis. To understand the basis for these differences we conducted analysis of several mouse models of inflammatory arthritis. The study of whether there are gender and symmetry differences in experimental arthritis expression in the mouse models may be of significance to the study of human rheumatoid arthritis. Our observations and statistical analyses on the incidence of arthritis in four different animal models incorporated relatively large numbers of mice allowing for robust conclusions. Our research showed that there is a sexual dimorphism for arthritis incidence and severity of arthritis in mice harboring specific genetic modifications. For F2 population the incidence of arthritis was 57.1% in female mice and 75.6% in male mice. There was a difference in severity related to sex in two populations: B6.DR1/ B6.DR4 (P < 0.001) and F2 (P = 0.023). Among these populations, scores for the right hindlimbs were significantly higher than those for the left hindlimbs in males (P<0.05). When examining disease manifestation using the collagen induced arthritis model with DBA/1 mice, sex-dimorphism did not reach statistical significance. However, left hindlimbs showed a tendency toward greater disease expression over the right. Our results suggest that sex difference of arthritis exists in animal models not only in terms of gender, but also of left and right limbs. Using animal models, this work has laid the foundation for future research on gender differences in rheumatoid arthritis. In conclusion, our studies on genetic and environmental factors regulating bone phenotypes and diseases have significant implications. Not only have they raised the concept of sexual dimorphism in disease, but they have highlighted the influence of genetic background on bone disease. The link between different sources of vitamins and immune-mediated disease is intriguing and warrants further research, as is the bone protective effect of BCP.

Single neonatal irradiation induces long-term gene expression changes in the thyroid gland, which may be involved in the tumorigenesis

Exposure to ionizing radiation in childhood has been recognized as a risk factor for thyroid cancer. We previously demonstrated that neonatal X-irradiation induced specific deformation of the thyroid follicles. Here, we further analyzed this model to understand the possible relationship with thyroid carcinogenesis. Wistar rats were subjected to cervical X-irradiation at different ages of 1–8 weeks old and at different doses of 1.5–12 Gy. For tumor promotion, rats were fed with an iodine-deficient diet (IDD). In cervically X-irradiated neonatal rats, the size of thyroid follicles decreased, accompanied by an increase in the number of TUNEL-positive cells. Fas and Lgals3 mRNA levels increased, while Mct8 and Lat4 expressions decreased. The co-administration of IDD induced the proliferation and the upregulation in Lgals3 expression, resulting in thyroid adenoma development at 28 weeks post-exposure. Our data demonstrated that single neonatal X-irradiation induced continuous apoptotic activity in the thyroid with the long-term alternation in Fas, Mct8, Lat4, and Lgals3 mRNA expressions. Some of these changes were similar to those induced by IDD, suggesting that neonatal X-irradiation may partially act as a thyroid tumor promoter. These radiation-induced thyroidal changes may be enhanced by the combined treatment with IDD, resulting in the early development of thyroid adenoma.

Upregulated expression of transforming growth factor-β receptor I/II in an endemic Osteoarthropathy in China

Background Kashin–Beck disease (KBD) is a chronic, deforming, endemic osteochondropathy that begins in patients as young as 2–3 years of age. The pathogenesis of KBD remains unclear, although selenium (Se) deficiency and T-2 toxin food contamination are both linked to the disease. In the present study, we evaluated transforming growth factor-β receptor (TGF-βR I and II) levels in clinical samples of KBD and in pre-clinical disease models. Methods Human specimens were obtained from the hand phalanges of eight donors with KBD and eight control donors. Animal models of the disease were established using Sprague–Dawley rats, which were fed an Se-deficient diet for 4 weeks and later administered the T-2 toxin. Cartilage cellularity and morphology were examined by hematoxylin and eosin staining. Expression and localization of TGF-βRI and II were evaluated using immunohistochemical staining and western blotting. Results In the KBD samples, chondral necrosis was detected based on cartilage cell disappearance and alkalinity loss in the matrix ground substance. In the necrotic areas, TGF-βRI and II staining were strong. Positive percentages of TGF-βRI and II staining were higher in the cartilage samples of KBD donors than in those of control donors. TGF-βRI and II staining was also increased in cartilage samples from rats administered T-2 toxin or fed on Se-deficient plus T-2 toxin diets. Conclusion TGF-βRI and II may be involved in the pathophysiology of KBD. This study provides new insights into the pathways that contribute to KBD development.