Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model

<p>Abstract</p> <p>Background</p> <p>Cardiac arrest, and the associated arrest of blood circulation, immediately leads to permanent brain damage because of the exhaustion of oxygen, glucose and energy resources in the brain. Most hippocampal CA1 neurons die during the first week post the insult. Molecular data concerning the recovery after resuscitation are sparse and limited to the early time period. Expression analysis of marker genes via quantitative real-time RT-PCR enables to follow up the remodeling process. However, proper validation of the applied normalization strategy is a crucial prerequisite for reliable conclusions.</p> <p>Therefore, the present study aimed to determine the expression stability of ten commonly used reference genes (<it>Actb</it>, actin, beta; <it>B2m</it>, beta-2 microglobulin;<it>CypA</it>, cyclophilin A; <it>Gapdh</it>, glyceraldehyde-3-phosphate dehydrogenase; <it>Hprt</it>, hypoxanthine guanine phosphoribosyl transferase; <it>Pgk1</it>, phosphoglycerate kinase 1; <it>Rpl13a</it>, ribosomal protein L13A; <it>Sdha</it>, succinat dehydrogenase complex, subunit a, flavoprotein (Fp); <it>Tbp</it>, TATA box binding protein; <it>Ywhaz</it>, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) in the rat hippocampus four, seven and twenty-one days after cardiac arrest. Moreover, experimental groups treated with the anti-inflammatory and anti-apoptotic drug minocycline have been included in the study as well.</p> <p>Results</p> <p>The microglial marker <it>Mac-1</it>, used as a target gene to validate the experimental model, was found to be upregulated about 10- to 20-fold after cardiac arrest.</p> <p>Expression stability of candidate reference genes was analyzed using geNorm and NormFinder software tools. Several of these genes behave rather stable. <it>CypA </it>and <it>Pgk1 </it>were identified by geNorm as the two most stable genes 4 and 21 days after asphyxial cardiac arrest, <it>CypA </it>and <it>Gapdh </it>at 7 days post treatment. <it>B2m </it>turned out to be the most variable candidate reference gene, being about 2-fold upregulated in the cardiac arrest treatment groups.</p> <p>Conclusion</p> <p>We have validated endogenous control genes for qRT-PCR analysis of gene expression in rat hippocampus after resuscitation from cardiac arrest. For normalization purposes in gene profiling studies a combination of <it>CypA </it>and <it>Pgk1 </it>should be considered 4 and 21 days post injury, whereas <it>CypA </it>and <it>Gapdh </it>is the best combination at 7 days. <it>CypA </it>is most favorable if restriction to a single reference gene for all time points is required.</p

Dysregulation of Rho GTPases in the αPix/Arhgef6 mouse model of X-linked intellectual disability is paralleled by impaired structural and synaptic plasticity and cognitive deficits

Mutations in the ARHGEF6 gene, encoding the guanine nucleotide exchange factor αPIX/Cool-2 for the Rho GTPases Rac1 and Cdc42, cause X-linked intellectual disability (ID) in humans. We show here that αPix/Arhgef6 is primarily expressed in neuropil regions of the hippocampus. To study the role of αPix/Arhgef6 in neuronal development and plasticity and gain insight into the pathogenic mechanisms underlying ID, we generated αPix/Arhgef6-deficient mice. Gross brain structure in these mice appeared to be normal; however, analysis of Golgi-Cox-stained pyramidal neurons revealed an increase in both dendritic length and spine density in the hippocampus, accompanied by an overall loss in spine synapses. Early-phase long-term potentiation was reduced and long-term depression was increased in the CA1 hippocampal area of αPix/Arhgef6-deficient animals. Knockout animals exhibited impaired spatial and complex learning and less behavioral control in mildly stressful situations, suggesting that this model mimics the human ID phenotype. The structural and electrophysiological alterations in the hippocampus were accompanied by a significant reduction in active Rac1 and Cdc42, but not RhoA. In conclusion, we suggest that imbalance in activity of different Rho GTPases may underlie altered neuronal connectivity and impaired synaptic function and cognition in αPix/Arhgef6 knockout mic

Phosphoprotein Associated with Glycosphingolipid-Enriched Microdomains Differentially Modulates Src Kinase Activity in Brain Maturation

Src family kinases (SFK) control multiple processes during brain development and function. We show here that the phosphoprotein associated with glycosphigolipid-enriched microdomains (PAG)/Csk binding protein (Cbp) modulates SFK activity in the brain. The timing and localization of PAG expression overlap with Fyn and Src, both of which we find associated to PAG. We demonstrate in newborn (P1) mice that PAG negatively regulates Src family kinases (SFK). P1 Pag1-/- mouse brains show decreased recruitment of Csk into lipid rafts, reduced phosphorylation of the inhibitory tyrosines within SFKs, and an increase in SFK activity of >/ = 50%. While in brain of P1 mice, PAG and Csk are highly and ubiquitously expressed, little Csk is found in adult brain suggesting altered modes of SFK regulation. In adult brain Pag1-deficiency has no effect upon Csk-distribution or inhibitory tyrosine phosphorylation, but kinase activity is now reduced (−20–30%), pointing to the development of a compensatory mechanism that may involve PSD93. The distribution of the Csk-homologous kinase CHK is not altered. Importantly, since the activities of Fyn and Src are decreased in adult Pag1-/- mice, thus presenting the reversed phenotype of P1, this provides the first in vivo evidence for a Csk-independent positive regulatory function for PAG in the brain

An intrinsic vasopressin system in the olfactory bulb is involved in social recognition

Many peptides, when released as chemical messengers within the brain, have powerful influences on complex behaviours. Most strikingly, vasopressin and oxytocin, once thought of as circulating hormones whose actions were confined to peripheral organs, are now known to be released in the brain where they play fundamentally important roles in social behaviours1. In humans, disruptions of these peptide systems have been linked to several neurobehavioural disorders, including Prader-Willi syndrome, affective disorders, and obsessive-compulsive disorder, and polymorphisms of the vasopressin V1a receptor have been linked to autism2,3. Here we report that the rat olfactory bulb contains a large population of interneurones which express vasopressin, that blocking the actions of vasopressin in the olfactory bulb impairs the social recognition abilities of rats, and that vasopressin agonists and antagonists can modulate the processing of information by olfactory bulb neurones. The findings indicate that social information is processed in part by a vasopressin system intrinsic to the olfactory system

Blunted HPA axis response in lactating, vasopressin-deficient Brattleboro rats

Adaptation to stress is a basic phenomenon in mammalian life that is mandatorily associated with the activity of the hypothalamic–pituitary–adrenal (HPA) axis. An increased resting activity of the HPA axis can be measured during pregnancy and lactation, suggesting that these reproductive states lead to chronic load in females. In this study, we examined the consequences of the congenital lack of vasopressin on the activity of the HPA axis during lactation using vasopressin-deficient Brattleboro rats. Virgin and lactating, homozygous vasopressin-deficient rats were compared with control, heterozygous rats. In control dams compared with virgins, physiological changes similar to those observed in a chronic stress state (thymus involution, adrenal gland hyperplasia, elevation of proopiomelanocortin mRNA levels in the adenohypophysis, and resting plasma corticosterone levels) were observed. In vasopressin-deficient dams, adrenal gland hyperplasia and resting corticosterone level elevations were not observed. Corticotropin-releasing hormone (Crh) mRNA levels in the hypothalamic paraventricular nucleus were elevated in only the control dams, while oxytocin (OT) mRNA levels were higher in vasopressin-deficient virgins and lactation induced a further increase in both the genotypes. Suckling-induced ACTH and corticosterone level elevations were blunted in vasopressin-deficient dams. Anaphylactoid reaction (i.v. egg white) and insulin-induced hypoglycemia stimulated the HPA axis, which were blunted in lactating rats compared with the virgins and in vasopressin-deficient rats compared with the controls without interaction of the two factors. Vasopressin seems to contribute to the physiological changes observed during lactation mimicking a chronic stress state, but its role in acute HPA axis regulation during lactation seems to be similar to that observed in virgins. If vasopressin is congenitally absent, OT, but not the CRH, compensates for the missing vasopressin; however, the functional restitution remains incomplete

Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model-0

different treatment groups together at 4 days (A), 7 days (B) and 21 days (C) after ACA treatment. is the least stable gene (highest M value) at all three time points. The combination of /, /and /are the most stable genes at 4 days, 7 days and 21 days, respectively.<p><b>Copyright information:</b></p><p>Taken from "Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model"</p><p>http://www.biomedcentral.com/1471-2199/9/53</p><p>BMC Molecular Biology 2008;9():53-53.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2430208.</p><p></p

Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model-5

Up the expression of the microglia marker is 9.3 ± 2.34 -fold upregulated four days after the insult in the asphyxial cardiac arrest group (p-value 0.006) and 14.9 ± 4.56 -fold in the ACA with minocycline treatment group (p-value 0.001) (A). Seven days after treatment is 10.3 ± 6.18 -fold (p-value 0.011) and 9.2 ± 4.84 -fold (p-value 0.001) upregulated in the ACA and in the ACA with minocycline treatment groups, respectively (B). The increased expression is detectable 21 days after the insult too (14.3 ± 9.17-fold, p-value 0.024 and 22.2 ± 9.93-fold, p-value 0.006) in the ACA and ACA with minocycline treatment groups, respectively (C). Calculations were done using REST-MCS software.<p><b>Copyright information:</b></p><p>Taken from "Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model"</p><p>http://www.biomedcentral.com/1471-2199/9/53</p><p>BMC Molecular Biology 2008;9():53-53.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2430208.</p><p></p

Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model-2

corresponding standard error were used to create the graph. The control group, i.e. sham operated, was assigned a value of 1.<p><b>Copyright information:</b></p><p>Taken from "Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model"</p><p>http://www.biomedcentral.com/1471-2199/9/53</p><p>BMC Molecular Biology 2008;9():53-53.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2430208.</p><p></p

Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model-3

Gulated due to asphyxial cardiac arrest 4 days (A) and 21 days (C) after treatment. Though not significant, there is a tendency for upregulation 7 days after ACA too (B). Compared to the sham treated group expression is 1.7 ± 0.30 -fold upregulated in the ACA group (p-value 0.038) and 2.0 ± 0.39 -fold upregulated in the ACA with minocycline treatment group (p-value 0.009), respectively four days after the injury. Twenty-one days after treatment is 2.4 ± 0.49 -fold upregulated in the ACA with minocycline treatment group (p-value 0.020) vs. sham operated group. Absolute gene regulation value, standard error and p-value are given as calculated using REST-MCS software.<p><b>Copyright information:</b></p><p>Taken from "Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model"</p><p>http://www.biomedcentral.com/1471-2199/9/53</p><p>BMC Molecular Biology 2008;9():53-53.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2430208.</p><p></p