Inbred C57BL/6J and DBA/2J Mouse Strains Exhibit Constitutive Differences in Regional Brain Fatty Acid Composition

Major behavioral and neurochemical features observed between inbred C57BL/6 and DBA/2 mouse strains can be reproduced within rodent strains following dietary-induced reductions in brain docosahexaenoic acid (DHA, 22:6n-3) composition. It was therefore hypothesized that C57BL/6 and DBA/2 mice exhibit constitutive differences in brain DHA composition that are independent of diet. To test this, adult C57BL/6J and DBA/2J prefrontal cortex, hippocampus, ventral striatum, and midbrain fatty acid composition was determined by gas chromatography. After correction for multiple comparisons, C57BL/6J mice exhibited significantly lower DHA composition in the hippocampus and ventral striatum, but not prefrontal cortex or midbrain, and significantly greater regional arachidonic acid (ARA, 20:4n-6):DHA ratios, relative to DBA/2J mice. C57BL/6J mice also exhibited significantly lower regional adrenic acid (ADA, 22:4n-6) composition, and a significantly smaller ADA:ARA ratio, relative to DBA/2J mice. C57BL/6J mice exhibited significantly smaller oleic acid:stearic acid ratio in the hippocampus and ventral striatum relative to DBA/2J mice. Among all mice, DHA composition was positively correlated with the ADA:ARA ratio and inversely correlated with the oleic acid:stearic acid ratio. These data demonstrate that inbred C57BL/6J and DBA/2J mouse strains exhibit constitutive and region-specific differences in fatty acid composition independent of diet, and suggest that heritable genetic factors are an important determinant of central fatty acid composition.     

Further Characterization of Intrastriatal Lipopolysaccharide Model of Parkinson’s Disease in C57BL/6 Mice

Parkinson’s disease (PD) is the most common movement disorder, characterized by progressive degeneration of the nigrostriatal pathway, which consists of dopaminergic cell bodies in substantia nigra and their neuronal projections to the striatum. Moreover, PD is associated with an array of non-motor symptoms such as olfactory dysfunction, gastrointestinal dysfunction, impaired regulation of the sleep-wake cycle, anxiety, depression, and cognitive impairment. Inflammation and concomitant oxidative stress are crucial in the pathogenesis of PD. Thus, this study aimed to model PD via intrastriatal injection of the inflammagen lipopolysaccharide (LPS)to investigate if the lesion causes olfactory and motor impairments, inflammation, oxidative stress, and alteration in synaptic proteins in the olfactory bulb, striatum, and colon. Ten µg of LPS was injected unilaterally into the striatum of 27 male C57BL/6 mice, and behavioural assessment was conducted at 4 and 8 weeks post-treatment, followed by tissue collection. Intrastriatal LPS induced motor impairment in C57BL/6 mice at 8 weeks post-treatment evidenced by reduced latency time in the rotarod test. LPS also induced inflammation in the striatum characterized by increased expression of microglial marker Iba-1 and astrocytic marker GFAP, with degeneration of dopaminergic neuronal fibres (reduced tyrosine hydroxylase immunoreactivity), and reduction of synaptic proteins and DJ-1 protein. Additionally, intrastriatal LPS induced inflammation, oxidative stress and alterations in synaptic proteins within the olfactory bulb, although this did not induce a significant impairment in olfactory function. Intrastriatal LPS induced mild inflammatory changes in the distal colon, accompanied by increased protein expression of 3-nitrotyrosine-modified proteins. This model recapitulated the major features of PD such as motor impairment and degeneration of dopaminergic neuronal fibres in the striatum, as well as some pathological changes in the olfactory bulb and colon; thus, this model could be suitable for understanding clinical PD and testing neuroprotective strategies.     

Impact of C57BL/6J and SV-129 Mouse Strain Differences on Ischemia-Induced Postnatal Angiogenesis and the Associated Leukocyte Infiltration in a Murine Hindlimb Model of Ischemia

Strain-related differences in arteriogenesis in inbred mouse strains have already been studied excessively. However, these analyses missed evaluating the mouse strain-related differences in ischemia-induced angiogenic capacities. With the present study, we wanted to shed light on the different angiogenic potentials and the associated leukocyte infiltration of C57BL/6J and SV-129 mice to facilitate the comparison of angiogenesis-related analyses between these strains. For the induction of angiogenesis, we ligated the femoral artery in 8–12-week-old male C57BL/6J and SV-129 mice and performed (immuno-) histological analyses on the ischemic gastrocnemius muscles collected 24 h or 7 days after ligation. As evidenced by hematoxylin and eosin staining, C57BL/6J mice showed reduced tissue damage but displayed an increased capillary-to-muscle fiber ratio and an elevated number of proliferating capillaries (CD31⁺/BrdU⁺ cells) compared to SV-129 mice, thus showing improved angiogenesis. Regarding the associated leukocyte infiltration, we found increased numbers of neutrophils (MPO⁺ cells), NETs (MPO⁺/CitH3⁺/DAPI⁺), and macrophages (CD68⁺ cells) in SV-129 mice, whereas macrophage polarization (MRC1^- vs. MRC1⁺) and total leukocyte infiltration (CD45⁺ cells) did not differ between the mouse strains. In summary, we show increased ischemia-induced angiogenic capacities in C57BL/6J mice compared to SV-129 mice, with the latter showing aggravated tissue damage, inflammation, and impaired angiogenesis.     

Treatment with the Bacterial Toxin CNF1 Selectively Rescues Cognitive and Brain Mitochondrial Deficits in a Female Mouse Model of Rett Syndrome Carrying a MeCP2-Null Mutation

Rett syndrome (RTT) is a rare neurological disorder caused by mutations in the X-linked MECP2 gene and a major cause of intellectual disability in females. No cure exists for RTT. We previously reported that the behavioural phenotype and brain mitochondria dysfunction are widely rescued by a single intracerebroventricular injection of the bacterial toxin CNF1 in a RTT mouse model carrying a truncating mutation of the MeCP2 gene (MeCP2-308 mice). Given the heterogeneity of MECP2 mutations in RTT patients, we tested the CNF1 therapeutic efficacy in a mouse model carrying a null mutation (MeCP2-Bird mice). CNF1 selectively rescued cognitive defects, without improving other RTT-related behavioural alterations, and restored brain mitochondrial respiratory chain complex activity in MeCP2-Bird mice. To shed light on the molecular mechanisms underlying the differential CNF1 effects on the behavioural phenotype, we compared treatment effects on relevant signalling cascades in the brain of the two RTT models. CNF1 provided a significant boost of the mTOR activation in MeCP2-308 hippocampus, which was not observed in the MeCP2-Bird model, possibly explaining the differential effects of CNF1. These results demonstrate that CNF1 efficacy depends on the mutation beared by MeCP2-mutated mice, stressing the need of testing potential therapeutic approaches across RTT models.     

Therapeutic Effects of Hydrogen Gas Inhalation on Trimethyltin-Induced Neurotoxicity and Cognitive Impairment in the C57BL/6 Mice Model

Oxidative stress (OS) is one of the causative factors in the pathogenesis of various neurodegenerative diseases, including Alzheimer’s disease (AD) and cognitive dysfunction. In the present study, we investigated the effects of hydrogen (H₂) gas inhalation in trimethyltin (TMT)-induced neurotoxicity and cognitive dysfunction in the C57BL/6 mice. First, mice were divided into the following groups: mice without TMT injection (NC), TMT-only injection group (TMT only), TMT injection + lithium chloride-treated group as a positive control (PC), and TMT injection + 2% H₂ inhalation-treated group (H₂). The TMT injection groups were administered a single dosage of intraperitoneal TMT injection (2.6 mg/kg body weight) and the H₂ group was treated with 2% H₂ for 30 min once a day for four weeks. Additionally, a behavioral test was performed with Y-maze to test the cognitive abilities of the mice. Furthermore, multiple OS- and AD-related biomarkers such as reactive oxygen species (ROS), nitric oxide (NO), calcium (Ca²⁺), malondialdehyde (MDA), glutathione peroxidase (GPx), catalase, inflammatory cytokines, apolipoprotein E (Apo-E), amyloid β (Aβ)-40, phospho-tau (p-tau), Bcl-2, and Bcl-2- associated X (Bax) were investigated in the blood and brain. Our results demonstrated that TMT exposure alters seizure and spatial recognition memory. However, after H₂ treatment, memory deficits were ameliorated. H₂ treatment also decreased AD-related biomarkers, such as Apo-E, Aβ-40, p-tau, and Bax and OS markers such as ROS, NO, Ca²⁺, and MDA in both serum and brain. In contrast, catalase and GPx activities were significantly increased in the TMT-only group and decreased after H₂ gas treatment in serum and brain. In addition, inflammatory cytokines such as granulocyte colony-stimulating factors (G-CSF), interleukin (IL)-6, and tumor necrosis factor alpha (TNF-α) were found to be significantly decreased after H₂ treatment in both serum and brain lysates. In contrast, Bcl-2 and vascular endothelial growth factor (VEGF) expression levels were found to be enhanced after H₂ treatment. Taken together, our results demonstrated that 2% H₂ gas inhalation in TMT-treated mice exhibits memory enhancing activity and decreases the AD, OS, and inflammatory-related markers. Therefore, H₂ might be a candidate for repairing neurodegenerative diseases with cognitive dysfunction. However, further mechanistic studies are needed to fully clarify the effects of H₂ inhalation on TMT-induced neurotoxicity and cognitive dysfunction.     

Differences in Diurnal Rhythm of Rod Outer Segment Renewal between 129T2/SvEmsJ and C57BL/6J Mice

In all mammalian species tested to date, rod photoreceptor outer segment renewal is a circadian process synchronized by light with a burst of outer segment fragment (POS) shedding and POS phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning at light onset. Recent reports show that RPE phagocytosis also increases shortly after dark onset in C57BL/6 (C57) mice. Genetic differences between C57 mice and 129T2/SvEmsJ (129) mice may affect regulation of outer segment renewal. Here, we used quantitative methods to directly compare outer segment renewal in C57 and 129 mouse retina. Quantification of rhodopsin-positive phagosomes in the RPE showed that in 129 mice, rod POS phagocytosis after light onset was significantly increased compared to C57 mice, but that 129 mice did not show a second peak after dark onset. Cone POS phagosome content of RPE cells did not differ by mouse strain with higher phagosome numbers after light than after dark. We further quantified externalization of the “eat me” signal phosphatidylserine by outer segment tips, which precedes POS phagocytosis. Live imaging of retina ex vivo showed that rod outer segments extended PS exposure in both strains but that frequency of outer segments with exposed PS after light onset was lower in C57 than in 129 retina. Taken together, 129 mice lacked a burst of rod outer segment renewal after dark onset. The increases in rod outer segment renewal after light and after dark onset in C57 mice were attenuated compared to the peak after light onset in 129 mice, suggesting an impairment in rhythmicity in C57 mice.     

Enhanced in vivo Optical Imaging of the Inflammatory Response to Acute Liver Injury in C57BL/6 Mice Using a Highly Bright Near-Infrared BODIPY Dye

Delving deeper is possible in whole-body in vivo imaging using a super-bright membrane-targeting BODIPY dye ( BD ). The dye was used to monitor homing of ex vivo fluorescently labelled neutrophils to an injured liver of dark-pigmented C57BL/6 mice. In vivo imaging system (IVIS) data conclusively showed an enhanced signal intensity and a higher signal-to-noise ratio in mice receiving neutrophils labelled with the BD dye relative to those labelled with a gold standard dye at 2 h post in vivo administration of fluorescently labelled cells. Fluorescence-activated cell sorting (FACS) confirmed that BD is nontoxic, and an exceptional cell labelling dye that opens up precision deep-organ in vivo imaging of inflammation in mice routinely used for biomedical research. The origin of enhanced performance is identified with the molecular structure and the distinct localisation of the dye within cells that enable remarkable changes in its optical parameters.     

Influence of Sex and Strain on Hepatic and Adipose Tissue Trace Element Concentrations and Gene Expression in C57BL/6J and DBA/2J High Fat Diet Models

The objective of this study was to determine the influence of sex and strain on the dysregulation of trace element concentration and associative gene expression due to diet induced obesity in adipose tissue and the liver. Male and female C57BL/6J (B6J) and DBA/2J (D2J) were randomly assigned to a normal-fat diet (NFD) containing 10% kcal fat/g or a mineral-matched high-fat diet (HFD) containing 60% kcal fat/g for 16 weeks. Liver and adipose tissue were assessed for copper, iron, manganese, and zinc concentrations and related changes in gene expression. Notable findings include three-way interactions of diet, sex, and strain amongst adipose tissue iron concentrations (p = 0.005), adipose hepcidin expression (p = 0.007), and hepatic iron regulatory protein (IRP) expression (p = 0.012). Cd11c to Cd163 ratio was increased in adipose tissue due to HFD amongst all biological groups except B6J females, for which tissue iron concentrations were reduced due to HFD (p = 0.002). Liver divalent metal transporter 1 (DMT-1) expression was increased due to HFD amongst B6J males (p < 0.005) and females (p < 0.004), which coincides with the reduction in hepatic iron concentrations found in these biological groups (p < 0.001). Sex, strain, and diet affected trace element concentration, the expression of genes that regulate trace element homeostasis, and the expression of macrophages that contribute to tissue iron-handling in adipose tissue. These findings suggest that sex and strain may be key factors that influence the adaptive capacity of iron mismanagement in adipose tissue and its subsequent consequences, such as insulin resistance.     

ILB®, a Low Molecular Weight Dextran Sulphate,Restores Glutamate Homeostasis,Amino Acid Metabolism and Neurocognitive Functions in a Rat Model of Severe Traumatic Brain Injury

In a previous study, we found that administration of ILB^®, a new low molecular weight dextran sulphate, significantly improved mitochondrial functions and energy metabolism, as well as decreased oxidative/nitrosative stress, of brain tissue of rats exposed to severe traumatic brain injury (sTBI), induced by the closed-head weight-drop model of diffused TBI. Using aliquots of deproteinized brain tissue of the same animals of this former study, we here determined the concentrations of 24 amino acids of control rats, untreated sTBI rats (sacrificed at 2 and 7 days post-injury) and sTBI rats receiving a subcutaneous ILB^® administration (at the dose levels of 1, 5 and 15 mg/kg b.w.) 30 min post-impact (sacrificed at 2 and 7 days post-injury). Additionally, in a different set of experiments, new groups of control rats, untreated sTBI rats and ILB^®-treated rats (administered 30 min after sTBI at the dose levels of 1 or 5 mg/kg b.w.) were studied for their neurocognitive functions (anxiety, locomotor capacities, short- and long-term memory) at 7 days after the induction of sTBI. Compared to untreated sTBI animals, ILB^® significantly decreased whole brain glutamate (normalizing the glutamate/glutamine ratio), glycine, serine and γ-aminobutyric acid. Furthermore, ILB^® administration restored arginine metabolism (preventing nitrosative stress), levels of amino acids involved in methylation reactions (methionine, L-cystathionine, S-adenosylhomocysteine), and N-acetylaspartate homeostasis. The macroscopic evidences of the beneficial effects on brain metabolism induced by ILB^® were the relevant improvement in neurocognitive functions of the group of animals treated with ILB^® 5 mg/kg b.w., compared to the marked cognitive decline measured in untreated sTBI animals. These results demonstrate that ILB^® administration 30 min after sTBI prevents glutamate excitotoxicity and normalizes levels of amino acids involved in crucial brain metabolic functions. The ameliorations of amino acid metabolism, mitochondrial functions and energy metabolism in ILB^®-treated rats exposed to sTBI produced significant improvement in neurocognitive functions, reinforcing the concept that ILB^® is a new effective therapeutic tool for the treatment of sTBI, worth being tested in the clinical setting.     

Influence of the Type of Reducing Agent (H2, CO, C3H6 and C3H8) on the Reduction of Stored NOX in a Pt/BaO/Al2O3 Model Catalyst

In this investigation, a comparative study for a NO _X storage catalytic system was performed focusing on the parameters that affect the reduction by using different reductants (H₂, CO, C₃H₆ and C₃H₈) and different temperatures (350, 250 and 150 °C), for a Pt/BaO/Al₂O₃ catalyst. Transient experiments show that H₂ and CO are highly efficient reductants compared to C₃H₆ which is somewhat less efficient. H₂ shows a significant reduction effect at relatively low temperature (150 °C) but with a low storage capacity. We find that C₃H₈does not show any NO _X reduction ability for NO _X stored in Pt/BaO/Al₂O₃ at any of the temperatures. The formation of ammonia and nitrous oxide is also discussed.