A Mouse Model of Hemorrhagic Transformation Induced by Acute Hyperglycemia Combined with Transient Focal Ischemia

Summary

This study presents a protocol for establishing a highly reproducible animal model of hemorrhagic transformation (HT) using middle cerebral artery occlusion/reperfusion (MCAO/R) in C57BL/6 mice with acute hyperglycemia.

Abstract

Hemorrhagic transformation (HT) is a serious complication that can occur as a result of thrombolytic therapy following ischemic stroke (IS), and it poses significant limitations on the clinical application of recombinant tissue plasminogen activator (rt-PA). Unfortunately, there are currently no effective interventions available for HT in clinical practice. Therefore, there is an urgent need for stable and reliable experimental animal models to elucidate the pathogenesis of HT and develop effective intervention strategies. This study presented a protocol for establishing a mouse model of HT induced by acute hyperglycemia combined with transient focal ischemia (tMCAO). Male C57BL/6J mice were injected with 30% glucose to induce hyperglycemia and then subjected to 60 min of tMCAO with reperfusion. The infarct volume, integrity of the blood-brain barrier (BBB), and degree of intracranial hemorrhage were assessed at 24 h after MCAO. The results showed that glucose injection led to transient hyperglycemia (14.3-20.3 mmol/L), which significantly increased both the infarct volume and the incidence of HT. Hematoxylin-eosin (H&E) staining indicated significant hemorrhagic lesions within the infarction zone in hyperglycemic mice. Additionally, hyperglycemic mice exhibited aggravated BBB disruption, as shown by more severe leakage of Evans blue (EB) and FITC-Dextran. In conclusion, acute hyperglycemia reliably and consistently resulted in macroscopic HT in a mouse model of tMCAO. This reproducible model offers a valuable tool for investigating the pathological mechanisms of HT and developing corresponding therapeutic interventions.

Introduction

Cerebral infarction is the primary cause of disability and the second leading cause of death in adults worldwide¹. The acute phase plays a crucial role in the progression of cerebral infarction, serving as a pivotal time point for disease treatment. Early and timely restoration of blood flow in the penumbra area is essential to prevent further brain cell death, with thrombolysis and interventional therapy representing the mainstays for acute cerebral infarction (ACI) treatment. However, hemorrhagic transformation (HT) poses a significant complication following thrombolysis and interventional therapy, occurring in 15%-30% of patients with ischem....

Protocol

The experimental protocol was approved by the Institutional Animal Care and Use Committee of Jianghan University (JHDXLL2024-080) and conducted in accordance with the Experimental Animal Ethical Guidelines issued by the Center for Disease Control of China. Adult male C57BL/6J mice weighing 21-26 g were used in this study. The details of the reagents and equipment used are listed in the Table of Materials.

1. Animal grouping and acute hyperglycemia inducing

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Discussion

The current protocol is designed to create a reliable animal model of hemorrhagic transformation following ischemic stroke, which can replicate the harmful effects of vessel revascularization under hyperglycemic conditions. Among the various risk factors for ischemic stroke, the blood glucose level within 24 h after the onset of stroke is positively correlated with the exacerbation of cerebral injury and increased mortality^3,18. Numerous clinical investigations h.......

Materials

Name	Company	Catalog Number	Comments
2,3,5-Triphenyltetrazolium Chloride (TTC)	Sigma-Aldrich	108380	The dye for TTC staining
24-well culture plate	Corning Incorporated	CLS3527	The vessel for TTC staining
30% glucose injection	Kelun Pharmaceutical	H42021188	Acute hyperglycemia induction
4% paraformaldehyde	Wuhan Servicebio Technology Co., Ltd.	G1101	Tissue fixation
5.0 Polyglycolic acid absorbable suture	Jinhuan Medical Co., Ltd	KCR531	Equipment for surgery
96-well culture plate	Corning Incorporated	CLS3596	EB content measuring
Anesthesia machine	Midmark Corporation	VMR	Anesthesia for animal
Antifade Mounting Medium with DAPI	Beyotime Biotech	P0131	Mount for tissue sections
Automation-tissue-dehydrating  machine	Leica Biosystems	TP1020	Dehydrate tissue
Confocal microscopy	Leica Biosystems	STELLARIS 5	Image acquisition
Diclofenac sodium gel	MaYinglong Pharmaceutical	H10950214	Analgesia for animal
Eosin staining solution	Servicebio Technology	G1001	The dye for H&E staining
Evans Blue	Aladdin	E104208	EB staining
Eye gel	Guangzhou Pharmaceutical	H44023098	Material for surgery
Fitc-dextran	Sigma-Aldrich	60842-46-8	BBB permeability assessing
Fluorescence microscope	Olympus	BX51	Image acquisition
Frozen microtome	Leica Biosystems	CM1900	Use for frozen sections
Glucometer	YuWell	580	Blood glucose measurement
Hematoxylin staining Solution	Servicebio	G1004	The dye for H&E staining
Iodine	Lircon	20020059	Material for surgery
Isoflurane	Rwd Life Science	R510-22-10	Anesthesia for animal
Laser doppler blood flow meter	Moor Instruments	moorVMS	Blood flow monitoring
MCAO Sutures	Rwd Life Science	907-00023-01	Material for surgery
Meloxicam	Boehringer-Ingelheim	J20160020	Analgesia for animal
Microsurgical instrument kit	Rwd Life Science	SP0003-M	Equipment for surgery
Microtome	Thermo Fisher Scientific	HM325	Tissue section production
Microtome blade	Leica Biosystems	819	Tissue section production
Mupirocin ointment	GlaxoSmithKline	H10930064	Anti-infection for animal
Neutral balsam	Absin Bioscience	abs9177	Seal for H&E staining
Paraffin embedding center	Thermo Fisher Scientific	EC 350	Produce paraffin blocks
Pentobarbital sodium	Sigma-Aldrich	P3761	Euthanasia for animal
Phosphate buffered saline	Beyotime Biotech	C0221A	Rinse for tissue section
Scanner	EPSON	V330	Tissue scanning
Shaver	Shenzhen Codos Electrical Appliances Co.,Ltd.	CP-9200	Equipment for surgery
Spectrophotometer	Thermo Fisher Scientific	1510-02362	EB content measuring
Sucrose solution	Shanghai Macklin Biochemical	57-50-1	Dehydration for tissue
Tissue-Tek O.C.T. Compound	Sakura	4583	Tissue embedding medium
Trichloroacetic acid	Sigma-Aldrich	T6399	EB content measuring