Use of Callistemon citrinus as a gastroprotective and anti-inflammatory agent on indomethacin-induced gastric ulcers in obese rats.

Background: Obesity leads to an elevated risk of developing gastrointestinal disease such as gastric ulcers. Callistemon citrinus leaf extract has shown antioxidant, antimicrobial, hepatoprotective, and chemoprotective effects against colon cancer. The aim of this study is to evaluate the gastroprotective effect of C. citrinus leaf extract on indomethacin-induced gastric ulcers in obese rats. Methods: Gastric ulcers were induced in female obese Wistar rats using a single oral dose of indomethacin (IND). In the first stage, the rats were fed with a high fat sugar diet (HFSD) for 15 weeks to induce obesity and, at the same time, the diet of the other group of animals included daily administration of ethanolic C. citrinus leaf extract (250 mg/kg) in addition to HFSD. In the second stage, gastric ulcers were induced with IND (30 mg/kg). The gastroprotective activity of C. citrinus, the inflammatory enzyme activities, and cytokines in the stomach were determined. Results: C. citrinus produced a reduction of gastric lesions caused by IND. Myeloperoxidase (MPO), cyclooxygenase-2 (COX-2), and 5-lipoxygenase (5-LOX) activities also decreased. Although inflammatory biomarkers such as TNFα, IL-6, AOPP, and leptin were significantly decreased by C. citrinus, adiponectin levels increased. Moreover, C. citrinus decreased weight gain and morphological and biochemical parameters. Conclusion: The use of indomethacin in rats fed with a high fat-sugar diet increased gastric ulcers. Gastroprotective effect of C. citrinus in obese rats is attributed to the reduction of pro-inflammatory cytokines and the inflammatory enzymes.

Early Life Stress Negatively Impacts Spatial Learning Acquisition and Increases Hippocampal CA1 Microglial Activation After a Mild Traumatic Brain Injury in Adult Male Rats.

Early life stress (ELS) affects neurogenesis and spatial learning, and increases neuroinflammation after a pediatric mild traumatic brain injury (mTBI). Previous studies have shown that ELS has minimal effects in juveniles but shows age-dependent effects in adults. Hence, we aimed to evaluate the effects of ELS in adult male rats after an mTBI. Maternal separation for 180 min per day (MS180) during the first 21 post-natal (P) days was used as the ELS model. At P110, the rats were subjected to a mild controlled cortical impact injury (2.6 mm) or sham surgery. Spatial learning was evaluated in the Morris water maze (MWM) 14 days after surgery and both microglial activation and neurogenesis were quantified. The results indicate that MS180 + mTBI, but not control (CONT) + mTBI, rats show deficiencies in the acquisition of spatial learning. mTBI led to comparable increases in microglial activation in both the hilus and cortical regions for both groups. However, MS180 + mTBI rats exhibited a greater increase in microglial activation in the ipsilateral CA1 hippocampus subfield compared with CONT + mTBI. Interestingly, for the contralateral CA1 region, this effect was observed exclusively in MS180 + mTBI. ELS and mTBI independently caused a decrease in hippocampal neurogenesis and this effect was not increased further in MS180 + mTBI rats. The findings demonstrate that ELS and mTBI synergistically affect cognitive performance and neuroinflammation, thus supporting the hypothesis that increased inflammation resulting from the combination of ELS and mTBI could underlie the observed effects on learning.

Incubation in shaded hatcheries biases sex-determination but preserves Lepidochelys olivacea hatchling physiology.

Some studies have associated ex situ conservation with cerebral and gonadal developmental delay, as well as decreased motor performance in Lepidochelys olivacea offspring. Ex situ management is also related to a more mature spleen and a differential leukocyte count in newly emerged Lepidochelys olivacea hatchlings. The physiological relevance of a more mature spleen is unknown in sea turtles, but studies in birds suggest an increased immune response. Because egg relocation to hatcheries is a common conservation practice, it is imperative to know its impact on hatchling physiology. Herein, plasma activity of superoxide dismutase, alkaline phosphatase and the alternative complement pathway, as well as total antioxidant capacity and hydrogen peroxide concentrations were quantified in hatchlings from in situ and ex situ nests under basal conditions at nest emergence. Toll-like receptor 4 (tlr4), heat shock proteins (hsp) 70 and hsp90 expression were quantified in the spleen and liver of the hatchlings. Hepatocyte density and nuclear area were quantified in histological sections of the liver and all turtles were sexed by histological sectioning of the gonads. Total antioxidant capacity and hydrogen peroxide concentrations in plasma were lower in turtles from ex situ nests, while tlr4 and hsp70 mRNA expression was higher in the spleen but not in the liver. Ex situ incubation produced 98% male hatchlings, whereas in situ incubation produced 100% females. There were no other differences in the attributes sampled between hatchlings emerging from ex situ and in situ treatments. The results suggest that ex situ relocated turtles may be less prone to oxidative stress than in situ incubated hatchlings and could have more mature splenic function. Together, the data suggest that ex situ relocation to shaded hatcheries biased sex determination but preserved the general physiological condition of sea turtle hatchlings.

Role of the major terpenes of Callistemon citrinus against the oxidative stress during a hypercaloric diet in rats.

1,8-Cineole, limonene and α-terpineol are the major terpenes present in Callistemon citrinus. This study reports for the first time that terpenes attenuate the oxidative stress in rats fed with high-fat-sucrose diet (HFSD) via antioxidant and anti-inflammatory mechanisms. Thirty-six male Wistar rats were divided into six groups (n = 6). Control (fed standard food, HFSD (fed with 41.7% fat and 16.6% sucrose), HFSD + 1,8-cineole (0.88 mg/kg body weight), limonene (0.43 mg/kg body weight), α-terpineol (0.32 mg/kg body weight) and a mixture of the three terpenes, given daily by gavage for 15 weeks. Morphometric and biochemical parameters were taken. Paraoxonase (PON1), reduced glutathione (GSH), lipid peroxidation products malondialdehyde (MDA) and hydroxyalkenals (HNE), advanced oxidation protein products (AOPP) and pro-inflammatory cytokines were measured in liver homogenates. All terpenes showed a remarkable reduction in weight gain, fat deposition, serum glucose and, triacylglycerol levels. However, terpenes presented different effects on the hepatic cell and the oxidative biomarkers. Conversely, the three terpenes and the mixture showed the same positive effect on the tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), leptin and adiponectin levels. Finally, 1,8-cineole, limonene and α-terpineol demonstrate significant anti-inflammatory effects and differential effects on the oxidative stress, suggesting the importance of these terpenes in Callistemon citrinus activities.

Short communication: Ex-situ conservation in hatcheries is associated with spleen development in Lepidochelys olivacea turtle hatchlings.

Ex-situ conservation in hatcheries is a successful strategy for the recovery of sea turtle populations. However, it alters the ontogenesis of the brain and gonads, as well as body size and locomotor performance at nest emergence. Relocation to hatcheries may alter immune system development, since this depends highly on the nest environment. We hypothesized that ex-situ brooding would negatively associate with immune traits of Lepidochelys olivacea. Splenic cytoarchitecture and leukocyte quantification were used as proxies for the immune configuration. Body size, gonadal sex and sand temperature during incubation were determined. Additionally, the success of nest hatching and emergence was quantified. Linear mixed models of splenic cytoarchitecture, leucocyte proportions and body size, using sex and nest type as explanatory variables, evaluated the effects of ex-situ brooding. Generalized linear mixed models using quasibinomial distributions (log link) analyzed effects on hatching and emergence success. Hatchlings from ex-situ nests were heavier, larger and showed a greater spleen-somatic index. They showed more and better defined splenic periarteriolar lymphoid sheaths, as well as a higher proportion of heterophils but less monocytes. Moreover, ex-situ brooding increased hatching and emergence success. Sand temperatures in hatcheries favored male sex determination, while the opposite occurred for in-situ incubation. Interestingly, the immune configuration and body size were independent of sex but associated with ex-situ conservation. Greater body size promotes early hatchling survival, while better spleen development is related to a greater antibody production and a better immune response to pathogens. Altogether, the results suggest that ex-situ incubation is associated with a better immune configuration and higher survival success.

Early life stress increases vulnerability to the sequelae of pediatric mild traumatic brain injury.

Early life stress (ELS) is a risk factor for many psychopathologies that happen later in life. Although stress can occur in cases of child abuse, studies on non-accidental brain injuries in pediatric populations do not consider the possible increase in vulnerability caused by ELS. Hence, we sought to determine whether ELS increases the effects of pediatric mild traumatic brain injury (mTBI) on cognition, hippocampal inflammation, and plasticity. Male rats were subjected to maternal separation for 180 min per day (MS180) or used as controls (CONT) during the first 21 post-natal (P) days. At P21 the rats were anesthetized with isoflurane and subjected to a mild controlled cortical impact or sham injury. At P32 the rats were injected with the cell proliferation marker bromodeoxyuridine (BrdU, 500 mg/kg), then evaluated for spatial learning and memory in a water maze (P35-40) and sacrificed for quantification of Ki67+, BrdU+ and Iba1+ (P42). Neither MS180 nor mTBI impacted cognitive outcome when provided alone but their combination (MS180 + mTBI) decreased spatial learning and memory relative to Sham controls (p < .01). mTBI increased microglial activation and affected BrdU+ cell survival in the ipsilateral hippocampus without affecting proliferation rates. However, only MS180 + mTBI increased microglial activation in the area adjacent to the injury and the contralateral CA1 hippocampal subfield, and decreased cell proliferation in the ipsilateral neurogenic niche. Overall, the data show that ELS increases the vulnerability to the sequelae of pediatric mTBI and may be mediated by increased neuroinflammation.

Investigation of the Antibacterial Activity and Subacute Toxicity of a Quercus crassifolia Polyphenolic Bark Extract for its Potential Use in Functional Foods.

Oak wood is used in barrels for wine aging. During aging, polyphenols are transferred from the barrels to the liquid. Although the bioactivity of oak polyphenols in wines has been extensively studied, no investigation exists on their toxicological properties, which limits their use as functional safe ingredients for other products. In this work, the chemical composition of a polyphenolic extract of Quercus crassifolia bark (QCBe) was studied by GC-MS. Its antibacterial properties on probiotic and pathogenic bacteria and its subacute-oral toxicity were determined as a way to understand the potential impact from its addition to fermented food as a functional ingredient. QCBe shows a selective inhibition of Escherichia coli compared with Lactobacillus bulgaricus and Streptococcus thermophylus. According to the toxicity evaluation, the subacute no-observed-adverse-effect-level was achieved at 11 mg/kg bw/day, whereas the subacute lowest-observed-adverse-effect-level for kidney damage was at 33 mg/kg bw/day. These results suggest that, given the fact an adverse effect was observed after subacute administration of this extract, further longer term toxicological studies are needed to provide sufficient safety evidence for its use in humans. PRACTICAL APPLICATION: Mexico's yogurt market is growing which creates opportunities for the development of some yogurt products as functional foods. As a first step to evaluate its potential use in yogurt formulation, the antibacterial effect of a Quercus crassifolia polyphenolic extract (QCBe) on probiotic bacteria and its subacute-oral toxicity in rats were studied. A low inhibition on probiotic bacteria growth was observed after QCBe addition to Lactobacillus bulgaricus and Streptococcus thermophylus cultures. Exposure to QCBe for a subacute duration resulted in renal injury in rats at dosages greater than or equal to 33 mg/kg/bw/day. This adverse effect indicates the importance of performing further long-term toxicological assessments prior to the addition of QCBe to a food like yogurt, which is regularly eaten by consumers.

Chemopreventive effect of Callistemon citrinus (Curtis) Skeels against colon cancer induced by 1,2-dimethylhydrazine in rats.

PURPOSE: Callistemon citrinus (Curtis) Skeels is a shrub native of Australia. In spite of containing an important number of bioactive compounds (1,8-cineole, limonene and α-terpineol) recognized as a potential chemotherapeutic agents, it is only used as an ornamental plant in Mexico. This study investigated the chemopreventive effect of C. citrinus leaves extract on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats. METHODS: Twenty-four rats were divided into 3 groups of eight rats. Group 1 served as negative control, groups 2 and 3 were given subcutaneous injections of DMH (65 mg/kg b.w.) twice a week the first 2 weeks, and then one the third week. In addition, group 3 was administrated with leaves extracts (250 mg/kg b.w., orally daily) during the 22 weeks of the experiment. Animals were killed and the presence of colon tumors and aberrant crypt foci (ACF) were scored for number and distribution pattern along the colon. The activity of two-phase II enzymes quinone reductase (QR) and glutathione S-transferase (GST) was determined in the liver and three segments of the colon: proximal, middle and distal. RESULTS: The results show that rats feed with C. citrinus leaves extract significantly reduced the size of tumors, the number of ACF and the crypt multiplicity. Additionally, C. citrinus leaves extract increased or maintained the activity of QR and GST in the different tissues as compared with DHM-treated group (p > 0.05). CONCLUSION: This study demonstrates that Callistemon citrinus extract could have a chemopreventive effect against colon carcinogenesis.

Antioxidant Properties of Polyphenolic Extracts from Quercus Laurina, Quercus Crassifolia, and Quercus Scytophylla Bark.

The objective of this work was to determine the concentration of total phenols, total flavonoids, hydroxycinnamic acids, and proanthocyanidins present in crude extracts of Quercus laurina, Q. crassifolia, and Q. scytophylla bark. They were extracted by ethanol (90%) maceration and hot water. The antioxidant capacity was determined by the ability to capture OH•, O2•−, ROO•, H2O2, NO•, and HClO. The hot water crude extract of Q. crassifolia was chosen to be concentrated and purified due to its higher extraction yield (20.04%), concentration of phenol compounds (747 mg gallic acid equivalent (GAE)/g, 25.4 mg quercetin equivalent (QE)/g, 235 mg ChAE/g, 25.7 mg chlorogenic acid equivalents (ChAE)/g), and antioxidant capacity (expressed as half maximal effective concentration (EC50, µg/mL): OH• = 918, O2•− = 80.5, ROO• = 577, H2O2 = 597, NO• ≥ 4000, HClO = 740). In a second stage, Q. crassifolia extracted with hot water was treated with ethyl acetate, concentrating the phenol compounds (860 mg GAE/g, 43.6 mg QE/g, 362 ChAE/g, 9.4 cyanidin chloride equivalents (CChE)/g) and improving the scavenging capacity (OH• = 467, O2•− = 58.1, ROO• = 716, H2O2 = 22.0, NO• ≥ 4000, HClO = 108). Q. crassifolia had the highest polyphenolic concentration and the better capacity for scavenging reactive species, being a favorable candidate to be considered in the development of new products.

Anterior chamber associated immune deviation used as a neuroprotective strategy in rats with spinal cord injury.

The inflammatory response is probably one of the main destructive events occurring after spinal cord injury (SCI). Its progression depends mostly on the autoimmune response developed against neural constituents. Therefore, modulation or inhibition of this self-reactive reaction could help to reduce tissue destruction. Anterior chamber associated immune deviation (ACAID) is a phenomenon that induces immune-tolerance to antigens injected into the eye´s anterior chamber, provoking the reduction of such immune response. In the light of this notion, induction of ACAID to neural constituents could be used as a potential prophylactic therapy to promote neuroprotection. In order to evaluate this approach, three experiments were performed. In the first one, the capability to induce ACAID of the spinal cord extract (SCE) and the myelin basic protein (MBP) was evaluated. Using the delayed type hypersensibility assay (DTH) we demonstrated that both, SCE and MBP were capable of inducing ACAID. In the second experiment we evaluated the effect of SCE-induced ACAID on neurological and morphological recovery after SCI. In the results, there was a significant improvement of motor recovery, nociceptive hypersensitivity and motoneuron survival in rats with SCE-induced ACAID. Moreover, ACAID also up-regulated the expression of genes encoding for anti-inflammatory cytokines and FoxP3 but down-regulated those for pro-inflamatory cytokines. Finally, in the third experiment, the effect of a more simple and practical strategy was evaluated: MBP-induced ACAID, we also found significant neurological and morphological outcomes. In the present study we demonstrate that the induction of ACAID against neural antigens in rats, promotes neuroprotection after SCI.

Speciation, phenotypic variation and plasticity: what can endocrine disruptors tell us?

Phenotype variability, phenotypic plasticity, and the inheritance of phenotypic traits constitute the fundamental ground of processes such as individuation, individual and species adaptation and ultimately speciation. Even though traditional evolutionary thinking relies on genetic mutations as the main source of intra- and interspecies phenotypic variability, recent studies suggest that the epigenetic modulation of gene transcription and translation, epigenetic memory, and epigenetic inheritance are by far the most frequent reliable sources of transgenerational variability among viable individuals within and across organismal species. Therefore, individuation and speciation should be considered as nonmutational epigenetic phenomena.

EphB4 is developmentally and differentially regulated in blood vessels throughout the forebrain neurogenic niche in the mouse brain: Implications for vascular remodeling.

Neurogenesis is a process influenced by environmental cues that create highly specific functional niches. Recently, the role of blood vessels in the maintenance and functioning of neurogenic niches during development and in adult life has been hallmarked. In addition to their trophic support for the highly demanding neurogenic process, blood vessels regulate neuroblast differentiation and migration and define functional domains. Since neurogenesis along the forebrain neurogenic niche (FNN) is a multistage process, in which neuroblast proliferation, differentiation and migration are spatially restricted to specific locations; we evaluated the structural features of vascular beds that support these processes during critical time points in their development. Additionally, we studied the molecular identity of the endothelial components of vascular beds using the expression of the venous marker EphB4. Our results show that blood vessels along the FNN: 1) are present very early in development; 2) define the borders of the FNN since early developmental stages; 3) experience constant remodeling until achieving their mature structure; 4) show venous features during perinatal developmental times; and 5) down-regulate their EphB4 expression as development proceeds. Collectively, our results describe the formation of the intricate vascular network that may support neurogenesis along the FNN and show that blood vessels along this neurogenic niche are dynamic entities that experience significant structural and molecular remodeling throughout development.

Brain plasticity, signal transduction and epigenesis: a missing link revealed

It has been long thought that the brain reorganizes itself in response to environmental needs. Sensory experiences coded in action potentials are the mean by which information on the surroundings is introduced into neuronal networks. The information approaching the brain in the form of electrochemical codes must then be translated in biochemical, epigenetic and genetic ones. Only until recently we have begun understanding the underpinning of such informational transformations and how this process is expressed as neuronal plastic responses. Central for our comprehension of this matter is the finding that signals transduction cascades can modify gene expression by remodeling the chromatin through epigenetic mechanisms. Hence, chromatin remodeling seems to be the process by which experiences are “imprinted”.

Semaphorin-3A and its receptor neuropilin-1 are predominantly expressed in endothelial cells along the rostral migratory stream of young and adult mice.

In the adult brain, neuroblasts originating in the subventricular zone migrate through the rostral migratory stream to the olfactory bulb. While migrating, neuroblasts undergo progressive differentiation until reaching their final locations and fates. Because molecules involved in migration may also exert differentiating effects on young neurons, the identification of factors that support migration could also shed light on the processes of adult neuroblast differentiation. This is the case for members of the family of semaphorins and of its cognate receptors, the neuropilins. Here, we have evaluated the presence of semaphorin-3A and of its receptor neuropilin-1 along the rostral migratory stream in young and adult mice by using immunocytochemical, histochemical, and in situ hybridization techniques. Our morphological studies show that semaphorin-3A and neuropilin-1 are both mainly expressed on endothelial cells along the rostral migratory stream during postnatal development. Our results suggest that endothelial cells constitute the primary source and target of semaphorin-3A along the rostral migratory stream. Moreover, the present work outlines the potential role of blood vessels on neuroblast migration in the postnatal rostral migratory stream.

Expression of secreted semaphorins and their receptors in specific neuromeres, boundaries, and neuronal groups in the developing mouse and chick brain.

Semaphorins constitute a family of signaling molecules with functions in axon pathfinding and neuronal migration. Neuropilins 1 and 2 have been identified as the ligand-binding component of semaphorin receptors. Both ligands and receptors are expressed in embryonic and adult organs in complementary and sometimes redundant patterns. In the present work, we compared the brain expression patterns of the class III semaphorins 3A, 3C, and 3F and neuropilins 1 and 2 between mouse and chick embryos at early developmental stages. Our studies revealed that expression of semaphorins is restricted in some cases to neuromeric transverse domains, to specific neuromeric boundaries, and to specific neuronal populations. Moreover, our studies also revealed coexpression of neuropilins and one or more semaphorins in some of the different expression sites. Comparison of the expression patterns between mouse and chick embryos showed large similarities, but important differences were also detected.

Diffusible signals and fasciculated growth in reticulospinal axon pathfinding in the hindbrain.

We have addressed the control of longitudinal axon pathfinding in the developing hindbrain, including the caudal projections of reticular and raphe neurons. To test potential sources of guidance signals, we assessed axon outgrowth from embryonic rat hindbrain explants cultured in collagen gels at a distance from explants of midbrain-hindbrain boundary (isthmus), caudal hindbrain, or cervical spinal cord. Our results showed that the isthmus inhibited caudally directed axon outgrowth by 80% relative to controls, whereas rostrally directed axon outgrowth was unaffected. Moreover, caudal hindbrain or cervical spinal cord explants did not inhibit caudal axons. Immunohistochemistry for reticular and raphe neuronal markers indicated that the caudal, but not the rostral projections of these neuronal subpopulations were inhibited by isthmic explants. Companion studies in chick embryos showed that, when the hindbrain was surgically separated from the isthmus, caudal reticulospinal axon projections failed to form and that descending pioneer axons of the medial longitudinal fasciculus (MLF) play an important role in the caudal reticulospinal projection. Taken together, these results suggest that diffusible chemorepellent or nonpermissive signals from the isthmus and substrate-anchored signals on the pioneer MLF axons are involved in the caudal direction of reticulospinal projections and might influence other longitudinal axon projections in the brainstem.