Inhibition of miR-29c ProteInhibition of miR-29c Protects the Brain in a Rat Model cts the Brain in a Rat Model of Prolonged Hypothermic of Prolonged Hypothermic

Circulatory ArrestCirculatory Arrest

Tianxiang Gu MD, PhDTianxiang Gu MD, PhDDepartment of Cardiac SurgeryDepartment of Cardiac Surgery

First Hospital of China Medical UniversityFirst Hospital of China Medical University

DISCLOSURESDISCLOSURES

There is no conflict of interest for all authors.There is no conflict of interest for all authors.

INTRODUCTIONINTRODUCTION Neurological deficit induced by deep hypothermia Neurological deficit induced by deep hypothermia

circulatory arrest (DHCA) remains circulatory arrest (DHCA) remains a major a major complication.complication.

Ischemia-reperfusion injury caused by circulation Ischemia-reperfusion injury caused by circulation arrest contributes mainly to the neurological injury arrest contributes mainly to the neurological injury after DHCA.after DHCA.

Oxidative stress and apoptotic neuronal death Oxidative stress and apoptotic neuronal death signaling pathways are involved in the brain signaling pathways are involved in the brain suffering from CPB and DHCA.suffering from CPB and DHCA.

Peroxisome proliferator-activated receptor-c Peroxisome proliferator-activated receptor-c (PPARc) coactivator-1α (PGC-1α) (PPARc) coactivator-1α (PGC-1α) is enriched in is enriched in brain and is a potent stimulator of mitochondrial brain and is a potent stimulator of mitochondrial biogenesis and respiration. biogenesis and respiration.

As an endogenous regulator of PGC-1As an endogenous regulator of PGC-1αα,, miR-29cmiR-29c plays dramatic roles in the brain development and plays dramatic roles in the brain development and neurite outgrowth.neurite outgrowth.

Journal of Cerebral Blood Flow & Journal of Cerebral Blood Flow & Metabolism (2011)31, 1897-07Metabolism (2011)31, 1897-07

OBJECTIVESOBJECTIVES

We tried to explore the possible neuroprotections We tried to explore the possible neuroprotections of using miRNA-29c as a regulator of PGC-1α in of using miRNA-29c as a regulator of PGC-1α in the brain after DHCA in the current study. the brain after DHCA in the current study.

Animals: Animals: Sprague-Dawley rats;Sprague-Dawley rats;

Lentivirus Vectors for AntagomiR-29cLentivirus Vectors for AntagomiR-29c Chemical modified antisense oligonucleotides of rat miR-29Chemical modified antisense oligonucleotides of rat miR-29

c (antagomiR-29c) lentivirus gene transfer vectors were conc (antagomiR-29c) lentivirus gene transfer vectors were constructed by Genechem (Shanghai, China).structed by Genechem (Shanghai, China).

The recombinant lentivirus vector of antagomiR-29c and thThe recombinant lentivirus vector of antagomiR-29c and the control lentivirus vector without antagomiR-29c were pree control lentivirus vector without antagomiR-29c were prepared and tittered to 1×10pared and tittered to 1×1099 TU/mL (transfection unit). TU/mL (transfection unit).

METHODSMETHODS

AntagomiR-29c transfection in vivoAntagomiR-29c transfection in vivo Lentivirus control vector transfection was performed by Lentivirus control vector transfection was performed by

intracerebroventricular injection;intracerebroventricular injection; Intracerebroventricular injection:Intracerebroventricular injection: a puncture needle was a puncture needle was

implanted into the lateral ventricle, the solutions were implanted into the lateral ventricle, the solutions were injected over 1 minute.injected over 1 minute.

CPB and DHCACPB and DHCA

The CPB circuit The CPB circuit apparatus for rats:apparatus for rats:

a venous reservoir, a venous reservoir,

a peristaltic pump,a peristaltic pump,

a custom-designed oxygenator.a custom-designed oxygenator.

Cannulation: right carotid artery and Cannulation: right carotid artery and jugular vein.jugular vein.

The tail artery was cannulated for blood The tail artery was cannulated for blood pressure monitoring and blood sampling.pressure monitoring and blood sampling.

CPB was conducted at a flow rate of 160 to CPB was conducted at a flow rate of 160 to 180 mL/kg/min and was consecutively 180 mL/kg/min and was consecutively decreased by half during the cooling period.decreased by half during the cooling period.

Circulatory arrest was conducted for 60 Circulatory arrest was conducted for 60 minutes when the pericranial temperature minutes when the pericranial temperature was cooled to 18 . ℃was cooled to 18 . ℃

The rats were re-warmed to a pericranial The rats were re-warmed to a pericranial temperature of 34 and then CPB was ℃temperature of 34 and then CPB was ℃weaned.weaned.

Experimental protocolExperimental protocol

Neurological assessmentNeurological assessment Cognitive functions:Cognitive functions: Modified hole board testModified hole board test. .

Vestibulomotor function:Vestibulomotor function: Beam balance taskBeam balance task. .

The duration it remained on the beam was recorded (maximum The duration it remained on the beam was recorded (maximum = 60s).= 60s).

Histologic Histologic examinationexamination

HHippocampus were stained withippocampus were stained with Hematoxylin-eosin (HE);Hematoxylin-eosin (HE);

Normal neurons and death neurons were counted using a Normal neurons and death neurons were counted using a defined rectangular field area with magnification of 200defined rectangular field area with magnification of 200××..

miR29-cmiR29-c、、 PGC-1αPGC-1α、、 caspase-3 and MDA expcaspase-3 and MDA expressionression

In a parallel series of experiments, rat brains were collecteIn a parallel series of experiments, rat brains were collected 2 hours after the surgical procedure from the four groups d 2 hours after the surgical procedure from the four groups (n=4 per group)(n=4 per group)..

qRT-PCRqRT-PCR: : miR-29cmiR-29c

Western BlotWestern Blot: : PGC-1α and caspase-3PGC-1α and caspase-3

MDA Assay KitMDA Assay Kit: : MDAMDA

RESULTSRESULTS

Mortality and exclusionMortality and exclusion A total of 37 rats were enrolled in the protocol for neurologiA total of 37 rats were enrolled in the protocol for neurologi

cal assessment. Complete data were obtained in the remainical assessment. Complete data were obtained in the remaining 32 rats (n=8, for each of the four groups).ng 32 rats (n=8, for each of the four groups).

Physiologic parametersPhysiologic parameters

Neurological assessmentNeurological assessment

•P<0.01,vs sham group; P<0.01,vs sham group;

•# P<0.05,vs DHCA gro# P<0.05,vs DHCA group.up.

Vestibulomotor function Vestibulomotor function .

* P<0.05,vs sham group; # P<0.05,vs DHCA group.* P<0.05,vs sham group; # P<0.05,vs DHCA group.

Cognitive functionCognitive function

miR-29c expression miR-29c expression in the hippocampusin the hippocampus

*P<0.05, vs DHCA group.*P<0.05, vs DHCA group.

MDA MeasurementMDA Measurement

* P<0.05,vs sham group; # P<0.05,vs DHCA group.* P<0.05,vs sham group; # P<0.05,vs DHCA group.

EExpressionxpression of of PGC-1αPGC-1α

Densitometric quantification of Densitometric quantification of PGC-1α (folds to the sham groPGC-1α (folds to the sham group). * P<0.01, vs sham group; # up). * P<0.01, vs sham group; # P<0.01,vs DHCA group.P<0.01,vs DHCA group.

EExpressionxpression of C of Caspase-3aspase-3

Densitometric quantification of casDensitometric quantification of caspase-3 (folds to the sham group). * pase-3 (folds to the sham group). * P<0.01, vs sham group; # P<0.01,vs P<0.01, vs sham group; # P<0.01,vs DHCA group.DHCA group.

Histological examinationHistological examination

×40

×200

×400

Sham DHCA DHCA+control vector

DHCA+antagomiR-29c

Coronal brain sections of the hippocampus Coronal brain sections of the hippocampus and the cerebellar vermis stained with HEand the cerebellar vermis stained with HE Pathological scoresPathological scores

* P<0.05, vs DHCA group.* P<0.05, vs DHCA group.

DISCUSSIONDISCUSSION

Salient findingsSalient findings Inhibition of miR-29c reduced the level of MDA and apoptoInhibition of miR-29c reduced the level of MDA and apopto

sis and increased the expression of PGC-1sis and increased the expression of PGC-1αα of the hippocam of the hippocampus after DHCA.pus after DHCA.

Pretreatment with antagomiR-29c improved the vestibulomPretreatment with antagomiR-29c improved the vestibulomotor and cognitive functions during the early postoperative otor and cognitive functions during the early postoperative period and attenuated histological injuries of the hippocamperiod and attenuated histological injuries of the hippocampus.pus.

miRNAs miRNAs and neurological injuryand neurological injury miRNAs have become a novel target for cerebral protection miRNAs have become a novel target for cerebral protection

against ischemia in recent days. against ischemia in recent days.

Important roles of miR-134, miR-30a and miR-592 in Important roles of miR-134, miR-30a and miR-592 in pathophysiological process of cerebral ischemia have been pathophysiological process of cerebral ischemia have been identified in different models.identified in different models.

(Neuroscience 2014;277:111-22. Neurochem Res.2014;39: 1279-91. (Neuroscience 2014;277:111-22. Neurochem Res.2014;39: 1279-91.

J Neurosci. 2014; 34:3419-28.)J Neurosci. 2014; 34:3419-28.)

The current data showed that miRNA-29c was a negative The current data showed that miRNA-29c was a negative mediator of cerebral protection against neurological mediator of cerebral protection against neurological injuries of DHCAinjuries of DHCA..

DISCUSSIONDISCUSSION

miR-29c and PGC-1αmiR-29c and PGC-1α

PGC-1α is vital for neuron survival under condition of ischePGC-1α is vital for neuron survival under condition of ischemia or hypoxia through a mitochondrial pathway.mia or hypoxia through a mitochondrial pathway.

The brain protective function of The brain protective function of PGC-1αPGC-1α have been identifie have been identified d iin collective studies.n collective studies.

((Nature. 2008;451:1008-12. Neuropharmacology.2010; 59: 70-6. Neuroscience. Nature. 2008;451:1008-12. Neuropharmacology.2010; 59: 70-6. Neuroscience. 2014; 281: 251-2572014; 281: 251-257..))

The current data indicated that tThe current data indicated that the expression of PGC-1α in he expression of PGC-1α in the hippocampus was markedly enhanced by inhibition of the hippocampus was markedly enhanced by inhibition of miRNA-29c.miRNA-29c.

Mechanism for the observed neuroprotectionMechanism for the observed neuroprotection

CONCLUSIONCONCLUSION Inhibition of miR-29c attenuates neurological injuInhibition of miR-29c attenuates neurological inju

ries induced by prolonged DHCA through a PGC-ries induced by prolonged DHCA through a PGC-1α pathway.1α pathway.

This is the first report indicated that regulation of This is the first report indicated that regulation of miRNAs may be a novel therapeutic strategy for miRNAs may be a novel therapeutic strategy for attenuation of neurological injuries after DHCA.attenuation of neurological injuries after DHCA.

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