MicroRNA-423-5p Inhibition is Associated with Reduced Infarct Severity After Permanent Cerebral Ischemia in Rats

Summary

Intracerebroventricular inhibition of microRNA-423-5p was associated with improved neurological scores, smaller infarct volume, reduced microRNA-423-5p abundance, and altered Bax/Bcl-2 protein expression after permanent middle cerebral artery occlusion in rats.

Abstract

Acute cerebral infarction causes irreversible injury in the infarct core and secondary damage in surrounding ischemic tissue, where apoptosis-related signaling may contribute to lesion progression. This study evaluated whether inhibition of microRNA-423-5p is associated with improved early outcomes in a rat model of permanent middle cerebral artery occlusion. Adult male Sprague-Dawley rats were assigned to Sham, permanent middle cerebral artery occlusion, permanent middle cerebral artery occlusion plus negative-control adenovirus, or permanent middle cerebral artery occlusion plus microRNA-423-5p-interfering adenovirus groups. A stereotactic intracerebroventricular injection was performed 72 h before occlusion. Neurological deficits were assessed 24 h after surgery using the Zea Longa score; infarct volume was quantified by 2,3,5-triphenyltetrazolium chloride staining; Bax and Bcl-2 protein expression were evaluated by Western blotting; and microRNA-423-5p expression was measured by quantitative real-time polymerase chain reaction. Bioinformatic prediction was also summarized to contextualize BAX as a potential microRNA-423-5p-associated apoptosis-related gene. microRNA-423-5p inhibition was associated with lower neurological deficit scores, reduced infarct volume, decreased Bax expression, increased Bcl-2 expression, and reduced microRNA-423-5p abundance in ischemic brain tissue. These findings support a preliminary association between inhibition of microRNA-423-5p and reduced injury severity after permanent cerebral ischemia. However, direct target validation and direct apoptosis assays are required to define the underlying mechanism.

Introduction

Cerebrovascular disease includes a broad group of disorders caused by impaired cerebral blood supply, vascular rupture, vascular wall injury, abnormal blood flow, or blood-related pathological changes. Ischemic stroke is the predominant subtype of stroke and remains a major cause of disability and death in China¹. Because ischemic stroke often occurs suddenly and is associated with long-term neurological impairment and recurrence, additional strategies are needed to reduce early brain injury and improve neurological outcomes. During acute cerebral infarction, the ischemic lesion includes an irreversibly injured core and a surrounding region of ....

Protocol

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Jiangxi Zhonghong Boyuan Biotechnology Co., Ltd. (Approval No. 2022071501) and were performed in accordance with the approved animal protocol and institutional guidelines for animal welfare and biosafety.

Animal preparation and experimental grouping

Adult specific-pathogen-free male Sprague-Dawley rats, approximately 8–10 weeks of age and weighing 280–350 g at the time of surgery, were used in this study. Male rats were selected to reduce biological variability associated with estro....

Results

Inhibition of miR-423-5p improves neurological outcomes and reduces cerebral infarct volume in rats subjected to permanent middle cerebral artery occlusion

Neurological function was assessed 24 h after permanent middle cerebral artery occlusion using the Zea Longa scoring system (Figure 1A). Sham-operated rats showed no detectable neurological deficits. Rats in the pMCAO and pMCAO + NC groups exhibited marked neurological impairment. The pMCAO + N.......

Discussion

This study evaluated the association between miR-423-5p inhibition and early ischemic injury in a rat model of permanent middle cerebral artery occlusion. Intracerebroventricular delivery of a miR-423-5p-interfering adenovirus reduced miR-423-5p abundance in ischemic brain tissue, improved neurological deficit scores, and decreased infarct volume 24 h after ischemic injury. The intervention was also associated with lower Bax expression and higher Bcl-2 expression in ischemic hemispheric tissue. Together, these findings s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% SDS-PAGE gel preparation kit	Beijing Solarbio Science & Technology Co., Ltd.	P1203	For preparation of 10% SDS-PAGE gels for Western blotting.
12% SDS-PAGE gel preparation kit	Beijing Solarbio Science & Technology Co., Ltd.	P1224	For preparation of 12% SDS-PAGE gels for Western blotting.
2,3,5-Triphenyltetrazolium chloride staining solution (TTC, 2%)	Beijing Solarbio Science & Technology Co., Ltd.	G3005	For TTC staining of coronal brain sections; prepare/use protected from light; 37 °C for 15–20 min.
75% ethanol	Sinopharm Chemical Reagent Co., Ltd.	10009218	Used for alternating surgical-area disinfection with povidone-iodine.
ABI 7500 Real-Time PCR System	Applied Biosystems, Thermo Fisher Scientific	4351105	Instrument used for quantitative real-time PCR.
Adult male Sprague-Dawley rats, SPF, 280–350 g	Jinan Pengyue Laboratory Animal Breeding Co., Ltd.	SCXK (Lu) 2019-0003	Experimental animals used for the pMCAO model.
Anti-Bax primary antibody	Proteintech Group, Inc.	50599-2-Ig	Western blot; use at 1:1,000 in this protocol.
Anti-Bcl-2 primary antibody	Proteintech Group, Inc.	60178-1-Ig	Western blot; use at 1:1,000 in this protocol.
BCA protein assay kit	Elabscience Biotechnology Inc.	E-BC-K318-M	For determination of total protein concentration before Western blotting.
Brain matrix, rat, coronal, 1 mm	Shenzhen RWD Life Science Co., Ltd.	68711	Used to prepare coronal brain slices; appropriate for rats around 300–600 g.
ChemiDoc MP Imaging System	Bio-Rad Laboratories, Inc.	17001402	Chemiluminescence imaging system for Western blot detection.
Enhanced chemiluminescence substrate	Thermo Fisher Scientific	34580	HRP chemiluminescent substrate for Western blot signal development.
Hamilton microsyringe, 10 µL	Hamilton Company	80300	For stereotactic intracerebroventricular adenovirus injection.
HRP-conjugated goat anti-mouse IgG secondary antibody	Servicebio Technology Co., Ltd.	GB23301	Western blot secondary antibody; use at 1:5,000–1:10,000 according to signal intensity.
HRP-conjugated goat anti-rabbit IgG secondary antibody	Servicebio Technology Co., Ltd.	GB23303	Western blot secondary antibody; use at 1:5,000–1:10,000 according to signal intensity.
Image-Pro Plus software	Media Cybernetics, Inc.	Version 6.0	Used for infarct-area and infarct-volume quantification.
Isoflurane	Shenzhen RWD Life Science Co., Ltd.	R510-22-8	Inhalation anesthetic; 3%–4% induction and 1.5%–2% maintenance in oxygen.
miR-423-5p and U6 quantitative PCR primers	General Biological Systems (Anhui) Co., Ltd.	https://www.generalbiol.com/	Sequence-specific primers used for miR-423-5p and U6 qPCR.
miR-423-5p-interfering adenovirus	Hanheng Biotechnology Co., Ltd.	https://www.hanbio.net/en	Replication-deficient E1/E3-deleted adenoviral vector; U6-driven antisense sequence 5′-AAAAGTCTCGCTCTCTGCCCCTCA-3′; CMV-EGFP reporter; working titer 1.0 × 10^10 PFU/mL.
miRDB database	miRDB	https://mirdb.org/	Used for bioinformatic prediction of candidate miRNA-target associations.
miRNA 1st strand cDNA synthesis kit	Vazyme Biotech Co., Ltd.	MR201-01	For reverse transcription of miRNAs before qPCR.
miRWalk database	miRWalk	http://mirwalk.umm.uni-heidelberg.de/	Used for bioinformatic prediction of miR-423-5p binding to BAX 3′UTR.
Negative-control adenovirus	Hanheng Biotechnology Co., Ltd.	https://www.hanbio.net/en	Replication-deficient adenoviral vector carrying scrambled non-targeting sequence 5′-TTCTCCGAACGTGTCACGT-3′; same backbone/reporter/titer as intervention virus.
Ophthalmic ointment	Alcon Laboratories, Inc.	Systane Lubricant Eye Ointment	Applied after anesthesia induction to prevent corneal drying.
Oxygen supply	Local certified supplier	Medical-grade oxygen	Carrier gas for isoflurane induction and maintenance.
Paraformaldehyde solution, 4%	Beijing Solarbio Science & Technology Co., Ltd.	P1110	For fixation of TTC-stained brain sections.
Phosphate-buffered saline (PBS), 1x, pH 7.2–7.4	Beijing Solarbio Science & Technology Co., Ltd.	P1020	Used for tissue rinsing and TTC solution preparation.
Povidone-iodine disinfectant	Beijing Solarbio Science & Technology Co., Ltd.	P8150	Used for alternating skin/scalp disinfection before stereotactic injection and pMCAO surgery.
Protease inhibitor cocktail	Applygen Technologies Inc.	P1265	Added to RIPA lysis buffer for protein extraction.
PVDF membrane	Millipore, Merck KGaA	IPVH00010	Protein transfer membrane for Western blotting.
Quantity One software	Bio-Rad Laboratories, Inc.	170-8601	Used for Western blot densitometric analysis.
RIPA lysis buffer	Applygen Technologies Inc.	C1053	For extraction of total protein from ischemic hemispheric brain tissue.
Silicone-coated MCAO monofilament suture	Beijing Cinontech Co., Ltd.	2636A4	Used for intraluminal-filament pMCAO; insertion depth approximately 18–20 mm from the carotid bifurcation.
Skim milk powder	Beijing Solarbio Science & Technology Co., Ltd.	D8340	Prepared as 5% skim milk in TBST for Western blot membrane blocking.
Small animal anesthesia machine	Shenzhen RWD Life Science Co., Ltd.	R500IE	For isoflurane anesthesia induction/maintenance in oxygen.
SPSS Statistics software	IBM Corp.	Version 23.0	Used for statistical analysis.
Stereotaxic apparatus, rat	Shenzhen RWD Life Science Co., Ltd.	68001	Used for intracerebroventricular adenovirus injection.
Sterile bone wax	Ethicon, Johnson & Johnson	W31G	Used to seal the burr hole after intracerebroventricular injection.
Surgical microscope	Suzhou Kangjie Medical Inc.	XTS-4A	Used for carotid artery exposure and separation during pMCAO surgery.
Surgical sutures	Ethicon, Johnson & Johnson	W1614	Used for skin/incision closure after stereotactic injection and pMCAO surgery.
SYBR Green qPCR master mix	Vazyme Biotech Co., Ltd.	Q711-02	SYBR Green chemistry used for quantitative real-time PCR.
TargetScan database	TargetScan	https://www.targetscan.org/	Used for bioinformatic prediction of candidate miRNA-target associations.
TBST buffer	Beijing Solarbio Science & Technology Co., Ltd.	P1033	Used for Western blot washing and antibody incubation buffer preparation.
Temperature-controlled heating pad	Shenzhen RWD Life Science Co., Ltd.	69023	Used to maintain body temperature near 37 °C during anesthesia, surgery, and early recovery.
TRIzol-like reagent	CWBIO, Jiangsu CoWin Biotech Co., Ltd.	CW0580S	Used to isolate total RNA, including small RNAs, from ischemic brain tissue.
Tween-20	Beijing Solarbio Science & Technology Co., Ltd.	T8220	Component of TBST for Western blot washing.
β-actin primary antibody	TransGen Biotech Co., Ltd.	HC201-01	Western blot loading control; use at 1:5,000 in this protocol.