Diurnal Variation Has Effect on Differential Gene Expression Analysis in the Hippocampus of the Pilocarpine-Induced Model of Mesial Temporal Lobe Epilepsy

Santos, Evelin Antonieli da Silva; Marques, Thalita Ewellyn Batista Sales; Matos, Heloisa de Carvalho; Leite, João Pereira; García-Cairasco, Norberto; Paçó-Larson, Maria Luísa; Gitaí, Daniel Leite Góes

doi:10.1371/journal.pone.0141121

Cited by 17 publications

(26 citation statements)

References 82 publications

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“…Curiously, although Ppia levels are close between 0h- and 6h-PMI ( Fig 2D ), the decreased Ppia transcript levels in acute group were significant only when compared to 6h-PMI ( Fig 3D ). Previous studies in epilepsy models indicated Ppia as a potential reference gene [ 16 , 49 ]. The fact that the acute group has no difference from its time-matched control group (0h-PMI) further corroborate the use of Ppia as a reference gene for epilepsy, and shows that controls with a longer postmortem interval (6h-PMI) could create false results.…”

Section: Discussionmentioning

confidence: 99%

“…All animal experiments were performed under a protocol approved by the Research Ethics Committee of the Federal University of Alagoas (Permit number: 27/2015) and were consistent with the International guidelines for the ethical use of animals, such as those from the Society for Neuroscience. The research staff monitored the rats’ health throughout the experimental period as described previously [ 16 ]. No animals presented clinical/behavioral signals of pain or unexpected distress used as humane endpoint criteria for euthanasia.…”

Section: Methodsmentioning

confidence: 99%

“…However, despite advances in knowledge of genes implicated in the epileptogenic process, these studies have generated some unexpected findings related to unpredictable variability sources in experimental design. In differential gene expression studies, two types of variation can affect the results: i) biological variation among individuals, such as age, gender, ethnicity, body mass index (BMI), lifestyle, and other individual characteristics [ 10 – 14 ]; ii) technical variation due to sample processing, such as pipetting errors, reverse transcription efficiency, RNA quality and other [ 15 , 16 ]. Several strategies can be adopted to minimize these factors, including the use of numerous biological replicates in matched-pairs design for biological variances and the use of suitable normalizers for technical variations [ 17 – 20 ].…”

Section: Introductionmentioning

confidence: 99%

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Using Postmortem hippocampi tissue can interfere with differential gene expression analysis of the epileptogenic process

et al. 2017

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Neuropathological studies often use autopsy brain tissue as controls to evaluate changes in protein or RNA levels in several diseases. In mesial temporal lobe epilepsy (MTLE), several genes are up or down regulated throughout the epileptogenic and chronic stages of the disease. Given that postmortem changes in several gene transcripts could impact the detection of changes in case-control studies, we evaluated the effect of using autopsy specimens with different postmortem intervals (PMI) on differential gene expression of the Pilocarpine (PILO)induced Status Epilepticus (SE) of MTLE. For this, we selected six genes (Gfap, Ppia, Gad65, Gad67, Npy, and Tnf-α) whose expression patterns in the hippocampus of PILO-injected rats are well known. Initially, we compared hippocampal expression of naïve rats whose hippocampi were harvested immediately after death (0h-PMI) with those harvested at 6h postmortem interval (6h-PMI): Npy and Ppia transcripts increased and Tnf-α transcripts decreased in the 6h-PMI group (p<0.05). We then investigated if these PMI-related changes in gene expression have the potential to adulterate or mask RT-qPCR results obtained with PILO-injected rats euthanized at acute or chronic phases. In the acute group, Npy transcript was significantly higher when compared with 0h-PMI rats, whereas Ppia transcript was lower than 6h-PMI group. When we used epileptic rats (chronic group), the RT-qPCR results showed higher Tnf-α only when compared to 6h-PMI group. In conclusion, our study demonstrates that PMI influences gene transcription and can mask changes in gene transcription seen during epileptogenesis in the PILO-SE model. Thus, to avoid erroneous conclusions, we strongly recommend that researchers account for changes in postmortem gene expression in their experimental design.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Using Postmortem hippocampi tissue can interfere with differential gene expression analysis of the epileptogenic process

et al. 2017

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“…Rats were kept at 22°C in groups of four per cage with free access to food and water, in a 12-h light/dark cycle (lights on at 08:00 am). Animal health was monitored throughout the experimental period as described previously [ 59 ]. No animals presented clinical/behavioral signals of pain or unexpected distress used as humane endpoint criteria for euthanasia.…”

Section: Methodsmentioning

confidence: 99%

Identification of microRNAs with Dysregulated Expression in Status Epilepticus Induced Epileptogenesis

et al. 2016

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The involvement of miRNA in mesial temporal lobe epilepsy (MTLE) pathogenesis has increasingly become a focus of epigenetic studies. Despite advances, the number of known miRNAs with a consistent expression response during epileptogenesis is still small. Addressing this situation requires additional miRNA profiling studies coupled to detailed individual expression analyses. Here, we perform a miRNA microarray analysis of the hippocampus of Wistar rats 24 hours after intra-hippocampal pilocarpine-induced Status Epilepticus (H-PILO SE). We identified 73 miRNAs that undergo significant changes, of which 36 were up-regulated and 37 were down-regulated. To validate, we selected 5 of these (10a-5p, 128a-3p, 196b-5p, 352 and 324-3p) for RT-qPCR analysis. Our results confirmed that miR-352 and 196b-5p levels were significantly higher and miR-128a-3p levels were significantly lower in the hippocampus of H-PILO SE rats. We also evaluated whether the 3 miRNAs show a dysregulated hippocampal expression at three time periods (0h, 24h and chronic phase) after systemic pilocarpine-induced status epilepticus (S-PILO SE). We demonstrate that miR-128a-3p transcripts are significantly reduced at all time points compared to the naïve group. Moreover, miR-196b-5p was significantly higher only at 24h post-SE, while miR-352 transcripts were significantly up-regulated after 24h and in chronic phase (epileptic) rats. Finally, when we compared hippocampi of epileptic and non-epileptic humans, we observed that transcript levels of miRNAs show similar trends to the animal models. In summary, we successfully identified two novel dysregulated miRNAs (196b-5p and 352) and confirmed miR-128a-3p downregulation in SE-induced epileptogenesis. Further functional assays are required to understand the role of these miRNAs in MTLE pathogenesis.

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“…It is also unknown how does circadian gene expression relate to induction of spontaneous seizures and epilepsy. In our previous study, we analyzed the temporal profiling of clock genes mRNA levels in the hippocampus of naïve rats ( 61 ). Here, we address this issue by studying these genes in a post-SE model of mTLE, a prototype showing similar epileptogenic and histopathological processes as human mTLE ( 62 ).…”

Section: Introductionmentioning

confidence: 99%

Rhythms of Core Clock Genes and Spontaneous Locomotor Activity in Post-Status Epilepticus Model of Mesial Temporal Lobe Epilepsy

et al. 2018

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The interaction of Mesial Temporal Lobe Epilepsy (mTLE) with the circadian system control is apparent from an oscillatory pattern of limbic seizures, daytime's effect on seizure onset and the efficacy of antiepileptic drugs. Moreover, seizures per se can interfere with the biological rhythm output, including circadian oscillation of body temperature, locomotor activity, EEG pattern as well as the transcriptome. However, the molecular mechanisms underlying this cross-talk remain unclear. In this study, we systematically evaluated the temporal expression of seven core circadian transcripts (Bmal1, Clock, Cry1, Cry2, Per1, Per2, and Per3) and the spontaneous locomotor activity (SLA) in post-status epilepticus (SE) model of mTLE. Twenty-four hour oscillating SLA remained intact in post-SE groups although the circadian phase and the amount and intensity of activity were changed in early post-SE and epileptic phases. The acrophase of the SLA rhythm was delayed during epileptogenesis, a fragmented 24 h rhythmicity and extended active phase length appeared in the epileptic phase. The temporal expression of circadian transcripts Bmal1, Cry1, Cry2, Per1, Per2, and Per3 was also substantially altered. The oscillatory expression of Bmal1 was maintained in rats imperiled to SE, but with lower amplitude (A = 0.2) and an advanced acrophase in the epileptic phase. The diurnal rhythm of Cry1 and Cry2 was absent in the early post-SE but was recovered in the epileptic phase. Per1 and Per2 rhythmic expression were disrupted in post-SE groups while Per3 presented an arrhythmic profile in the epileptic phase, only. The expression of Clock did not display rhythmic pattern in any condition. These oscillating patterns of core clock genes may contribute to hippocampal 24 h cycling and, consequently to seizure periodicity. Furthermore, by using a pool of samples collected at 6 different Zeitgeber Times (ZT), we found that all clock transcripts were significantly dysregulated after SE induction, except Per3 and Per2. Collectively, altered SLA rhythm in early post-SE and epileptic phases implies a possible role for seizure as a nonphotic cue, which is likely linked to activation of hippocampal–accumbens pathway. On the other hand, altered temporal expression of the clock genes after SE suggests their involvement in the MTLE.

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Diurnal Variation Has Effect on Differential Gene Expression Analysis in the Hippocampus of the Pilocarpine-Induced Model of Mesial Temporal Lobe Epilepsy

Cited by 17 publications

References 82 publications

Using Postmortem hippocampi tissue can interfere with differential gene expression analysis of the epileptogenic process

Using Postmortem hippocampi tissue can interfere with differential gene expression analysis of the epileptogenic process

Identification of microRNAs with Dysregulated Expression in Status Epilepticus Induced Epileptogenesis

Rhythms of Core Clock Genes and Spontaneous Locomotor Activity in Post-Status Epilepticus Model of Mesial Temporal Lobe Epilepsy

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