VOC Profiles of Saliva in Assessment of Halitosis and Submandibular Abscesses Using HS-SPME-GC/MS Technique.

Halitosis and submandibular abscesses are examples of mouth-related diseases with the possible bacterial origin. Salivary volatile organic compounds (VOCs) are potential biomarkers of them, once they can be addressed as metabolites of bacterial activity. Healthy patients (n = 15), subjects with submandibular abscesses located in fascial deep space (n = 10), and subjects with halitosis (n = 5) were enrolled in the study. Saliva samples were subjected to headspace solid-phase microextraction (HS-SPME) and gas chromatography coupled to mass spectrometry (GC/MS) analysis. A total number of 164 VOCs was detected by the developed methodology, 23 specific for halitosis and 41 for abscess. Halitosis' profiles were characterized by a larger number of sulfur compounds, while for abscess they had a higher variety of alcohols, aldehydes, and hydrocarbons-biomarkers of inflammatory processes. Principal components analysis allowed visualization of clusters formed according to the evaluated conditions. Kruskal-Wallis test indicated that 39 VOCs presented differentiated responses between the studied groups, with statistical relevance (p < 0.05). Random forest was applied, and a prediction model based on eight VOCs (2-butanone, methyl thioacetate, 2-methylbutanoic acid, S-methyl pentanethioate, dimethyl tetrasulfide, indolizine, pentadecane, and octadecanal) provided 100% of sensitivity, 82% of specificity, and 91% of balanced accuracy, indicating the specific presence of submandibular abscess.

Assessment of adult neurogenesis in mice.

Adult neurogenesis is a lifelong developmental process that occurs in two discrete regions in the adult mammalian brain: the subgranular zone of the dentate gyrus (DG) and the subventricular zone (SVZ) along the lateral ventricles. Despite immense interest in the therapeutic potential of adult neural stem cells (aNSCs) residing along these two neurogenic regions, molecular and cellular mechanisms regulating this process are not fully defined. Defining the regulatory mechanisms responsible for the genesis of new neurons in the adult brain is integral to understanding the basic biology of aNSCs. The techniques described here provide a basic blueprint to isolate, culture, and perform experiments using aNSCs in vitro as well as providing methods to perform immunohistochemistry on brain sections. Curr. Protoc. Toxicol. 56:12.20.1-12.20.16. © 2013 by John Wiley & Sons, Inc.

Assessment of the effects of virus-mediated limited Oct4 overexpression on the structure of the hippocampus and behavior in mice.

Recently, pluripotency induction or cellular reprogramming by introducing critical transcription factors has been extensively studied, but has been demonstrated only in vitro. Based on reports that Oct4 is critically involved in transforming neural stem cells into pluripotent cells, we used the lentiviral vector to introduce the Oct4 gene into the hippocampal dentate gyrus (DG) of adult mice. We examined whether this manipulation led to cellular or behavioral changes, possibly through processes involving the transformation of NS cells into pluripotent cells. The Oct4 lentivirus-infused group and the green fluorescent protein lentivirus-infused group showed a similar thickness of the DG and a comparable level of synaptophysin expression in the DG. Furthermore, our behavioral analyses did not show any differences between the groups concerning exploratory activity, anxiety, or memory abilities. This first trial for pluripotency induction in vivo, despite negative results, provides implications and information for future studies on in vivo cellular reprogramming.

Assessment of the role of MAP kinase in mediating activity-dependent transcriptional activation of the immediate early gene Arc/Arg3.1 in the dentate gyrus in vivo.

Different physiological and behavioral events activate transcription of Arc/Arg3.1 in neurons in vivo, but the signal transduction pathways that mediate induction in particular situations remain to be defined. Here, we explore the relationships between induction of Arc/Arg3.1 transcription in dentate granule cells in vivo and activation of mitogen-activated protein (MAP) kinase as measured by extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation. We show that ERK1/2 phosphorylation is strongly induced in dentate granule cells within minutes after induction of perforant path long-term potentiation (LTP). Phospho-ERK staining appears in nuclei within minutes after stimulation commences, and ERK phosphorylation returns to control levels within 60 min. Electroconvulsive seizures, which strongly induce prolonged Arc/Arg3.1 transcription in dentate granule cells, induced ERK1/2 phosphorylation in granule cells that returned to control levels within 30 min. Following 30, 60, and 120 min of exploration in a novel complex environment, Arc/Arg3.1 transcription was activated in many more granule cells than stained positively for p-ERK at all time points. Although Arc/Arg3.1 transcription was induced in most pyramidal neurons in CA1 following exploration, very few pyramidal neurons exhibited nuclear p-ERK1/2 staining. Local delivery of U0126 during the induction of perforant path LTP blocked transcriptional activation of Arc/Arg3.1 in a small region near the injection site and blocked Arc/Arg3.1 protein expression over a wider region. Our results indicate that activation of Arc/Arg3.1 transcription in dentate granule cells in vivo is mediated in part by MAP kinase activation, but other signaling pathways also contribute, especially in the case of Arc/Arg3.1 induction in response to experience.

Alterations in hippocampal calcium-binding neurons induced by stress models of depression: a preliminary assessment.

In this study, the neuropathological changes induced by chronic unpredictable stress (CUS) and chronic mild stress (CMS) in calbindin D-28K (CB) and parvalbumin (PV) immunoreactive neurons in the rat hippocampus were demonstrated. We used immunohistochemical techniques to quantify the numerical density and morphological changes of PV immunoreactive and CB immunoreactive neurons in the dentate gyrus (DG) and the CA1 and CA3 regions of the hippocampus. We also assessed cell proliferation (Ki-67) and apoptotic processes (active caspase-3) in the DG. We found a significant decrease (16.6% for CUS and 13.3% for CMS) in the numerical density of granule cells (GC), alterations in the CB immunoreactive cells of the GC in the DG and an impairment of mossy fiber CB immunolabelling in the CA3. These changes were not accompanied by a decrease in Ki-67 labeling or the level of caspase-3 in the DG. These data indicate a stress-induced reduction of calcium binding neuron parameters, which may be related to the behavioral paradigms exhibited in these models.

Temporal assessment of histone H3 phospho-acetylation and casein kinase 2 activation in dentate gyrus from ischemic rats.

Hippocampal dentate gyrus possesses an exceptional capacity of adaptation to ischemic insults. Recently, using a transient global ischemic model in the adult rat, we identified a neuroprotective signalling cascade in the dentate gyrus involving calcium/calmodulin-dependent protein kinase IV (CaMKIV), cyclic AMP response element (CRE)-binding protein (CREB) and brain-derived neurotrophic factor (BDNF), a major regulator of survival. We have shown that intracerebroventricular injections of anti-BDNF and anti-CREB are sufficient to cause substantial tissular damages and apoptotic deaths in late periods (48-72 h) after ischemia. Herein, we provide immunohistochemical and biochemical evidence that antibody-induced impairment of the protective CaMKIV/CREB/BDNF pathway induces an apparent duality of response in the dentate gyrus. The experimental protocol is performed as follows: (a) rats are anesthetized and vertebral arteries are occluded by electrocauterization; (b) on the following day, transient global ischemia is produced by occlusion of carotid arteries for 25 min; (c) finally, rats are infused with the pharmacologic agents into the left cerebral ventricle and then perfusion-fixed at different time points after ischemia for immunohistochemical and immunoblotting analyses. After infusion with anti-CaMKIV, phosphorylation of mitogen-activated protein kinases (MAPK) MKK3, MKK6 and p38 and phospho-acetylation of histone H3 occur at 6 h after ischemia without presence of any caspase-9 activation and cellular injuries. In contrast, infusion of anti-BDNF or anti-CREB surprisingly results in a remarkable stimulation of casein kinase 2 (CK2) and caspase-9 activities at 48-72 h post-insult. This is accompanied by the disappearance of phosphorylation of MKK(3/6) and p38 and phospho-acetylation of histone H3. These results suggest that: (1) activation of a MKK(3/6)/p38/H3 cascade at early periods post-ischemia may be capable of causing a short transient protective effect in the dentate gyrus; (2) CK2 might be implicated in inhibition of activity of molecules such as MKK(3/6), p38 and deacetylases at late periods post-insult, thereby promoting injuries and cell deaths in the dentate cell layer.